9:50a – 11:59p
Ph Project Cell staining
- hot wash post-hybe
- PBS wash
- Block 1 hour in 2% BSA
- primary stain rb a-Ph for 2 hrs RT, 6 hrs 4C
- rinse, wash in PBS, reblock,
- secondary stains O/N at 4C (m a-lamin 488 + dk a-rb A647)
- rinse
STORM
- record max power from all primary laser lines on STORM2
- imaging dual color F05-A647 F06-A750.
- initial spots started quite bright, by 11:30p are pretty dim. Probably buffer issues
- not sure why buffer issues always seem worse / last for less long in dual color imaging. Maybe the low BME?
Exploring feducial drift correction
It appears averaging the beads and doing no smoothing on the positions gives the finest results.
Integrating on top of 60 frame averaging makes these beads worse (previously thought it was worse without this).
Averaging all bead movies in latest data set (136 movies) at 60 frames combined.
Code changes
- substantially modified drift correction
- only integrates frames if the sampling rate = 1
- if there are more than 2 feducials, it uses the maximumally auto-correlated one as the guide dot, and it’s next most autocorrelated one to estimate drift error
- if there are only 2 feducials, it uses the bead that moves the least.
This version aligns beads quite tightly.
Aligning beads in sequential images
Align so that two beads match as best as possible (assuming these are the beads that didn’t move at all and compare to these other beads).
Compare best 2 match vs. best affine match:
Really does seem like the beads move.