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I would like to thanks everyone for FRAP comments. I think I’ve nearly got
the answer.
Regards,
Ghafar
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]On
Behalf Of Kevin Braeckmans
Sent: Thursday, 29 March 2007 3:41 PM
To: [log in to unmask]
Subject: Re: Photo bleaching (FRAP)
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Maybe just one addition, I think it is the saturation of the triplet level
that is important in photobleaching studies, rather then the singlet level.
I am not a photochemist, but as far as I understand it is the long lived
triplet state from which photobleaching usually proceeds. This is also what
we have found in an extensive study on the photobleaching of fluorescein
under typical FRAP conditions.
Jim, I know you are not a FRAP fan ;) but I also think that, if one
understands how to do quantitative FRAP experiments, not so much intensity
is needed as one might think. I sometimes have the impression that the idea
exists that the FRAP ROI has to be completely 'black' after photobleaching
and hence, as much laser power should be uses ad possible. This is not at
all the case. About 30% of photobleaching can be sufficient in many cases.
For fluorescein we typically use 1-2mW of 488 nm at the sample, and only
0.5-1mW for R-Phycoerythrin and GFP (and a single photobleaching scan of
course). And this is for a low NA lens with a focused spot of 1 micron
radius. How many times I have seen people doing imaging of weak samples at
near 100% laser intensity with high NA lenses ...
For those who want to avoid FRAP, fluorescence correlation spectroscopy
(FCS) and single particle tracking (SPT) are alternative methods for
measuring molecule dynamics which do not require high photon doses.
Best regards,
Kevin
Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
E-mail: [log in to unmask]
> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]] Namens James Pawley
> Verzonden: donderdag 29 maart 2007 1:42
> Aan: [log in to unmask]
> Onderwerp: Re: Photo bleaching (FRAP)
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Ghafar,
>
> I think that you have generated a lot of good replies but one
> thing that seems missing is "What is the relation between
> bleach rate and the light intensity?"
>
> Early FRAP calculations assumed that bleaching was
> proportional to photons absorbed.
>
> If you use point illumination (like a laser) then (about) the
> same number of excitation photons passed through every plane
> above and below focus plane but the area of the circle they
> passed though is proportional to the square of the distance
> from the focus plane.
> Therefore, the intensity of the light (photons/s/cm2) also
> drops off with the square of the distance from focus plane.
> If you don't move this bleaching cone, then the story is over
> but if you scan a raster, then to some extent, the overlap of
> the disks in the out-of-focus planes compensates for the
> lower intensity and you get the same number of photons
> absorbed for some distance above and below the focus plane.
>
> But does same number of photons absorbed equal same amount of
> bleaching?
>
> This is a very contentious area. With a mW of laser power in
> a beam about 0.5 microns across, you have enough intensity to
> keep a fair fraction (20-60%) of most dye molecules near the
> focus in the excited state (singlet-state saturation).
>
> Does it make a difference? The new Handbook has two chapters
> on bleaching and I won't try to summarize it all here. But I
> will say that when using light this bright (>1mW in 0.25
> square microns), with many dyes you do see non-linear
> bleaching: specifically more dead molecules/excitation than
> is produced at lower power intensity levels. In other words,
> you can have some enhancement of bleaching in the focus
> plane, but it depends on the size of the spot (a 1-micron
> spot will have 4x the area and 1/4th the intensity) and the
> excitation cross-section, decay constant and preferred
> failure mechanism of the dye.
>
> Then there is the matter of what this extreme light intensity
> does to the biological or other organic "structures" present
> in the area you have "blasted". Notwithstanding the
> popularity of FRAP, you should not assume that bleaching is
> the "only" thing that happens in a cell or a gel just because
> it is the only thing that you want to happen.
>
> At the very least, bleaching usually seems to involve the
> production of a lot of singlet-oxygen. This interacts with
> the dye molecules and inactivates them by turning them into
> different, non-fluorescent (and possibly poisonous)
> molecules. But the singlet oxygen doesn't only react with
> dyes. It can damage any organic material within about a
> micron of its site of generation. One should assume that any
> simple analysis of a FRAP response may be complicated by
> cytotoxicity produced by having created singlet oxygen in the
> cell or gel.
>
> You cannot approach singlet-state saturation with the
> intensity available from a Hg arc source. But bleaching cells
> in media containing O2, still often involves the production
> singlet-oxygen.
>
> Cheers,
>
> 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
> [log in to unmask]
> 3D Microscopy of Living Cells Course, June 17-28, 2007, UBC,
> Vancouver Canada
> Info: http://www.3dcourse.ubc.ca/ Applications due
> by March 15, 2007
> "If it ain't diffraction, it must be statistics." Anon.
>
>
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Dear Confocalist,
> >Can you help me out on this FRAP question. Is it right in
> thinking that
> >if you focus bleaching, on a small ROI on a 1 micron thick
> section of
> >the membrane, the parts of the membrane above and below that section
> >are not bleached. In other word when you do the bleaching is
> that focal
> >plain of the cell or does it bleach throughout the cell from
> top to bottom in that area?
> >Any advice you can give would be most appreciated.
> >With regards,
> >Ghafar
> >***********************************************
> >Ghafar T Sarvestani, PhD
> >Hanson Institute for Medical Research
> >Hanson Building level 2, Room 2-42-N
> >IMVS, Frome Road, Adelaide SA 5000
> >
> >Affiliate Lecturer
> >Department of Medicine
> >The University Of Adelaide
> >Tel: +61 8 82223603
> >Fax: +61 8 82223162
> >Email:[log in to unmask]
>
>
> --
> **********************************************
> Prof. James B. Pawley, Ph.
> 608-263-3147
> Room 223, Zoology Research Building,
> FAX 608-265-5315
> 1117 Johnson Ave., Madison, WI, 53706
> [log in to unmask]
> 3D Microscopy of Living Cells Course, June 17-28, 2007, UBC,
> Vancouver Canada
> Info: http://www.3dcourse.ubc.ca/ Applications due
> by March 15, 2007
> "If it ain't diffraction, it must be statistics." Anon.
>
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