Wednesday 01/30/13

9:30 A – 6:30P, 9:00P – 11:20P

STORM

Samples:

BX-C cy5. Imaged 4 loci before 3P. Turned microscope over to Doory. Will setup to image more loci overnight
Fab7 405, cy5. Image tomorrow night?
Ubx Cy5. Store at 4C in 2x SSCT

Shuffling some 400 GB folders around between different 2Tb external hard-drives via usb2 is slow and requires notes:
Move 2012-11-06_inv from Alistair7 to Alistair5 (keep all embryo resin data together on Alistair5, keep cell staining together on Alistair7)
Move 2013-01-24-BXC to from Alistair5 to Alistair8 (to make space for _06_inv data) then move it to Alistair7 (to consolidate)
move existing 01-29 and 01-30 data off of STORM2 onto Alistair8

comparing run times on test data 647_HP1_storm_002.dax (102,723 frames) on Tuck. insightM runs in 100.2 minutes. Found 199,476
GPU on Monet ran in 24.5 minutes, found 440,053 (without linking frames or drift correction). Max overlapping = 4 molecules.
DaoSTORM with 2 iterations ran on Tuck in 55 minutes, found 261,000 molecules
DaoSTORM with recommended 20 iterations runs on Tuck still running after 7+ hours. update tomorrow? currently frame 70,000, 1.36 million molecules. (threshold too low. Cancelled, reset threshold to match insight, rerunning O/N).
DaoSTORM completes on Tuck, found 544,793 molecules in 139.7 min
GPU on Tuck seems to have crashed in matlab, finding consistently 0 molecules. (should optimize parameters specifically for this dataset though). Need to get GPU running on Tuck again.
Started writing GPU3D function (inside SR_demo directory since it has several dependencies).
Starting to parse/translate Z-fitting parameters. Still need to test on beads the quality of fit.

Collect virgin 2x mat alpha tub
Flipped hs-en, SCM/TM3, and Pc[1]/TM1 into new bottles.
screen males for esc[6]/Sp — actually selecting for Sp, some 2nd generation flies in here have lost the Sp allele? (only alleles are esc, CyO, and Sp, so everybody who’s not CyO should be esc or Sp, except for 2nd generation recombinants who have linked esc and Sp and made a wildtype chromosome? Anyway most males still look good, we should get some appropriate Esc nulls). Tossed all vials of Esc[6]/CyO x Sp/CyO.
set up two vials of crosses of esc[6]/Sp x Df/esc[2],CyO

checked 3 coverslips of embryos sections with DAPI. all have only fragments of embryos — problem with curling and fracturing of sections during cutting. Very few embryos have large enough sections in tact to clearly distinguish posterior and anterior. But at least we can see if the BX-C probe sticks and makes foci. Can later try co-staining with Ubx / abdB protein…
etching previously embedded 330 nm resin-sectioned embyros to test DAPI staining.
1 uL of 100 uM 2x-cy5 secondary + 2 uL of 48 uM BX-C cy5, mixed in 80 uL of DFISH hybe mix and added 25 uL per slide.
48 uM = 48 pico-mol/uL 100 uM = 100 pico-mol/uL. ~ 33 picomole/slide of BX-C and ~33 picomole/slide of 2xCy-5 secondary.