STORM2 dye and buffer testing

optimizing buffer conditions for deep 3D imaging of A647 and A750 labeled chromatin

First buffer

  • 750 and 647_storm_0001 – 0004
  • 1/2x BME (new)
  • 2 mM COT (new, reg strength)
  • 350-380 mW 647 laser (2960 mA)
  • 2100 mA of A750
  • 1% 561 laser (35 mW) for 750 imaging
  • up to 3% 561 (35 mW) for for 647 imaging

Observations

  • A750 switches dark quickly, (this makes getting good conv images difficult)
  • beads stay alive easily throughout imaging

second buffer

  • 750_G1_storm_0005
  • 0.2 mM MEA (1-2 mM is standard)
  • 2 mM COT
  • 2200 mA of A750. Increased to 2350 mA (~max power)
  • 3-4%

Observations

  • A750 switches dark quickly, (this makes getting good conv images difficult)
  • 540 beads not very bright with 1% laser power. Up to 3% laser power to see
  • beads bleach much more easily
  • A750 MUCH brighter. More switching (or at least detected switching too).
  • A750 very responsive to 405, clearer response.
  • A647 defintely much slower turning off, even at 500 mW.
  • suspect more A647 dyes bleach without being detected in this mode.
  • A647 also seems less responsive
  • Overall though this seems to give better image pairs.

Sample observations

  • lots of A647 spots on slide F11 don’t have 750 counterparts. G1 looks better. Labeling balance between dyes not so good…
  • Maybe P3 / S3 is worse combination? I think it is worth trying out other Primaries.
  • F11 A750 spots stained without A647-P1 look great in conventional imaging (undetectable on most cells when stained together).
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