TITLE: Confocal imaging on Nikon AXR confocal microscope
AUTHORS: Andrew Hunter

[DESCRIPTION]
This protocol provides a step by step protocol to acquire images on Nikon AXR confocal set-up. 

[STEPS]
SECTION: Confocal set-up
1. In this order: Turn on power to microscope stand (15seconds to power up), power on the controller by pushing the button and waiting for the standby lights to go green, then provide power to the lasers by turning the activation key
SECTION: Confocal set-up
2. If using CAM, begin reservation to receive access to the computer and software
SECTION: Confocal set-up
3. Ensure connection to R drive for data storage
SECTION: Confocal set-up
4. Select and load the NIS-Elements AR 5.41.02 64-bit software
SECTION: Confocal set-up
5. Set lightpath to Eyepiece – EPI in order to locate brain slice
SECTION: Confocal set-up
6. Turn on epifluorescent light to 50-75% intensity, look through eyepiece in order to see brain slices
SECTION: Confocal set-up
7. Once slice is observable, change lightpath to AX
SECTION: Confocal set-up
8. Check for lasers of interest and make sure that they are highlighted and in use (488: 499-551) (561: 571-625) (640: 663-738)
SECTION: Confocal set-up
9. Check the pinhole size (aperture) make sure it is set to 1.0 AU
SECTION: Confocal set-up
10. Scanner set to Galvano with scattered sampling Channel Series for fixed tissue imaging
SECTION: Confocal set-up
11. For scanning set resolution to lowest/fastest value at 256x256 with maximum field of view to make identifying slices and finding target regions as fast as possible
SECTION: Confocal set-up
12. Adjust Z using knob to find slices and find depth of greatest intensity
SECTION: Confocal set-up
13. Open up “Look Up Table” to view saturation histograms for each channel
SECTION: Confocal set-up
14. Locate the highest intensity field in your region of interest
SECTION: Confocal set-up
15. Use AutoSignal.ai (set to Fixed/Robust for mounted slides)
SECTION: Confocal set-up
16. Use LUTs to determine saturation of image, adjust the power and the gain of the lasers to adjust saturation; try to make the laser power as low as possible while preserving image, do not let gain exceed 50-60
SECTION: Confocal set-up
17. Once the histograms for all lasers are within acceptable ranges, begin to design scan
SECTION: Confocal set-up
18. “Acquire” -> “Scan Large Image” to pull up the large image overview user interface (UI)
SECTION: Confocal set-up
19. Using the controls, set the far left right, top, and bottom borders of the slice in order to identify the size of the image, ensure that the proper objective lens is selected in “objective” in the top left corner
SECTION: Confocal set-up
20. Note the size of the image (will be ___ x ___ fields) as well as the expected size of the image, close “Scan Large Image”
SECTION: Confocal set-up
21. Use the rendered image of the scan size and place the microscope in the center of the generated grid (if 8x8, move cursor to the point between square 4 and square 5 on both the X and Y axes, double click to move camera to center)
SECTION: Confocal set-up
22. When ready to capture image: sample at Nyquist value and 1024x1024 or 512x512 pixel resolution
SECTION: Confocal set-up
23. Go back into “Scan Large Image” the number of fields will have updated to match the new resolution
SECTION: Confocal set-up
24. Scan an additional time to ensure that the camera shifted to the center of your ROI
SECTION: Confocal set-up
25. On the side tab, select “ND acquisition – Define/Run Experiment”
SECTION: Confocal set-up
26. Select XY channel -> “Custom” will pull up the custom image size pull-out
SECTION: Confocal set-up
27. Select “Large Image” and enter scan area __ x __ fields, select the objective, and insert preferred overlap (10-15%), press “Finish”
SECTION: Confocal set-up
28. Once the ND acquisition screen has populated, set “redefine Reference Z after Auto Focus, and select “Use PFS”
SECTION: Confocal set-up
29. If Z-scanning, also check off the Z tab
SECTION: Confocal set-up
30. Use the Z adjustment to locate the top and bottom of the slice and set these boundaries using the “Top” and “bottom” buttons – WRITE DOWN THESE Z-VALUES FOR STEROLOGY PURPOSES
SECTION: Confocal set-up
31. Set the Z step value, ideally use the step suggested by the software (0.122um [or 0.2um])
SECTION: Confocal set-up
32. Press “Run Now” to begin scanning
SECTION: Confocal set-up
33. Once image is produced, it will generate a deck of images that are not scanned, save the file as “XXXXXX_RAW”
SECTION: Confocal set-up
34. Saving criteria: mouseID_genotype_hemisphere_series#_Primary_Objective#_SamplingResolution_Slice (ex m8535_Gabrr3Cre_RightHem_series3_NeuN_Nyq1024_Slice7)
SECTION: Confocal set-up
35. To construct stitched image go to “Image” -> ND Processing -> “Stitch Multipoint to Large Image”
SECTION: Confocal set-up
36. Save image with same name as the RAW file but delete “_RAW”