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Re: Fluorophore bleaching
by excitation light sources
There is of course nothing
magic about photons produced by LEDs rather
than Hg or other arc lamps, so any apparent
reduction in photobleaching when using LEDs
for illumination must presumably relate to
the spectral characteristics of the excitation
light. The spectral characteristics
of Hg (as opposed to Xe) arc lamps are very
uneven, so very effective filtering may be
necessary to block out-of-band spectral peaks
that might cause increased phtobleaching.
The
Donnert et al paper is very interesting.
Basically their suggestion is that
a small proportion of excited flurophores
goes to a much longer-lived triplet state,
with a lifetime of around a microsecond,
rather than undergoing "normal" fluorescence
emission with a lifetime of a few nanoseconds.
Therefore, as the light intensity is
increased, more and more of the fluorophores
are pushed into the triplet state on account
of its much greater lifetime. If (as
is likely) the triplet state fluorophores
are destroyed (i.e. bleached) if they absorb
a second photon, you then have a nice model
to explain why high light intensities cause
disproportionately greater photobleaching
(it's a square-law effect, as two photons
are involved).
The Donnert et al paper
used pulsed illumination as a rather neat
way of showing this effect, but in our opinion
pulsed illumination is unlikely to offer
any SIGNIFICANT benefit over continuous illumination
of the same average intensity. The
important criterion is that the illumination
intensity should be such that on average
any one fluorophore molecule is excited at
a rate that is low compared with the lifetime
of its triplet state, so that it never gets
the chance to absorb a second photon. Whether
all the fluorophores are excited at the same
time by a very brief pulse, or asynchronously
by the same average light intensity, is unlikely
to make a big difference in our opinion,
but of course it would be worth checking
this out experimentally. Our OptoLED
product is capable in principle of being
switched at these sort of rates, but with
pulsed illumination of any LED source (i.e.
the LED off most of the time) you probably
wouldn't get enough light, so in practice
we think that you'd need laser illumination
to check this one out.
Cairn OptoLED - http://www.cairn-research.co.uk/Products
Dr. Martin Thomas
Managing
Director
Cairn Research Ltd
Graveney
Road
Faversham
Kent, ME13 8UP
UK
www.cairn-research.co.uk
[hidden email]
Tel: + 44 (0)1795 590140
Fax:
+ 44 (0)1795 594510
>Search the CONFOCAL archive
at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Re: Fluorophore
> bleaching by excitation
light sources
>Dave,
>
>Depends on what you mean by easy.
To narrow the problem a bit, the important
>
issue is how much UV reaches the sample.
Any other filter is more or less
>
irrelevant with respects to cell viability,
unless there is so muchback
> scattered
UV that extra background noise must be averaged
out with extra
> exposures.
>
>Ignoring the latter, one might deal
with the former by using a test object that
>
is excited by UV. There are quite a few options
including UV sensitive
> inorganic
phosphors that can be purchased as fine powders
or disks.The latter
> compounds can
have luminescent lifetimes that range from
as short as 40 nsec.
> to milliseconds.
The latter often use a lanthanide such as
europium (red) or
> terbium (blue,
green, red) with sharp emission lines. Some
have broad
> emissions such as P31,
a ZnS based phosphor. Another possibility
would be some
> organic fluorophores
including AMCA or well saturated nuclei stained
with DAPI
> or Hoechst (say 3 ug dye
/ml of cells).
>
>A slide
made with a phosphorescent disk will last
for years. As I recall, some
> EM suppliers
sell phosphorescent disks and related materials
for use in
> cathodoluminescent detectors.
I have used or made all of the above options
in
> one form or another. A further
option is to use a fiber optic couple micro
>
spectrofluorometer. I like the USB ported
version from Ocean Optics, Fl. (no
>
financial interest). They can provide a cable
that is capped with a collection
>
lens that carries the excitation light from
the microscope objective to the
> spectrometer
which uses a grating and linear CCD. As I
recall, there are a few
> grating options
one of which provides sensitivity down near
220 nm on up to
> 700 nm.
>
>Anyway, some of these approaches
are crude but they will tell you if there
is
> significant UV bleed through.
>
>Mario
>
>
Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I have used the
> CoolLED system, and
it is not operating in strobe mode. It
is continuous
> illumination. The
UV question is interesting. Is there
aneasy way to test if
> there is UV
leaking through the epi filter set with the
mercury burner? Dave
> On Mar
12, 2008, at 4:27 PM, Stanislav Vitha wrote:
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
> Regarding the difference between
LED illumination and Hg lamp -
> Perhaps
if the illumination is not continuous but
is set up for strobe
> operation of
the LED, this could allow dark state relaxation
an prolong
> the life of the fluorophore
(I am not sure the same would apply to the
>
life of the cell). - The time between light
pulses should be more than one
> microscecond.
>
reference:
> Gerald Donnert,
Christian Eggeling & Stefan W Hell: Major
signal increase
> in fluorescence microscopy
through dark-state relaxation. Nature Methods
-
> 4, 81 - 86 (2007)
>
>
> Stan Vitha
>
> Dr.
David Knecht Department of Molecular
and Cell Biology Co-head Flow
> Cytometry
and Confocal Microscopy Facility U-3125 91
N. Eagleville Rd.
> University of Connecticut
Storrs, CT 06269 860-486-2200 860-486-4331
(fax)
>
----------------------- Original
Message -----------------------
Date: Wed, 12 Mar 2008 17:17:55
-0700
Subject: Re: Fluorophore
bleaching by excitation light sources
Search the CONFOCAL
archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dave,
Depends on what you mean by easy. To
narrow the problem a bit, the important issue
is how much UV reaches the sample. Any other
filter is more or less irrelevant with respects
to cell viability, unless there is so much
back scattered UV that extra background noise
must be averaged out with extra exposures.
Ignoring the latter, one might deal
with the former by using a test object that
is excited by UV. There are quite a few options
including UV sensitive inorganic phosphors
that can be purchased as fine powders or
disks. The latter compounds can have luminescent
lifetimes that range from as short as 40
nsec. to milliseconds. The latter often use
a lanthanide such as europium (red) or terbium
(blue, green, red) with sharp emission lines.
Some have broad emissions such as P31, a
ZnS based phosphor. Another possibility would
be some organic fluorophores including AMCA
or well saturated nuclei stained with DAPI
or Hoechst (say 3 ug dye /ml of cells).
A slide made with a phosphorescent disk
will last for years. As I recall, some EM
suppliers sell phosphorescent disks and related
materials for use in cathodoluminescent detectors.
I have used or made all of the above options
in one form or another. A further option
is to use a fiber optic couple micro spectrofluorometer.
I like the USB ported version from Ocean
Optics, Fl. (no financial interest). They
can provide a cable that is capped with a
collection lens that carries the excitation
light from the microscope objective to the
spectrometer which uses a grating and linear
CCD. As I recall, there are a few grating
options one of which provides sensitivity
down near 220 nm on up to 700 nm.
Anyway, some of these approaches are
crude but they will tell you if there is
significant UV bleed through.
Mario
Search the
CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I have used the CoolLED system, and it is
not operating in strobe mode. It is
continuous illumination. The UV question
is interesting. Is there an easy way
to test if there is UV leaking through the
epi filter set with the mercury burner? Dave
On Mar 12,
2008, at 4:27 PM, Stanislav Vitha wrote:
Search the
CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Regarding
the difference between LED illumination and
Hg lamp -
Perhaps if the illumination
is not continuous but is set up for strobe
operation
of the LED, this could allow dark state relaxation
an prolong
the life of the fluorophore
(I am not sure the same would apply to the
life
of the cell). - The time between light pulses
should be more than one
microscecond.
reference:
Gerald Donnert, Christian
Eggeling & Stefan W Hell: Major signal
increase
in fluorescence microscopy through
dark-state relaxation. Nature Methods -
4,
81 - 86 (2007)
Stan Vitha
Dr. David
Knecht
Department
of Molecular and Cell Biology
Co-head Flow
Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville
Rd.
University
of Connecticut
Storrs, CT
06269
860-486-2200
860-486-4331
(fax)
--
________________________________________________________________________________
Mario M. Moronne, Ph.D.