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Hi,
I am trying to image GFP labelled viral-like particles on a high sensitivity
level with a Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat
63x/1.2 W Corr. Obj., and the sample is excited with Lumencor Spectra light
engine (Cyan selected) with back focal plane of the objective filled. I have
the following filters on the path
-Semrock FF01-475/35-25 (in the Lumencore Spectra)
-Semrock FF01-469/35-25 (added later on in the slider on excitation path)
-Chroma TRF49904-ZE TIRF Filter Cube
-Semrock FF01-525/39-25 (added later on in the slider on emission path)
but somehow I get fair amount of counts even when there is no sample on the
microscope by just turning on the light source alone. I added extra filters
on the path but didn't helped. I looked at the spectra of the light with an
Acton Spectrograph 2150i and it is overlapping with the emission filter band.
Can this happen due to non-sample autofluorescence? if yes, what are the
main elements that can create non-sample autofluorescence? any ideas on how
to avoid it?
I found this article from semrock
(
http://www.semrock.com/Data/Sites/1/semrockpdfs/spectral_modeling_in_fluorescence_microscopy.pdf)
but i still wanted to see if are there any others experienced and solved
that kind of issue.
Thanks
Serkan
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