10:30 A – 6:00P
- Testing HP1 STORM imaging. 647 switching extremely well, extremely dense movies once optics properly aligned, no 405 required
- Had 405 base power ~30 mW instead of 1.5, might have bleached out 750 movies.
- testing 488 +/- Na Borhydride. maybe helps in reducing background, make STORM image match features / contrasts of conv image better. Need to do some more careful spot fitting to be sure. might exclude first 7-14K frames and compare. New antibody also seems to be doing better — need still to compare to old antibody stained in parallel, it might be the labeling and imaging before formamide treatment.
- Overall, all 488 images seem better than previous — maybe formamide treated cells get more background in 488?
- 750 images still not a lot of switching, not very dense image coverage. Nothing like the 647. Maybe Graham and Josh’s new approach will fix this?
- Possible fix?: stain cells with proteins in 647. Image. Stain with formamide DNA-cy5, image again. Should work out a control for alignment: maybe doing the DNA label twice on same cells and check alignment after second round of treatment. Or brief foramide treatment and successive protein stains (might work less well because of perturbed proteins by formamide, on the other hand, this might be just the control for seeing how perturbed they get.
Cell culture
- Prep S2, S2R+ and Kc167 cells for freezing. Slow feeze 24 hrs.