Transcript
Table of Contents 1. General Procedures 1. Nikon A1R Startup Procedure 2. Nikon A1R Shutdown Procedure 3. Startup Procedure 4. Shutdown Procedure
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2. Introduction to Nikon Elements GUI
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3. General Operating Procedure for the Nikon A1R 1. Initialize the Nikon Elements GUI 2. Select the appropriate objective 3. Verify the presence of your fluorophores and focus your sample 4. Adjust the power of each laser line and PMT 5. Register your changed settings to the 4Ch + DIC configuration 6. OPTIONAL: Adjust xy pixel size to improve image quality 7. Channel Series 8. Capture and save your image
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4. Multidimensional Acquisitions: ND Acquisition 1. Select optical configuration: Lambda (λ) 2. Z-‐Stacks 3. Multipoint XY 4. Time-‐Lapse Imaging 5. Large Image 6. ND Acquisition Order of Experiment
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5. Perfect Focus System 1. The PFS and Z-‐stacks
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6. Tips and Tricks 1. Scan Large Image 2. Move to Center, Mouse XY, and Mouse Z 3. Regions Of Interest and Line Profile 4. Ratio View 5. Magnifying Glass 6. Reuse System Settings
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7. Trouble Shooting 1. No Excitation Light: Widefield 2. No Excitation Light: Laser 3. Scan Large Image does appear correct 4. Mouse XY moves erratically 5. When in doubt, contact Joe Dragavon
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1. General Procedures 1. Nikon A1R Startup Procedure
1. 2. 3. 4. 5. 6. 7. 8.
Turn on the two power strips (1 and 2). Turn key on the laser box to the On position (3). Turn on the Computer (4). Turn on the LED light source, BACK then FRONT (5). Start Nikon Elements. When prompted, select “Nikon Confocal” and press ok. There is no password. When prompted, select “Nikon Confocal” and press ok. Make beautiful images. When in doubt, contact Joe Dragavon: Office: C315 Phone: (303) 735-‐6988
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2.
Nikon A1R Shutdown Procedure 1. Save your beautiful images and transfer them to your shared drive. 2. Exit Nikon Elements and disconnect from your shared drive. 3. Remove your sample from the stage and clean up the microscope (remove oil from the objective, shutdown the environmental chamber…). 4. Open the reservation calendar. If there is a user within 1 hour of your session, proceed to Step 9. If the next user is more than 1 hour after you, proceed to Step 5. 5. Turn the key on the laser box to the Off position (3). 6. Turn off the Computer (4). 7. Turn off the LED light source, FRONT then BACK (5). 8. Once the laser fan has stopped, you can turn off the two power strips (1 and 2). 9. Publish an awesome article. When in doubt, contact Joe Dragavon: Office: C315 Phone: (303) 735-‐6988
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3. Environmental Chamber Startup Procedure
1. If necessary, fill the humidifying bottle with DI water, at most to the MAX FILL line. DI water can be found in the hall faucet. Then, secure the cap assembly into position. 2. Turn on the LiveCell Controller Unit (on back). 3. Validate the settings on the appropriate controllers by pushing the small green SEL button below each of the digital readouts. Adjust the settings by using the arrows. Push the SEL button to register the settings. 4. Initialize the appropriate controllers by pushing the green square button next to the digital readout. The green button will illuminate when selected. 5. Open the appropriate gas cylinders if necessary. 6. Place the environmental chamber securely into position above your sample. ATTENTION! Be sure to use the appropriate stage insert. 7. Allow the environmental chamber settings to equilibrate for 30 minutes. All of the digital readouts should show the appropriate input values. 8. Acquire awesome live cell images!
ATTENTION! NEVER RUN THE SYSTEM WITHOUT WATER IN THE HUMIDIFYING BOTTLE IF THE HUMIDIFYING CONTROL IS ENABLED!
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4. Environmental Chamber Shutdown Procedure
1. Remove the environmental chamber from the sample stage and carefully set it to the side. 2. If the humidifying chamber was used, disengage the humidifying controller by pushing the appropriate illuminated green square button. The system must be allowed to run until the humidity level drops below 25%. 3. After the humidity has dropped below 25%, disengage the remaining controllers by pushing the appropriate green square buttons. 4. Close the gas cylinders. 5. Turn off the LiveCell Controller Unit (on back). 6. Publish awesome live cell images!
ATTENTION! NEVER RUN THE SYSTEM WITHOUT WATER IN THE HUMIDIFYING BOTTLE IF THE HUMIDIFYING CONTROL IS ENABLED!
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2. Introduction to Nikon Elements GUI
The Nikon Elements graphical user interface (GUI) appears to be much more intimidating than it really is. There are many buttons that can be pushed, and options to be selected, but most Users (you) will interact with a subset of these possibilities. The standard view of the GUI appears on the next page. It can be broken down into 8 sections, each of which is outlined below. 1. Image Viewing Panel The acquired images will be shown here. 2. OC Panel The OC Panel contains the pre-‐defined configurations for both laser scanning confocal (Confocal) and widefield (Eyepiece) modes. These presets can be modified to fit your imaging needs. 3. Ti Pad The Ti Pad is an interactive panel that allows for the direct control of the microscope. In general, the only portion of this window that will be used with any regularity is the DIA lamp. This icon and slide bar controls the halogen lamp, allowing you to find your sample with your eyes. 4. Filters, Shutters, and Switchers This panel allows you to open and close the epifluorescence shutter (Epi). 5. Histogram and Look Up Table (LUT) This two-‐page panel provides numerical information regarding the distribution of pixel intensities across your live or acquired images (Histogram). You can also adjust the brightness and contrast of your image to highlight specific features (LUT). This can be done manually or automatically. 6. A1plus Compact GUI Within this panel are all of the necessary controls for the laser scanning confocal such as which lasers to use, laser power, and PMT gain. 7. A1Plus Scan Area Here you can control the size of your scan area and the step size of the galvanometer (pixel size). 8. ND Acquisition This panel is used to set up and perform multidimensional acquisitions including time lapses, z-‐stacks, multi-‐points, and large images (also known as mosaic or tiled images). A1R User Manual
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Figure 1. Overview of the Nikon Elements GUI. A1R User Manual
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3. General Operating Procedure for the Nikon A1R
1. Initialize the Nikon Elements GUI
-‐Press the 4Ch + DIC configuration in the OC Panel The Nikon Elements GUI opens automatically following the launch of the software. All of the pre-‐defined optical configurations within the OC Panel are reset to their default values. However, the laser settings (in the A1plus Compact GUI) and the ND Acquisition window will remain in their previously used state. To reset the GUI, simply press the 4Ch + DIC configuration found in the OC Panel (Figure 2). Once this is done, the laser configuration should change to show that the 405, 488, 561, and 638nm lasers and DIC imaging are all active (Figure 3).
Figure 2. The OC Panel with the 4Ch + DIC configuration selected.
Figure 3. After starting the software, the lasers open with the previously used settings (Top). Selecting the 4Ch + DIC configuration will reset the A1plus Compact GUI (Bottom).
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2. Select the appropriate objective
-‐Press on the appropriate objective in the Ti Pad panel First, verify that the appropriate sample stage is mounted to the motorized stage of the microscope. Change it if necessary. There are five objectives available on the A1R and they are outlined in the table below. Click on the appropriate objective in the Ti Pad panel to select it (Figure 4). The microscope will automatically rotate the objective turret into position. Add immersion oil if necessary. PLEASE DO NOT PUT OIL ON AN AIR OBJECTIVE (10x and 20x). Regarding oil, do not put too much onto the objective. A small drop is sufficient. If too much oil is used, it can enter into the objective and render it useless. Objectives are expensive, ranging from $2,000 -‐ $11,000. Once the immersion oil is placed, place your sample and raise the objective to its appropriate position. Be careful not to touch the objective to the coverslip or sample stage. The objective may scratch or bend. If an oil immersion objective is used, raise the objective until you observe the oil touching the coverslip. At this point you are close to the focal plane. Table 1. Objectives found on the Nikon A1R Laser Scanning Confocal. Working Position Magnification NA Immersion Distance (µm) Characteristics 1 10x 0.5 Air 4000 Plan Apo λ 2 20x 0.8 Air 1000 Plan Apo λ 3 Empty 4 40x 1.3 Oil 200 Plan Fluor DIC H N2 5 100x 1.5 Oil 130 Plan Apo λ 6 100x TIRF 1.5 Oil 130 Plan Fluor DIC H N2
Figure 4. The 40x objective has been selected.
3. Verify the presence of your fluorophores and focus your sample
-‐Press the 488 Widefield Eyes configuration in the OC Panel -‐Press Eye Port in the A1plus Compact GUI To observe the fluorescence with your eyes through the oculars you must put the A1R into the widefield configuration. This is done by pressing first one of the Eyepiece predefined configurations within the OC Panel followed by selecting Eye Port from the A1plus Compact GUI. In Figure 5, the 488 Widefield Eyes configuration was selected. These steps will automatically select the appropriate filter cube and allow for the epifluorescence (widefield) excitation to occur. You samples may be found by adjusting the z position. Once the appropriate focal plane
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has been found, verify all of your fluorophores that should be present, and that correspond to the three available configurations. There is no filter set that corresponds to far-‐red dyes (i.e. Cy5.5) because these tend to be difficult to observe by eye.
Figure 5. The 488 Widefield Eyes and Eye Port options have been selected. -‐Press Eye Port and then 4Ch + DIC to re-‐enter confocal mode It is necessary to re-‐enter the confocal mode in order to observe your samples through Nikon Elements. First de-‐select Eye Port and then select 4Ch + DIC. This will automatically reconfigure the microscope for confocal microscopy.
4. Adjust the power of each laser line and PMT
-‐Deselect all of the laser lines except for one Deselect all of the lasers except for one by clicking the √ next to the lasers you wish to disengage in the A1plus Compact GUI. It is best to manipulate the settings for one laser at a time as to reduce the chance of photobleaching your sample. Start with your brightest fluorophore. The 488nm laser is selected in Figure 6. Then, press the Scan button at the top of the A1plus Compact GUI.
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Figure 6. Deselect the laser lines leaving only one active. Then, press the Scan button (Left). This will produce your first image in the Image Viewing Panel (Above). Be sure to press the continuous AutoScale button on the top of viewing panel, indicated by the red arrow. Pressing the Scan button will initiate the A1R and begin real-‐time confocal scanning. The system will continue to scan until the Scan button is pushed again. Be sure to select the continuous Autoscale button at the top of the viewing panel (indicated by the red arrow in Figure 6). Adjust the height of your objective to find the focal plane as it may differ from what was observed by eye. You should be able to distinguish your sample as you approach the focal plane. Once the appropriate focus has been found, check for pixel saturation. -‐Check for pixel saturation Pixel saturation can be identified in two areas (Figure 7). One is location is within the Histogram and LUT panel. Select the Histogram tab. This feature will show the frequency of all the possible intensity values found within the acquired image. If a circle appears in the upper right corner of the histogram plot, then there are saturated pixels present. Another method for identifying pixel saturation is to press the Pixel Saturation Indication button above the actively acquired image (or press Ctrl+Shift+S). Any saturated pixels will appear as a complimentary color (i.e, pink in green, white in blue, teal in red).
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Figure 7. Saturated pixels can be indicated by the presence of a circle in the upper right corner of the Histogram tab (Left) or as a complimentary color in the image if the Pixel Saturation Indication button has been selected (red arrow, Below).
-‐Adjust the pinhole, laser power, and PMT gain to maximize signal while avoiding pixel saturation The pinhole needs to be set to 1 airy unit (AU) in order for a truly confocal image to be acquired. Nikon suggests a pinhole diameter of 1.2 AU as a good compromise between confocality and signal. The physical diameter of the pinhole depends on the designated laser (Figure 8). The Nikon A1R uses one pinhole for all of the lasers. Thus, it is recommended that you set the pinhole to the laser that will provide you with the most relevant/important information. In general, designating the 488nm or 561nm laser for the pinhole calculation will prove to be a good fit for all the necessary laser lines. The designated laser can be selected from the AU calculated for: drop box (1 in Figure 8). Set the appropriate pinhole before adjusting the PMT gain (noted as
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HV by the software; 2 in Figure 8) and the laser power (3 in Figure 8). The suggested workflow is as follows: 1. Select the laser for the pinhole calculation and adjust the pinhole. 2 Set the gain (HV) to 100. 3. Adjust the laser power until no saturated pixels are observed. In general it is best to work with a higher gain and a lower laser power than the inverse. The gain should be set between 80 and 120. Above 120 you run the risk of amplifying noise and artifacts. The laser power should be between 1 and 5, though this depends on the strength of your fluorophore. 1
2 4 3 Figure 8. An example of modified pinhole (1), PMT gain (2) and laser power settings (3) to avoid any saturated pixels. The Offset (4) is used to reduce the reported value of background pixels.
The Offset setting (4 in Figure 8) is used to reduce the reported value for background/empty pixels. This can be adjusted to bring the background values as close to 0 as possible, though this is usually necessary. A pixel value of 0 is not realistic and should be avoided since such a value indicates that real data is being removed. If two images are to be mathematically treated (i.e. a ratio calculated, see §7.4), a pixel value of 0 will lead to erroneous results (unless you are Chuck Norris, who can divide by 0). Similarly to saturated pixels, undersaturated pixels (those with a value of 0) will be indicated in the upper left corner of the Histogram plot and/or appear blue within the image (Figure 9). A1R User Manual
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Figure 9. Undersaturated pixels will be indicated by a circle in the upper left corner of the Histogram plot (Left) and/or by the presence of blue pixels if the Pixel Saturation Indication is selected (red arrow, Bottom).
5. Register your changed settings to the 4Ch + DIC configuration Any modifications to a given laser line that you wish to use must be registered to the modified Confocal configuration. In the example shown here, the settings of the 488nm channel have been modified. When any modification is made, a red exclamation point (!) appears next to the modified configuration (Figure 10). To register the modification, press the small white arrow just to the right of the configuration. The ! will disappear and the changes saved. If this step is not carried out, then any changes will disappear if a second configuration is selected. A1R User Manual
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Figure 10. The presence of a red exclamation point (!) indicates that the predefined configuration has been modified (Left). Pressing the small right arrow to the right of the modified configuration will assign the current settings to the configuration (Right). Notice that the color representation of the configuration also reflects the currently selected channel. -‐Repeat the above steps for each laser line to be used Each of the above steps should be repeated for each laser line that is to be used. This will ensure that your combined image will be optimally illuminated and the highest contrast achieved while avoiding any saturated pixels. Be sure to register your modifications for each individual laser line used. Finally, activate all the appropriate lasers, register the modifications to the configuration, and acquire an image.
6. OPTIONAL: Adjust xy pixel size to improve image quality The Nikon A1R uses a 512 pixel x 512 pixel image frame by default. While this is adequate for most users and applications, the apparent image quality can appear to be lacking. To improve upon this it is useful to increase your sampling rate, or your xy step size. This can be increased in two ways. The first is to select the Nyquist XY in the A1plus Scan Area (Figure 11). Pressing this button will tell the computer to calculate the optimal sampling step size in xy for the given objective. The Nyquist sampling rate represents allows for the optimal spatial resolution (in xy) for your sample to be achieved. As such, your sample will be properly sampled. Once the Nyquist XY button is selected, the scan area will decrease substantially as the pixel size is reduced (green box in Figure 11). Increasing the number of scan steps taken will increase the area of the scan while maintaining the Nyquist sampling rate. Alternatively, the scan area can be manually changed by modifying the Zoom factor or by dragging the corner of the scan area. The pixel size will adjust automatically to the size of the modified scan area. The indicator box will become red, and you will need to Right click on the box to accept the modification. Similarly, Left-‐clicking and dragging the box into the desired location can move the location of the A1R User Manual
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scan area. Again, the indicator box will become red and you will need to Right click on the box to accept the modification (Figure 13). Figure 12. The size of the scan area (green box) in the A1plus Scan Area panel will automatically adjust if the Nyquist XY button is selected. The size of the scan area is dependent upon the number of steps taken in x and y (labeled as Scan size) and the objective used as this last variable is used to calculate the optimal pixel size. Fewer steps (i.e. 512; Left) will result in a smaller scan area compared to more steps (i.e. 2048; Right). A1R User Manual
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Figure 13. Clicking and dragging the corners of the green box, adjusting the Zoom factor, or by clicking and dragging the box itself can manually modify the location of the scan area. The modified box will appear in red (Left). Any modifications need to be accepted by Right-‐clicking on the box (Right).
7. Channel Series By default Nikon Elements will acquire all of the activated channels simultaneously. This optimizes the acquisition rate of the microscope. There are instances where this can be a problem due to the existence of spectral bleed through. For example, imagine a scenario where your DAPI signal is very strong but your GFP/Alexa488 signal is very weak. As a result you use a high gain setting for your GFP. DAPI has a very broad emission spectrum, with its tail extending out to nearly 600nm. As a result of your high GFP gain, you can actually observe the DAPI emission within your GFP channel. To overcome such an obstacle it is necessary to acquire each channel independently. To do this, select the icon labeled Ch Series in the A1plus Compact GUI (Figure 14, highlighted in green). This will open a new window titled Line Channel Series Setup. Here you can set the order of the line scans for each channel. The system is flexible in that you can have each channel acquired independently, or you can have them paired. Often it is A1R User Manual
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sufficient to have channel 1 (DAPI) acquired separately from the others, which are acquired simultaneously.
Figure 14. Channel Series setup. Using the Channel Series allows you to avoid spectral bleed through and creates optically cleaner images.
8. Capture and save your image
Pressing the Scan button within the A1plus GUI will allow for the continuous acquisition of your confocal image. To stop the scan, simply press the Scan button a second time. Press the Capture button if a single acquisition is desired (Figure 15). This will generate an independent acquisition window. Captured images are not automatically saved.
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Figure 15. Scan and Capture controls from the A1plus Compact GUI.
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4. Multidimensional Acquisitions: ND Acquisition
Multidimensional acquisitions such as multiple pre-‐defined configurations (λ), z-‐stacks, multipoint, time series, and large scans are configured within the ND Acquisition panel (panel 8) located underneath the Image Viewing Panel. Select the check box next to the corresponding multidimensional acquisition to activate the configuration tab.
1. Select optical configuration: Lambda (λ)
It is possible to define and use multiple optical configurations within the Lambda (λ) ND Acquisition tab (Figure 16). At least one optical configuration must be identified if the Lambda (λ) tab is activated. Using this tab will ensure that the multidimensional acquisition uses the channel settings that you defined. Further, this tab will only use the registered configurations. Thus, if your preset has been modified such that a red ! is present next to your configuration (i.e. Figure 10), those modifications will not be used within the multidimensional acquisition. Additional optical configurations may be added by checking the grey box below the defined optical configuration (Figure 16, Top) or selecting the + Add button. It is recommended that you complete the Path and the Filename for your acquisition, as, under this mode and contrary to using the single Capture control, the images will be automatically saved (Figure 16, Bottom). The Path represents the location that your file will be saved to. This can be completed manually or by selecting the Browse… button. Once this information is entered, press the Run Now button (bottom right corner of the ND Acquisition Panel) to acquire the image.
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Figure 16. Individual or multiple optical configurations can be used for a single acquisition by selecting the Lambda (λ) tab within the ND Acquisition panel. At least one optical configuration must be selected if this tab is used and additional configurations added by selecting either the grey box underneath the already entered preset or by pressing the + Add button. The file Path and Filename should be entered into the appropriate fields to ensure that the acquired images are saved to the correct location.
2. Z-‐Stacks A z-‐stack takes advantage of the confocality of the A1R to acquire 3D images of your samples. There are multiple ways to define the parameters of the z-‐stack (Figure 17): 1. Defined top and bottom 2. Symmetric z-‐stack 3. Asymmetric z-‐stack A1R User Manual
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Figure 17. The parameters of the z-‐stack can have a defined top and bottom (start and end; Top), be symmetric around a central plane (Middle), or be asymmetric around a given plane (Bottom). A1R User Manual
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While each z-‐stack mode has its differences, there are some parameters that will remain the same for each, including the z Step size (Step), the number of z Steps to be acquired (Steps), and the Z Device used (either the Ti ZDrive, Step-‐by-‐step Nikon A1 Piezo Z, and the Nikon A1 Piezo Z Drive). Step: The size in µm that the Z Device will move in between each acquisition Steps: The number of total Z steps to be acquired Z Device: The z direction controller. The Ti ZDrive will use the objective turret to control the z position (this is most commonly used). The Step-‐by-‐Step Nikon A1 Piezo Z and the Nikon Piezo Z Drive can only be used if the piezo Z stage insert (the one for the environmental chamber) has been mounted to the microscope. This will give greater z control and stability but is usually not necessary for most users. In between the Step and Steps fields lies an active button with a z distance (noted as 0.200µm in Figure 17). This is akin to the Nikon XY button found within the A1plus Scan Area but it applies to Z. Pressing this button will automatically adjust your Step size and the number of Steps necessary to complete a z stack while ensuring the proper sampling frequency of your object. Most users will want to define the top and bottom of their sample, thus this configuration will be discussed in detail. The remaining methods follow similar principles and are relatively straightforward to figure out once the defined top and bottom is understood. For the following protocol, please use Figure 17 (Top) as your reference: A1R User Manual
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1. Select the left-‐most z stack icon allowing you to define the top and bottom of your z stack. 2. Press the Reset button, located between the Top and Bottom icons. This will remove all the previously used settings. 3. Select the appropriate Z Device (Ti ZDrive typically). 4. Find a central, in focus plane for your sample. 5. Deselect all of the different laser lines (channels) within the A1plus Compact GUI except for one. It is recommended that you leave the channel of most importance, or the one that will most help you define the top and bottom (i.e. a membrane-‐ bound fluorophore) active. DO NOT REGISTER THE CHANGE TO THE PRESET. 6. Select the single Autoscale button (to the Right of continuous Autoscale, Figure 6). 7. Press Scan in the A1plus Compact GUI (Figure 15). 8. Adjust the z focus up until the image is barely visible. Mark this position by selecting Top in the z stack panel. If you are not sure which way you are going (up or down), you can look at the LCD panel on the microscope as the z position is displayed. 9. After selecting the top, move the z wheel in the opposite direction, passing through your central focal plane, until the image again is barely visible. Mark this position by selecting Bottom in the z stack panel. 10. Press Scan (A1 Compact GUI) to stop the lasers. Reactivate all of the relevant laser lines (the red ! next to your optical configuration should disappear). 11. Define the Step size or the number of desired Steps. 12. Press the Run Now button to start the z stack. A progress bar and estimated acquisition time indicator should appear.
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3. Multipoint XY
For time-‐lapse imagery it is usually of interest to follow multiple points (i.e. multiple cells) throughout the course of the experiment. The Nikon Elements software with the motorized XY stage allows you to save multiple regions of interest. This is done under the XY tab of the ND Acquisition panel (Figure 18). This panel will show the physical location (x and y) of each point. Selecting Include Z under the point list will show the z position. Additional points may be added by moving the stage to a new position and then clicking + Add (Please see § 6.2). Press the Run Now button to begin the acquisition.
Figure 18. Multiple XY positions can be entered and sequentially imaged by using the ND Acquisition panel.
4. Time-‐Lapse Imaging
Time-‐lapse imaging is extremely useful for the observation of cellular and subcellular kinetics. The parameters found within the Time tab allow for significant control over the acquisition rate (Figure 19). The tab is broken down into 4 sections: Phase, Interval, Duration, and Loops: Phase: The time sequence to be followed. Multiple phases can be established such that after one is completed the next ensues. This can be useful if the kinetics you are trying to observe change over time.
Interval: The length of time of one image cycle.
Duration: The total amount of time for the Phase.
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The four fields allow for significant flexibility. For example, if the phenomenon you want to observe happens at some point between 1 and 2 hours post stimulation, you can start with a Phase that includes an acquisition Interval of 15 minutes for a Duration of 1 hour, followed by a second Phase with an acquisition Interval of 5 minutes for a Duration of 1 hour. Thus your sample will be exposed at a higher frequency only when your event of interest is occurring, reducing the phototoxicity experienced by your sample. Selecting the down arrow of the Interval and Duration fields will allow you to change the units of the time (or you can type 10s for 10 seconds, h for hours, m for minutes, ms for milliseconds). Further, you can select No delay (Interval) and Continuous (Duration) in order to acquire images as fast as possible until you tell the acquisition to end (select Finish within the acquisition window). Selecting No Acquisition will indicate the software to proceed with the time but to not acquire any images during this Phase. You can also have the software perform a Time Measurement (intensity over time) by checking the Perform Time Measurement box in the lower right corner of the Time tab. Additional Phases may be added by clicking + Add. Press the Run Now button to begin the acquisition.
Figure 19. The Time tab within the ND Acquisition Panel.
5. Large Image The Large Image tab allows you to acquire a large set of images around a central defined point (Figure 20). The Scan Area can be defined by the number of fields or in size (mm). Here, 1 field is 1 acquisition. Thus a 4 x 4 field size is actually a 4 x 4 matrix of acquisitions (16 in total). The images can have a defined amount of Overlap, typically 10%. The overlap is necessary to mathematically align the images, called A1R User Manual
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Stitching. Stitching works best when there is a lot of detail (i.e. many cells). It is difficult to properly align images that lack significant detail or have large portions with no information. A stitched image will be represented by a single acquisition where as a non-‐stitched image (Do Not Stitch) will result in an image file with each acquisition. These images can be stitched later using other software packages such as ImageJ/Fiji. Large Images can be used in conjunction with multipoint XY to acquire substantial amounts of data similar to High Content Screening (HCS) technologies.
Figure 20. The Large Image tab within the ND Acquisition panel.
6. ND Acquisition Order of Experiment
The individual tabs within the ND Acquisition panel can be used in essentially any combination that is useful for your experiment. As such it is possible to acquire z stacks over time for large images at multiple points using multiple optical configurations. In other words, you can generate a lot of data. Selecting the Order of Experiments tab can modify the order of which the tabs are accessed depending on what is most ideal for your experiment (Figure 21). The order flows like a mathematical operation from the inner most parentheses outward (or, more simply, from right to left). For example, in Figure 21, the Order of Z series(Lambda(Large Image)) will first do a large image, then change the lambda, and finish with a z stack. The XY position and time series are not listed as the time will be continuously progressing while the stage moves from point to point. Also worth noting is that step for each active tab is indicated above the Path and Filename. T: Time lapse steps M: XY positions Z: Z stack steps λ: Lambda; Optical Configurations L: Large Image matrix size
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Figure 21. Possible Order of Experiments within the ND Acquisition panel.
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5. Perfect Focus System
The loss of focus during a long time-‐lapse acquisition is a common occurrence and greatly hinders your capability to do longitudinal studies. Nikon’s Perfect Focus System (PFS) uses a near-‐infrared LED and a line CCD to find the surface of the coverslip, ensuring that the distance between the coverslip and the objective remains constant throughout the course of the experiment (Figure 22). This greatly reduces z-‐drift for time lapse and large scan images. The PFS is ideally suited for live samples that are kept in low refractive index media such as cell culture media or PBS. Fixed samples in high refractive index mounting media (such as Prolong Gold) tend not to be compatible with the PFS. This is due to the fact that little to no reflection of light occurs between a coverslip and the mounting medium because their respective refractive indices are too similar (1.5255 for glass, 1.41 – 1.49 for many mounting media) whereas the refractive index of water and cell culture media (DMEM + 10% FBS) are 1.33 and 1.34, respectively. Without the significant difference of in the refractive index, the light from NIR LED does not reflect back to the line CCD. While true for high power immersion objectives (40x, 100x), this does not appear to be such an issue for low power air objectives (10x, 20x). The Nikon PFS can be activated by pressing the appropriate button on the microscope itself or within the Ti Pad in the Nikon Elements software (Figure 23). Upon activation, the PFS will attempt to locate the coverslip by detecting the reflected light on the line CCD. The line CCD is a traditional CCD camera, but with only 1 array of pixels. If the reflected light returns to the CCD, the z position of the objective will automatically be adjusted to bring the reflected signal to the center of the CCD. At this point the Z wheel of the microscope is no longer activated and the PFS offset controller (Figure 24) must be used to adjust the z position of the objective. This is because you are manually adjusting the position of the Offset Lens (Figure 22). As you adjust this offset, the position of the objective will change in order to bring the reflected LED light back to the center of the Line-‐CCD. The working distance of the Offset Lens is fixed, meaning that the PFS will not be compatible with very thick samples. A very useful java applet can be found at: http://www.microscopyu.com/tutorials/flash/focusdrift/perfectfocus Or, just put Nikon Perfect Focus into Google and you will find it quickly.
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Figure 22. A schematic representation of the Nikon Perfect Focus System. The light emitted by a NIR LED is put into the optical path. The reflected signal from the coverslip returns and is detected by a Line-‐CCD. The PFS Offset controller adjusts the Offset Lens, which, in turn, causes the objected to adjust its position in z.
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Figure 23. The PFS can be activated by pushing the appropriate button on the microscope itself (Top, red circle), or on the Ti Pad within Nikon Elements (Bottom, red circle). Upon selecting the PFS, the button will beep and immediately illuminate (green). However, if the coverslip is not found the diode will begin to blink. Adjust the z focus using the z wheel on the microscope until the PFS engages (there will be a second beep and the PFS indicator will stop blinking and remain illuminated). At this point the z wheel on the microscope will be deactivated and the PFS controller will need to be used (Figure 24). A1R User Manual
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Figure 24. The PFS controller is located next to the microscope is used to control the PFS Offset Lens. The speed of the offset can be adjusted by pressing the blue button. The In position will have a fine adjustment, while out will be coarse.
1. The PFS and Z-‐stacks
The PFS can be used in combination with z-‐stacks. However, the symmetric z-‐stack (§4.2 Figure 17, Middle) must be used as the absolute z position will be lost due to the PFS. During a z-‐stack, the PFS will disengage, and then reengage once the acquisition is completed. This is useful if z stacks over time are to be acquired.
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6. Tips and Tricks 1. Scan Large Image The Nikon Elements software will allow you to scan a large image without the need to run through the ND Acquisition panel. This is useful when you are looking for a cell expressing a particular protein or in a given state. Similar to the Large Image acquisition within the ND Acquisition panel, you can decide the number of frames to acquire and if your current position should be the center or the top left corner of the Large Image. (This last feature is not available from the ND Acquisition panel, and I do not know why) This works very well with the PFS as your entire view will remain in focus. The Scan Large Image feature can be found under Acquire within the tool bar (Figure 25).
Figure 25. The Scan Large Image feature can be found under Acquire within the tool bar (highlighted in green). Upon selecting Scan Large Image, the Scan Large Image control panel will appear (Figure 26). Here you will be able to define the size of your large image (2), the stitch percentage (3), and if your current location should be the center or the top left corner (2). BE SURE TO VERIFY THAT THE OBJECTIVE IN THE SCAN LARGE IMAGE CONTROL PANEL A1R User Manual
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IS THE SAME THAT YOU HAVE SELECTED FOR YOUR ACQUISITION WITHIN THE Ti PAD (Figure 26, 1). If this is not done, the microscope will automatically adjust to use the objective that is selected within the Scan Large Image control panel. This can be very annoying. Once you have verified all of the settings select Scan (Figure 26, 4). Once the scan is complete, close the control panel. A full stitched image will appear (Figure 27). The individual fields are realigned to correctly indicate the overlap between each, resulting in blue areas on the outside of the Large Image. This does not imply that data has been lost, but that the individual images have been aligned.
1 2
3
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Figure 26. The Scan Large Image control panel will appear once the option is selected from Acquire within the tool bar. Be sure to verify the objectives to be used (1), define the number of fields to be selected (2) and if the current position is the center or the top left corner of the scan (2), the percentage of overlap (typically 10%, 3). Finally, press Scan (4) to start the acquisition. As an added benefit, the resulting Large Image is interactive since the xy coordinates of the image are registered. If you physically move the sample the xy registration will be lost. This will be expanded upon next.
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2. Move to Center, Mouse XY, and Mouse Z -‐Move to Center Right-‐clicking on the Large Image or the active Scan window will bring up many options. One of the most relevant is Move this Point to Center (Figure 27, in green). By selecting Move this Point to Center, the motorized stage will automatically adjust its position to bring the selected position to the center of your scan area. This is very powerful when combined with a Large Image as you can easily redirect the microscope to center on the region of interest that you have identified without using the joystick. You also have the option to Add this Point to ND Acquisition for multipoint XY (see § 4.3). You do not need to move the point to the center for this option.
Figure 27. By right-‐clicking on a region of interest you can bring up a number of controls including Move this Point to Center (highlighted in green). A1R User Manual
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-‐Mouse XY You can manually move the stage in xy during a live Scan by selecting the Mouse XY icon (Figure 28, in red). Activating this option will turn the mouse arrow into a different icon, indicating that you can control the stage. Left click and hold on a given area on the screen, and then drag the mouse in any direction. The stage will move correspondingly. This is useful when you need to fine-‐tune the position of the stage.
Figure 28. The Mouse XY icon (in red) can be found above the active Scan window and will allow you to control the stage movements from the computer instead of using the joystick. -‐Mouse Z The z position of the objective can also be modified from the active Scan window. Left click on the screen to activate the interaction, and then slowly scroll using the mouse wheel and the objective will move accordingly. If you move the mouse wheel too quickly the software can have trouble tracking its movements. Using the mouse wheel to adjust Z will allow you to fine tune your focal plane with potentially finer control than using the z wheel.
3. Regions Of Interest and Line Profile -‐Regions Of Interest Regions of Interest (ROIs) can be added to your Scan image and will carry over onto your ND Acquisition for real-‐time analysis of the fluorescence intensity. ROIs may be added in two ways. The first is to press and hold the Right click on the mouse. After a brief period a set of options will appear around the mouse curser. You can then release the Right click and select any of the available options. From here you can create Rectangular, Polygonal, and Circular ROIs (Figure 29, Left). A1R User Manual
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Alternatively, you can press the down arrow next to the second square on the acquisition window side bar (Figure 29, Right). Similar ROI options will become available.
Figure 29. Multiple ROI options will appear by pressing and holding the Right-‐click for a brief period (Left). Alternatively, you can select the down arrow next to the second square on the side bar of the acquired image (Right). Similar ROI options will be available. -‐Line Profile The 7th icon from the top (the one that looks like a sine wave) will generate a line profile of your image (Figure 30, highlighted in green). A yellow arrow will appear representing the orientation and direction of the line profile. Clicking and dragging on the ends of the arrow can modify the location and orientation of the line profile. The corresponding intensity profile plot will automatically be generated and can be exported to multiple formats.
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Figure 30. Selecting the indicated icon from the image side panel will generate a line profile. Clicking and dragging the ends of the arrow will modify the orientation and location of the intensity profile.
4. Ratio View
It is possible to observe a real-‐time pixel-‐to-‐pixel intensity ratio image during a time series acquisition. Right click in an empty area on the bottom information bar of an active acquisition to bring up the Ratio View options (red x, Figure 31, Top). Select Ratio View to add this additional viewing option. Selecting Ratio Properties opens a second window where the numerator and denominator can be designated, as well as the color scale set (Figure 31, Bottom).
x
Figure 31. Right clicking on an empty space on the bottom information bar of the acquisition window will activate a ratio view of a time sequence (red x, Top). From the same command window you can select ratio properties in order to define the numerator and denominator of the ratio view.
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5. Magnifying Glass A local zoom can be produced on an image by selecting the magnifying icon from the left-‐side tool bar (Figure 32) or press “z” on the keyboard (you can switch back to the pointer by pressing “p”. Left click on the image to activate the magnification. This is very useful to examine Large Scan images (§4.5 and 6.2).
Figure 32. The Magnifying Glass tool allows you to easily zoom in on your image. This feature can be found on the left-‐side tool bar or by pressing “z” on the keyboard. Left click on the image to activate the magnification. You can switch back to the pointer by pressing “p”.
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6. Reuse System Settings You can reuse your system settings from previously acquired images by opening the files within Nikon Elements. Right click on the opened image to view the list of secondary options. Near the bottom will be several Reuse options: Reuse Camera Settings, Reuse Device Settings, and Reuse ND Setup (Figure 33). Unfortunately there is not a “Reuse All” option so you will have to right click on the image up to 3 times to recall all of the appropriate settings.
Figure 33. Several Reuse options can be found on previously acquired images. Right click on the image to find the options near the bottom of the list.
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7. Trouble Shooting 1. No Excitation Light: Widefield Be sure the LED lamp is powered on, Front and Back. Be sure the Epifluorescence shutter is open (Filters, Shutters, and Switchers Panel, Epi). The LED lamp will not power on if the liquid waveguide (the black cable that is coming out of the front of the power box) is not properly positioned. The waveguide slips out of position from time to time. Ensure it is properly in place by gently pushing the cable back into the box. Do not push too hard as it is possible to crack the waveguide.
2. No Excitation Light: Laser Be sure that the lasers are turned on (check the position of the key). Be sure that you are in the confocal mode (using a pre-‐defined optical configuration in the OC Panel) and that the light path indicates the L100 port (Ti Pad).
3. Scan Large Image does appear correct Sometimes the objectives can lose their calibration settings indicating. This can lead to the stage moving inappropriately and renders the Large Image useless. If this occurs, contact Joe Dragavon.
4. Mouse XY moves erratically This occurs when the objective loses its calibration settings. If this occurs, contact Joe Dragavon.
5. When in doubt, contact Joe Dragavon
Office: Phone:
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