Re: Fluorophore bleaching by excitation light sources
Posted by
Ignatius, Mike on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluorophore-bleaching-by-excitation-light-sources-tp592521p592530.html
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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.
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
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:
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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)