Some Atto-532 In A MiSeq
Today I decided to throw some Atto-532 in my MiSeq as see if I could use it as a regular epi-fluorescent microscope. I slapped some Atto-532 on some slide glass roughly cut to the right size and stuck some cover glass on it. Then jammed it in a MiSeq flowcell plastic carrier:
I tried to scratch up the surface a bit, so it wasn’t just totally unstructured. The cover class I’m using is 100 micron. Ideally this should be 300 micron to work with the MiSeq objective. I’m told a nice person should be bringing me some 300 micron cover glass next week. So perhaps I’ll get to try that too.
Anyway. I was able to manually focus on the surface, and take images in the Y direction. Took some work figuring out how to do this…
The images I took are at the top of the post. But just for fun here are some more from a different part of the slide:
So images appear brightest in T, slightly dimmer in G and almost nothing registers in A and C. G and T being close and on the same emission (green) source is consistent with statements from Illumina.
Atto-532 is a good match for green LED used in the MiSeq as we previously saw. We’re also expecting some cross talk between dyes as we1 previous showed in our independent image analysis/base calling pipeline:
In particular we’re expecting the G dye to leak into the T channel. So I think this is consistent with the Atto-532 being a good match for the G dye… I think…
This would make Alexa 568 the T dye? Want me to try that next? Consider a paid subscription.
For some unfathomable reason I seem to have started using the royal we? At least in this case I generated this plot and wrote most of the software…