I acquired this snap with full pinhole opened, and no samples on stage. Laser used 647nm. Can anyone comment on this?
Hi @Deepak_Jangra1 ,
I have no actual concrete idea what’s going on, but here’s what I’m thinking for what it’s worth.
The pattern seems to appear independently of the pinhole size (img 1 vs img 2)
The pattern looks like an XY cross section of an out-of-focus PSF. So an unfocused point particle somewhere in the light path? It looks like an out-of-focus point sample in front of the objective. Is there something on the lens? Are you using a really high NA lens with accidentally some impure oil residue on it?
The image noise also has an unusual (to me) grid pattern. It reminds me of a stitched image… Don’t know how using a point scanner on a single FOV would cause this grid!
What if you raise/lower the transmitted light condenser above the stage, is there some kind of reflection happening? Are you using the correct beam splitter (probably it has to have this laser wavelength in the name)?
Does it appear in all objectives / does it move when you slightly turn the objective? What if you use a different dichroic/beam splitter if available (unlikely cause I think)? Is the system using an emission filter or some other way of selecting the spectral band, which could have a piece of dust on it?
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