Fluorescence filter problem

[Kathryn Hedges-2]

This isn't about C. elegans per se, but I got the filters to view
CFP/YFP in worms, and I know there are a lot of other folks in the C.
elegans community using fluorescence microscopy. I'm probably going to
cross-post this to some photography, C. elegans, and microscopy groups
because I need help quickly.

I bought a set of used dualband fluorescence filters for CFP/YFP
(Chroma 51017) on eBay, installed them in a filter cube per the mfr's
instructions, and put the cube in the scope. (Zeiss Axioplan 2, the
old style with 5 cubes instead of 8.) Instead of seeing a dark field
with just the fluorescent cells glowing cyan or yellow, I saw a solid
yellow-green field. Same thing if I looked at plain glass slide
without anything to autofluoresce, so it isn't just the agarose pad or
something.

The most likely culprit was the dichroic, so I flipped it over in case
I'd installed it wrong. No help. I went through all the combinations
of flipping filters and dichroics, exchanging filters, and the only
thing that happened is that when I exchanged the excitation and
emission filters, the light coming out the lens changed from violet to
yellow. (I'm presuming violet is correct because the excitation
wavelength needs to be shorter than what the fluorophore emits.)

The "outside" face of the exciter looks like someone left a smear when
cleaning it, although when I look through it, it looks fine (so the
coating is probably OK).

Since the set is used, and the seller got it with a used microscope
his store purchased from a customer, there's a possibility the
dichroic isn't the right one. I hope not...

If anyone can help, I'd truly appreciate it. I'm behind schedule for
some research I'm trying to present at Model Organisms on Jan. 5-8
(and I have a batch of dual-color worms ready to observe).

I forgot to mention this in my other versions of this posting, but Jeff
at Chroma has been fantastic. On Thursday I told him about getting this
used filter cube from someone else that didn't fit my scope, and he got
me the right cube by Saturday--and I'm on the opposite coast from
Chroma.

--Kathryn



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