Monday 03/31/14

9:45a – 12:25a

Chromatin Project

Microscopy sessions!

STORM2 Lib2 mutlicolor imaging.

  • make fresh MEA
  • make fresh Glox
  • check stains, find a good stain to image Wed or Thur night.
  • if cells and spots are dense 10 regions can be a good start.
  • substantial Microscope Alignment issues
    • using an iris appeture in place of the objective and the spot on the ceiling, it is clear that the main column is way out of line.
    • the camera is well off of line relative to the main objective, because after aligning the iris and the roof spot so we have a clean aligned column coming up from the dichroic mirror through the objective, the field looks pretty flat but is cut almost in half by the apetures in the quadview.
  • The TIRF controller is crazy on this setup and needs to be replaced – it starts moving of its own accord at random.
  • imaging sample F7-6 F6-7
    • spots very bright and strong in 647 channel
    • cells very sparse on this coverslip 🙁
    • 750 spots hardly detectable in conventional image, very high background. I swear I have gotten much much brighter 750 spots.
    • STORM of 750, do see spots, just also have lots of background. Tons of switching, sometimes bright. On average way less bright than I was getting earlier with this MEA COT .5:1x buffer mix. * Not sure what that’s about. Maybe this COT aliquot is dead.
    • need to compare G2G3 control
    • G2G3 control conventional images still pretty faint, readily detectable but not too strong. Not like previous 750 images.
  • For comparison, here’s a 750 conventional selected at random from the 2/15 dataset. Not sure if that day was particularly good anyway, but the spots are clearly brighter (max = 1000 in this image).

A750_spots_140215

STORM3 imaging with Pallav

STORM1 single-color imaging Lib3 E01

  • checked alignment of back-optic, looks reasonable. Adjusted somewhat to center on roof spot. (should have cranked the pinhole before the defocusing mirror down to shrink the beam spot for greater sensitivity).
  • checked alignment of dual-view in bypass mode to camera, also looks reasonable.
  • Conventional images look great
  • STORM not as bright or switching as fast as desired.
  • Lots of swtiching, easily 200K frames. molecules not as sprightly as desired. spotfitter seems to make these look smaller than they look on the screen based on the frequency of overlapping dots. This sample is also definetely one for DaoSTORM. Overlapping double activations quite frequent.
  • Potential issue with EO1 identity — locus region and name don’t match properly in library table online. need to check this.
  • Position 3 or so loses focus in during scan. This O/N is probably not going to work too well :(.
  • Maybe I should try STORM 3 for more laser power and just use the blead-through beads (I think I can get these to show up just fine).

STORM4

  • continue running project 2 experiments and controls
  • see notes

Commitments

  • Accepted paper to Review. Due in 10 days.

Fly work

  • Flip fly stocks! (postponed today).
This entry was posted in Summaries and tagged , . Bookmark the permalink.