On Sun, 28 May 1995, Michal Opas wrote:
> Difficult so far.  Filter modification might help a bit BUT an
> improvement, if any, is unlikely to justify costs.
> Different fluorochrome should help such as Bodipy FL (Mol.Probes) that
> excites at ca. 500 nm and emits at 512 nm might be a candidate. Before
> buying a year's supply of the fluorochrome I would ask Mol Probes for a
> comparison chart or even better, LEGIBLE graphs showing shapes of emission
> curves of both products (i.e., FITC and Bodipy FL) to see how far they
> trail into red AND if the difference justifies switching to a new
> flourochrome.
> If anyone gets more illuminated on this topic, I would appreciate to know!
> Thanks
> michal
>
> Dr. Michal Opas
> Department of Anatomy & Cell Biology
> University of Toronto
> Medical Sciences Building
> Toronto, Ontario, M5S 1A8 Canada
>
> tel 416 978-8947
> fax 416 978-3954
> e-mail [log in to unmask]
>
 
In sincere hopes of not sounding commercial...
 
Use of a bandpass emission filter rather than a longpass should help
eliminate most of the red tail problem that many people here seem to have.
A good source of filters for this is Omega Optical in Brattleboro, VT.
A bandpass filter will let through plenty of green signal and remove
much of the red.
 
Rhodamines, while bright and fairly photostable, are much less desireable
for multicolor work because their excitation is much closer to that of
fluorescein (the peak excitation usually being somewhere around 546 nm,
but having tails that overlap into the fluorescein excitation range).
Texas Red has much less of a tail in the fluorescein excitation range and
is a much better choice for multicolor work for this reason.
 
I have made several multicolor photographs on color 35 mm film using
fluorescence microscopy with a mercury arc lamp using BODIPY FL and Texas
Red or BODIPY TR fluorophores (antibodies, streptavidins, phalloidins,
etc) and find that using bandpass fluorescein and Texas Red filter sets
eliminate most of the crosstalk between channels.  (BODIPY TR is
Molecular Probes' BODIPY analog of Texas Red; they are spectrally quite
similar.)
 
BODIPY FL is a fluorophore similar to fluorescein in excitation and
emission with two advantages:  It is pH-insensitive, and the emission
spectra is indeed narrower than fluorescein.  Molecular Probes does
indeed have spectra for fluorescein and BODIPY FL and would be happy to
fax you any information about these dyes.  You can reach their technical
assistance department by phone at 503-465-8300; fax at 503-344-6504;
email at [log in to unmask]  I think that the combination of
a BODIPY FL fluorophore with a bandpass emission filter set, and use of
a Texas Red fluorophore as opposed to a rhodamine, will give you a good
result with little crosstalk.
 
I hope this has helped you.
 
Sincerely,
 
Jennifer Kramer
Molecular Probes, Inc.