9:30 A – 7:30P, 8:30P- 12:00A
- Software install on STORM2. Hal can’t detect drivers for national instruments panel.
- Overview of software install: see post
- Collect virgins
- Sort and cross Espl/sim flies
- Clear MTDs into 2 new bottles, move existing bottles to virgin collection
- esc flies from Vivek stocks arrived today, thankyou Kasia. Moved to stocks for expansion.
- Order fly stocks
- Fix embryos
- collect PM virgins
embryo DNA FISH
- preliminary test of post washed Ubx-cy5 embryos shows no evident staining on turnkey.
- counterstain with Hoechst and WGA-488
- labeled cent-dig with rb-K27ac, m-Dm01, shp a-dig
- # check on confocal
STORM imaging of s2 cells
- 2D imaging in glox, 40,000 frames H3 still lots of switching (it’s all H3 in the cell all in the same view (well from a optical TIRF section), so this probably isn’t going to stop.
- switching is extremely dense in 647, first 1000 frames have substantial amount of overlapping emission. Try this on multi-fitter? Also need a higher threshold — some non-switching background spots (these should help the drift correction though). About frame 15K hitting single molecule emission more often (maybe 20K). Still no 405.
- clean over 100,000 frames, still no activator, all spontaneous switching
- 200,000 frames, still no activator needed. suspect switching looks sluggish?
- 561 extremely sluggish switching, suspect laser power issue, must check. Okay, speeding up a bit after 10,000 frames. Probably buffer rundown, been driving glox pretty hard, container is completely open
- 488 okay switching? some reasonable background, probably better than embryonic tissue though. should do a double label control or something to test this better.
- Powers; 405 = .88mW, 488=100mW, 561=90mW 657=130mW, 750=60mW
- bead calibration. new bead slide, 1.7 vis to 1 uL IR beads. A little too dense on the vis, try 1.5 to 1. 1:20,000 a little dense. Try 1:10,000 and 1:15,000
- make pyranose oxidase for longer switching. 2.9 mg in 50uL im buffer + 50uL catalase for 100x stock
- Move camera back. Realign exit path optics: using 512-quadview bead field center for brightness and focus.
- 3 rounds of automated runs using parameters from first run. Try Pyranose — seems to be switching better, should figure out some way to compare in parallel. (or add to identical samples, seal, and check after longer period of time).
- Microscope off, 11:45PM