9:50 A -7:40P, 8:40P – 12:30 A
yari lab meeting
- De-noising: use conventional image as low pass filter to remove non-blinking background
- Use conventional alignment for drift correction, translate all STORM movies to match multi-color conventional image.
- Use field of dense beads to provide flat-field correction
Visit to Wu lab at HMS
- Need dextran sulfate in hybe buffer — “concentrates” the probe by reducing usable volume
- Pick up frozen stock of Kc167 and S2R+ cells.
- en cell-specific ChIP-seq? Ask Bender. Write to Bulyk lab?
Cell Culture Work
- Thaw and plate Kc167 and S2R+ cells.
- Let recover a little too long (2hrs) in resupension media + DMSO from freezing.
- Incubating in drying oven at 28C
Parallel cell and section labeling with cent-AATAT dye
- incubate in m-HP1a (sole primary).
- secondaries for cell culture: anti-m 488, anti m-Cy3B (best for confocal?), anti m-750, dual label m-488,750.
- for sections, m-750.
- cells ready for STORM. HP1a staining looks good on Turnkey. Should get some confocal images tomorrow (780 time in evening).
- embryo section coverslip has bacteria on it. Need to screen for what these come from. Some bacteria also detectable in cell culture, but not as many. Let’s post fix this after staining STORM imaging.
STORM
- centromeres not as bright as in previous staining, but still switching okay. Need 405 to get switching, probably only 30K good sections with full 405 activation (compared to 100K+ frames at 250mW 642 and no 405 needed in previous runs). Not sure if that is an issue with this round of staining
- 750 switching 300 times better double check that cylindrical lens is still passing light properly? (doing first pass with 2D data).
- 488 also switching much better, much lower background. Switching might not be horribly specific? Further judgement needs to await a complete image reassembly.
- TIRF 14.35. Get focused on coverslip (following bead imaging?) then choose dots with smallest offset from coverslip.
- 750 switching betterin 750 only channel (not competing with rb 488). Imaging at 1650 mA.
- Test 488 switching in unlabeled well. Substantial single molecule switching still observed. Maybe more quenching with sodium borohydride should reduce this? Should do a carefully controlled +/- borohydride experiment on fixed cells with 488 imaging in particular, and image beofre post fix. Meanwhile lets compare these movie stats.
- some 647 images get 60-80K frames of good switching. Still need 405 from early on though.
- image background not nearly what it has been, need to reset all thresholds for Spot Counter though.
Set up PCRs to repeat cross deletion PCR amplification for screening sim and Espl deletions (Espl L8R10 and simL2R1).
Flies in large cages dead. Larvae don’t look dense enough to be worth saving. Need to do some dedicated fly work. It would be really nice if the deletion PCRs check out, and we could start testing those lines.