Posted by
Jeremy Adler-4 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-deep-can-you-see-in-a-brain-slice-tp7256661p7269108.html
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In fluorescence emitted photons are often the limiting factor so in
widefield fluorescence filter blocks need to be optimised.
A possible problem is that for a molecule with a Stokes shift of say
20nm the detector will only detect the shortest emitted photons it
includes a range that starts at less than the Stokes shift, say
excitation + 15nm. But looking at the width of excitation filters used
in widefield fluoresence the excitation range, perhaps 460/40, then
many of the emitted photons will not even reach a detector with an
emission filter set to exclude excitation light, perhaps a 490nm Long
pass.
This argues that if photon detection efficiency is the primary concern
then excitation ranges need to be narrow. In the real world the power
output of widefield light sources is limited and an excitation range
is needed to effective produce fluorescence is useful amounts and the
emission spectrum of most fluorophores has a very long tail - but
should consideration be given to the the excitation range with respect
to the Stokes shift ?
Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden
0046 (0)18 471 4607