Volocity Standard Imaging Procedure Take 2

1. Turn on the hardware, turn on the computer (windows), then launch Velocity
2. Save as new library (yyyy_mm_dd_Volocity).
3. Calibrate the stage in the XY stage view before putting slide on:
1. lower the stage away from the objective.
2. stage-> calibrate stage
4. Look through the eyepiece of the scope to find region of interest under Fluor light and appropriate filter. Focus.
1. control of fluorescent shutter is on the computer as well as on the box #3
5. Switch light path to camera (pull out post). Make sure camera is set to monochrome, Volocity is set to Mono and Fluor (2 different pulldown menus)
   Starts off in black and white until you choose a channel to pass the light through
6. click on a channel, choose light path appropriate to the fluorophore
7. double click on the lime-green rectangular box (to the right of the snowflake) to open “Acquisition set-up”
1. change name (yyyy_mm_dd_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
8. make sure you keep the shutter closed as much as possible to avoid bleaching
9. in Live View, do a quick focus if necessary, then click Autoexpose to start to find the appropriate exposure
   a. start with DAPI channel
   b. look for brightest area or most intense staining when using Autoexposure
   i. Try to keep exposure between 50 and 250 ms (not always possible)
   ii. If exposure is too long, increase gain to reduce time
   iii. Offset controls black level, generally positive (+) value, should show some tissue 
   features while leaving ventricle black
10. click the floppy disk on the top right to save the channel settings
11. switch to the XY stage view at 5x and select an area with marquee tool, 'scan selected area' to get a sense of where we are in the section
12. switch back to Live view to correct exposure
13. re-check the acquisition set-up
14. in XY stage view, select desired region and hit record (usually at 10x or 20x)

   15.  Fill out Image Acq Log for each image before taking the next
                a. most relevant info for image, like total acq time, summarized in the "experiment log" tab
   16. Multichannel: add channels in acq setup
                a. Use a different light path for each channel
                b. Set exposure for each separately
                     i. use brightest area for each
                     ii. save before moving to the next channel