9:00 A – 8:00 P, 9:00 P – 10:45 P
General
- (8:00 A allergy test)
STORM analysis
- Getting images of relative background levels in 3-Cy5 vs 405-cy5
- Drift correction makes sparse images look horrible (even the rather long integration of 2000 frames).
- Some images look sharper without drift correction (generally true if image is detectably blurry). Some sharp images look a little more blurry without the drift correction
- Overall STORM-activator summary. Results look nice by design has some confusing aspects. Needs to be repeated.
- Try with index-matching media (60% sucrose, 5-10% glucose, Tris + Glox + BME). Hopefully focus lock still works. May not get any gain from TIRF without index mismatch
- Summary of initial Ph results
Coding
- QuickLoad on STORMrender not working because hObject is different for each function, and guidata(hObject,handles) needs to write to the correct structure.
- fixed. Need to call in opening function after QuickLoad:
handles = guidata(hObject); guidata(hObject, handles); - Added analyze region to STORMfinderBeta. (Needs STORMrender to open it of course). A bit slow to cut out region of big video. Whole cut-out still needs to be read into memory in order to save as a dax with current ReadDaxBeta / WriteDAXfiles approach.
Meeting with XZ
- Doesn’t like the idea of 3D imaging 1 um from surface.
- SHOULD have said: 3D imaging is actually necessary, if only to have a better idea of which molecules to discard. in 2D imaging molecules 600 nm above the focal plane are (A) substantially spherically abberated and (B) still appear in the image.
- Drift is an issue with sparse images. XZ recommends feducials. Problem when out of focus.
- Test my two proposals: Feducials stuck to cells and same channel image biological feducial like nuclear lamina.
- does not remember suggestion about photo-switchable oligmerization mutant at all. relation to ‘new’ project?
- Probe prep improving. Yes, heard about exonuclease, assymetric PCR, gel-free electrolysis etc (did not discuss).