Monday 05/05/14

9:00a – 5:00p

Lab Meeting

  • Guest speaker, see post.

Multicolor STORM more trouble-shooting

  • observed that 647 images look particularly weak, especially given the brightness of these spots.
  • 15 min of max 405 completely bleaches the 647 spot. (~50K frames).
  • 561 laser at 10% is .45-5 mW at back objective at 35 mW power source.
  • 405 laser at max power is .45 mW at back objective
  • 40 min of 461 at .45 mW (10% AOTF, 35mW source set), results in about a 2/3rds reduction in brightness of the spots.
  • should test 488.
  • imaging depth — 750 now appears above instead of below the 647 channel? (at least in test image, 750 spot visible in same x-y position when offset is 200 above the 647 position). This is very weird, the beads don’t seem to have this offset. maybe a

Lessons

  • area bleaching is stronger / larger destroyed region in 2 color samples. space out the fields of view more.
  • 647 greatly-greatly diminished after 750 imaging. Long time at max 405 power appears to be the cause.
  • can’t excite simultaneously — too much blead-through.
  • just keep the 405 low and image for longer.

Project 2,

  • see protected post

Data Analysis

  • Start processing of data on Cajal and Tuck for L3F07, L3F08, L3E04, L3E04E05, and L3E06E07

Coding

  • resumed work on integrating new ChromeWarp method into STORMfinder
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