- 750: 104 mW
- 647 : 150 mW
- 561: 150 mW
- 488: 150 mW
- 405: 1.5 mW
- 750 switching nicely.
- 647 very bright conventional images. Under high laser power image still inverts — low nuc singal high background. Background does not remain high, but not sure that localizations are coming from nuclei.
- 561 much like 647. poor localizations
- not fully etched from 647 / 561 view.
- lower laser power doesn’t excite the ‘resin?’ background as much and can still switch maybe.
- Etched new slide rad21 for 3 hrs, some of sample beginning to detach. Identically treated 2H (had more 70% EtOH rinse at the end but otherwise identical), is completely gone after 3 hrs.
O/N run of H2. (K27ac-750, H3-647, K27me3-Cy3B ).
- 75o channel behaves as expected.
- Not sure about H3 and K27 in this sample. Still worried about resin.
- Dyes do photoswitch in all channels, lets see how it looks tomorrow.
new slide rad21
- 647 keeps switching for quite a while (51K frames and counting) We’ll see how clustered it is. 750 looked alright but didn’t switch very long. But I think I exhausted the 750 H3 secondary with repeated reuse.
- 561 Lots of bright switching events but not sure pattern is correct. Dot shape is more irregular.
single section of young embryo Dm01 (nice round nuclei).
- 647 very strong for finding embryos.
- 750 looks good but sparse number of localizations.
- 647 going strong, good for 20K frames
- 561 didn’t look very good by confocal in resin block. Briefly looks like some dye is localized during initial activation. plenty of switching. Dots irregular size and shape though — might be background. Higher threshold contrast looks better.
- 488 looked strong by confocal in resin block. Let’s see if we can turn this into a reaonsable channel.