Thursday 07/24/14

9:45 am – 11:00 pm


  • XZ meeting prep
    • updated plots of y-internal and b-internal
    • multicolor update
    • low photons
    • sequential method I
    • sequential shadows
    • toe hold probes
  • Sequential staining
    • test S3 first stain
    • test wash out with toehold S3toe5
    • test restain with S1
  • Fig prep / data analysis
    • crop y-internal regions for fig
  • comments on manuscript (see email)

Sequential Staining

  • heating objective
  • heating wash buffer
  • adhere beads to sample
  • make new STORM buffer
  • Imaged ~8 regions of E09-P3
  • Piezo not on — some notable drift may have happened 🙁
  • attempting to remove S3 probe with S3toe5 at 2:1000 hybe dil buffer for 30 min at 37C (imaged cells during incubation)
  • most of the decrease in signal appears to be due to bleaching, judging by near-by non-imaged regions.
  • attempting to remove S3 probe with S3toe5 at 10:1000 in hybe dil buffer for 30 min at 37C
  • second treatment at 1:100 dilution works! Took some images.
  • washout with 50% formamide 2x SSCT at 37C ~10min
  • hybe S1 at 2:1000 hybe dil buffer at 37C
  • washout stain, 20 min
  • add new STORM buffer and image. Dots are nice and bright
  • Dots are perfectly clear in previously imaged regions, but a bit dimmer. I think the high (330 mW 647 laser) may not be ideal.
  • spots not imaged don’t actually STORM that much better (imaged after rnd 2 imaging completed).
  • maybe just a longer hybe and higher secondary concentration would be better?

Analysis of Sequential Stain data

  • L2F03F04 from 2014-05-29
    • hybe2 mostly failed (due to laser damage?)
    • some images have weak staining

F03F04seqHybe hybe2laserDamage


  • treating 1 PH-M 1 PH-Wt and 2 S2 coverslips
  • 30 min in 5% FA + 1/2x PBT for post fix
  • PBS wash
  • 30 min in PBS + RNase A
  • wash in 2X SSCT
  • prehybe in 50% formamide + 2X SSCT
  • stain PH-M, PH-Wt, and S2 with 750-BXC
  • stain second S2 with 750 L3E09

Data analysis

  • Merging old and new BXC data
  • be careful, need to change source-folder for internal scaling data for BX-C back to old
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