Transcript
M626E14.9.Nx.4
Super-resolution Microscope N-SIM INSTRUCTIONS
Introduction Thank you for purchasing a Nikon product. This instruction manual is written for users of the N-SIM function of Nikon NIS-Elements. To ensure correct usage, read this manual carefully before operating this product. • No part of this manual may be reproduced or transmitted in any form without prior written permission from Nikon. • The contents of this manual are subject to change without notice. • Although every effort has been made to ensure the accuracy of this manual, errors or inconsistencies may remain. If you note any points that are unclear or incorrect, please contact your nearest Nikon representative. • Some of the equipment described in this manual may not be included in the set you have purchased. • If you intend to use any other equipment with this product, read the manual for that equipment too. • If this equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. • Sample images used in this manual are for reference only and may differ from the actual images.
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Contents Introduction ............................................................................................................................................. i Chapter 1
Before Using ...................................................................................................................... 1
1.1 Starting and Exiting the N-SIM System ................................................................................... 1 1.1.1
Starting the N-SIM System ....................................................................................... 1
1.1.2
Starting the NIS-Elements AR................................................................................... 3
1.1.3
Exiting the N-SIM System ......................................................................................... 5
1.2 Configuration of the N-SIM Pad Tab Sheet ............................................................................. 6 1.3 Help and Version Display ........................................................................................................ 7 1.4 HASP License ......................................................................................................................... 9 1.5 PC Settings ............................................................................................................................. 9 Chapter 2
Basic Operations ............................................................................................................ 10
2.1 N-SIM Operation Flow........................................................................................................... 10 2.2 Selecting a SIM Mode ........................................................................................................... 12 2.3 Selecting the Diffraction Grating ........................................................................................... 14 2.4 Live Image Acquisition .......................................................................................................... 16 2.4.1
Selecting an Illumination Mode ............................................................................... 16
2.4.2
Starting and Stopping Live Image Acquisition ........................................................ 17
2.5 Setting Image Acquisition Parameters .................................................................................. 18 2.5.1
Laser Power Setting ............................................................................................... 18
2.5.2
Camera Setting ....................................................................................................... 19
2.6 SIM Image Acquisition........................................................................................................... 21 2.6.1
Image Capture Execution ....................................................................................... 21
2.6.2
Image Acquisition on the ND Acquisition Tab Sheet ............................................... 22
2.7 Setting Reconstruction Parameters ...................................................................................... 23 2.8 SIM Image Reconstruction .................................................................................................... 29 Chapter 3
Image Acquisition ........................................................................................................... 30
3.1 Settings for SIM Image Acquisition ....................................................................................... 30 3.2 Live Image Acquisition .......................................................................................................... 32 3.2.1
Selecting an Illumination Mode ............................................................................... 32
3.2.2
Starting and Stopping Live Image Acquisition ........................................................ 33
3.3 SIM Image Acquisition........................................................................................................... 34 3.3.1
Selecting a SIM Mode............................................................................................. 34
3.3.2
Image Capture Execution ....................................................................................... 36
3.3.3
Image Acquisition on the ND Acquisition Tab Sheet ............................................... 37
3.4 Setting Image Acquisition Parameters .................................................................................. 38 3.4.1
OC (Optical Configuration) Settings ....................................................................... 38
3.4.2
Laser Power Setting ............................................................................................... 39
3.4.3
Camera Setting ....................................................................................................... 40
3.5 Acquiring SIM Images for Stack Reconstruction ................................................................... 43 3.5.1
Selecting the 3D-SIM Mode .................................................................................... 43
3.5.2
Z-Stack Setting ....................................................................................................... 44
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Contents
3.5.3
ND Acquisition Setting ............................................................................................ 46
3.5.4
Multi-channel Image Acquisition Setting ................................................................. 47
3.6 Acquiring SIM Images in the Dual Mode............................................................................... 48 3.6.1
Dual Camera Mode Setting .................................................................................... 48
3.6.2
Dual Laser Mode Setting ........................................................................................ 53
3.6.3
SIM Image Acquisition in the Dual Mode ................................................................ 54
3.6.4
Position Alignment of Camera Images ................................................................... 55
3.6.5
Position Shift Correction between Camera Images ................................................ 58
3.7 Grating Focus Adjustment ..................................................................................................... 63 3.7.1
Grating Focus Adjustment Procedure..................................................................... 63
3.7.2
Starting Grating Focus Adjustment ......................................................................... 64
3.7.3
Grating Focus (Selection) Window ......................................................................... 64
3.7.4
Grating Focus Image Acquisition ............................................................................ 66
3.7.5
Grating Focus Adjustment ...................................................................................... 67
3.7.6
Grating Focus Adjustment Result ........................................................................... 68
3.7.7
Grating Focus (Result Application Confirmation) Dialog Box ................................. 71
3.7.8
Saving the Result of Grating Focus as a File ......................................................... 71
3.7.9
Correction Ring Adjustment Navigation .................................................................. 72
3.7.10 Grating Focus Help ................................................................................................. 73 Chapter 4
Image Reconstruction .................................................................................................... 74
4.1 SIM Image Reconstruction Setting ....................................................................................... 74 4.2 Slice Reconstruction ............................................................................................................. 76 4.2.1
Setting Slice Reconstruction Parameters ............................................................... 76
4.2.2
Executing Slice Reconstruction .............................................................................. 83
4.3 Slice Reconstruction Preview ............................................................................................... 88 4.4 Stack Reconstruction ............................................................................................................ 93 4.4.1
Setting Stack Reconstruction Parameters .............................................................. 93
4.4.2
Executing Stack Reconstruction ............................................................................. 96
4.5 Batch Reconstruction ............................................................................................................ 97 4.5.1
Batch Reconstruction Setting ................................................................................. 97
4.5.2
Executing Batch Reconstruction ........................................................................... 102
4.6 FFT Image Display .............................................................................................................. 104
Chapter 5
4.6.1
FFT Image Display Setting ................................................................................... 104
4.6.2
FFT Image Display ............................................................................................... 105
Setting and Status ........................................................................................................ 106
5.1 Option Settings and Status Display..................................................................................... 106 5.2 N-SIM Settings Window ...................................................................................................... 107 5.2.1
Selecting the Diffraction Grating ........................................................................... 110
5.2.2
Laser Display Switchover Setting ......................................................................... 111
5.2.3
Capture Option Display Setting ............................................................................ 114
5.2.4
Param Button Display Setting ............................................................................... 115
5.2.5
Reconstruction Parameter Value Display Setting ................................................. 116
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5.2.6
FFT Image Display Setting ................................................................................... 117
5.3 Equipment Status Display ................................................................................................... 118 5.3.1
Normal-status Display........................................................................................... 118
5.3.2
Error-status Display .............................................................................................. 119
5.3.3
Laser Interlock State ............................................................................................. 120
5.3.4
LU5 Disconnected State ....................................................................................... 121
5.4 Communication Status Display ........................................................................................... 122 5.5 Help and Version Display .................................................................................................... 123
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1
Before Using
The N-SIM software is used as one of the functions of the N-SIM system and the NIS-Elements AR (or C). It is not allowed to be used solely except when used with an Offline license only. This chapter describes how to start and exit the N-SIM system and the NIS-Elements AR, including the structure of the N-SIM software window.
1.1
Starting and Exiting the N-SIM System
This section describes the starting and exiting the N-SIM system.
1.1.1
Starting the N-SIM System
When powering on the N-SIM system, be sure to power on each device in the following order. * When using with an Offline license: This procedure is not required as the software is used solely. Start from 1.1.2, “Starting the NIS-Elements AR.”
1
Turn on the power to the piezo stage controller for the N-SIM illuminator.
2
Turn on the power to the Z stage controller for vertical movement of the specimen.
3
Turn on the power to the motorized stage controller.
4
Turn on the power to the HG precentered fiber illuminator.
5
When using the dia pillar illuminator, turn on the TI-PS100 W/A power supply. * For image acquisition with the N-SIM, it is recommended that the power supply of the dia pillar illuminator be turned off or the lowest voltage be selected in order to prevent the rise of equipment temperature.
6
Turn on the power to the HUB controller A at the back of the microscope.
7
Turn on the power to the laser. * For the system with the LU5 N-SIM 5 Laser Unit, turn on the power to each laser head controller and press the OUTPUT button to ON. * For the system with the LU-NV series laser unit, switch the main power switch on the laser unit rear panel to ON, and then press the laser On/Off button for each laser on the front panel to ON.
8
Turn on the power to the PC and the monitor to start the PC. * When Andor DU-897 is used, the camera is also turned on in conjunction with the PC power supply. * When Hamamatsu ORCA Flash4.0 is used, you need to turn on the power to the camera. * When the REMOTE switch on the laser unit rear panel is switched to ON, the laser unit is also turned on in conjunction with the PC power supply.
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Chapter 1 Before Using
9
Switch the power switch on the laser unit rear panel to ON. This operation is necessary only when the REMOTE switch is switched to OFF and the power to the laser unit is not coupled with the PC power supply.
10
Start the NIS-Elements AR. * Warm-up after the N-SIM system power on To ensure proper performance, warm up the system for at least three hours.
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Chapter 1 Before Using
1.1.2
Starting the NIS-Elements AR
Double click the NIS-Elements AR icon on the desktop.
Figure 1.1-1 NIS-Elements icon
The NIS-Elements title window is displayed. Then, the title window closes and the NIS-Elements AR starts.
Figure 1.1-2 Title window
Then, the Driver selection window is displayed. (The Driver selection window may not be displayed depending on the operating environment.) To use the regular N-SIM system, select [ANDOR with N-SIM] or [Hamamatsu with N-SIM].
Figure 1.1-3 Driver selection window - ANDOR with N-SIM
When using with an Offline license, select [No Grabber].
Figure 1.1-4 Driver selection window - No Grabber
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Chapter 1 Before Using
As the NIS-Elements AR starts, the N-SIM Pad tab sheet is displayed automatically as well.
Figure 1.1-5 Initial NIS-Elements window and the N-SIM Pad tab sheet
However, when the NIS-Elements was exited with the N-SIM Pad tab sheet closed during the previous use, the window is not displayed automatically at the next startup because the NIS-Elements reproduces the status at the previous exiting. To manually display the N-SIM Pad tab sheet, right-click on the gray area (without any dialog box and setting window displayed) to display a menu as shown below. Select [Acquisition Controls] o [N-SIM Pad] in the menu.
Figure 1.1-6 To display the N-SIM Pad tab sheet
* Other display method Select [View] on the menu bar then select [Acquisition Controls] o [N-SIM Pad] to display the N-SIM Pad tab sheet.
Figure 1.1-7 To display the N-SIM Pad tab sheet
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Chapter 1 Before Using
1.1.3
Exiting the N-SIM System
To exit the N-SIM system, be sure to power off each device in the following order. * When using with an Offline license: Just exit the NIS-Elements AR. No other procedures are necessary.
1
Remove the specimen from the stage and clean the tip of the immersion objective.
2
Exit the NIS-Elements AR. When the NIS-Elements closes, the N-SIM Pad tab sheet will also close. A layout of the NIS-Elements AR is saved prior to closing it down.
3
Switch the power switch on the laser unit rear panel to OFF. This operation is not necessary when the REMOTE switch is switched to ON; because, the power to the laser unit is also turned off in conjunction with the PC shutdown.
4
Shut down the PC to turn off the power. * When Andor DU-897 is used, the camera is also turned off in conjunction with the PC shutdown. * When Hamamatsu ORCA Flash4.0 is used, you need to turn off the power to the camera. When the REMOTE switch on the laser unit rear panel is set to ON, the laser unit is turned off in conjunction with the PC shutdown.
5
Turn off the power to the laser. * For the system with the LU5 N-SIM 5 Laser Unit, turn off the power to each laser head controller. * For the system with the LU-NV series laser unit, switch the main power switch on the laser unit rear panel to OFF.
6
Turn off the power to the HUB controller A at the back of the microscope.
7
Turn off the power to the HG precentered fiber illuminator.
8
When using the dia pillar illuminator, turn off the power to the TI-PS 100 W/A power supply.
9
Turn off the power to the motorized stage controller.
10
Turn off the power to the Z stage controller for vertical movement of the specimen.
11
Turn off the power to the piezo stage controller for the N-SIM illuminator.
This is the end of exiting the N-SIM system.
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Chapter 1 Before Using
1.2
Configuration of the N-SIM Pad Tab Sheet
The N-SIM Pad tab sheet can be used to make a series of settings for using the SIM Super-resolution Microscope, including SIM image acquisition settings, laser settings, and SIM image reconstruction settings.
1
2
3
Figure 1.2-1 N-SIM Pad tab sheet
Table 1.2-1 Functional overview of the N-SIM Pad tab sheet Name
Function
1
Acquisition area
Make settings for acquiring a SIM image, including an image acquisition mode and options.
2
Laser Control area
Select a laser to excite or specify laser power settings.
3
Reconstruct area
Make settings of the reconstruction parameters to reconstruct a SIM image and execute the reconstruction process.
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Chapter 1 Before Using
1.3
Help and Version Display
On the N-SIM Pad tab sheet, the N-SIM software’s help and version can be displayed. Click the [?] button on the N-SIM Pad tab sheet to display a pop-up menu.
Figure 1.3-1 N-SIM Pad tab sheet - Help button
To display the N-SIM software’s help, select “Help” from the pop-up menu.
Figure 1.3-2 N-SIM Help window
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Chapter 1 Before Using
To display version information of the N-SIM software, select “Version” from the pop-up menu.
Figure 1.3-3 N-SIM Version Info window
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Chapter 1 Before Using
1.4
HASP License
The N-SIM HASP license is required to start the N-SIM system. Functions available with the N-SIM HASP license are as shown below. Table 1.4-1 Overview of functions available with the HASP license HASP license
SIM image acquisition
SIM image reconstruction
Laser control
Normal image acquisition
N-SIM
Available
Available
Available
Available
N-SIM Offline
Not available
Available
Not available
Available
Normal
Not available
Not available
Not available
Available
1.5
PC Settings
When the PC is in a sleep mode, USB communication with the microscope or a laser unit is disconnected, and the N-SIM system can be no longer used normally. Therefore, do not use the screen saver or power management settings which may allow the PC to enter the sleep mode. Note that as a factory default, the sleep mode is disabled for those settings. Therefore, pay attention to make any change of settings for the screen saver or power management.
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2
Basic Operations
This chapter describes the procedures for basic operations of the N-SIM software according to the N-SIM operation flow.
2.1
N-SIM Operation Flow
The N-SIM operation flow is described below. System start up
Objective selection
2D-SIM
SIM mode selection
TIRF-SIM
3D-SIM Set diffraction grating block
Set diffraction grating block
Set diffraction grating block
Select multi-mode optical fiber. Adjust optical axis.
Select multi-mode optical fiber. Adjust optical axis.
Select single-mode optical fiber. Adjust optical axis.
Grating focus adjustment
Set a specimen on the microscope
Visual observation for imaging position
If an OC (optical configuration) has been registered, switch to the OC for SIM.
Select an illumination mode from the Live option
Start “Live Image”
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Chapter 2 Basic Operations
Parameter configuration with the Live image (Laser power and camera exposure time, etc.)
Select a SIM mode from the Capture option ND Acquisition (Time, Z, lambda) Acquire the SIM image set by [Capture] button or [1 time loop] button on the ND Acquisition tab sheet
Set the reconstruction parameters
Automatic SIM image generation (“Reconstruction”) No
Yes
Acquire image again?
No
Is the SIM image OK? Yes
Save the SIM image and original image (nd2 format recommended)
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Note: Z-feed amount is restricted depending on modes.
Chapter 2 Basic Operations
2.2
Selecting a SIM Mode
Select a SIM mode from the Capture option menu on the N-SIM Pad tab sheet.
Figure 2.2-1 N-SIM Pad tab sheet - Capture option Table 2.2-1 Outline of SIM Mode SIM Mode
2D-SIM
Description Automatically captures 9 images, 3 phases (3 grid shifts) and 3 grid angles, as SIM data. Theoretical XY resolution is same as 3D, but 2D has more contrast. This mode uses the multi-mode optical fiber. As the resolution in the Z direction is same as conventional fluorescence microscopes, this mode is suitable for thin specimens. Automatically captures 15 images, 5 phases (3 grid shifts) and 3 grid angles, as SIM data.
3D-SIM
Theoretical XY resolution is same as 2D, but 3D-SIM can acquire optical sectioning images on the Z-axis. Use the multi-mode optical fiber in this mode. 3D-SIM has two types of reconstruction method, “Slice 3D-SIM” and “Stack 3D-SIM.” Basically, the quality of the reconstructed images is the same. Slice 3D-SIM can acquire Z stack images of any number of layers, which enables live-cell capture. Since Stack 3D-SIM processes volume data collectively, images with less noise than Slice 3D-SIM can be reconstructed. In addition, Stack 3D-SIM is suitable for fixed specimens because it has a long capture time. Slice 3D-SIM
Slice 3D-SIM can acquire a 1-layer SIM image by only capturing 1 layer (15 images) of images in the Z direction. For volume data (Z-Stack), Slice 3D-SIM reconstructs SIM images for each layer in the Z direction.
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SIM Mode
Description Number of Z-Stacks Stack 3D-SIM can reconstruct is 16 to 512 layers. Reconstruction can be possible with layers less than 16, but it may cause deterioration in image quality. Split reconstruction is possible by executing reconstruction twice for layers more than 512, but this may cause a discontinuous seam.
3D-SIM
Stack 3D-SIM
TIRF-SIM
Automatically captures 9 images, 3 phases (3 grid shifts) and 3 grid angles, as SIM data. (This is same as 2D-SIM) Typical TIRF images (non-SIM TIRF images) cannot be acquired. This mode uses the single mode optical fiber.
WideField
Captures normal images, not SIM images. Images illuminated with zeroth-order light from the diffraction grating block can be acquired.
Number of Z-Stacks has restriction depending on the feed pitch. For the appropriate relationship, refer to Tables 3.5-2 and 3.5-3 in 3.5.2, “Z-Stack Settings.” Stack 3D-SIM reconstruction needs to capture an image with at least a few steps of the Z directional offset including the area to be observed.
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2.3
Selecting the Diffraction Grating
After changing the diffraction grating block, it is necessary to change the setting on the N-SIM Pad tab sheet. Click the [Settings] button on the N-SIM Pad tab sheet. The N-SIM Settings dialog box to change the diffraction grating is displayed.
Figure 2.3-1 N-SIM Pad tab sheet - Settings button
Figure 2.3-2
N-SIM Settings window
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Table 2.3-1 Diffraction Grating Types Diffraction Grating
Optical Fiber
Description
100EX V-G
Multi-mode
100x magnification, 405 - 561 nm wavelength supported
100EX V-R
Multi-mode
100x magnification, 405 - 640 nm wavelength supported
60EX V-R
Multi-mode
60x magnification, 405 - 640 nm wavelength supported
TIRF 488
Single mode
100x magnification, TIRF 488 nm wavelength supported
TIRF 561
Single mode
100x magnification, TIRF 561 nm wavelength supported
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Chapter 2 Basic Operations
2.4
Live Image Acquisition
This section describes how to select an illumination mode for Live image acquisition and how to execute Live image acquisition. 2.4.1
Selecting an Illumination Mode
Select an illumination mode from the Live option menu on the N-SIM Pad tab sheet.
Figure 2.4-1 N-SIM Pad tab sheet - Live option Table 2.4-1 Outline of Illumination Modes Illumination Mode
Description
Fixed
The specimen is illuminated by a structured illumination pattern using ±1-st order light. Since the illumination pattern is fixed, fluorescence photobleaching in a stripe pattern may occur with the specimen. Not recommended as the illumination mode for SIM image acquisition. (Normal of the previous version)
Moving
The specimen is illuminated by a structured illumination pattern using ±1-st order light. Since the illumination pattern is moving (phase shift), photobleaching in a stripe pattern with the specimen is avoided. Recommended as the illumination mode for SIM image acquisition, because it gives a real TIRF image in the Live preview. (Sine Wave Drive of the previous version)
WideField
Illuminates using zeroth-order light from the diffraction grating block. This is similar to epi-fluorescence observation illumination and is used to capture the comparative non-SIM image. Since it is not structured illumination, the brightness differs from the time of SIM image acquisition. Use this illumination mode for optical axis adjustment for optical fiber. Use this illumination mode when the performing auto calibration for the objective. The auto calibration cannot be performed in other illumination modes. (Full Illumination of the previous version)
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Chapter 2 Basic Operations
2.4.2
Starting and Stopping Live Image Acquisition
To start Live image acquisition, click the [Live] button on the N-SIM Pad tab sheet.
Figure 2.4-2 N-SIM Pad tab sheet - [Live] start button
To stop Live image acquisition, click the [Live] button on the N-SIM Pad tab sheet during Live image acquisition.
Figure 2.4-3 N-SIM Pad tab sheet - [Live] stop button
The NIS-Elements menu can also be used to start and stop Live image acquisition.
Figure 2.4-4 NIS-Elements Menu - Live image acquisition start and stop
* The image displayed in the Live window is the epi-fluorescence image using the N-SIM illuminator, not SIM image.
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Chapter 2 Basic Operations
2.5
Setting Image Acquisition Parameters
This section describes how to set individual parameters for image acquisition. 2.5.1
Laser Power Setting
Select the laser wavelength to be used on the N-SIM Pad tab sheet.
Figure 2.5-1
N-SIM Pad tab sheet - Laser selection
Adjust the laser power with the slider bar or input box of the selected laser wavelength.
Figure 2.5-2 N-SIM Pad tab sheet - Laser power adjustment
* The N-SIM Settings window can be used to change the settings of each channel of the laser wavelengths shown on the N-SIM Pad tab sheet.
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2.5.2
Camera Setting
When using Andor DU-897 Set the camera parameters on the DU-897 Settings tab sheet. To display the DU-897 Settings tab sheet, select [View] o [Acquisition Controls] o [DU-897 Settings] on the NIS-Elements.
1 2 3
4 5 6
Figure 2.5-3 DU-897 Settings window
Table 2.5-1 Setting Items and Recommended Settings for Andor DU-897 Setting Item
Recommended Settings
1
Format For Live
No Binning (Binning cannot be used in SIM image acquisition.)
2
Format For Capture
No Binning (Binning cannot be used in SIM image acquisition.)
3
Exposure Time
Any can be set. The data acquisition time varies depending on the exposure time.
4
5
6
Readout Mode
2D/ Slice 3D-SIM
For moving specimen, such as a live-cell, select “EM Gain 10MHz at 14-bit.” Although the acquisition time becomes longer, for image acquisition with better S/N ratio, select “EM Gain 1MHz at 16-bit.”
Stack 3D-SIM
Select “Normal 1 MHz at 16-bit” or “EM Gain 1 MHz at 16-bit.”
EM Gain Multiplier
Conversion Gain
Any (less than 300) can be set.
2D/ Slice 3D-SIM
1x (Any can be set.)
Stack 3D-SIM
1x or 2.4x (Higher gain may increase noise.)
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Chapter 2 Basic Operations
When using Hamamatsu ORCA Flash4.0 Set the camera parameters on the Flash4.0 Settings tab sheet. To display the Flash4.0 Settings tab sheet, select [View] o [Acquisition Controls] o [Flash4.0 Settings] on the NIS-Elements.
1 2 3 4
Figure 2.5-4 Flash4.0 Settings window
Table 2.5-2 Setting Items and Recommended Settings for Hamamatsu ORCA Flash4.0 Setting Item
Recommended Settings
1
Format For Live
16-bit No Binning (Binning cannot be used in SIM image acquisition.)
2
Format For Capture
16-bit No Binning (Binning cannot be used in SIM image acquisition.)
3
Exposure Time
Any can be set. The data acquisition time varies depending on the exposure time.
4
Scan Mode
2D/ Slice 3D-SIM Stack 3D-SIM
For moving specimen, such as a live-cell, select “Fast.” Although the acquisition time becomes longer, for image acquisition with better S/N ratio, select “Slow.” Select “Slow.”
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2.6
SIM Image Acquisition
Use the [Capture] button or the ND Acquisition window to acquire SIM images. 2.6.1
Image Capture Execution
Click the [Capture] button on the N-SIM Pad tab sheet to execute SIM image acquisition. The SIM image that is captured is the image of the SIM mode selected in the Capture option menu.
Figure 2.6-1 N-SIM Pad tab sheet - Capture button
The NIS-Elements menu can also be used to execute image capture.
Figure 2.6-2 NIS-Elements menu - Image capture execution
* Only one-frame SIM image is acquired through image capture. To acquire multi-frame images, use the ND Acquisition window.
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2.6.2
Image Acquisition on the ND Acquisition Tab Sheet
Use the ND Acquisition tab sheet to acquire SIM images. To display the ND Acquisition tab sheet, select [View] o [Acquisition Controls] o [ND Acquisition] on the NIS-Elements. The SIM image that is captured is the image of the SIM mode selected in the Capture option menu on the N-SIM Pad tab sheet. To change the SIM mode, change the Capture option selection on the N-SIM Pad tab sheet.
1 3
2
4
5 Figure 2.6-3 ND Acquisition tab sheet Table 2.6-1 Outline of ND Acquisition Setting Control
Description
1
Timelapse setting tab
Use to set Timelapse. Specify items including interval, duration, and loops.
2
Z-Stack setting tab
Use to set Z-Stack. Specify items including the number of Z-Stack, step size, and position.
3
Lambda setting tab
Use to set multi-channels. Set up channels and allocate OC.
4
1 time loop button
Executes the loop of the image acquisition settings for ND Acquisition only once.
5
Run now button
Use to fully execute the image acquisition settings for ND Acquisition.
* For details on ND Acquisition settings, refer to the NIS-Elements AR User’s Guide.
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2.7
Setting Reconstruction Parameters
You can set the reconstruction parameters on the N-SIM Slice Reconstruction window or the N-SIM Stack Reconstruction window. This section describes the N-SIM Slice Reconstruction window. To display the N-SIM Slice Reconstruction window, click the [Param] button for Reconstruct Slice on the N-SIM Pad tab sheet.
Figure 2.7-1 N-SIM Pad tab sheet - [Param] button for Reconstruct Slice
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The N-SIM Slice Reconstruction (parameters setting) window is shown below. 1 8 2 3 9
4
10 5 11 6 12 7 13
Figure 2.7-2 N-SIM Slice Reconstruction (parameters setting) window
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Table 2.7-1 Outline of N-SIM Slice Reconstruction Parameter Setup Control 1
2
3
SIM mode change combo box
Channel setting checkbox & combo box
Reconstruction with good contrast components only checkbox
Description Change the settings of the reconstruction parameters retained for each SIM mode. The settings of the selected SIM mode are shown in the dialog box. Checking the checkbox enables the settings of each channel, and retains the parameters for each laser wavelength. In this case, parameter settings of the laser channel selected with the combo box are shown. Unchecking the checkbox disables the combo box, and the same parameters are used for all the channels. Setting to specify whether to execute the reconstruction processing with only the good contrast components when the illumination contrast is judged “low.” With this checkbox checked, the reconstruction processing is executed with only the signals in the directions with good contrast. This setting is enabled only when [Illumination Modulation Contrast] is set to “Auto.” Otherwise this setting is disabled. Parameter for adjusting the effect of artifacts of reconstructed SIM images and contrast-emphasis. ([Structured illumination Contrast] of the previous version) The adjustable range is “Auto” and 0.01 to 1.00 (0.01 to 5.00 only for 3D-SIM). Clicking the [Auto] button changes this parameter to “Auto.” With the parameter set to “Auto,” the optimal parameters for reconstruction are automatically calculated based on the acquired images. However, the image cannot be normally reconstructed with the parameters automatically derived from these images and may have much noise when the illumination contrast of the acquired images is low. In that case, check the Reconstruction with good contrast components only checkbox. Reconstruction process is executed with only the signals in the directions with good contrast, and you can obtain the reconstructed image with less noise.
4
Illumination Modulation Contrast parameter adjustment control
Changing the parameter changes the intensity ratio of each component caused by structured illumination as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. Entering small value Conventional MTF component
ļ
Entering large value MTF
2D-SIM Super-resolution component Spatial frequency
Spatial frequency
3D-SIM
Spatial frequency
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Spatial frequency
Chapter 2 Basic Operations
Control
Description Parameter for adjusting the noise level of reconstructed SIM images. ([Apodization Filter Parameter] of the previous version) The adjustable range is 0.10 to 5.00. Changing the parameter changes the intensity of high-frequency wave area as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment.
5
High Resolution Noise Suppression parameter adjustment control
Entering small value
ļ
Entering large value MTF
MTF
Spatial frequency
Spatial frequency
Parameter for adjusting the optical sectioning thickness of reconstructed SIM images. ([Width of 3D-SIM Filter] of the previous version) The adjustable range is 0.01 to 0.50. Changing the parameter adjusts the spread of intensity in the Z direction as shown below. The sectioning image of the result is displayed on the screen as rough indication for adjustment.
6
Out of Focus Blur Suppression parameter adjustment control
Entering small value
ļ
Entering large value Z
Z
X,Y
Spread of intensity distribution of luminescent point in real space
26
X,Y
Chapter 2 Basic Operations
Control
Description At the reconstruction process, the K vector information (described below), necessary for reconstruction, is read from the acquired images for each wavelength and is calculated and processed. By checking this checkbox, the most reliable and the least noise K vector value is selected from the K vector information read from each frame and is applied to a set of images for a set of acquired images (nd2) for Timelapse and Z-Stack etc. (ND Optimize of the previous version)
Position where to compose super-resolution components
K vector: Vector from origin to center of super-resolution components Position of conventional component (Origin)
7
K vector optimum value selection checkbox & combo box
A set of K vectors (in three directions for each wavelength) is selected and applied as the Z-Stack data. Corresponding to the ND Acquisition setting, three settings are provided for Timelapse. Select “File” in the selection combo box to enable the file specification combo box for specifying the saved SIM reconstruction image files. The following modes are selectable on the selection combo box: All Frames
Selects a set of the optimum K vectors from all frames and applies it to the reconstructed image.
Each Time-Phase
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each Phase of Time schedule for ND Acquisition and applies it to the reconstructed image.
Each Time-Loop
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each acquisition time (Loop) of Time schedule for ND Acquisition and applies it to the reconstructed image.
File
Reads the K vector of the specified SIM reconstruction image files and applies it to the reconstructed image. The specified files must contain the wavelengths that are used for the acquired SIM image to be reconstructed.
Button for loading the saved user parameters and for saving or clearing the user parameters. Clicking the ź button to select the process changes the name of the button. This button is the Load User Params button at the default. When the user parameters are not saved, the default reconstruction parameters are set. 8
Load User Params button
The selectable processes are as follows: Load User Parameters
Loads the saved user parameters and sets the values to each control of the reconstruction parameters.
Save User Parameters
Saves the values set to each control of the reconstruction parameters as the user parameters.
Clear User Parameters
Clears the saved user parameters and sets the default reconstruction parameters.
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Chapter 2 Basic Operations
Control
Description
9
Reuse button
Acquires reconstruction parameters of specified reconstructed images to set to the control of each parameter.
10
Reconstruct without Registration button
Newly opens the reconstruction window and displays a reconstructed SIM image (all frames) without correcting position shift between channels. The position shift between channels is not corrected even when reconstructing the SIM image acquired in the Dual mode. Newly opens the reconstruction window and displays a reconstructed SIM image (all frames).
11
Reconstruct button
12
Apply button
Applies changed parameters. The present window remains displayed.
13
Close button
Closes the window. When the parameters have been changed, a confirmation window appears asking whether to save the changed value.
Corrects the position shift between channels using the correction parameter saved on the image to reconstruct the SIM image acquired in the Dual mode.
* For the N-SIM Stack Reconstruction parameter setup window, refer to “Stack Reconstruction” in Chapter 4, “Image Reconstruction.”
Clicking the ź button of the user parameters displays the pop-up menu as shown below.
Figure 2.7-3 N-SIM Slice Reconstruction (parameters setting) window - User parameters menu
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Chapter 2 Basic Operations
2.8
SIM Image Reconstruction
For SIM image reconstruction processing, Slice reconstruction, Slice preview, Stack reconstruction, and Batch reconstruction are available. This section describes Slice reconstruction. To execute Slice reconstruction, make an acquired SIM image active, then click the [Reconstruct Slice] button on the N-SIM Pad tab sheet.
Figure 2.8-1 N-SIM Pad tab sheet - Reconstruct Slice button
* If an image other than acquired SIM images is made active, a warning message appears and reconstruction processing is not executed. * For reconstruction processing, the parameters set on the N-SIM Slice Reconstruction (parameters setting) window are applied. * To reconstruct the SIM image acquired in the Dual mode, the position shift between channels is corrected with the correction parameter saved on the image.
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3
Image Acquisition
This chapter describes the N-SIM software operations for SIM image acquisition.
3.1
Settings for SIM Image Acquisition
Use the Acquisition area and Laser Control area on the N-SIM Pad tab sheet to specify the settings for SIM image acquisition.
1 10
2
9
3
8 4 7 6
5
Figure 3.1-1 N-SIM Pad tab sheet Table 3.1-1 Outline of SIM Image Acquisition Setting Control Control
Description
1
Live button
Starts and stops Live image acquisition. Live image acquisition is executed using the mode selected in the Live option menu.
2
Capture button
Executes image capture. Image capture is executed using the mode selected in the Capture option menu.
3
Live acquisition option combo box
Select a Live acquisition option. The selections are “Fixed,” “Moving,” and “WideField.”
4
Capture option combo box
Select a capture option. The selections are 1D-SIM, 2D-SIM, 3D-SIM, TIRF-SIM, and WideField.
5
Laser wavelength selection buttons
Select a laser wavelength.
6
Laser power control
Specify the laser power with the slider bar or the input box. The settable range: 0 to 100%
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Chapter 3 Image Acquisition
Control
Description Fits the state (In or Out) of TuCam-dedicated 2nd DM to the actual setting in TuCam. (This button is hidden for the system without TuCam.)
7
TuCam-dedicated DM state setting button
As given below, the SIM image acquired in either channel is eliminated according to the state of TuCam-dedicated 2nd DM when only one laser is output and the Dual mode is selected: In:
The SIM image acquired in the channel on which a laser is not output is eliminated.
Out: The SIM image acquired in the reflection-side channel (CH1) is eliminated. 8
Camera Registration button
Creates the position shift correction parameter for camera image captured in the Dual mode. (This button is hidden for the system without TuCam.)
9
Z-Stack setting button
Displays the Z-Stack window. Enabled only when 3D-SIM is selected in the Capture option menu.
10
Grating focus button
Displays the Grating Focus (selection) dialog box.
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Chapter 3 Image Acquisition
3.2
Live Image Acquisition
Live image acquisition can be executed by selecting an illumination mode.
3.2.1
Selecting an Illumination Mode
Select an illumination mode from the Live option menu on the N-SIM Pad tab sheet.
Figure 3.2-1 N-SIM Pad tab sheet - Live option Table 3.2-1 Outline of Illumination Modes Illumination Mode
Description
Fixed
The specimen is illuminated by a structured illumination pattern using ±1-st order light. Since the illumination pattern is fixed, fluorescence photobleaching in a stripe pattern may occur with the specimen. Not recommended as the illumination mode for SIM image acquisition. (Normal of the previous version)
Moving
The specimen is illuminated by a structured illumination pattern using ±1-st order light. Since the illumination pattern is moving (phase shift), photobleaching in a stripe pattern with the specimen is avoided. Recommended as the illumination mode for SIM image acquisition because it gives a real TIRF image in the Live preview. (Sine Wave Drive of the previous version)
WideField
Illuminates using zeroth-order light from the diffraction grating block. This is similar to epi-fluorescence observation illumination and is used to capture the comparative non-SIM image. Since it is not structured illumination, the brightness differs from the time of SIM image acquisition. Use this illumination mode for optical axis adjustment for optical fiber. Use this illumination mode when the performing auto calibration for the objective. The auto calibration cannot be performed in other illumination modes. (Full Illumination of the previous version)
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Chapter 3 Image Acquisition
3.2.2
Starting and Stopping Live Image Acquisition
To start Live image acquisition, click the [Live] button on the N-SIM Pad tab sheet.
Figure 3.2-2 N-SIM Pad tab sheet - [Live] start button
To stop Live image acquisition, click the [Live] button on the N-SIM Pad tab sheet during Live image acquisition.
Figure 3.2-3 N-SIM Pad tab sheet - [Live] stop button
The NIS-Elements menu can also be used to start and stop Live image acquisition.
Figure 3.2-4 NIS-Elements Menu - Live image acquisition start and stop
* The image displayed in the Live window is the epi-fluorescence image using the N-SIM illuminator, not SIM image.
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Chapter 3 Image Acquisition
3.3
SIM Image Acquisition
SIM image acquisition can be executed by selecting a SIM mode. Use the [Capture] button or the ND Acquisition tab sheet to acquire SIM images.
3.3.1
Selecting a SIM Mode
Select a SIM mode from the Capture option menu on the N-SIM Pad tab sheet.
Figure 3.3-1 N-SIM Pad tab sheet - Capture option
Table 3.3-1 Outline of SIM Mode SIM Mode
2D-SIM
Description Automatically captures 9 images, 3 phases (3 grid shifts) and 3 grid angles, as SIM data. Theoretical XY resolution is same as 3D, but 2D has more contrast. This mode uses the multi-mode optical fiber. As the resolution in the Z direction is same as conventional fluorescence microscopes, this mode is suitable for thin specimens. Automatically captures 15 images, 5 phases (3 grid shifts) and 3 grid angles, as SIM data.
3D-SIM
Theoretical XY resolution is same as 2D, but 3D-SIM can acquire optical sectioning images on the Z-axis. Use the multi-mode optical fiber in this mode. 3D-SIM has two types of reconstruction method, “Slice 3D-SIM” and “Stack 3D-SIM.” Basically, the quality of the reconstructed images is the same. Slice 3D-SIM can acquire Z stack images of any number of layers, which enables live-cell capture. Since Stack 3D-SIM processes volume data collectively, images with less noise than Slice 3D-SIM can be reconstructed. In addition, Stack 3D-SIM is suitable for fixed specimens because it has a long capture time.
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Chapter 3 Image Acquisition
SIM Mode
Description
Slice 3D-SIM
Slice 3D-SIM can acquire a 1-layer SIM image by only capturing 1 layer (15 images) of images in the Z direction. For volume data (Z-Stack), Slice 3D-SIM reconstructs SIM images for each layer in the Z direction.
Stack 3D-SIM
Number of Z-Stacks Stack 3D-SIM can reconstruct is 16 to 512 layers. Reconstruction can be possible with layers less than 16, but it may cause deterioration in image quality. Split reconstruction is possible by executing reconstruction twice for layers more than 512, but this may cause a discontinuous seam. Number of Z-Stacks has restriction depending on the feed pitch. For the appropriate relationship, refer to Tables 3.5-2 and 3.5-3 in 3.5.2, “Z-Stack Settings.” Stack 3D-SIM reconstruction needs to capture an image with at least a few steps of the Z directional offset including the area to be observed.
3D-SIM
TIRF-SIM
Automatically captures 9 images, 3 phases (3 grid shifts) and 3 grid angles, as SIM data. (This is the same as 2D-SIM) Typical TIRF images (non-SIM TIRF images) cannot be acquired. This mode uses the single mode optical fiber.
Fixed
Normal-image acquisition mode in which a structured illumination pattern using ±1-st order light is fixed for illumination. This mode can be selected when the option setting for “Show the Fixed and Moving Capture Option” is enabled.
Moving
Normal-image acquisition mode in which a structured illumination pattern using ±1-st order light is moved for illumination. This mode can be selected when the option setting for “Show the Fixed and Moving Capture Option” is enabled.
WideField
Normal-image acquisition mode in which zeroth-order light from the diffraction grating block is used for illumination.
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Chapter 3 Image Acquisition
3.3.2
Image Capture Execution
Click the [Capture] button on the N-SIM Pad tab sheet to execute SIM image acquisition. The SIM image that is captured is the image of the SIM mode selected in the Capture option menu.
Figure 3.3-2 N-SIM Pad tab sheet - Capture button
The NIS-Elements menu can also be used to execute image capture.
Figure 3.3-3 NIS-Elements menu - Image capture execution
* Only one-frame SIM image is acquired through image capture. To acquire multi-frame images, use the ND Acquisition tab sheet.
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Chapter 3 Image Acquisition
3.3.3
Image Acquisition on the ND Acquisition Tab Sheet
Use the ND Acquisition tab sheet to acquire SIM images. To display the ND Acquisition tab sheet, select [View] o [Acquisition Controls] o [ND Acquisition] on the NIS-Elements. The SIM image that is captured is the image of the SIM mode selected in the Capture option menu on the N-SIM Pad tab sheet. To change the SIM mode, change the Capture option selection on the N-SIM Pad tab sheet.
1 3
2 4
5 Figure 3.3-4 ND Acquisition tab sheet
Table 3.3-2 Outline of ND Acquisition Setting Control
Description
1
Timelapse setting tab
Use to set Timelapse. For each phase, specify items including interval, duration, and loops.
2
Z-Stack setting tab
Use to set Z-Stack. Specify items including the number of Z-Stack, step size, and position.
3
Lambda setting tab
Use to set multi-channels. Set up each channel and allocate OC.
4
1 time loop button
Executes the loop of the image acquisition settings for ND Acquisition only once.
5
Run now button
Use to fully execute the image acquisition settings for ND Acquisition.
* For details on ND Acquisition settings, refer to the NIS-Elements AR User’s Guide.
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Chapter 3 Image Acquisition
3.4
Setting Image Acquisition Parameters
The parameter settings for image acquisition include OC (optical configuration) settings, laser power settings, and camera settings.
3.4.1
OC (Optical Configuration) Settings
By registering currently set SIM image acquisition setting information in an OC (optical configuration), the information can be applied to the N-SIM system merely by calling the OC. To register the currently set SIM image acquisition setting information in an OC, specify a name for the OC on the New Optical Configuration window and then register it. To display the New Optical Configuration window, select [Calibration] o [New Optical Configuration] on the NIS-Elements.
Figure 3.4-1 New Optical Configuration window
The NIS-Elements menu can be used to call an OC.
Figure 3.4-2
NIS-Elements menu - OC call
* For details on OC settings, refer to the NIS-Elements AR User’s Guide.
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Chapter 3 Image Acquisition
3.4.2
Laser Power Setting
Use each control in the Laser Control area on the N-SIM Pad tab sheet to specify the laser power setting. With the [Laser wavelength selection] buttons, select the laser to be used.
Figure 3.4-3
N-SIM Pad tab sheet - Laser selection
Adjust the laser power with the slider bar or input box of the selected laser wavelength.
Figure 3.4-4 N-SIM Pad tab sheet - Laser power adjustment
The N-SIM Settings window can be used to change the settings of each channel of the laser wavelengths shown on the N-SIM Pad tab sheet. If the “Show the Active Laser only” option on the N-SIM Settings window is enabled, the [Laser wavelength selection] button is changed to a combo box as shown below.
Figure 3.4-5 N-SIM Pad tab sheet - Laser control
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Chapter 3 Image Acquisition
3.4.3
Camera Setting
When using Andor DU-897 Set the camera parameters on the DU-897 Settings tab sheet. To display the DU-897 Settings tab sheet, select [View] o [Acquisition Controls] o [DU-897 Settings] on the NIS-Elements.
1 2 4 3
5
6
Figure 3.4-6 DU-897 Settings tab sheet
Table 3.4-1 Setting Items and Recommended Settings for Andor DU-897 Setting Item
Recommended Settings
1
Format For Live
No Binning (Binning cannot be used in SIM image acquisition.)
2
Format For Capture
No Binning (Binning cannot be used in SIM image acquisition.)
3
Exposure Time
Any can be set. The data acquisition time varies depending on the exposure time.
4
5
6
Readout Mode
2D/ Slice 3D-SIM
For moving specimen, such as a live-cell, select “EM Gain 10MHz at 14-bit.” Although the acquisition time becomes longer, for image acquisition with better S/N ratio, select “EM Gain 1MHz at 16-bit.”
Stack 3D-SIM
Select “Normal 1 MHz at 16-bit” or “EM Gain 1 MHz at 16-bit.”
EM Gain Multiplier
Conversion Gain
Any (less than 300) can be set.
2D/ Slice 3D-SIM
1x (Any can be set.)
Stack 3D-SIM
1x or 2.4x (Higher gain may increase noise.)
* For using two cameras, advanced settings are necessary. Refer to 3.6.1, “Setting the Dual Camera Mode.”
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Chapter 3 Image Acquisition
When using Hamamatsu ORCA Flash4.0 Set the camera parameters on the Flash4.0 Settings tab sheet. To display the Flash4.0 Settings tab sheet, select [View] o [Acquisition Controls] o [Flash4.0 Settings] on the NIS-Elements.
1 2
3 4 Figure 3.4-7 Flash4.0 Settings tab sheet Table 3.4-2 Setting Items and Recommended Settings for Hamamatsu ORCA Flash4.0 Setting Item
Recommended Settings
1
Format For Live
16-bit No Binning (Binning cannot be used in SIM image acquisition.)
2
Format For Capture
16-bit No Binning (Binning cannot be used in SIM image acquisition.)
3
Exposure Time
Any can be set. The data acquisition time varies depending on the exposure time.
4
Scan Mode
2D/ Slice 3D-SIM Stack 3D-SIM
For moving specimen, such as a live-cell, select “Fast.” Although the acquisition time becomes longer, for image acquisition with better S/N ratio, select “Slow.” Select “Slow.”
* When using Hamamatsu ORCA Flash4.0, perform the advanced camera settings as shown below. To perform the advanced camera settings, Select [Advanced Setup...] from the Commands menu on the Flash4.0 Settings tab sheet.
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Chapter 3 Image Acquisition
Figure 3.4-8 Flash4.0 Settings tab sheet – Commands menu
Check the checkbox of [Rotate 180°] on the Advanced Setup window.
Figure 3.4-9 Advanced Setup (for Flash4.0) window
* For using two cameras, the different advanced settings are necessary. Refer to 3.6.1, “Setting the Dual Camera Mode.”
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Chapter 3 Image Acquisition
3.5
Acquiring SIM Images for Stack Reconstruction
To acquire SIM images for stack reconstruction, set the Z-Stack information on the ND Acquisition tab sheet from the Z-Stack window. The format of SIM images for stack reconstruction is Z-Stack image in the 3D-SIM mode.
3.5.1
Selecting the 3D-SIM Mode
Select the 3D-SIM mode from the Capture option menu on the N-SIM Pad tab sheet. The [Z-Stack setting] button is shown in yellow.
Figure 3.5-1 N-SIM Pad - With 3D-SIM selected
With the [Z-Stack setting] button shown in yellow on the N-SIM Pad tab sheet, click the button to display the Z-Stack window. * SIM images for stack reconstruction can only be acquired when the NIS-Elements is connected to the MCL Nano-Drive. When the NIS-Elements is not connected to the MCL Nano-Drive, even if the 3D-SIM mode is selected, the [Z-Stack setting] button is disabled and the Z-Stack window cannot be displayed. In this case, connect to the MCL Nano-Drive on the Manage devices window of the NIS-Elements. To display the Manage devices window, select [Devices] then [Manage devices...] on NIS-Elements. * EM Gain 1MHz at 16-bit is recommended for camera readout mode when acquiring SIM images for stack reconstruction. * When combined with A1 or N-STORM, the NIS-Elements may not be connected to the MCL Nano-Drive. In this case, connect the MCL Nano-Drive on the Manage devices window of the NIS-Elements. Note that the MCL Nano-Drive, conversely, has to be disconnected for capturing A1 images or N-STORM images.
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Chapter 3 Image Acquisition
3.5.2
Z-Stack Setting
Use the N-SIM Z-Stack window to make settings for acquiring Z-Stack images for stack reconstruction. 1 3 2 4 6 5 Figure 3.5-2 N-SIM Z-Stack window
Table 3.5-1 Outline of N-SIM Z-Stack Window Control Control
Description
1
Magnification display
Shows the objective magnification currently set.
2
Z-Stack pitch size setting combo box
Pitch sizes selectable for the objective magnification currently set are listed in the combo box as selections. Choose a pitch size from the selections.
3
Z-Stack number-of-steps setting input box
Setting a pitch size automatically sets the maximum settable number of steps into the box. If a value larger than the number of steps automatically set is entered and the [Set ND Acquisition] button is clicked, a warning window will appear disabling the setting. The settable range is 7 to 511 (when the pitch size is 0.12 µm).
4
Z-Stack Range setting input box
Sets a Z-Stack range. Setting a range automatically updates the number of steps. Also shows a Z-Stack range suitable for the pitch size and number of steps.
5
Set ND Acquisition setting button
Applies the Z-Stack setting information to each control in the Z-Stack setting tab on the ND Acquisition window.
6
Cancel button
Cancels the Z-Stack settings and closes the window.
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Chapter 3 Image Acquisition
Table 3.5-2 Number of Steps and Pitch Sizes Settable on N-SIM Z-Stack Window (Magnification: 100x) Step Pitch 0.03 µm 0.06 µm 0.12 µm 0.24 µm 0.48 µm 0.96 µm 1.92 µm 3.84 µm 7.68 µm
7
8 to 15
16 to 31
32 to 63
64 to 127
128 to 255
256 to 511
Unsettable
Unsettable
Settable
Settable
Settable
Settable
Settable
Unsettable
Deprecated
Settable
Settable
Settable
Settable
Settable
Deprecated
Deprecated
Settable
Settable
Settable
Settable
Settable
Deprecated
Deprecated
Settable
Settable
Settable
Settable
Unsettable
Deprecated
Deprecated
Settable
Settable
Settable
Unsettable
Unsettable
Deprecated
Deprecated
Settable
Settable
Unsettable
Unsettable
Unsettable
Deprecated
Deprecated
Settable
Unsettable
Unsettable
Unsettable
Unsettable
Deprecated
Deprecated
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
Deprecated
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
Table 3.5-3 Number of Steps and Pitch Sizes settable on N-SIM Z-Stack Window (Magnification: 60x) Step Pitch 0.04 µm 0.08 µm 0.16 µm 0.32 µm 0.64 µm 1.28 µm 2.56 µm 5.12 µm 10.24 µm
7
8 to 15
16 to 31
32 to 63
64 to 127
128 to 255
256 to 511
Unsettable
Unsettable
Settable
Settable
Settable
Settable
Settable
Unsettable
Deprecated
Settable
Settable
Settable
Settable
Settable
Deprecated
Deprecated
Settable
Settable
Settable
Settable
Settable
Deprecated
Deprecated
Settable
Settable
Settable
Settable
Unsettable
Deprecated
Deprecated
Settable
Settable
Settable
Unsettable
Unsettable
Deprecated
Deprecated
Settable
Settable
Unsettable
Unsettable
Unsettable
Deprecated
Deprecated
Settable
Unsettable
Unsettable
Unsettable
Unsettable
Deprecated
Deprecated
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
Deprecated
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
Unsettable
* When a value outside the settable range is entered for the number of steps, a warning message appears and settings are disabled on the ND Acquisition tab sheet.
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Chapter 3 Image Acquisition
3.5.3
ND Acquisition Setting
The Z-Stack setting information specified on the N-SIM Z-Stack window is applied to each control in the Z-Stack setting tab on the ND Acquisition tab sheet.
1 4
2
3
5 6
7
Figure 3.5-3 ND Acquisition tab sheet - Z-Stack setting
Table 3.5-4 Outline of ND Acquisition Setting Control
Description
1
Order of Experiment button
Changes the Z-Stack and Ȝ-Series execution order.
2
Timelapse setting tab
Use to set Timelapse. Specify items including interval, duration, and loops.
3
Lambda setting tab
Use to set multi-channels. Set up each channel and allocate OC.
4
Z-Stack setting tab
Use to set Z-Stack. Specify items including the number of Z-Stack, step size, and position.
5
Z-Stack setting information
Sets a pitch size, number of steps, and Z-Stack range. The settings on the N-SIM Z-Stack window are applied.
6
1 time loop button
Executes the loop of the image acquisition settings for ND Acquisition only once.
7
Run now button
Use to fully execute the image acquisition settings for ND Acquisition.
* If the Z-Stack setting information specified on the N-SIM Z-Stack window is changed on the ND Acquisition window, stack reconstruction of acquired images may fail. When executing stack reconstruction, do not change the Z-Stack setting information on the ND Acquisition window.
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Chapter 3 Image Acquisition
3.5.4
Multi-channel Image Acquisition Setting
For sequentially (meaning the acquisition by channel, not the Dual mode (simultaneous acquisition of two channels)) acquiring Z-Stack images for stack reconstruction with multiple channels, first execute Z-Stack image acquisition for all the layers with one channel, then move to another channel for Z-Stack image acquisition. The [Order of Experiment] button on the ND Acquisition tab sheet can be used to change the execution order setting. Using the [Order of Experiment] button, make the setting so that Lambda (Z series) is selected. Note that Large Image cannot be executed with N-SIM.
Figure 3.5-4 ND Acquisition tab sheet - Execution order change
After changing the execution order, check that the order is “Ȝ to Z” as below.
Figure 3.5-5 ND Acquisition tab sheet - Execution order confirmation
* For details on ND Acquisition settings, refer to the NIS-Elements AR User’s Guide.
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Chapter 3 Image Acquisition
3.6
Acquiring SIM Images in the Dual Mode
To acquire SIM images in the Dual mode (simultaneous acquisition of two channels), the system needs to contain two cameras. Cameras suitable for the Dual mode are Andor DU-897 or Hamamatsu ORCA Flash4.0. In the Dual mode, laser beams of two wavelengths are simultaneously output and each channel of laser enter each camera via the N-SIM 488/561 Dual DM Filter Cube to acquire SIM images.
3.6.1
Dual Camera Mode Setting
To acquire SIM images in the Dual mode, set the NIS-Elements to the Dual camera mode. When using Andor DU-897 Select [Select ANDOR
…] from the Acquire pull-down menu on the NIS-Elements. Set the Dual mode on the Select HW Unit And Camera window.
Figure 3.6-1 Select ANDOR
… menu
Select “Dual DU-897” on the Select HW Unit And Camera window.
Figure 3.6-2 Select HW Unit And Camera window - Dual DU-897
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Chapter 3 Image Acquisition
When “Dual DU-897” is selected, the Dual DU-897 Settings tab sheet appears for the camera setting.
Figure 3.6-3 Dual DU-897 Settings tab sheet
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Chapter 3 Image Acquisition
Select [Advanced Setup...] from the Commands menu on the Dual DU-897 Settings tab sheet to perform the advanced camera settings. • Check that if the serial numbers displayed in [Master Camera] and [Slave Camera] on the Advanced Camera Settings window are correct. • Check the checkbox of [Flip] under [Slave Camera]. • Select “Slave,Master” in [Channel Order].
Figure 3.6-4 Select HW Unit And Camera window - Dual DU-897
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Chapter 3 Image Acquisition
When using Hamamatsu ORCA Flash4.0 Select [Select Hamamatsu
…] from the Acquire pull-down menu on the NIS-Elements. Set the Dual mode on the Select HW Unit And Camera window.
Figure 3.6-5 Select Hamamatsu
… menu
Select “Dual Flash4.0” on the Select HW Unit And Camera window.
Figure 3.6-6 Select HW Unit And Camera window - Dual Flash4.0
When “Dual Flash4.0” is selected, the Dual Flash4.0 Settings tab sheet appears for the camera setting.
Figure 3.6-7 Dual Flash4.0 Settings window
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Chapter 3 Image Acquisition
Select [Advanced Setup...] from the Commands menu on the Dual Flash4.0 Settings tab sheet to perform the advanced camera settings. Check that if the serial numbers displayed in [Master Camera] and [Slave Camera] on the Advanced Settings window are correct. • Check the checkbox of [Rotate 180°] under [Master Camera]. • Check the checkboxes of [Flip] and [Rotate 180°] under [Slave Camera]. • Select “Slave,Master” in [Channel Order].
Figure 3.6-8 Advanced Setup (for Dual Flash4.0) window
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Chapter 3 Image Acquisition
3.6.2
Dual Laser Mode Setting
To acquire SIM images in the Dual mode, the settings in [Laser Control] on the N-SIM Pad tab sheet need to be changed to the Dual Laser mode to output two lasers. To set the Dual Laser mode, select the Show these Dual Lasers radio button under [Channel Setup] on the N-SIM Settings window.
Figure 3.6-9 N-SIM Settings window - Show these Dual Lasers
Two lasers are displayed in [Laser Control] on the N-SIM Pad tab sheet. You can select two lasers. The selected two lasers are simultaneously output at the Live or the Capture operation.
Figure 3.6-10 N-SIM Pad tab sheet - Dual Laser mode
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Chapter 3 Image Acquisition
3.6.3
SIM Image Acquisition in the Dual Mode
The SIM Image acquired in the Dual mode is basically a two-channel image. (Except that the image is acquired as Ȝ-Series with OC.) Each channel set on the N-SIM Pad tab sheet corresponds the channel used for acquiring the SIM image.
3
1 2 Figure 3.6-11 N-SIM Pad tab sheet - Dual Laser mode
1
2
Figure 3.6-12
Acquired SIM image - Dual mode
* When a two-channel SIM image is acquired in the Dual mode by only one channel of laser, the channel in which no laser is output generates the dark image. Therefore, the channel in which no laser is output is automatically eliminated. (The SIM image acquired is not eliminated when a laser is output on both the channels.) The channel to automatically be eliminated is determined according to the state of theTuCam-dedicated DM state setting button (indicated as 3 in Figure 3.6-11). In:
The SIM image acquired in the channel on which a laser is not output is eliminated.
Out: The SIM image acquired in the reflection-side channel (CH1) is eliminated.
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Chapter 3 Image Acquisition
3.6.4
Position Alignment of Camera Images
When using two Hamamatsu ORCA Flash4.0 cameras, you can align the image captured by each camera. To align the camera image, select [Advanced Setup] from the [Commands] pull-down menu on the Dual Flash4.0 Settings tab sheet.
Figure 3.6-13 Dual Flash4.0 Settings tab sheet - Commands menu
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Chapter 3 Image Acquisition
Clicking the [Start] button under [Alignment] on the Advanced Setup window displays the Live window.
Figure 3.6-14
Advanced Setup window
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Chapter 3 Image Acquisition
By clicking [Ÿ] buttons, shift the image captured by the Master camera or the Slave camera to the desired directions while checking the position of each camera image on the Live window. When the camera images are aligned, click the [Stop] button to stop the Live window. Click the [OK] button to close the Advanced Setup window. The amount of image shift of each camera is saved.
Figure 3.6-15
Advanced Setup window - Camera image alignment display
* You can align the camera image on the reconstructed image by using the camera image correction function of the color drift correction tool. For the camera image correction function, refer to the manual for the color drift correction tool. * When using two Andor DU-897 cameras, align the camera images by using the camera image correction function of the color drift correction tool * For details on the Advanced Setup window, refer to the NIS-Elements AR User’s Guide.
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3.6.5
Position Shift Correction between Camera Images
The position shift between camera images can be corrected in the XY direction, rotation, and scale. To correct the position shift between camera images, set multi-color beads as a specimen for the microscope and click the [Camera Reg] button on the N-SIM Pad tab sheet.
Figure 3.6-16 N-SIM Pad tab sheet - Camera Registration button
An image is acquired by two cameras in the WideField mode and the Image Setting window appears.
3 1
4 2 6
5 Figure 3.6-17 Image Setting window Table 3.6-1 Outline of Image Setting Window Control Control
Description
1
Base Image setting radio button
Select a channel to be used as a base by clicking either button.
2
Channel name combo box
The channel name is displayed in the combo box of each channel.
3
Objective setting combo box
The magnification of the currently-set objective is displayed in this combo box.
4
Wavelength display box
The currently-set laser wavelength of each channel is displayed in this box.
5
Registration Camera button
Displays the Registration Point window for specifying correction points.
6
Cancel button
Cancels the position shift correction between camera images and closes the window.
The channel on the long wavelength side (CH2) is selected as default.
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Clicking the [Registration Camera] button on the Image Setting window displays the Registration Point window. 1
5
2
6
3
7
8
4
9
10
Figure 3.6-18 Registration Point window Table 3.6-2 Outline of Registration Point Window (for Position Shift Correction between Camera Images) Control Control
Description
1
Image captured by Camera 1
The WideField-mode image of the Base Image channel is displayed.
2
Image captured by Camera 2
The WideField-mode image of the non-Base Image channel is displayed.
3
Intensity control button
The intensity of each camera image is automatically adjusted by enabling this button.
4
Combo box for changing magnification
Each camera image is displayed at selected magnification. Selectable magnifications are 50%, 100%, 150%, and 200%. The image displayed at magnification of 100% or larger can be scrolled. 5
Correction point registration button
6
Correction point detection button
7
Correction point deletion button
8
Correction point list
Registers the specified correction point to the correction point list. Automatically detects up to ten correction points. This button is enabled when three or more correction points are registered. Deletes the unchecked correction point from the correction point list. The XY coordinate of each registered correction point of each camera image is displayed in the list. The check box of the valid correction point is checked.
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Control
Description
9
Run Registration button
Performs the position shift correction between camera images and displays the corrected image confirmation window.
10
Close button
Cancels the position shift correction between camera images and closes the window.
Make an adjustment so that the beads image is visible by operating the Intensity control button and magnification displayed in the combo box for changing magnification on the Registration Point window. Select the same bead by clicking the bead with the mouse on the images captured by Camera 1 and Camera 2. Click the correction point registration button to register the selected beads as the correction points. Repeat the same procedure two more times and add three points to the correction point list.
Figure 3.6-19 Registration Point window – Correction points selection
Up to 10 correction points are automatically detected by clicking the correction point detection button.
Figure 3.6-20 Registration Point window – Correction points detection
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Change the image display magnification to 50% to have the entire image displayed. The areas at the four corners and at the center of the nine-divided entire image must contain one or more correction point. If there are the areas that do not contain a correction point, add correction points after deleting unnecessary correction points in the other areas. To delete a correction point, uncheck the check box in the correction point list and click the correction point deletion button.
Figure 3.6-21 Registration Point window – Correction points adjustment
When specifying the correction points, click the [Run Registration] button to execute the correction.
Figure 3.6-22 Registration Point window – Correction execution
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When the Registration Image View window appears, check whether the correction has been executed normally while selectively switching the [All], [Camera 1], and [Camera 2] buttons. When the [All] is selected, the images of Camera 1 and Camera 2 are displayed one over the other. When the result of the correction has been confirmed normal, click the [Apply] button.
Figure 3.6-23 Registration Image View window
When the confirmation dialog box for registering correction parameters appears, click the [Yes] button to register the correction parameters.
Figure 3.6-24 Confirmation dialog box for registering correction parameters
The registered correction parameters are saved on the captured SIM image (nd2 file) at SIM image capture and used for position shift correction at Reconstruction.
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3.7
Grating Focus Adjustment
To use the 3D-SIM mode, perform grating focus adjustment if the diffraction grating block has been replaced. Even if the diffraction grating block has not been replaced, perform grating focus adjustment every six months.
3.7.1
1 2
Grating Focus Adjustment Procedure Place a 100-nm fluorescent bead specimen on the microscope, and select the fluorescent bead specimen. Click the Grating focus button on the N-SIM Pad tab sheet. The Grating Focus (selection) dialog box is displayed.
3 4
Following the navigation, adjust the objective correction ring. Click the [Measure Now] button on the Grating Focus (selection) dialog box. A confirmation dialog box for execution of grating focus adjustment is displayed.
5
Click the [Yes] button on the Grating Focus (adjustment confirmation) dialog box. Z-Stack information is automatically set to the ND Acquisition tab sheet, and Z-Stack image acquisition is executed. When Z-Stack image acquisition is completed, the Grating focus Adjustment window is displayed.
6
Specify a ROI on the Grating focus Adjustment window, and click the [Adjustment] button. The Grating Focus Result window is displayed.
7
Check the graph on the Grating Focus Result window. Following the check points for the grating focus result graph, check the result graph.
8
Click the [Apply] button on the Grating Focus Result window. A confirmation window for applying the result of grating focus is displayed.
9
Select the wavelength to be applied, and click the [OK] button. The result of grating focus is saved in the setting file.
* Perform the grating focus for every wavelength.
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3.7.2
Starting Grating Focus Adjustment
To start grating focusing, click the Grating focus button on the N-SIM Pad tab sheet.
Figure 3.7-1 N-SIM Pad tab sheet - Grating focus button
3.7.3
Grating Focus (Selection) Window
You can execute grating focus adjustment and check execution dates on the Grating Focus (selection) window.
1 2 5
3
6
4 Figure 3.7-2 Grating Focus (selection) window Table 3.7-1 Outline of Grating Focus (Selection) Window Control Control
Description
1
Diffraction grating display
Shows the diffraction grating currently set.
2
Grating focus adjustment execution date
Shows grating focus adjustment execution date for each wavelength. When the grating focus result of other wavelength is applied, “*** (adjustment executed wavelength) Apply” is displayed.
3
Measure Now button
Executes grating focus adjustment.
4
Use Current button
Uses the grating focus adjustment value currently set.
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Control
Description
5
Load from File
… button
Loads the specified grating focus adjustment result file.
6
Correction Ring Navi button
Displays the navigation window for supporting the correction ring adjustment.
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3.7.4
Grating Focus Image Acquisition
To acquire grating focus images, first click the [Measure Now] button on the Grating Focus (selection) dialog box. Then, click the [Yes] button on the displayed Grating Focus (adjustment confirmation) dialog box.
Figure 3.7-3 Grating Focus (adjustment confirmation) dialog box
Grating focus images are composed of 5 SIM images (5 phases of 1 grid shift), and Z-Stack processing is executed for 101 layers. When grating focus image acquisition is in progress, the ND Progress window and grating focus images are displayed.
Figure 3.7-4 ND Progress window and grating focus image
When grating focus image acquisition is completed, the Grating focus Adjustment window is displayed.
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3.7.5
Grating Focus Adjustment
Perform grating focus adjustment on the Grating focus Adjustment window which is displayed after grating focus image acquisition.
1
2
4
3
Figure 3.7-5 Grating focus Adjustment window Table 3.7-2 Outline of Grating Focus Adjustment Window Control Control
Description
1
ROI
Specify a ROI for a fluorescent bead. Enclose the whole fluorescent bead including the blurry light. Do not include the light from other fluorescent beads in the field of view in the ROI.
2
Recommend Size checkbox
Specify whether to lock the ROI to the recommended size. With the checkbox checked, the ROI size is locked to the recommended value, so that the size cannot be changed. With the checkbox unchecked, the ROI size can be changed freely.
3
Z-Stack position specification slider
Slider for changing the Z-Stack position. Specify a position (frame 90 to 100) at which a fluorescent bead is defocused.
4
Adjustment button
Executes the adjustment. Auto fitting processing to PSF is executed.
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3.7.6
Grating Focus Adjustment Result
The result of grating focus adjustment is displayed as a grating focus result graph.
3 1 5
4
6
2
9
7
10
8
Figure 3.7-6 Grating Focus Result window Table 3.7-3 Outline of Grating Focus Result Window Control
Description
1
Ordinate
Relative intensity
2
Abscissa
Z step
3
Grating focus result
Result of grating focus, calculated with noise levels of acquired images. Appropriateness of grating focus results is judged with the value of [Measurement Precision].
4
Straight line of Original
Original diffraction grating position in the space of the specimen
5
Straight line of Target
Target position for overlaying the conjugate plane of the diffraction grating on the specimen
6
Graph curve
Z profile of a fluorescent bead specified with the ROI. Appropriateness of grating focus results is judged with the lateral symmetry of this curve and peak position.
7
Wavelength-to-apply and grating positions confirmation display
The laser wavelength used for executing grating focus adjustment and the grating positions of the previous adjustment and new adjustment result are displayed.
8
Help button
Displays Help text which describes how to read grating focus adjustment result graphs.
9
Save button
Saves the grating focus result in a file (gfr file).
10
Apply button
Displays the Grating Focus (result application confirmation) dialog box.
* Clicking the [OK] button on the Grating Focus (result application confirmation) dialog box applies the grating focus adjustment results and saves the result information in the setting file as diffraction grating block position information. The grating block position information is retained in the PC even after the power to the equipment is turned off.
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Items to be checked to judge the appropriateness of grating focus adjustment result graphs are described below: [Check Point 1] Right-left symmetry of graph curve (Z profile of fluorescent bead specimen) Table 3.7-4 Grating Focus Adjustment Result Check Point 1 State
Judgment
Cause and Countermeasure
Normally distributed symmetric shape
OK
Ყ
Asymmetric
NG
The setting of the correction ring may be wrong. Adjust the correction ring again, and execute grating focus adjustment again.
NG
Saturation may have occurred. Switch the illumination mode to Moving and check the Live image, then adjust the amount of light intensity to a degree where the maximum value of the Histogram window does not exceed 4000, and execute grating focus adjustment again.
NG
The ROI specified on the Grating focus Adjustment window may not be an isolated bead, but multiple gathered beads. Specify an isolated bead with the ROI, and execute grating focus adjustment again.
Hitting a ceiling
Wide width of the crest
[Check Point 2] Grating focus adjustment result (Measurement Precision values) Table 3.7-5 Grating Focus Adjustment Result Check Point 2 State
Judgment
Cause and Countermeasure
Less than 2
OK
Ყ
NG
There is a lot of noise in the image, and it is impossible to read the Current position and the distance to Target. Floating beads may be mixed or the image may be too dark. Use the fluorescent bead specimen fixed on the cover glass, and execute grating focus adjustment again with an amount of light intensity that makes the maximum value of brightness of the Histogram window around 4000.
2 or more
[Check Point 3] Peak position of graph curve (Z profile of fluorescent bead specimen) Table 3.7-6 Grating Focus Adjustment Result Check Point 3 State
Judgment
Cause and Countermeasure
Within the blue background area
OK
Ყ
NG
The drift around the specimen may be strong such as on the motorized stage, the PFS motorized nosepiece, etc., or the focusing before acquisition of the image may be inadequate. Wait a sufficient amount of time for the device to warm up and reach a state of thermal equilibrium, readjust the focus, then execute grating focus adjustment again.
Outside of the blue background area
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[Check Point 4] Shape of graph curve (Z profile of fluorescent bead specimen) Table 3.7-7 Grating Focus Adjustment Result Check Point 4 State
Judgment
Cause and Countermeasure
Smooth Gaussian distribution shape
OK
Ყ When the curve of the grating focus adjustment result graph becomes jagged sawtooth shape, a trouble may have occurred with the specimen PZT stage. Contact your nearest Nikon representative.
Jagged sawtooth shape
NG
Clicking the [Apply] button on the Grating Focus Result window displays the Grating Focus (result application confirmation) dialog box. Click the [OK] button to apply the result and finish the grating focus adjustment. To exit the graph window, click the [x] button on the upper right of the window.
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3.7.7
Grating Focus (Result Application Confirmation) Dialog Box
Clicking the [Apply] button on the Grating Focus Result window displays the Grating Focus (result application confirmation) dialog box.
Figure 3.7-7 Grating Focus (result application confirmation) dialog box
Select the wavelength to be applied and click the [OK] button. The grating block position information of the grating focus adjustment result is saved as a setting file. If 405 is used for the adjustment, the result cannot be applied to other wavelengths. If a wavelength other than 405 is used for the adjustment, the result can be applied to other unadjusted wavelengths.
3.7.8
Saving the Result of Grating Focus as a File
Clicking the [Save] button on the Grating Focus Result window saves the result of grating focus as a file.
Figure 3.7-8 File save window for the result of grating focus
Specify a file name on the File save window and click the [Save] button. The result of grating focus is saved as a file (.gfr). The saved result file can be loaded by selecting the [Load from File...] button on the Grating Focus (selection) window.
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3.7.9
Correction Ring Adjustment Navigation
Clicking the [Correction Ring Navi] button on the Grating Focus (selection) dialog box displays the navigation windows for correction ring adjustment.
Figure 3.7-9 Grating Focus (selection) dialog box
Navigation for correction ring adjustment operations is provided. Click the [Next] button to move to the next step.
Figure 3.7-10 Correction Ring Adjustment Navigation Step 1 window
Windows for Steps 1 to 14 are displayed. When the adjustment is completed, click the [OK] button in Step 14.
Figure 3.7-11 Correction Ring Adjustment Navigation Step 14 window
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3.7.10
Grating Focus Help
Clicking the Help] button on the Grating Focus Result window displays the help windows for grating focus.
Figure 3.7-12 Grating Focus Result window
Figure 3.7-13 Help window for grating focus
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4
Image Reconstruction
This chapter describes N-SIM software operations for SIM image reconstruction.
4.1
SIM Image Reconstruction Setting
Use the Reconstruct area on the N-SIM Pad tab sheet to specify the settings for SIM image reconstruction. When performing 2D-SIM, TIRF-SIM, or Slice 3D-SIM (reconstruct acquired images of 3D-SIM by Z layer), use slice reconstruction. When performing Stack 3D-SIM (reconstruct acquired images of 3D-SIM as a volume data), use stack reconstruction.
1
3 4
2
6 5
7 Figure 4.1-1 N-SIM Pad tab sheet Table 4.1-1 Outline of SIM image Acquisition Setting Control
Control 1
2
Description
Preview Slice button
Displays the N-SIM Slice Reconstruction Preview window.
Reconstruct Slice button
Reconstructs acquired images of 2D-SIM, TIRF-SIM, or Slice 3D-SIM. To reconstruct the SIM image acquired in the Dual mode, the position shift between channels is corrected with the correction parameter saved on the image. Displays the N-SIM Slice Reconstruction (parameters setting) window when the “Show the Reconstruction Parameter Setup Buttons” checkbox under [Options] on the N-SIM Settings window is not checked.
3
Reconstruct Stack button
Reconstructs acquired images of Stack 3D-SIM. To reconstruct the SIM image acquired in the Dual mode, the position shift between channels is corrected with the correction parameter saved on the image. Displays the N-SIM Stack Reconstruction (parameters setting) window when the “Show the Reconstruction Parameter Setup Buttons” checkbox under [Options] on the N-SIM Settings window is not checked.
4
Reconstruct Batch button
Displays the N-SIM Batch Reconstruction setup window.
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Control
Description
5
Param button for Reconstruct Slice
Displays the N-SIM Slice Reconstruction (parameters setting) window. This button is not shown when the “Show the Reconstruction Parameter Setup Buttons” checkbox under [Options] on the N-SIM Settings window is not checked.
6
Param button for Reconstruct Stack
Displays the N-SIM Stack Reconstruction parameter setup window. This button is not shown when the “Show the Reconstruction Parameter Setup Buttons” checkbox under [Options] on the N-SIM Settings window is not checked.
7
FFT Image button
Displays the FFT image of a reconstructed SIM image.
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4.2
Slice Reconstruction
Slice reconstruction is a reconstruction method to execute reconstruction processing for each frame regardless of the previous and subsequent frames. To execute slice reconstruction, first set slice reconstruction parameters.
4.2.1
Setting Slice Reconstruction Parameters
Click the [Param] button for Reconstruct Slice on the N-SIM Pad tab sheet. The N-SIM Slice Reconstruction (parameters setting) window is displayed.
Figure 4.2-1 N-SIM Pad tab sheet – Param button for Reconstruct Slice
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Set reconstruction parameters on the N-SIM Slice Reconstruction (parameters setting) window. 1 8 2 3 9 4
10
5
11
6 12 7
13
Figure 4.2-2 N-SIM Slice Reconstruction (parameters setting) window
Table 4.2-1 Outline of N-SIM Slice Reconstruction Parameter Setup Control 1
2
3
SIM mode change combo box
Channel setting checkbox & combo box
Reconstruction with good contrast components only checkbox
Description Reconstruction parameters have been set for each SIM mode. Changing the SIM mode in this box causes the settings of the mode to appear in the dialog box. Checking the checkbox enables the settings of each channel, and retains the parameters for each laser wavelength. In this case, parameter settings of the laser channel selected with the combo box are shown. Unchecking the checkbox disables the combo box, and the same parameters are used for all the channels. Setting to specify whether to execute the reconstruction processing with only the good contrast components when the illumination contrast is judged “low.” With this checkbox checked, the reconstruction processing is executed with only the signals in the directions with good contrast. This setting is enabled only when [Illumination Modulation Contrast] is set to “Auto.” Otherwise this setting is disabled.
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Control
Description Parameter for adjusting the effect of artifacts of reconstructed SIM images and contrast-emphasis. ([Structured illumination Contrast] of the previous version) The adjustable range is “Auto” and 0.01 to 1.00 (0.01 to 5.00 only for 3D-SIM). Clicking the [Auto] button changes this parameter to “Auto.” With the parameter set to “Auto,” the optimal parameters for reconstruction are automatically calculated based on the acquired images. However, the image cannot be normally reconstructed with the parameters automatically derived from these images and may have much noise when the illumination contrast of the acquired images is low. In that case, check the Reconstruction with good contrast components only checkbox. Reconstruction process is executed with only the signals in the directions with good contrast, and you can obtain the reconstructed image with less noise.
4
Illumination Modulation Contrast parameter adjustment control
Changing the parameter changes the intensity ratio of each component caused by structured illumination as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. Entering small value
ļ
Conventional MTF component
Entering large value MTF
2D-SIM Super-resolution component Spatial frequency
Spatial frequency
3D-SIM
Spatial frequency
Spatial frequency
Parameter for adjusting the noise level of reconstructed SIM images. ([Apodization Filter Parameter] of the previous version) The adjustable range is 0.10 to 5.00. Changing the parameter changes the intensity of high-frequency wave area as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. 5
High Resolution Noise Suppression parameter adjustment control
Entering small value
ļ
Entering large value MTF
MTF
Spatial frequency
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Chapter 4 Image Reconstruction
Control
Description Parameter for adjusting the optical sectioning thickness of reconstructed SIM images. ([Width of 3D-SIM Filter] of the previous version) The adjustable range is 0.01 to 0.50. Changing the parameter adjusts the spread of intensity in the Z direction as shown below. The sectioning image of the result is displayed on the screen as rough indication for adjustment.
6
Out of Focus Blur Suppression parameter adjustment control
Entering small value
ļ
Entering large value Z
Z
X,Y
X,Y
Spread of intensity distribution of luminescent point in real space
At the reconstruction process, the K vector information (described below), necessary for reconstruction, is read from the acquired images for each wavelength and is calculated and processed. By checking this checkbox, the most reliable and the least noise K vector value is selected from the K vector information read from each frame and is applied to a set of images for a set of acquired images (nd2) for Timelapse and Z-Stack etc. (ND Optimize of the previous version)
Position where to compose super-resolution components
K vector: Vector from origin to center of super-resolution components
7
Position of conventional component (Origin)
K vector optimum value selection checkbox & combo box
A set of K vectors (in three directions for each wavelength) is selected and applied as the Z-Stack data. Corresponding to the ND Acquisition setting, three settings are provided for Timelapse. Select “File” in the selection combo box to enable the file specification combo box for specifying the saved SIM reconstruction image files. The following modes are selectable on the selection combo box: All Frames
Selects a set of the optimum K vectors from all frames and applies it to the reconstructed image.
Each Time-Phase
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each Phase of Time schedule for ND Acquisition and applies it to the reconstructed image.
Each Time-Loop
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each acquisition time (Loop) of Time schedule for ND Acquisition and applies it to the reconstructed image.
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Control
7
K vector optimum value selection checkbox & combo box
Description
File
Reads the K vector of the specified SIM reconstruction image files and applies it to the reconstructed image. The specified files must contain the wavelengths that are used for the acquired SIM image to be reconstructed.
Button for loading the saved user parameters and for saving or clearing the user parameters. Clicking the ź button to select the process changes the name of the button. This button is the Load User Params button at the default. When the user parameters are not saved, the default reconstruction parameters are set. 8
Load User Params button
9
Reuse button
10
Reconstruct without Registration button
The selectable processes are as follows: Load User Parameters
Loads the saved user parameters and sets the values to each control of the reconstruction parameters.
Save User Parameters
Saves the values set to each control of the reconstruction parameters as the user parameters.
Clear User Parameters
Clears the saved user parameters and sets the default reconstruction parameters.
Acquires reconstruction parameters of specified reconstructed SIM images to set to the control of each parameter. Newly opens the reconstruction window and displays a reconstructed SIM image (all frames). The position shift between channels is not corrected even when reconstructing the SIM image captured in the Dual mode. Newly opens the reconstruction window and displays a reconstructed SIM image (all frames).
11
Reconstruct button
To reconstruct the SIM image acquired in the Dual mode, the position shift between channels is corrected with the correction parameter saved on the image.
12
Apply button
Applies changed parameters. The present window remains displayed.
13
Close button
Closes the window. When the parameters have been changed, a confirmation window appears asking whether to save the changed value.
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Clicking the ź button of the user parameters displays the pop-up menu as shown below.
Figure 4.2-3 N-SIM Slice Reconstruction (parameters setting) window - User parameters menu
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The following describes the mode for selecting the optimum K vector with an ND Acquisition setting example.
Figure 4.2-4 ND Acquisition tab sheet - Example of Phase & Loops definitions
With the ND Acquisition setting above, Z-Stack acquisition for each wavelength is respectively repeated three times, five times, and two times in each Phase. To reconstruct the SIM image acquired with this setting, the K vectors are selected according to the mode for selecting the optimum K vector (as shown in the table below) and are applied. Table 4.2-2 K Vector Selection and Application Examples Setting Mode
Selection and Application Examples
All Frame
For each wavelength, a set of the optimum K vectors is selected from all frames and applied. Two wavelengths are set for this setting; two sets of K vectors are selected for this case.
Each Time-Phase
For each wavelength, a set of the optimum K vectors for each Phase is selected and applied. Two wavelengths and three phases are set for this setting; six sets (2 x 3) of K vectors are selected for this case.
Each Time-Loop
For each wavelength, a set of the optimum K vectors for each Loop is selected and applied. Two wavelengths and ten loops are set for this setting; 20 sets (2 x 10) of K vectors are selected for this case.
File
For each wavelength, a set of K vectors is read from the specified SIM reconstruction image files and applied. Two wavelengths are set for this setting; two sets of K vectors are read for this case.
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4.2.2
Executing Slice Reconstruction
To execute slice reconstruction, make an acquired SIM image active, and then click the [Reconstruct Slice] button on the N-SIM Pad tab sheet. The SIM mode for the acquired SIM image is recognized automatically.
Figure 4.2-5 Acquired SIM image (2D-SIM mode)
Figure 4.2-6 N-SIM Pad tab sheet - Reconstruction Slice button
The [Reconstruct] button on the N-SIM Slice Reconstruction (parameters setting) window can also be used to execute slice reconstruction.
Figure 4.2-7 Reconstructed SIM image
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When slice reconstruction is executed with the Illumination Modulation Contrast parameter set to “Auto,” the following reconstruction result message is displayed.
Figure 4.2-8 Reconstruct Message dialog box
The 3D-SIM reconstructed image is generated by composing the 1 order diffraction component and the 2 order diffraction component with the conventional component in each direction. When the Reconstruction with good contrast components only checkbox is checked, the score (judged by 8 levels) displayed in the Reconstruct Message dialog box indicates that with which signals of the component the reconstructed image was generated. The following table shows the description of each score. Table 4.2-3 Score of Reconstruction Result – 3D-SIM Score
Description
FFT Image Example
8
Reconstruction is executed normally. Every separation component is judged as an effective signal and is reflected to the reconstructed image.
7
The 2 order diffraction component in one direction is judged as a defective signal; the reconstructed image is generated without the component.
6
The 2 order diffraction component in two directions are judged as a defective signal; the reconstructed image is generated without the components.
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Score
Description
FFT Image Example
5
The 1 order and the 2 order diffraction components to one direction are judged as a defective signal; the reconstructed image is generated without the components.
4
The 2 order diffraction component in three directions are judged as a defective signal; the reconstructed image is generated without the components.
3
The 1 order diffraction component in one direction and 2 order diffraction component in other direction are judged as a defective signal; the reconstructed image is generated without the components.
2
The 1 order diffraction component in one direction and 2 order diffraction component in other two directions are judged as a defective signal; the reconstructed image is generated without the components.
1
The 1 order and the 2 order diffraction components in two directions or more are judged as a defective signal; the reconstructed image is generated without the components.
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The 2D-SIM reconstructed image is generated by composing the 1 order diffraction component with the conventional component in each direction. Since the 2D-SIM reconstructed image does not composed with the 2 order diffraction component, the score displayed for the 2D-SIM reconstructed image is only 1, 5, or 8. Table 4.2-4 Score of Reconstruction Result – 2D-SIM Score
Description
FFT Image Example
8
Reconstruction is executed normally. Every separation component is judged as an effective signal and is reflected to the reconstructed image.
5
The 1 order diffraction component in one direction is judged as a defective signal; the reconstructed image is generated without the component.
1
The 1 order diffraction component in two directions or more are judged as a defective signal; the reconstructed image is generated without the components.
When executing slice reconstruction for an acquired SIM image with multi frames, the detail of the frame with the highest score is displayed on the Reconstruct Message dialog box. To check the detail of the other frames, click the [Message File] button on the Reconstruct Message dialog box. The file that contains the reconstruction result of every frame is displayed.
Figure 4.2-9 Reconstruction Result List file
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The following table describes the notation such as “Time=1” or “Ch=1” shown at the beginning of a message. Table 4.2-5 Notations of Reconstruction Result List file Notation Time
Description Frame of Timelapse
Ch
Channel of Ȝ series or Dual mode
XY
Point of Multi Point
Z
Frame of Z-Stack
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4.3
Slice Reconstruction Preview
Use slice reconstruction preview to execute reconstruction with a specified single frame of an acquired SIM image. Click the [Preview Slice] button on the N-SIM Pad tab sheet. The N-SIM Slice Reconstruction (preview) window is displayed.
Figure 4.3-1 N-SIM Pad tab sheet - Preview Slice button
To execute slice reconstruction preview, click the [New Preview] button or [Update Preview] button on the N-SIM Slice Reconstruction (preview) window. 1
8
2 3 9
4
10 5
11
6
12
7 13 Figure 4.3-2 N-SIM Slice Reconstruction (preview) window
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Table 4.3-1 Outline of N-SIM Slice Reconstruction Preview Control 1
SIM mode display
2
Channel setting checkbox & combo box
3
Reconstruction with good contrast components only checkbox
Description Shows the SIM mode of the active acquired SIM image. Checking the checkbox enables the settings of each channel, and retains the parameters for each laser wavelength. In this case, parameter settings of the laser channel selected with the combo box are shown. Unchecking the checkbox disables the combo box, and the same parameters are used for all the channels. Setting to specify whether to execute the reconstruction processing with only the good contrast components when the illumination contrast is judged “low.” With this checkbox checked, the reconstruction processing is executed with only the signals in the directions with good contrast. This setting is enabled only when [Illumination Modulation Contrast] is set to “Auto.” Otherwise this setting is disabled. Parameter for adjusting the effect of artifacts of reconstructed SIM images and contrast-emphasis. ([Structured illumination Contrast] of the previous version) The adjustable range is “Auto” and 0.01 to 1.00 (0.01 to 5.00 only for 3D-SIM). Clicking the [Auto] button changes this parameter to “Auto.” With the parameter set to “Auto,” the optimal parameters for reconstruction are automatically calculated based on the acquired images. However, the image cannot be normally reconstructed with the parameters automatically derived from these images and may have much noise when the illumination contrast of the acquired images is low. In that case, check the Reconstruction with good contrast components only checkbox. Reconstruction process is executed with only the signals in the directions with good contrast, and you can obtain the reconstructed image with less noise.
4
Illumination Modulation Contrast parameter adjustment control
Changing the parameter changes the intensity ratio of each component caused by structured illumination as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. Entering small value Conventional MTF component
ļ
Entering large value MTF
2D-SIM Super-resolution component Spatial frequency
Spatial frequency
3D-SIM
Spatial frequency
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Chapter 4 Image Reconstruction
Control
Description Parameter for adjusting the noise level of reconstructed SIM images. ([Apodization Filter Parameter] of the previous version) The adjustable range is 0.10 to 5.00. Changing the parameter changes the intensity of high-frequency wave area as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment.
5
High Resolution Noise Suppression parameter adjustment control
Entering small value
ļ
Entering large value MTF
MTF
Spatial frequency
Spatial frequency
Parameter for adjusting the optical sectioning thickness of reconstructed SIM images. ([Width of 3D-SIM Filter] of the previous version) The adjustable range is 0.01 to 0.50. Changing the parameter adjusts the spread of intensity in the Z direction as shown below. The sectioning image of the result is displayed on the screen as rough indication for adjustment.
6
Out of Focus Blur Suppression parameter adjustment control
Entering small value
ļ
Entering large value Z
Z
X,Y
Spread of intensity distribution of luminescent point in real space
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Chapter 4 Image Reconstruction
Control
Description At the reconstruction process, the K vector information (described below), necessary for reconstruction, is read from the acquired images for each wavelength and is calculated and processed. By checking this checkbox, the most reliable and the least noise K vector value is selected from the K vector information read from each frame and is applied to a set of images for a set of acquired images (nd2) for Timelapse and Z-Stack etc. (ND Optimize of the previous version)
Position where to compose super-resolution components
K vector: Vector from origin to center of super-resolution components Position of conventional component (Origin)
7
K vector optimum value selection checkbox & combo box
A set of K vectors (in three directions for each wavelength) is selected and applied as the Z-Stack data. Corresponding to the ND Acquisition setting, three settings are provided for Timelapse. Select “File” in the selection combo box to enable the file specification combo box for specifying the saved SIM reconstruction image files. The following modes are selectable on the selection combo box: All Frames
Selects a set of the optimum K vectors from all frames and applies it to the reconstructed image.
Each Time-Phase
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each Phase of Time schedule for ND Acquisition and applies it to the reconstructed image.
Each Time-Loop
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each acquisition time (Loop) of Time schedule for ND Acquisition and applies it to the reconstructed image. Reads the K vector of the specified SIM reconstruction image files and applies it to the reconstructed image.
File
The specified files must contain the wavelengths that are used for the acquired SIM image to be reconstructed.
Button for loading the saved user parameters and for saving or clearing the user parameters. Clicking the ź button to select the process changes the name of the button. This button is the Load User Params button at the default. When the user parameters are not saved, the default reconstruction parameters are set. 8
Load User Params button
The selectable processes are as follows: Load User Parameters
Loads the saved user parameters and sets the values to each control of the reconstruction parameters.
Save User Parameters
Saves the values set to each control of the reconstruction parameters as the user parameters.
Clear User Parameters
Clears the saved user parameters and sets the default reconstruction parameters.
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Control
Description
9
Reuse button
Acquires reconstruction parameters of specified reconstructed images to set to the control of each parameter.
10
New Preview button
Newly opens a window to display a reconstructed preview image (one frame only) of the specified frame.
11
Update Preview
Displays a reconstructed preview image (one frame only) of the specified frame on the last reconstructed SIM reconstruction window.
12
Reconstruct button
Newly opens the reconstruction window and displays a reconstructed SIM image (all frames), then closes the window.
13
Close button
Closes the window.
Clicking the ź button of the user parameters displays the pop-up menu as shown below.
Figure 4.3-3 N-SIM Slice Reconstruction (preview) window - User parameters menu
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4.4
Stack Reconstruction
Stack reconstruction is a reconstruction method to execute reconstruction processing not only by frame but also in the Z direction. To execute stack reconstruction, first set stack reconstruction parameters.
4.4.1
Setting Stack Reconstruction Parameters
Click the [Param] button for Reconstruct Stack on the N-SIM Pad tab sheet. The N-SIM Stack Reconstruction (parameters setting) window is displayed.
Figure 4.4-1 N-SIM Pad tab sheet – Param button for Reconstruct Stack
Set reconstruction parameters on the N-SIM Stack Reconstruction (parameters setting) window.
4
1
5 2 6 3
7 8 9 Figure 4.4-2 N-SIM Stack Reconstruction (parameters setting) window
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Table 4.4-1 Outline of N-SIM Stack Reconstruction Parameter Setup Control
1
Channel setting checkbox & combo box
Description Checking the checkbox enables the settings of each channel, and retains the parameters for each laser wavelength. In this case, parameter settings of the laser channel selected in the combo box are shown. Unchecking the checkbox disables the combo box, and the same parameters are used for all the channels. Parameter for adjusting the effect of artifacts of reconstructed SIM images and contrast-emphasis. ([Structured illumination Contrast] of the previous version) The adjustable range is “Auto” and 0.01 to 5.00. Clicking the [Auto] button changes this parameter to “Auto.” With the parameter set to “Auto,” the optimal parameters for reconstruction are automatically calculated based on the acquired images.
2
Illumination Modulation Contrast parameter adjustment control
Changing the parameter changes the intensity ratio of each component caused by structured illumination as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. Entering small value
ļ
MTF
Entering large value MTF
Kz plane
Spatial frequency
Spatial frequency
Parameter for adjusting the noise level of reconstructed SIM images. ([Apodization Filter Parameter] of the previous version) The adjustable range is 0.10 to 5.00. Changing the parameter changes the intensity of high-frequency wave area as shown below. The effective MTF graph curve of the result is displayed on the screen as rough indication for adjustment. 3
High Resolution Noise Suppression parameter adjustment control
Entering small value
ļ
Entering large value MTF
MTF
Spatial frequency
Spatial frequency
Button for loading the saved user parameters and for saving or clearing the user parameters. Clicking the ź button to select the process changes the name of the button. This button is the Load User Params button at the default. When the user parameters are not saved, the default reconstruction parameters are set. 4
Load User Params button
The selectable processes are as follows: Load User Parameters
Loads the saved user parameters and sets the values to each control of the reconstruction parameters.
Save User Parameters
Saves the values set to each control of the reconstruction parameters as the user parameters.
Clear User Parameters
Clears the saved user parameters and sets the default reconstruction parameters.
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Control
Description
5
Reuse button
Acquires reconstruction parameters of specified reconstructed SIM images to set to the control of each parameter.
6
Reconstruct without Registration button
Newly opens the reconstruction window and displays a reconstructed SIM image (all frames) without correcting position shift between channels. The position shift between channels is not corrected even when reconstructing the SIM image acquired in the Dual mode. Newly opens the reconstruction window and displays a reconstructed SIM image (all frames).
7
Reconstruct button
To reconstruct the SIM image acquired in the Dual mode, the position shift between channels is corrected with the correction parameter saved on the image.
8
Apply button
Applies changed parameters. The present window remains displayed.
9
Close button
Closes the window. With parameters changed, a confirmation window appears asking whether to apply.
Clicking the ź button of the user parameters displays the pop-up menu as shown below.
Figure 4.4-3 N-SIM Stack Reconstruction (parameters setting) window - User parameters menu
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4.4.2
Executing Stack Reconstruction
To execute stack reconstruction, make an acquired SIM image active, and then click the [Reconstruct Stack] button on the N-SIM Pad tab sheet. Images which can be reconstructed with this processing are only Z-Stack images in the 3D-SIM mode.
Figure 4.4-4 Z-Stack SIM image acquired in the 3D-SIM mode
Figure 4.4-5 N-SIM Pad tab sheet - Reconstruct Stack button
The [Reconstruct] button on the N-SIM Stack Reconstruction parameter setup window can also be used to execute stack reconstruction.
Figure 4.4-6 Reconstructed SIM image
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4.5
Batch Reconstruction
Batch reconstruction is helpful to execute multiple reconstruction processing later at a time. To execute batch reconstruction, first make settings on the N-SIM Batch Reconstruction window.
4.5.1
Batch Reconstruction Setting
Click the [Reconstruct Batch] button on the N-SIM Pad tab sheet. The N-SIM Batch Reconstruction window is displayed.
Figure 4.5-1 N-SIM Pad tab sheet – Reconstruct Batch button
Make necessary settings on the N-SIM Batch Reconstruction window.
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2
1
8
3
4
9
5
10
Figure 4.5-2 N-SIM Batch Reconstruction window
98
6
7
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Chapter 4 Image Reconstruction
Table 4.5-1 Outline of N-SIM Batch Reconstruction Setup Control
Description
1
Image specification button
Specify a file of an acquired SIM image.
2
Select-all button
Selects all the items in the batch reconstruction list.
3
Unselect button
Cancels the selection of an acquired SIM image.
4
Upward button
Shifts a selected, acquired SIM image upward in the batch reconstruction list.
5
Downward button
Shifts a selected, acquired SIM image downward in the batch reconstruction list.
6
Delete button
Deletes a selected, acquired SIM image from the batch reconstruction list.
7
Delete-all button
Deletes all the items in the batch reconstruction list.
8
9
Suffix specification input box
If multiple acquired SIM images with the same name have been specified in the batch reconstruction list, the file name format of reconstructed SIM images is the name of an acquired “SIM image_suffix character string # batch reconstruction list No.nd2.”
Param button
Displays the N-SIM Batch Reconstruction Parameter setup window. This button is enabled when an acquired SIM image in the batch reconstruction list is selected. Specify reconstruction parameters to be used for batch reconstruction for each SIM image acquired on the displayed setup window.
#
Item No. in the batch reconstruction list. Reconstruction processing is executed in the ascending order of numbers.
Status * Shows reconstruction processing status for each item. 10
none
Reconstruction processing is not being executed because the SIM mode is not available.
Processing
Reconstruction processing is in progress.
Done
Reconstruction processing is completed properly.
Skip
Reconstruction processing is not completed for such a reason that the file does not exist.
Failure
Reconstruction processing failed.
File Path
Shows the file path of an acquired SIM image. A reconstructed SIM image is saved in the file path of an acquired SIM image.
File Name
Shows the file name of an acquired SIM image.
Mode
Shows the SIM mode of an acquired SIM image.
Parameter
Shows reconstruction parameters which have been set.
Use best
Shows the mode of “Use best Reconstruction Parameters found in.”
File
Shows a specified file name when “Use best” is “Type4.”
Status Msg
Shows an error message when “Status” is “Skip” or “Failure.”
Start button 11
Specify a suffix character string to be added to a file name of a reconstructed SIM image. The file name format of a reconstructed SIM image is the name of an acquired “SIM image_suffix character string.nd2.”
* The button changes depending on the processing.
Start
Starts batch reconstruction processing.
Pause
Temporarily interrupts batch reconstruction processing being executed.
Resume
Resumes batch reconstruction processing being interrupted.
Close
Closes the setup window when batch reconstruction processing is completed.
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After selecting an acquired SIM image from the batch reconstruction list, click the [Param] button. The N-SIM Batch Reconstruction Parameter window is displayed. Set reconstruction parameters to be used for reconstruction processing on the setup window.
2
1
3 4 5 6
7
8
9 Figure 4.5-3 N-SIM Batch Reconstruction Parameter window
Table 4.5-2 Outline of N-SIM Batch Reconstruction Parameter Setup Control
1
SIM mode setting combo box
2
Reconstruction with good contrast components only checkbox
Description Shows the SIM mode of an acquired SIM image which has been selected in the batch reconstruction list. When the acquired SIM image is a 3D-SIM, “Slice 3D-SIM” or “Stack 3D-SIM” can be selected. Selecting “Slice 3D-SIM” executes the slice reconstruction. Selecting “Stack 3D-SIM” executes the stack reconstruction. Setting to specify whether to execute the reconstruction processing with only the good contrast components when the illumination contrast is judged “Low.” With this checkbox checked, the reconstruction processing is executed with only the signals in the directions with good contrast. This setting is enabled only when [Illumination Modulation Contrast] is set to “Auto.” Otherwise this setting is disabled.
3
Illumination Modulation Contrast setting control
Parameter for adjusting the effect of artifacts of reconstructed SIM images and contrast-emphasis. ([Structured illumination Contrast] of the previous version) The adjustable range is “Auto” and 0.01 to 1.00 (0.01 to 5.00 only for 3D-SIM)
4
High Resolution Noise Suppression setting control
Parameter for adjusting the noise level of reconstructed SIM images. (Apodization Filter Parameter of the previous version) The adjustable range: 0.10 to 5.00
5
Out of Focus Blur Suppression setting control
Parameter for adjusting the optical sectioning thickness of reconstructed SIM images. (Width of 3D-SIM Filter of the previous version) The adjustable range: 0.01 to 0.50
6
Load User Params button
Sets the saved user parameters to the control of each parameter.
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Control
Description By checking this checkbox, the most reliable and the least noise K vector value is selected from the K vector information read from each frame and is applied to a set of images for a set of acquired images (nd2) for Timelapse and Z-Stack etc. (ND Optimize of the previous version) Checking the checkbox makes the settings effective. Select “File” in the selection combo box to enable the file specification combo box for specifying SIM reconstruction image files. The following modes are selectable on the selection combo box:
7
K vector optimum value selection checkbox & combo box
Type1: All Frames
Selects a set of the optimum K vectors from all frames and applies it to the reconstructed image.
Type2: Each Time-Phase
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each Phase of Time schedule for ND Acquisition and applies it to the reconstructed image.
Type3: Each Time-Loop
Based on the difference of time of acquisition, selects a set of the optimum K vectors for each capture time (Loop) of Time schedule for ND Acquisition and applies it to the reconstructed image.
Type4: File
Reads the K vector of the specified SIM reconstruction image files and applies it to the reconstructed image. The specified files must contain the wavelengths that are used for the acquired SIM image to be reconstructed.
8
OK button
Applies the specified reconstruction parameters and closes the window.
9
Cancel button
Discards the specified reconstruction parameters and closes the window.
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4.5.2
Executing Batch Reconstruction
To execute batch reconstruction processing, click the [Start] button on the N-SIM Batch Reconstruction window.
Figure 4.5-4 N-SIM Batch Reconstruction window - When executed
To temporarily interrupt reconstruction processing being executed, click the [Pause] button on the N-SIM Batch Reconstruction window.
Figure 4.5-5 N-SIM Batch Reconstruction window - While being executed
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To resume reconstruction processing being interrupted, click the [Resume] button on the N-SIM Batch Reconstruction window.
Figure 4.5-6 N-SIM Batch Reconstruction window - While being interrupted
When batch reconstruction processing is completed, click the [Close] button or the [X] button to close the N-SIM Batch Reconstruction window.
Figure 4.5-7 N-SIM Batch Reconstruction window - When completed
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4.6
FFT Image Display
An FFT image indicates the signal of a component with which the reconstructed image is constructed. An FFT image can be displayed at the reconstruction process. An FFT image can also be displayed by selecting a reconstructed image that has already been reconstructed.
4.6.1
FFT Image Display Setting
When the checkbox of [Show FFT Images after Reconstruction] is checked, FFT image is displayed at the reconstruction process. Selecting “All frames” displays the FFT images of all frames. Selecting “Only Current frame” displays the FFT image of the frame currently displayed on the acquired SIM image.
Figure 4.6-1
N-SIM Settings window – FFT image display setting
The FFT image is displayed as well as the reconstructed image at the reconstruction process.
Figure 4.6-2 FFT image
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4.6.2
FFT Image Display
Making a reconstructed image active and clicking the [FFT Image] button on the N-SIM Pad tab sheet displays the FFT image of the activated reconstructed image. Whether the FFT images of all frames are displayed or the only FFT image of the frame currently displayed is displayed is determined according to the FFT image display setting on the N-SIM Settings window.
Figure 4.6-3 N-SIM Pad tab sheet - FFT Image button
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5
Setting and Status
This chapter describes N-SIM software operations for option settings and status display.
5.1
Option Settings and Status Display
For option settings and status display, use the individual buttons in the Acquisition area on the N-SIM Pad tab sheet.
1
3
2
4
5
Figure 5.1-1 N-SIM Pad tab sheet Table 5.1-1 Outline of SIM Image Acquisition Setting Control Control
Description
1
Equipment status display
Shows the current equipment status and error status. When in normal status, diffraction grating, fiber and laser interlock status are shown.
2
Interlock Release button
The release button becomes effective depending on the laser interlock status. When not in connection with the equipment, the [Connect] button is shown instead.
3
Help button
Displays the Help/Version pop-up menu. With Help selected, shows Help text. With Version selected, shows the version No. of the software.
4
Option setting button
Displays the option setting window.
5
Communication status display
Shows communication status with the SIM controller.
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5.2
N-SIM Settings Window
To make option settings, make settings on the N-SIM Settings window. To display the N-SIM Settings window, click the [Option setting] button on the N-SIM Pad tab sheet.
Figure 5.2-1 N-SIM Pad tab sheet - Option setting button
Make each setting on the N-SIM Settings window.
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1 2 3
4 5 6 7 8 9 10
13
11
12
14 Figure 5.2-2
N-SIM Settings window
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Table 5.2-1 Outline of N-SIM Settings Window Control Control
Description
1
Diffraction grating selection combo box
Select a diffraction grating. If the selected diffraction grating does not match with the current objective, wavelength, and fiber mode, a warning telling a diffraction grating selection error is shown for “Status” on the N-SIM Pad tab sheet.
2
Show the Active Laser only radio button
Sets the display in [Laser Control] on the N-SIM Pad tab sheet to the Active laser mode. When selected, the controls of only active lasers are displayed in [Laser Control] on the N-SIM Pad tab sheet.
3
Channel Setup (Show these Lasers radio button & channel wavelength setting combo boxes)
Sets the display in [Laser Control] on the N-SIM Pad tab sheet to the Channel laser mode. When selected, the Channel check boxes and the Laser combo boxes are enabled and the control of set lasers are displayed in [Laser Control] on the N-SIM Pad tab sheet.
4
Channel Setup (Show these Dual Lasers radio button & channel wavelength setting combo boxes)
Sets the display in [Laser Control] on the N-SIM Pad tab sheet to the Dual laser mode. The Dual laser mode is used for the dual camera system. When selected, the control of selected lasers as Channel1 and Channel2 are displayed in [Laser Control] on the N-SIM Pad tab sheet.
5
Reconstruction processing automatic execution after image acquisition option
Specify whether to automatically execute reconstruction processing after image acquisition. With the checkbox checked, automatically executes reconstruction processing after image acquisition and displays a reconstructed SIM image. The acquired SIM image closes automatically.
6
Capture option display mode 1 checkbox
Specify whether to show “1D-SIM” as a Capture option on the N-SIM Pad tab sheet. With the checkbox checked, “1D-SIM” is shown as a Capture option.
7
Capture option display mode 2 checkbox
Specify whether to show “Fixed” and “Moving” as Capture options on the N-SIM Pad tab sheet. With the checkbox checked, “Fixed” and “Moving” are shown as Capture options.
8
Param button display option
Specify whether to display the Param button on the N-SIM Pad tab sheet. With the checkbox checked, the Reconstruction parameter setup window is displayed.
9
Reconstruction parameter value display option
Specify whether to show reconstruction parameter values with % on the Reconstruction parameter setup window. With the checkbox checked, reconstruction parameter values are shown with %.
10
FFT Image Display Option
Specifies whether to display FFT image at the reconstruction process. When selected to ON, also select to display FFT image of all the frames or FFT image of the displayed frame only. With these buttons, set the mode to be applied for grating focus value at the image capture.
11
Capture Mode selection radio button
Select a mode from the three modes: “Contrast Priority,” “Speed Priority,” and “Show Confirmation Dialog.” When “Show Confirmation Dialog” is selected, a confirmation dialog box appears at the start of the image capture allowing you to select either “Contrast Priority” or “Speed Priority.”
12
OK button
Applies the settings and closes the window.
13
Cancel button
Discards the settings and closes the window.
14
Apply button
Applies the settings. This button does not close the window.
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5.2.1
Selecting the Diffraction Grating
After changing the diffraction grating block in the N-SIM illuminator, it is necessary to change the setting on the N-SIM Settings window.
Figure 5.2-3
N-SIM Settings window - Diffraction grating selection Table 5.2-2 Diffraction Grating Types
Diffraction Grating
Optical Fiber
100EX V-G
Multi-mode
100x magnification, 405-561 nm wavelength supported
100EX V-R
Multi-mode
100x magnification, 405-640 nm wavelength supported
60EX V-R
Multi-mode
60x magnification, 405-640 nm wavelength supported
TIRF 488
Single mode
100x magnification, TIRF 488 nm wavelength supported
TIRF 561
Single mode
100x magnification, TIRF 561 nm wavelength supported
Description
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5.2.2
Laser Display Switchover Setting
If the laser display change setting on the N-SIM Settings window is changed, the display of the Laser Control area on the N-SIM Pad tab sheet is also switched. Show the Active Laser only
Figure 5.2-4 N-SIM Settings window - Active Laser display setting
The display of the Laser Control area on the N-SIM Pad tab sheet is narrowed to a single wavelength so that wavelengths can be switched using a combo box.
Figure 5.2-5 N-SIM Pad tab sheet - Active Laser display
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Show these Lasers Check the checkboxes of necessary wavelength.
Figure 5.2-6 N-SIM Settings window - Show these Lasers setting
The display of the Laser Control area on the N-SIM Pad tab sheet shows each wavelength so that adjustment can be made with the controls of each wavelength.
Figure 5.2-7 N-SIM Pad tab sheet - Laser Control setting
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Show these Dual Lasers Use the Dual Laser mode for the dual camera system.
Figure 5.2-8 N-SIM Settings window - Show these Dual Lasers display setting
Two lasers are displayed in [Laser Control] on the N-SIM Pad tab sheet. You can select two lasers. The selected two lasers are output simultaneously in the Dual Laser mode.
Figure 5.2-9 N-SIM Pad tab sheet – Dual lasers displayed
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5.2.3
Capture Option Display Setting
If the Capture option display setting on the N-SIM Settings window is changed, the Capture option menu selections on the N-SIM Pad tab sheet is also changed. Check the checkboxes for “Show 1D-SIM Capture Option” and “Show Fixed and Moving Capture Options” on the N-SIM Settings window.
Figure 5.2-10 N-SIM Settings window - Capture option setting
“1D-SIM,” “Fixed,” and “Moving” are added to the combo box of the Capture options on the N-SIM Pad tab sheet.
Figure 5.2-11 N-SIM Pad tab sheet - Capture option display
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5.2.4
Param Button Display Setting
If the Param button display setting is changed on the N-SIM Settings window, the display of the Param buttons on the N-SIM Pad tab sheet is also changed. Uncheck the checkbox for “Show the Reconstruction Parameter Setup Buttons” on the N-SIM Settings window.
Figure 5.2-12 N-SIM Settings window - Param button display setting
The Param buttons on the N-SIM Pad tab sheet become invisible.
Figure 5.2-13 N-SIM Pad tab sheet - Param buttons non-display
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5.2.5
Reconstruction Parameter Value Display Setting
If the reconstruction parameter value display setting on the N-SIM Settings window is changed, the display of the reconstruction parameters on each Reconstruction parameter setup window is also changed. Check the checkbox for “Show the Reconstruction Parameters as a %” on the N-SIM Settings window.
Figure 5.2-14 N-SIM Settings window - Reconstruction parameter value display setting
The reconstruction parameter values on the N-SIM Slice Reconstruction (parameters setting) window are shown with %.
Figure 5.2-15 N-SIM Slice Reconstruction (parameters setting) window Reconstruction parameter value display
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5.2.6
FFT Image Display Setting
When the checkbox of [Show FFT Images after Reconstruction] is checked, FFT image is displayed at the reconstruction process. Selecting “All frames” displays the FFT images of all frames. Selecting “Only Current frame” displays the FFT image of the frame currently displayed on the acquired SIM image.
Figure 5.2-16 N-SIM Settings window - FFT image display setting
The FFT image is displayed as well as the reconstructed image at the reconstruction process.
Figure 5.2-17 FFT image
* FFT image can be also displayed by making a reconstructed image active and clicking the [FFT Image] button on the N-SIM Pad tab sheet.
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5.3
Equipment Status Display
The current equipment status is shown on the N-SIM Pad tab sheet.
5.3.1
Normal-status Display
When in normal state, diffraction grating, fiber mode, and laser interlock statuses are shown with gray background.
Figure 5.3-1 N-SIM Pad tab sheet - Normal-status display
The table below lists equipment statuses to be shown when in normal state. Table 5.3-1 List of Equipment Status to be Shown in Normal State Item
Grating
Fiber Laser
Character string to be shown
State
100EX V-G
100x magnification, 405 - 561 nm wavelength supporting diffraction grating
100EX V-R
100x magnification, 405 - 640 nm wavelength supporting diffraction grating
60EX V-R
60x magnification, 405 - 640 nm wavelength supporting diffraction grating
TIRF 488
100x magnification, TIRF 488 nm wavelength supporting diffraction grating
TIRF 561
100x magnification, TIRF 561 nm wavelength supporting diffraction grating
Multi (2D/3D-SIM)
Multi mode fiber
Single (TIRF-SIM)
Single mode fiber
All Laser Locks Closed
All lasers are in non-interlock state
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5.3.2
Error-status Display
If an error occurs, the error state of the equipment is displayed in yellow with a red background.
Figure 5.3-2 N-SIM Pad tab sheet - Error-status display
The table below lists equipment statuses to be shown when in error state. Table 5.3-2 List of Equipment Status to be Shown in Error State Character string to be shown
State
PC is not connected with LU5
Disconnection with LU5 detected
PC is not connected with LU-NV. Please restart NIS-Elements
Disconnection with LU-NV detected
One of the Laser Locks is open
Laser interlock detected
Wrong Fiber: 2D/3D-SIM requires the Multi Mode Fiber
Selecting multi-mode fiber is required.
Wrong Fiber: TIRF-SIM requires the Single Mode Fiber
Selecting single-mode fiber is required.
The SIM mode cannot be combined with the selected grating
Inappropriate diffraction grating for laser wavelength
TIRF-SIM cannot be combined with the Dual lasers mode
TIRF-SIM is unselectable in Dual Laser mode.
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The table below shows the selectable combination of diffraction grating and laser wavelength. Table 5.3-3 Selectable Combination of Diffraction Grating and Laser Wavelength Laser Wavelength
Diffraction Grating 100EX V-G
100EX V-R
60EX V-R
TIRF 488
TIRF 561
405
Selectable
Selectable
Selectable
Unselectable
Unselectable
445/457
Selectable
Selectable
Selectable
Unselectable
Unselectable
488
Selectable
Selectable
Selectable
Selectable
Unselectable
514
Selectable
Selectable
Selectable
Unselectable
Unselectable
561
Selectable
Selectable
Selectable
Unselectable
Selectable
640/647
Unselectable
Selectable
Selectable
Unselectable
Unselectable
* 100EX V-G, 100EX V-R, TIRF 488, or TIRF 561 is unselectable when an objective of 60x magnification has been chosen. 60EX V-R is unselectable when an objective of 100x magnification has been chosen.
5.3.3
Laser Interlock State
When being in laser interlocked, the display of the equipment status shows the error state, and the [Interlock Release] button turns red and becomes effective. In that case, eliminate the cause of the interlock, and click the [Interlock Release] button to release the interlock.
Figure 5.3-3 N-SIM Pad tab sheet - Laser interlock state display
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5.3.4
LU5 Disconnected State
When LU5 and the PC are disconnected, the display of the equipment status shows an error state, and the [Connect] button turns red and becomes effective. In that case, eliminate the cause of the disconnection, and click the [Connect] button to restore the connection with LU5.
Figure 5.3-4 N-SIM Pad tab sheet - Equipment disconnection state display
* However, the [Connect] button is not displayed when LU-NV is disconnected. In that case, restart the NIS-Elements.
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5.4
Communication Status Display
Communication status with the SIM controller is shown at the bottom of the N-SIM Pad tab sheet.
Figure 5.4-1 N-SIM Pad tab sheet - Communication-status display
When in normal communication state, nothing is shown. If an error occurs, a message is shown. The table below lists communication statuses to be shown when in error state. Table 5.4-1 List of Communication Status to be Shown in Error State Character string to be shown
State
DEVICE_ERROR
Communication command parameter error, parameter out-of-range, etc.
TRANSMITFAIL_ERROR
Communication command transmission error
NOTCONNECTED_ERROR
USB communication connection error
TIMEOUT ERROR
Communication command time-out
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5.5
Help and Version Display
On the N-SIM Pad tab sheet, N-SIM software’s help and version can be displayed. Click the [?] button on the N-SIM Pad tab sheet to display a pop-up menu.
Figure 5.5-1 N-SIM Pad tab sheet - Help button
To display the N-SIM software’s help, select “Help” from the pop-up menu.
Figure 5.5-2
N-SIM Help window
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To display version information of the N-SIM software, select “Version” from the pop-up menu.
Figure 5.5-3 N-SIM Version Info window - LU5 connected
Figure 5.5-4 N-SIM Version Info window - LU-NV connected
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