[Protocol] Mounting and imaging

Monday, March 25, 2013 9:21 AM

Before mounting

Measure the power coming through the objective with the power meter, and record it Mount 15 or so embryos Use a big enough piece of paper to put the embryos on You shouldn't need to fold the paper to get the embryos off onto it When you put bleach on the embryos, try de-clumping them gently with the tweezers Try lighting the water bath with embryos in it from the side with the flexible light like Hernan does o Try using the small brush, sweeping up one embryo a time from the water, and aligning the embryos as soon as you put them down on the membrane Really, try aligning the embryos on a grid o Keep sucking water off of the membrane so that it won't cling to the embryos, etc. Put four drops on the coverslip, and blow on the slide to get rid of any air bubbles Put the coverslip on slightly off-center so that you can suck oil away from the edges (Play around with this - I couldn't figure out from Hernan why this should work) o Try setting the coverslip on the embryos very quickly so that the embryos don't have a chance to float around! Don't drop it, though Use a Kimwipe to suck oil from the sides of the coverslip, flattening the embryos; suck from multiple sides until you can't suck anymore or until the embryos are about to burst Unless you've decided otherwise, put the PMT gains on the new settings [for testing the red cups, put the sensitive PMT on ch2 and set it to 830 mV, I'd say] Before starting the stack acquisition, take a whole-embryo image to compare to the one that you take post-imaging, so you can see whether there was drift Try to image as close to the anterior pole as you can Don't average [2P1] Re-initialize all variables in the stack window in order to get them to be applied [2P1] When changing z-height, change the listed height in the coordinates window and then redefine position 1, which is the position the stage will go to first when taking the stack of 10 frames Next time, measure the time it takes the embryo to pass through *every* cycle Always compare your image to the NCReference page so you can be sure what nc it's in! Crank the power up (not too high, however, lest the embryo explode) when you acquire the final images so that they won't misstitch o Take them at zoom 2.0 (or as low of a zoom as you can get while still having fluorescence at the edges) o Make sure the final images are at the same rotation angle as the movie o Make sure that, by eye, it looks like the left and the right sides of the embryo stitch together in ch2! Now, investigate the other embryos on the slide to see if you can take any movies of them; when you're done, take one set of flat-field images for every rotation angle that you imaged at

Mounting

Imaging

Taking the final images

After you're done taking those images, then take two flat-field images at the same rotation angle as the other images! Turn the PMT gains back down to zero before turning them off Measure the power coming through the objective with the power meter, and record it