Fluorophore bleaching by excitation light sources
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Gerard Whoriskey-3 |
Fluorophore bleaching by excitation light sources
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Commercial interest. We have recently had preliminary feedback from a number of independent sources that show much reduced bleaching when a sample is excited using an LED source rather than a Hg bulb. These tests, carried out with identical powers in the excitation bandpass region, showed that imaging could be carried out for up to three times longer. On live tests cells were seen to be still living happily after being exposed for twice the time it took to kill the cells under Hg excitation. We are still gathering information on this and intend to publish in due course. In the meantime I will be happy to discuss offline and would be interested in hearing from anyone who has seen similar results. Best Regards, Gerry Gerard Whoriskey Development Engineer CoolLED Ltd CIL House Charlton Road Andover Hampshire SP10 3JL Mob: 07789535762 Tel: +44 (0) 1264 321321 Dir: +44 (0)1264 320984 web site: www.coolled.com |
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Mark Cannell |
Re: Fluorophore bleaching by excitation light sources
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I don't understand this. The only explanation I can think of is that that the excitation wavelengths are _not_ the same in the two cases.(if they were and the power is the same the photon flux is the same). Any other comments/views? Regards. Gerard Whoriskey wrote: > Commercial interest. > > We have recently had preliminary feedback from a number of independent > sources that show much reduced bleaching when a sample is excited using an > LED source rather than a Hg bulb. These tests, carried out with identical > powers in the excitation bandpass region, showed that imaging could be > carried out for up to three times longer. > On live tests cells were seen to be still living happily after being > exposed for twice the time it took to kill the cells under Hg excitation. > We are still gathering information on this and intend to publish in due > course. > |
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Ignatius, Mike |
Re: Fluorophore bleaching by excitation light sources
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal My understanding is that it is critical that all UV light be removed/blocked as Hg bulbs produce lots of UV. Diodes don't have this concern. Not all scopes can claim 100% UV block produced by Hg or xenon bulbs. But when it is done (for example AP/Delta Vision Scopes), cell viability along with dye stability is greatly enhanced. Mike Ignatius -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell Sent: Tuesday, March 11, 2008 2:02 PM To: [hidden email] Subject: Re: Fluorophore bleaching by excitation light sources Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I don't understand this. The only explanation I can think of is that that the excitation wavelengths are _not_ the same in the two cases.(if they were and the power is the same the photon flux is the same). Any other comments/views? Regards. Gerard Whoriskey wrote: > Commercial interest. > > We have recently had preliminary feedback from a number of independent > sources that show much reduced bleaching when a sample is excited using an > LED source rather than a Hg bulb. These tests, carried out with identical > powers in the excitation bandpass region, showed that imaging could be > carried out for up to three times longer. > On live tests cells were seen to be still living happily after being > exposed for twice the time it took to kill the cells under Hg excitation. > We are still gathering information on this and intend to publish in due > course. > |
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In reply to this post by Mark Cannell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I think we'd need to be told a whole lot more - what the dye was, what the bandpass was, etc. A mercury lamp has very powerful spectral lines, so (especially in the green) the measured power is not spread across the spectral range but concentrated at one wavelength. If this happens to hit a particular transition the effect on the fluorochrome could be different from excitation by a broad band. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell Sent: Wednesday, 12 March 2008 8:02 AM To: [hidden email] Subject: Re: Fluorophore bleaching by excitation light sources Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I don't understand this. The only explanation I can think of is that that the excitation wavelengths are _not_ the same in the two cases.(if they were and the power is the same the photon flux is the same). Any other comments/views? Regards. Gerard Whoriskey wrote: > Commercial interest. > > We have recently had preliminary feedback from a number of independent > sources that show much reduced bleaching when a sample is excited > using an LED source rather than a Hg bulb. These tests, carried out > with identical powers in the excitation bandpass region, showed that > imaging could be carried out for up to three times longer. > On live tests cells were seen to be still living happily after being > exposed for twice the time it took to kill the cells under Hg excitation. > We are still gathering information on this and intend to publish in > due course. > No virus found in this incoming message. Checked by AVG. Version: 7.5.518 / Virus Database: 269.21.7/1324 - Release Date: 10/03/2008 7:27 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.518 / Virus Database: 269.21.7/1325 - Release Date: 11/03/2008 1:41 PM |
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James Pawley |
Re: Fluorophore bleaching by excitation light sources
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In reply to this post by Mark Cannell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >I don't understand this. The only explanation I can think of is that >that the excitation wavelengths are _not_ the same in the two >cases.(if they were and the power is the same the photon flux is the >same). Any other comments/views? > >Regards. > >Gerard Whoriskey wrote: >>Commercial interest. >> >>We have recently had preliminary feedback from a number of >>independent sources that show much reduced bleaching when a sample >>is excited using an LED source rather than a Hg bulb. These tests, >>carried out with identical powers in the excitation bandpass >>region, showed that imaging could be carried out for up to three >>times longer. >>On live tests cells were seen to be still living happily after >>being exposed for twice the time it took to kill the cells under Hg >>excitation. We are still gathering information on this and intend >>to publish in due course. Need to check that both systems have UV absorption filters or optics... If you have quartz optics, and no UV filter, then you are relying on the blocking in the barrier filter, which is not always enough. LEDs have no UV but Hg does. Jim Pawley -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008 "If it ain't diffraction, it must be statistics." Anon. |
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Stanislav Vitha |
Re: Fluorophore bleaching by excitation light sources
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In reply to this post by Gerard Whoriskey-3
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 |
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Knecht, David |
Re: Fluorophore bleaching by excitation light sources
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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 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) |
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Hi Dave,
Got the RFP. Thanks. Any chance of getting together at the end of next week (Thu. or Fri.) to discuss CSU options? Gary |
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Ignatius, Mike |
Re: Fluorophore bleaching by excitation light sources
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In reply to this post by Knecht, David
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Pulsed
Illumination: Prior to Stefan
Hell's truly elegant paper this paper discussed making a cheaper version of
pulsed diode to reduce photo-fading/toxicity.
Stroboscopic
illumination using light-emitting diodes reduces phototoxicity in fluorescence
cell imaging.
by Nishigaki T, Wood CD, Shiba K, Baba SA, Darszon A. in <A
href="javascript:AL_get(this, 'jour', 'Biotechniques.');">Biotechniques. 2006
Aug;41(2):191-7.
The effects weren't as
dramatic, but the benefits were very clear and at a fraction of the cost.
It is worth noting as
well that the spinning discs, fast scanners, fast galvos, etc, effectively
deliver a decent form of the T-Relaxation light form that Dr.
Hell recommends in his paper.
As for UV leak, J.
Nordberg at U Mass had an interesting ASCB abstract/poster in 2006. UV was
effecting his experiments, so he looked at the amount of leak. My
apologies to Dr. Nordberg if I misrepresent his findings, but my notes say that
i) photon flux from UV light vs tungsten was 30,000 times greater. (Any of
us failing to have filter block in place when we look through the eye
pieces with an Hg lamp on know this
personally.) Still from
my notes, so these numbers need verification from the pros out there, but
standard filters block 99% or 2 orders of magnitude of light while optimized
filters can cut another 4 orders more. I liken this to watching
soccer/football under the lights at night - like a tungsten lamp on a
slide. Imagine then if the light was turned up 300 times (30,000 less
99%), this is what the extent of UV leak might look like to our cells. We
don't see it as it is UV, but they (and the dyes) feel it.
Mike
Ignatius,
Molecular
Probes/Invitrogen From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Wednesday, March 12, 2008 2:56 PM To: [hidden email] Subject: Re: Fluorophore bleaching by excitation light sources On Mar 12, 2008, at 4:27 PM, Stanislav Vitha wrote: Search the CONFOCAL archive at 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) |
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In reply to this post by Gary Laevsky-2
Fascinating...
-----Original Message----- From: Confocal Microscopy List on behalf of Gary Laevsky Sent: Wed 3/12/2008 6:23 PM To: [hidden email] Cc: Subject: Meet about specs Hi Dave, Got the RFP. Thanks. Any chance of getting together at the end of next week (Thu. or Fri.) to discuss CSU options? Gary |
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In reply to this post by Knecht, David
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:
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.
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Cairn research ltd |
Re: Fluorophore bleaching by excitation light sources
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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. Cairn OptoLED - http://www.cairn-research.co.uk/Products Managing
Director www.cairn-research.co.uk > 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) > > -- >_______________________________________________________________________________ >_ >Mario M. Moronne, Ph.D. > >cell (510) 367-8497 > > [hidden email] > [hidden email] > [hidden email] ----------------------- Original
Message -----------------------
From: Mario Moronne [hidden email]
To: [hidden email]
Date: Wed, 12 Mar 2008 17:17:55
-0700
Subject: 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 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:
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.
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