General Procedure for Image Acquisition using Volocity

• clean the slide before use to avoid refracting light if necessary
  
  

  (gently wipe with chemwipe, do not use cleaning solution)

  
  
  

  
  

1. Turn on the scope, turn on the computer (windows)
2. Open Volocity once the scope AND camera are on.
3. Save as new library (2013_04_02 Volocity).
4. Calibrate the stage in the XY stage view:
1. lower the stage away from the objective.
2. stage-> calibrate stage
5. Look through the eyepiece of the scope to find the tissue under transmitted light and focus.
1. control of fluorescent shutter is on the computer as well as on the box #3
6. Starts off in black and white until you choose a channel to pass the light through
7. click on a channel
8. double click on the lime-green rectangular box (to the right of the snowflake) to open “Acquisition set-up”
1. change name (2013_04_02_001_primary_fluor)
2. select the number of channels
3. change focus using (NONE) if not taking z step
4. stitching
1. change XY using Ludl XY stage
2. dont correct for shading
3. auto align field
4. correct for brightness
5. save raw tiles
6. 12% overlap
9. make sure you keep the shutter closed as much as possible to avoid bleaching
10. click autoexpose to start to find the appropriate exposure (you can also change gain and offset to improve the signal)
11. click the floppy disk on the top right to save the channel settings
12. switch to the XY stage view and select an area, scan selected area to get a sense of where we are in the section
13. switch back to Live view to correct exposure
14. re-check the acquisition set-up
15. in XY stage view, select desired region and record