Transcript
Leica SP2-AOBS Confocal Microscope (revised 10/01/08)
The Leica SP2 confocal microscope is controlled via software LCS (Leica Confocal Software). Bellow is a brief step-by-step guide to help new users get started smoothly.
Check your specimen under a conventional fluorescent microscope and confirm the staining. Plan in advance which lasers you are going to use before you go to the confocal microscope: If you do not need the Argon Laser (GFP, FITC or YFP), please do not switch on this laser and skip points 3 and 4. The other lasers are less critical and can be switched on or off later as needed.
start-up procedure
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7 7’ 1. Turn on the Microscope Box Control 2. Switch on the Mercury lamp (if you need to check your fluorescence) 3. Turn on the Argon laser fan and the Argon laser itself (3’.) (only if you need it!) 4. Switch on the computer and wait until Windows appears (about 2 minutes) Login with your personal login During this time: Fill out the Logbook 1
5. Switch on the Scanner 6. Click on the Leica LCS software icon. select ‘personal’ and click ‘start’
Now you must wait 2 more minutes until setup and system tests are finished. During this time, do not yet work with the microscope stand. 7. If needed switch on the other laser lines 405, 543, 594 and 633 nm 8. Turn Argon laser level to 10 o’clock
IMPORTANT Before inserting your specimen, check the microscope to make sure it is clean and free of oil. You are responsible for leaving it this way. Before using an unknown objective, be sure to use the correct immersion medium. It is very easy to damage an objective by putting for example oil on a dry objective. Please, ask if you are in doubt. Always lower stage before starting software and inserting specimen
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The microscope stand
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1. Eyepieces - magnifies the image 10X 2. Field Diaphragm – restricts the illumination light to the observation area 3. Polarizer Filters 4. DIC slider – to improve contrast in transmitted light 5. LCD display - lens information, filters, and stepsize (z-control) 6. Mercury lamp control box 7. Revolving nosepiece - rotates objective lenses into viewing position 8. Stage with slide holder 9. Fine focus wheel - brings the specimen into focus with the with the selected stepsize 10. Condenser - focuses the light on the specimen; can be raised and lowered to achieve Köhler illumination 11. Scan Head – Entry for lasers 12. Incandescent light wheel – Controls the brightness of the transmission illumination (halogen lamp) 13. Shutter – Opens the fluorescence pathway 14. FLUO BUTTONS – Blue – Green – Red – controls filter cubes for fluorescence with the mercury lamp 15. PORTS – switches between VIS – SIDE – BOTTOM ports 16. Wollaston prism wheel 2 – Places the correct prism for DIC 17. Wollaston prism 1 – Places the correct prism for DIC 18. Objective switch – switches between objectives
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Filters cubes: (1) for UV excitation, blue emission (Dapi, Hoechst, etc,…) (2) for green excitation, red emission (Cy3, dsRed, Rhodamine, …) (3) for blue excitation, green emission (FITC, Alexa 488, GFP, …) (4) Transmission
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These filters are not used when scanning; they are only for the mercury lamp 1
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Filters cube switcher (A = Blue, N21=Red, I3=Green, --= Transmission and Scan) 3
Objectives Objectives (in rotation order) : 63X 1.30 GLYCEROL 10X 0.30 DRY 20X 0.7 MULTI IMMERSION (oil, glycerol or water) 40X 1.25 – 0.75 (with internal diaphragm) OIL 63X 1.4 – 0.6 (with internal diaphragm) OIL 100X 1.4 – 0.7 (with internal diaphragm) OIL
The 40X, 63X and 100X OIL immersion objectives have a variable aperture. This should be fully open (turned fully clockwise) for maximum resolution and intensity. The 63X GLYCEROL immersion objective has a cover slip correction collar. All cover slips vary but generally a #1 cover slip is .14 mm and a #1 1/2 is .17 mm. Adjust the collar for the best image: while looking at the specimen by eye, adjust the collar in small increments, refocusing continuously with the microscope's fine focus. Table 1 LENS N.A.
IMMERSION MEDIUM
WORKING Z XY RESOLUTION DISTANCE RESOLUTION
Confocal Optical Section with Pinhole = 1 A.U.
10X
0.3
DRY
11 mm
0.651 um
4.768 um
9.83 μm
20 X
0.7
MULTI IMM
260 um
0.279 um
0.768 um
2.65 μm
40 X 1.25
OIL
100 um
0.156 um
0.334 um
0.70 μm
63 X 1.40
OIL
100 um
0.140 um
0.236 um
0.52 μm
100 X 1.40
OIL
90 um
0.140 um
0.236 um
0.56 μm
N. A. = Numerical Aperture A.U. = Airy Unit Confocal Optical Sections for our objectives were calculated at 514nm with the following formula:
Where:
λem = emission wavelength PH = pinhole diameter (μm) n = refractive index of the immersion liquid
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WORKING DISTANCE: The working distance of a lens is the space between the front element of the objective and the top of the cover slip. Any space between the cover slip and the tissue adds to the working distance.
Changing Objectives: Use the two square buttons on the left side of the microscope 18 Touching the upper button changes to the next highest magnification objective. Touching the lower button changes to the next lower magnification objective. When either key is held in longer than 0.3 seconds then the display jumps to the next magnification every 0.5 seconds. The nosepiece doesn’t turn until the key is released. The objective in the light path is indicated on the front panel of the microscope. Be careful not to put the 63X GLYCEROL objective into an oil drop on your slide.
Setting the Focus Step Size The focus step size (S0, S1, S2, S3) is indicated on the front of the microscope. The "STEP" button on the front of the microscope changes between the fine step sizes (S0-S3)
About the piezo-stage The galvanometer driven stage (outlined in red) is accurate to <1 nm. The minimum step size of the "galvo stage" is 40 nanometers (nm). The galvo stage has a limited travel of 170 micrometers. Check the working distance (table 1) of the objective lens in use first. Many of our objectives only have working distances of 100 microns. The weight limit of the galvo stage is 200 grams. If using a chamber that weighs over 200 grams then remove the stage using the two screws shown with arrows in the left figure. The nosepiece focus (z-wide) will then need to be used. XZ imaging can not be done with the nosepiece focus. IMPORTANT: Do not apply any pressure to the stage or the slide holder. Be particularly careful if the slide 5
holder is removed. The galvo stage can be easily damaged when the slide holder is not in place. The microscope head must be lowered when scanning to open the laser shutter. Otherwise you get the message ‘Check safety interlock’.
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Starting with LCS (software) and setting up the microscope stand LCS has several function modules. The default module at start is the Acquisition module. It is the module you need for acquiring new images.
1. Click Beam
to launch the Beam Path Setting window
Before setting up beam path parameters, you need to choose the field of view you want to image. You can either use the halogen lamp for transmission or the mercury lamp for fluorescence illumination (the latter is often easier with very low contrast samples such as cultures cells).
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2. Click and press microscope control icon MicCtrl, choose visual from the pull-down menu
3. Verify that the stage has been lowered 4. Put your slide UPSIDE DOWN on the microscope stage 5. Select the objective with the two buttons on the left side of the stand Objectives can also be changed from LCS software by OBJ icon
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6. Choose your filter-set for conventional fluorescence
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7. Open fluorescence shutter microscope stand 12 )
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or turn on halogen lamp (wheel on left side of the
8. Find focal plane and adjust focus
In addition to the focus knobs 9 on the sides of the microscope stand there are two buttons on the right side of the stand for fast focusing
9. Define focal plane as 0 by clicking on the UP button for 3 seconds. It will ask you “set?” and then “0um”. Z position is reset.
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10. Choose the field you wish to scan 11. If you plan to use the transmission PMT, turn the condenser knob on Scan
12. Click and press microscope control icon MicCtrl again, choose Scan from the pull-down menu to switch to the scan mode 13. You are ready to scan
Working inside the beam window 3
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Using pre-settings In the LCS window, double click one of the pre-settings from the list 1 for different fluorescent dyes. The upper part of the list under Leica (with the red L) is protected and cannot be changed. The lower part of the list under User holds user-modified and saved settings. By double clicking on a pre-setting, you apply all basic acquisition-related parameters allowing you to scan and obtain an image. All parameters like laser power, detector gain and offset, emission window, scan speed, image format,... can be changed later once you have obtained your first image (see below). In the Beam window you have the possibility to regulate up to 4 PMT detection channels, and up to 9 different laser lines 3 .
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(photomultiplier)
Make sure that your detection window is at least 8 nm apart from the laser lines you are using. Otherwise you might have reflection (from 413 with 405 excitation for ex.) The easiest is to open one of the Leica predefined settings (double-click) then manually change laser power, PMT gain and offset, and detection window as necessary. You can switch between original color and under/overflow-coloring with the Q-Lut icon on the display screen.
Use the Glow-U scale to regulate gain and offset of the PMT (see below).
Scanning your first image Press the continuous button
The image of your specimen will appear on the right screen.
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Most often you will need to adjust now the following three parameters: - Gain - Offset - Z-level (focus) These parameters are the key players when you get a black image screen These and other parameters can be easily changed through the 7-knob-control-box
Gain
Offset
Zoom Z Pos
The function of each knob can be defined. The most useful setup is the smart gain/smart offset pre-defined one (as shown here). To choose it, click on the little middle icon down right on the left screen and select Hardware Control.
Q-LUT or under/overflow-colouring The table uses a range of green-white-red-blue for the pixel intensity (0-255). Using Q-LUT do the following adjustments: Adjust PMT gain so that blue (detector overload) pixels just disappear (a few blue pixels in the brightest zones are OK) Adjust offset so that green (detector underload) pixels just disappear except in the blackest zones. Offset is the black level of an image and may be viewed by the QLUT as green. The image should not contain any green in areas where there is a signal and the green should be adjusted for most imaging such that it is speckled and not solid. Setting the offset too high will cause the loss of low-intensity signal and should be avoided. IMPORTANT It is not the Gain alone that is important to get the correct signal, but the couple Gain-Laser intensity. You can get the same amount of signal by setting a low laser power/high gain than with a high laser/low gain setting. But: High laser/low gain = clear image but cells damaged and fluorophores bleached Low laser/high gain = noisy image but cells and fluorescence preserved So there is a compromise to find: - With living samples and easily quenched/weak fluorophores: use low laser/high gain (Line averaging (see below) will greatly reduce the noise in the image). - With fixed sampled and strong fluorescence: you can use more laser power and reduce 11
the gain. In all cases, you should not use a gain higher than 850 as the signal gets really too noisy.
Z-Position The specimen may simply be out of the confocal plane, adjusting the Z Pos knob to move it may make the live preview much brighter. IMPORTANT Clearly focus visually on the sample before switching to the scan mode Line Average Usually it is difficult to get a clear background simply by adjusting gain and offset alone at the cost of signal intensity. Instead, you can tolerate some background noise to maintain reasonable intensity and use image average to improve SNR (signal to noise ratio) and quality of your image. Noise reduction is proportional to the square root on the number of lines or frames which are averaged. Line Average should be used to capture all final images with reduced noise. Setting the line average to either 4 or 8 lines seems to be adequate for most use. While it has not been looked at quantitatively we think that the 16 line average doesn't provide much improvement on 8 lines, especially when the time require for acquisition is considered.
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*Alternately, Frame Averaging may also be used; in doing so the microscope scans the full frame multiple times and averages those full frames. Zoom Zooming in on the image will allow the user to capture an image with much greater detail and seeing the smaller structures in a sample. It is important that one doesn't zoom beyond the optical resolution of the scope.
Using Zoom In, you can make a region of interest (ROI) and scan only in this specific region.
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Leica AOBS Confocal Maximum and "Optimum" Zoom settings Why this matters? Collecting optical information to the maximum resolution is often desired. "Oversampling" is known to cause sample destruction while not yielding additional information. Not sampling to the maximum resolution is commonly called "Undersampling". While this does not destroy the sample, the maximum resolution is not attained. Should maximum resolution be necessary, optimal settings must be achieved. Some people will not demand maximum resolution and should not concern themselves with this other than to note that they have no need to zoom beyond the maximum optimal zoom. These calculations are only important if you demand the optimum sampling size with minimal sample destruction. Why not Oversample? Oversampling will not yield increased resolution, and will simultaneously destroy the sample to a greater degree than is necessary. While oversampled images may sometimes look better, they are collected at the cost of excessive bleaching and without the gain of additional data. It is important to emphasize that oversampling with the confocal microscope is not beneficial to users demanding both high resolution and minimal sample degradation. Is undersampling always bad? Features that are clearly visible while undersampled may not necessitate the use of optimal zoom settings. Lower zoom settings may in fact be considered optimal even while the image is undersampled. A lower zoom exposes the sample to a lower amount of light and is consequently less damaging. If the lower setting achieves the desired image resolution then it is not necessary to increase the zoom in an effort to attain the maximum possible resolution. These conditions and approximate zoom settings were calculated for the Leica AOBS. The value to consider for optimal zoom is the “distance of raster points”. It is worth 1/3 of the optical resolution because 3 pixels are necessary to best display this maximum resolution.
N.A 63x and 100x 405 458 476 488
1.4 1.4 1.4 1.4 1.4
smallest optically resolvable distance (nm)
115.71 130.86 136.00 139.43
distance of raster points (nm)
38.57 43.62 45.33 46.48
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Zoom @ 512*512
12.05 10.66 10.25 9.99
Zoom @ 1024*1024
6.02 5.32 5.12 5.00
Zoom @ 2048*2048
3.01 2.66 2.56 2.50
514 543 633 40x 405 458 476 488 514 543 633 20x 405 458 476 488 514 543 633
1.4 1.4 1.4 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7
146.86 155.14 180.86
48.95 51.71 60.29
9.48 8.98 7.70
4.75 4.50 3.86
2.37 2.25 1.93
129.60 146.56 152.32 156.16 164.48 173.76 202.56
43.20 48.85 50.77 52.05 54.83 57.92 67.52
16.93 14.97 14.40 14.08 13.37 12.64 10.84
8.47 7.49 7.20 7.03 6.67 6.31 5.42
4.23 3.74 3.60 3.51 3.33 3.16 2.71
231.43 261.71 272.00 278.86 293.71 310.29 361.71
77.14 87.24 90.67 92.95 97.90 103.43 120.57
18.96 16.77 16.13 15.75 14.95 14.12 12.11
9.48 8.38 8.07 7.87 7.47 7.07 6.07
4.74 4.19 4.03 3.93 3.73 3.54 3.03
Changing scan related parameters If you wish you can set additional scan related parameters: scan Mode, scan Speed, scan Format, Pinhole size, … If this is the first scan after you launch the program, there are default values for these parameters: Scan mode: xyz Scan speed : 400 Hz Scan format: 512 x 512 Pinhole: 1 Airy unit (Most users should not change the pinhole diameter. Opening it will allow a brighter signal but will reduce the z resolution If you change these parameters you often need to re-adjust gain and offset to the new conditions (see above) Click Single scan or Series scan to get the final data acquired.
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Working in the image screen The images will appear on the image viewer at the right-side monitor (display screen). You can toggle image display mode among single channel, tiled (two or more channels), OVL (overlay), Gallery, or play series (if you have acquired z- or time-series). This can be done by clicking the respective icon on the vertical tool bar on the left side of the image viewer window. Scale bar, grids and coordinates can be superimposed on the image by checking the function overview panel on the lower left side of the LCS main window. Export has to be used to fix these markers on the image. See more at the item 18 (data saved)
If you need to print or save a snapshot (see below) for a good resolution the display window must be 1:1 and not AUTO.
TIPS When multiple labeling, you should check for spill over from any shorter wavelength channel into any longer wavelength channel (especially from the green channel into the red channel). You must use the sequential imaging mode if there is spill over (see page 20). If you can't get any laser through the system, make sure the illumination pillar is in its upright position. When it is tilted back, the laser illumination path is shut. Otherwise you get the message ‘Check safety interlock’. 16
Saving your files All images acquired are listed under Experiment overview.
Continuous scan is used for image optimization thus images acquired through it are constantly updated without fixed entry under experiment overview, and cannot be saved except for the latest one. Images are always saved as .tif files and are combined in an experiment folder called experiments *.lei. Using the save as function allows you to save all images and image series so far acquired: When you click save, you will be prompted to enter a name. It is the folder name for the experiment and it is also the first common part of file name for all images under the same *.lei entry. After the first part, there are image-name, series number, channel number, TIFF extension. All these can make very long file name if not named carefully. So, the first part doesn’t need to be specific, just use a category name and as short as possible. 17
After the experiment is saved, you can modify the second part “image-xx” to a short specific name. Other parts cannot be modified. We ask you to follow the following rules for saving: Data are saved in hard-disk D in a folder called users: In this folder every user has his/her own folder where all files are saved. For easier access of saved data we recommend to create a date folder for each session at the microscope (example: 080627 for the 27th June 2008). Inside this folder you can add several experiment folders during your session (for example on called treat the other control). After saving one experiment (for example treat) you have to open a new experiment folder by clicking the new icon in the upper left corner of the screen. The image overlay, scale bar and grids are not saved by default. To save them, you have to export: - select them by right mouse-clicking onto the image, choose export: SAVING AN IMAGE FROM THE VIEWER: Right click on the viewer and select “Send to”, which offers you three possibilities as follows: a. Selection(raw) save raw data of selected image without the addition of supplemental layers such as scale bars b. Selection (snapshot) save all information of selected image with additional layers such as scale bars. c. All (snapshot) save all images plus additions inside the viewer window. Click “Save” button to save the change of the experiment. IMPORTANT: Display Zoom affects the final resolution of Snapshots: select “1:1” and not “auto” before sending images. After saving files on the hard disk, you can turn off the system in the reversed order as you turn it on.
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Shutdown procedures
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Standby (if another user will come within 1 hour) - Turn the Argon intensity knob to zero (8) - Save images, quit Leica software, and log off from Windows. - Only if next user comes more than 30 min later: Turn off HBO mercury lamp (2) - Turn off scanner (5) - Turn off all lasers except Argon krypton (3 + 3’) - Clean all microscope objectives you have used and check also if the stage is clean - Fill out the Log Book - Lower stage before switching off the Leica control box (1) Shutdownn (if no user within next our) - Turn the Argon intensity knob to zero (8) - Save images, quit Leica software, and log off from Windows. - Turn off HBO mercury lamp (2) - Turn off scanner (5) - Turn off all lasers (3’, 7, 7’) - Clean all microscope objectives you have used and check also if the stage is clean - Fill out the Log Book - Lower stage before switching off the Leica control box (1) - Wait 15 more minutes before turning off the Argon laser cooling fan (3)
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Sequential imaging mode When multiple labeling, you should check for spill over from any shorter wavelength channel into any longer wavelength channel (especially from the green channel into the red channel). You must use the sequential imaging mode if there is spill over. First Channel: First, you have to set all parameters for the first recording method (first channel) and save the settings as an instrument parameter setting. For this purpose, click on the «Acquire» arrow symbol and on the «Beam» button to open the «Beam Path Setting» dialog window.
Choose the excitation wavelength and set the laser intensity.
Move the slider or double-click on the number and enter a value for the laser intensity. Select a «PMT» detection channel and set fluorescence dye and detection bandwidth.
Click on the list box of a detection channel, for example «PMT2», and select the fluorescence dye to be used, for example «TRITC». The emission curve of the fluorescence dye is displayed in the spectrum. 20
Position the slider below the emission curve.
Note on positioning the detection band When recording fluorescence light, take care that you do not position the detection band directly below the laser line, since thus reflected light will also be recorded. The distance between laser line and detection band should be at least 5 nm in the blue spectral range (excitation line 488 nm), 8-10 nm in the green spectral range (excitation line 543 nm) and 15 nm in the red spectral range (excitation line 633 nm).
Double-click on the slider of the selected detection channel. In the «Range Properties» dialog window, enter a wavelength for the beginning and the end of the detection band.
Click on the symbol for the color look-up table. 21
Chose a color in the color look-up table. Start the continuous scanning mode with the «Continuous» button to optimize image quality during image recording.
Set the focal plane (z-position), the detector parameters and the detection pinhole. In the «Beam Path Setting» dialog window, click on the «Save» button to save the settings of the first recording method as an instrument parameter setting.
Give the instrument parameter setting a name and click on OK.
The new instrument parameter setting appears in the list box below «U». Second channel: Next, all instrument parameters for the second recording method are set and saved as an instrument parameter setting. Choose the excitation wavelength and set the laser intensity. Select an other «PMT» detection channel and set the fluorescence dye and detection bandwidth.
Best method to save sequential scan methods It is judicious that the mechanical parts move as little as possible when switching from one channel to the other (especially with “between lines” switching, see below). So a good method is to define the position of each PMT slider from the beginning (of course without overlap) and then make, lets say, PMT2 active for saving the first method and then PMT 1 and 3 active for saving the second method. That way, the sliders won’t move when switching from one method to the other. 22
Click on the list boxes of the detection channels, for example «PMT1» and «PMT3», and select the fluorescence dye to be used in each channel, for example «FITC» and «CY5». The emission curves of the fluorescence dyes are displayed in the spectrum. Position the sliders below the respective emission curves.
Double-click on the slider of the selected detection channel. In the «Range Properties» dialog window, enter a value for the beginning and the end of the detection band.
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Click on the symbol for the color look-up table. Click on the color look-up table you want to use and on «OK». Start the continuous scanning mode with the «Continuous» button to optimize image quality during image recording.
In the «Beam Path Setting» dialog window, click on the «Save» button to save the settings for the second recording method as an instrument parameter setting.
Give the instrument parameter setting a name and click on OK.
The new instrument parameter setting appears in the list box below «U». 24
In the «Beam Path Setting» dialog window, click on the «Seq. » button in the bottom right corner.
The dialog window needed for configuring a sequential image recording appear. In the «Beam Path Setting» dialog window, copy the instrument parameter settings into the «Sequential scan settings» list box.
Highlight the instrument parameter setting in the list box in the upper right corner of the dialog window.
Click on the «Add» button in the «Sequential scan settings» field in the lower left corner of the dialog window. or Click on the instrument parameter setting, keep the left mouse button pressed and move the IPS to the «Sequential scan settings» list box (drag and drop). Click on the «Remove» button to remove the instrument parameter setting from the list. Click on the «Save» button to save the instrument parameter settings together as a sequential recording method.
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Use the «Load» button to load this sequential recording method again. In the «Mode» list box, select one of the three sequential scan modes. This selection defines when the sequential recording methods are changed.
Select «between lines» if you want to change the sequential recording method after a line has been recorded. Select «between frames» if you want to change the sequential recording method after an image has been recorded. Select «between stacks» if you want to change the sequential recording method after an image stack has been recorded. In the «Parameter» list box, check the parameters which will be used for the recording of all sequential recording methods.
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Click on the check box of the desired parameters. Next, adjust all other instrument parameters which differ depending on the type of recording: spatial image series, time image series or spectral image series. Click on the «Series» button or on the «Single Scan» button to start the sequential image recording.
If you have selected the sequential mode «between lines», you can also use the «Continuous» button to start the sequential recording. The advantage of the continuous scanning mode is that you can modify many parameters during image recording.
Click on the «Gallery» button to display the single images in the Viewer window already during image recording.
You can save an image of a single detection channel as a new data set in the currently active experiment. Click on the image in the selected detection channel. Hold the mouse pointer over the Viewer window and click the right mouse button to open the context menu.
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Choose «Selection (raw)» to save the image data of the selected image in a new file. Choose «Selection (snapshot)» to save a snapshot of the selected image in a new file. Choose «All (snapshot)» to save a snapshot of all images in the Viewer window in a new file. The new files are displayed in the «Experiment Overview» window where you have access to the following commands in the context menu:
Highlight a file of your choice and click the right mouse button to open the context menu. Select «Activate» to display the image data of the file in the Viewer window. Select «New Window» to open the currently displayed recording in a second Viewer window. This is useful, for example, when you want to visualize the same image data with another color look-up table. Select «Delete» to remove the file from the list. Select «Rename» to give the file a new name. Select «Export» to save a recording in the *.avi file format. Select «Browse Images» to display all images of an experiment as thumbnails images on the user interface. Select «Properties» to document important properties of the recording in a dialog window. To move single files from one experiment to the other (drag and drop) click on the file, keep the left mouse button pressed and drag the file to the target folder.
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Spatial image series Click on the «Acquire» arrow symbol and then on the «Mode» button to select the «xyz» or «xzy» scan mode.
Select the «xyz» scan mode if you want to record a stack of horizontal images (orthogonal to the optical axis). Select the «xzy» scan mode if you want to record a stack of vertical images (parallel to the optical axis). Select or define a setting for the beam path.
Multiple channels can be used, even sequentially (see previous chapter). Start the continuous scanning mode with the «Continuous» button to optimize image quality during image recording.
While in Continuous mode, click on the «Series» button and open the «Series Scan Overview» dialog window to define begin point and end point of the spatial image series.
Scan mode: xyz
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Scan mode: xzy To set the begin point, move the yellow square to the desired z-position (xyz scan mode) or y-position (xzy scan mode) and click the «Begin» check box. To set the end point, move the yellow square to the desired z-position (xyz scan mode) or yposition (xzy scan mode) and click the «End» check box. Or Use the right knob on the 7-knob-control-box to define the begin and end positions
Z Pos
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Click on the «Sections» button and determine the number of recordings between begin point and end point.
Select one of the values in the list or «Others». When you select «Others», the «Z/Y-Configuration» dialog window appears where you can calculate the number of recordings «#» or the distance between the recordings «Step size» depending on the total height of the image stack «Image dim.z».
When you change the number of recordings and click on «Calculate» next to «#», the step size is calculated with the priority of leaving the height of the image stack unchanged. When you change the number of recordings and click on «Calculate» next to «Step size», the step size is calculated with the priority of leaving the number of recordings unchanged. When you change the step size and click on «Calculate» next to «Step size», the number of recordings is calculated with the priority of leaving the height of the image stack unchanged. 31
When you change the step size and click on «Calculate» next to «#», the number of recordings is calculated with the priority of leaving the step size unchanged. Click on the «Series» button to start recording the spatial image series.
To improve the signal-to-noise ratio click on the «Average» button. Define how often the recording of an image is to be repeated. The average value is displayed in the result image.
or Click on the «Line Average» button. Define how often the recording of a line is to be repeated. The average value is displayed in the result image.
Click on the «Gallery» button to display the single images in the Viewer window already during image recording.
Note It has a negative impact on the system performance when you record large image data sets and use the «Gallery» function.
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For a statistical evaluation of the spatial image series click on the «Quantify» arrow symbol and then, for example, on the «Profile» button.
Clicking this button opens the «Profile» dialog window and displays a measurement section on the image.
The measurement section can be positioned anywhere on the image. In the «Profile» dialog window where an evaluation graph and various statistical data are displayed, you have access to the following additional commands:
Hold the mouse pointer over the diagram and click the right mouse button to open the context menu. Select «Export» to save the evaluation data in an ASCII text file. Select «Send to» and «Document» to save a snapshot of the evaluation graph as a new file in the currently active experiment. Select «Print» to print out the evaluation graph. Click on the «View» arrow symbol and the «Play» button to run the spatial image series as a movie. Simultaneously, the evaluation graph and evaluation data of each single image are displayed.
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You can save an image of a single detection channel as a new data set in the currently active experiment. Click on the image in the selected detection channel. Hold the mouse pointer over the Viewer window and click the right mouse button to open the context menu.
Choose «Selection (raw)» to save the image data of the selected image in a new file. Choose «Selection (snapshot)» to save a snapshot of the selected image in a new file. Choose «All (snapshot)» to save a snapshot of all images in the Viewer window in a new file. The new files are displayed in the «Experiment Overview» window where you have access to the following commands in the context menu:
Highlight a file of your choice and click the right mouse button to open the context menu. Select «Activate» to display the image data of the file in the Viewer window. Select «New Window» to open the currently displayed recording in a second Viewer window. This is useful, for example, when you want to visualize the same image data with another color look-up table. Select «Delete» to remove the file from the list. Select «Rename» to give the file a new name. Select «Export» to save a recording in the *.avi file format. Select «Browse Images» to display all images of an experiment as thumbnails images on the user interface. Select «Properties» to document important properties of the recording in a dialog window. To move single files from one experiment to the other (drag and drop) click on the file, keep the left mouse button pressed and drag the file to the target folder.
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