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).