Add CC2-DMPE aliquots directly to medium for final concentration of 5µM (1:1000).
Vortex to distribute dye evenly.
Use DiBAC4(3) at 47.5 µM (1:4000) for cell culture, and 0.95 µM (1:2000) for whole organisms (embryos, tadpoles). Pipette double the amount of dye needed into a microcentrifuge tube. Add the same amount of DMSO and vortex. Add at least the same volume of medium and vortex again. Centrifuge at 14,000 rpm for at least 10 min. Pipette off half the contents, being careful not to disturb the pellet, and add it to the medium. Centrifuge at 5000 rpm for 10 min. Use the supernate.
Incubate in CC2-DMPE in the dark for 30 to 60 min.
Wash the cells in plain medium once or twice.
Incubate in DiBAC4(3) for at least 30 min; do not remove the dye solution. For long term imaging (e.g., time lapse), fill the agarose viewing chamber with DiBAC4(3) solution. Add the specimens, then fill the dish until the meniscus is above the rim. Carefully seal the dish with the large coverslip.
Select the specimens that are expected to have the brightest DiBAC4(3) emission to set the exposure times. These will be the ones expected to be most depolarized.
Use a brightfield image to find, and do the initial focus of, the specimen.
Determine the proper exposure for DiBAC4(3) and for CC2-DMPE.
Move the slide a little to get away from the probably bleached section.
Correct the focus using the CC2-DMPE image (the DiBAC4(3) will bleach.)
Take the picture set (one picture each of the two dyes, and any other desired images, such as brightfield or differential interference contrast). If the exposures are good, continue. Otherwise, repeat Steps 10 to 12.
Once certain of the exposures, close the shutter so no light can reach the specimen and take a picture set at the chosen exposures. These are your DARKFIELD (DF) images.
Re-open the shutter and start a live image of the CC2-DMPE.
While watching the screen, move the stage so that there is no longer a specimen in view, then lower the stage until the image is out of focus – the result is a field with nothing in it, i.e. a “flat” field. Take a picture set. These are your FLATFIELD (FF) images. (You can actually take these images at any time; you will need an FF image for every exposure you use.)
Using brightfield, return to your specimen and refocus.
Focus again using the CC2-DMPE filter set.
Take a picture set.
Image correction (Step 19) can be postponed to a later time, but should be done with the same software that was used to create the images. That is because the image (i.e. the data) might be altered when moving between software packages.
Correct the images. Each raw data image needs to be corrected using the image arithmetic function (meaning corresponding pixels in two images will be added, subtracted, etc., and a new image of the sum, difference, etc. will be generated; that is the corrected image.) Raw CC2 image – DF image = DF corrected CC2 image FF CC2 image – DF image = DF corrected FF CC2 image DF corrected CC2 image ÷ DF corrected FF CC2 image = CC2- data image Repeat Steps 19.i through 19.iii using DiBAC4(3) images. CC2 data image ÷ DiBAC4(3) data image = RATIO IMAGE or DATA
