Training Guide for Carl Zeiss AxioZoom V16 Stereo Microscope

Transcription

1 Training Guide for Carl Zeiss AxioZoom V16 Stereo Microscope ZEN 2012 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017)

2 Power ON Routine 1 2 If you require fluorescence imaging, turn ON HXP 200 C epi-fluorescence light source. 2 AxioZoom V16 Training Guide 3 If you require reflected light imaging, turn ON CL 9000 LED located directly behind AxioZoom microscope. Turn ON EMS 3 box from power switch on left side.

3 Power ON Routine Turn ON HP Z820 PC. Wait for PC to boot into Windows OS and login to zeiss user account. From the desktop, double click on ZEN application. Wait for ZEN software to complete its initialization routine. 3 AxioZoom V16 Training Guide

4 Power ON Routine On the SYCOP 3 remote, the system will prompt you to calibrate the stage. Please verify that you have NO SAMPLE mounted on the stage prior to running the calibration. Click Yes to perform stage calibration. Click OK. 4 AxioZoom V16 Training Guide The stage will move to the extreme ends of its travel range during the calibration so it is important that no sample be mounted to avoid any potential damage to the sample and/or the system. Wait for the SYCOP to complete the stage calibration before mounting sample.

5 Getting Started with ZEN 2012 ZEN 2012 is separated into three distinct areas. Image Acquisition Tools Represented by a group of blue bars each containing a series of tools for sample observation, image acquisition, image processing and system maintenance. Locate tab also contains primary image capture functions. Image Display This area is where each newly captured image will appear. Settings here allow the user to control how the image is viewed on screen. Catalog of Open Images This list displays each image that is currently open within the ZEN software. This area contains tools for saving data sets. 5 AxioZoom V16 Training Guide

6 Getting Started with ZEN AxioZoom V16 Training Guide

7 Mounting Sample on the Microscope Once the system has been fully powered on and calibrated, we can mount our specimen on the stage below the microscope objective. 1. Place sample underneath microscope objective (roughly in the center of the field of view). 2. Push eyepiece plunger IN to send light to eyepieces. 3. From the Locate tab in ZEN, select viewing mode: A. Fluorescence - from the Favorites shortcuts in ZEN, click the shortcut button. DAPI generic UV fluorescence filter set. FITC - generic green fluorescence filter set. TxRED - generic red fluorescence filter set. B. Fiber reflected light via fiber optic. Position the LED fiber optics from the CL9000 manually for optimal illumination. C. BF transmitted light mode. Note: ensure glass reflected light base is inserted in stage prior to using transmitted light functions. 4. Using stage and focus controls, position (X,Y) and focus (Z) your sample via the eyepieces. 7 AxioZoom V16 Training Guide

8 Mounting Sample on the Microscope 5. When suitable location has been found, pull eyepiece plunger OUT to Camera. 6. Switch ZEN from the default Locate tab to Acquisition tab. 7. Change camera plunger to match camera selected in software. IN = AxioCam IC (Color Camera) OUT = AxioCam HR R3 (B&W camera for fluorescence) 8 AxioZoom V16 Training Guide

9 Using AxioZoom to Collect Fluorescence Images 1. From the Experiment Manager choose 3ColorBFMono from the dropdown list. Note: The experiment 3ColorBFMono is designed for 3 fluorescence colors (red, green & blue) plus a brightfield image. Choose only the tracks which match the fluorophores you want to image. 2. From the Channels dialog, place a check next to each of the tracks you wish to use. In this example, we will use Track 1 (Texas Red) and Track 2 (EGFP). 3. Pass your mouse over Track 1 and click to highlight it. 4. Click Set Exposure to measure the correct exposure time for that particular channel. Note: Set Exposure is an automatic exposure measurement taken directly from the area of the sample exposed. Generally speaking, if you have reasonably bright fluorescence then this function works well but it may need to be fine-tuned. If your exposures are dimmer than required you can either manually increase the exposure Time slider or increase the Shift % and run Set Exposure again. If your exposures are brighter than required you can either manually decrease the exposure Time slider or decrease the Shift % and run Set Exposure again. 5. Repeat steps 3 & 4 for each Track (or color) you have. 6. When the exposure time has been measured for all Tracks, click Snap to collect an image with all colors. 9 AxioZoom V16 Training Guide

10 Using AxioZoom to Collect Color Images 1. From the Experiment Manager choose Brightfield Color (AxioCam IC) from the drop-down list. Note: The experiment Brightfield Color (AxioCam IC) is designed to only control the camera. It requires that you define your lighting, etc. manually and leave it on when switching over from the eyepieces to the camera. 2. Click Set Exposure to measure the correct exposure time. Note: Set Exposure is an automatic exposure measurement taken directly from the area of the sample exposed. Generally speaking, if your lighting intensity is reasonably bright then this function works well but it may need to be fine-tuned. If your exposures are dimmer than required you can either manually increase the exposure Time slider or increase the Shift % and run Set Exposure again. If your exposures are brighter than required you can either manually decrease the exposure Time slider or decrease the Shift % and run Set Exposure again. If after setting the correct exposure the colors in the image do not look correct, it may be necessary to correct the White Balance of the image. 3. Click Live to begin collecting a new image. 4. From the Acquisition Mode dialog, click Auto from the White Balance settings. Note: The Auto feature works reasonably well on most samples. You may need to use the Color Temperature slider to optimize the image to taste. 5. Click Snap to collect a final image. 10 AxioZoom V16 Training Guide

11 Using AxioZoom to Collect Z Stacks To collect serial sections (Z) we need to first activate the Z-Stack tools in ZEN which are hidden by default. 1. Check Z-Stack from the experiment options. This will activate the Z Stack dialog in the Multidimensional Acquisition settings. 2. Click Live to begin collecting an actively updated image. 3. Focus the drive in one direction (does not matter which direction you start with) until the image of your sample just goes out of focus. 4. From the Z-Stack dialog, click Set First to mark the start position. 5. While continuing to image, focus the drive in the opposite direction until your sample comes back into focus, then continue focusing until it goes out of focus again. This time on the other side of the sample. 6. Click Set Last. 7. Click Optimal to set the interval size to the optimal size based on the selected objective. 8. Click Start Experiment to initiate your Z-Stack. Note: The focus drive will always operate against gravity for maximum reproducibility. On a stereo microscope, if you mark the point furthest from the objective as Last the system will automatically start from Last and go to First. 11 AxioZoom V16 Training Guide

12 Using AxioZoom for Tiling & Stitching To collect tiled images, we need to first activate the Tile tools in ZEN which are hidden by default. 1. Check Tiles from the experiment options. This will activate the Tiles dialog in the Multidimensional Acquisition settings. 2. From the Tiles dialog, click Advanced Setup to begin defining your tile regions. Note: The Advanced Setup will open a preview window that allows you to interactively define your tile regions from the Live image display. The idea here is that we will define the tile boundaries from which ZEN will calculate the number of tiles required to cover the area selected. 12 AxioZoom V16 Training Guide

13 Using AxioZoom for Tiling & Stitching 3. Using the XY joystick, move your stage position to the left most area of the sample. Position the left edge into the center of the live image field of view. 4. Using the Tile Region Setup tools located directly below the image display, activate Contour. 5. Select the square contour tool. 6. In the image display, click and drag to draw a yellow box around the live preview image. 13 AxioZoom V16 Training Guide

14 Using AxioZoom for Tiling & Stitching 7. Using the XY joystick, move your stage position to the right most area of the sample. Position the right edge into the center of the live image field of view. Note: Use the F8 key on the keyboard to zoom the display out so you can see both the current location AND the original left marked location. (F7 to zoom back in) 8. Click & Drag the right edge of the yellow bounded box over to the opposite side of the right edge live image. 9. Using the XY joystick, move your stage position to the top most area of the sample. Position the top edge into the center of the live image field of view. 14 AxioZoom V16 Training Guide

15 Using AxioZoom for Tiling & Stitching 10. Click & Drag the top edge of the yellow bounded box over to the opposite side of the top edge live image. 11. Using the XY joystick, move your stage position to the bottom most area of the sample. Position the bottom edge into the center of the live image field of view. 12. Click & Drag the bottom edge of the yellow bounded box over to the opposite side of the bottom edge live image. 15 AxioZoom V16 Training Guide

16 Using AxioZoom for Tiling & Stitching Note: Once you have completed all four edges your total tile area has been defined. You can fine tune this by double-clicking each independent image area (marked in red) to move the live image to that location. Use this to verify your sample is within the boundaries of the tile area. 13. Once tile locations have been verified, close the Advanced Setup imaging window. 14. From the Options menu in the Tiles dialog, set your Tile Overlap to between 10%- 20% to start. Note: The more detail you have in the overlap seams, the lower you can set your tile overlap percentage. 15. Ensure that Stitching During Acquisition is disabled. 16. Click Start Experiment to begin tile acquisition. Notes for Optimal Tiling & Stitching When creating tiled images, you may notice that each tile region has distinct intensity differences around the periphery of each tile, resulting in an undesirable checkboard pattern in the final image. Shading corrections are used to eliminate this problem but these corrections must be applied to the camera BEFORE the tiled image is acquired. For Fluorescence Imaging shading corrections can be done with a uniform dye slide (ask OiVM staff for sample). For Color Brightfield Imaging shading correction can be acquired from an area void of any sample. Defocus slightly to eliminate any dust or debris from the stage. It is also critical that white balance be accurate and done before tiling is begun. 16 AxioZoom V16 Training Guide

17 Using AxioZoom for Tiling & Stitching When the tile scan has completed, we will need to stitch the result together to form a single seamless image. 1. From the Processing tab, select Stitching from the method tab. 2. Select New Output from the method parameters. 3. Check Fuse Tiles. 4. Choose stitching parameters: Edge Detector if your sample has strong, contrasted edges then setting this function to Yes may help the quality of the stitching. Minimal Overlap set to the same Tile Overlap percentage as image was collected. Max Shift set to twice the Tile Overlap. Comparer Use Best to get most elaborate comparison between overlap regions. This will slow down the stitching but provide the best stitch. Global Optimizer set to Best so all overlap regions are utilized during stitching. 5. Click Apply to begin stitching image. 17 AxioZoom V16 Training Guide

18 Saving Images Once your image collection is complete you can save your data to the local hard drive. Along the right-hand side of the ZEN software (Region 3, page 6) you will find the list of images currently opened in ZEN. 1. Select the image you wish to save from the list of open images on the right-hand side of the screen. 2. Click to save the image. 3. Navigate to the local D:\ and select the folder with your name. If there is not one please create one. 4. Choose the Save as Type to CZI. Note: The default format for the ZEN system is CZI. This file format is essentially a TIFF file that contains all the requisite system information in the file header. This will allow functions such as Reuse to work correctly. 5. Give your file a name and click Save. 18 AxioZoom V16 Training Guide

19 Shutting Down the System 1. Close ZEN 2012 application. 2. Shutdown Windows OS. 3. Press Power button on EMS 3 box and wait for microscope to shutdown (see Power ON Routine, Step 3). 4. Turn off CL 9000 LED (if applicable) (see Power ON Routine, Step 2). 5. Turn off HXP 200 C (if applicable) (see Power ON Routine, Step 1). 19 AxioZoom V16 Training Guide