Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Signal-bleaching-and-toxicity-vs-exposure-time-for-constant-total-light-dosage-tp7580528p7580530.html
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My experience (without much data to support it) is this:
At low illumination intensities, photobleaching seems to depend mostly on the dose of light (illumination intensity x exposure time). I explain this by the fact that, under this regime, a fluorescent molecule has a defined probability of getting destroyed at each excitation/emission cycle, so a population of fluorophores will decay as a function of the amount of exposure received.
At high illumination intensities, several things can happen:
(a) fluorophores can go into a dark state (fluorescence emission goes down, but recovers quickly once exposure stops, as opposed to photobleaching). We can see this on some of our widefield microscopes where illumination intensity is fairly high (e.g Deltavision Elite where we have about 20-30 mW of power focused on the center of the field of view in critical illumination mode). If I do a time series (no delay) at full illumination intensity, image brightness goes quickly down, but recovers to original values if I wait a few seconds and repeat the experiment (i.e I get a saw pattern)
(b) fluorophores can go into an irreversible inactive state forever (photobleaching). This tends to happen on point scanners where local illumination intensity (per area unit) can be significantly higher that on widefield systems. If we do a photobleaching curve at different laser intensities on a point scanner, response is not linear (fluorophores bleach much faster at higher illumination intensities, rather than proportionately). The danger zone (when things start bleaching quite fast) in our experience is around and above 10-30 micro-Watts. Depends on the specimen.
Recently, it has been reported that using resonant scanners in confocal microscopy generates higher signal (i.e integrating ten images at fast speed gives significantly brighter image than collecting 1 image at 10x lower speed). A discussion of the possible mechanisms can be found on the Leica web site:
http://www.leica-microsystems.com/science-lab/brighter-fluorescence-by-resonant-scanning/(c) other things may be happening I am not aware of. Which fluorophore you use, mounting medium, etc, may have an influence.
A discussion of photobleaching mechanisms can be found in Jim Pawley's Confocal handbook (e.g. Chapter 39 of 3rd Edition, by Alberto Diaspro)
The bottom line is that several mechanisms may be taking place at the same time, the relative importance of which which may depend on the specimen and on the specific microscope used (based on mode of illumination- widefield vs point confocal-, and the intensity of the illumination). When we work with critical samples, we just run a few quick tests to see what works best on any given microscope system.
Julio Vazquez
Fred Hutchinson Cancer Research Center2
Seattle, WA 98109
http://www.fhcrc.org/en.html==
On Jun 19, 2013, at 11:42 AM, Andrew York wrote:
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>
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>
> Simple question:
> I take two 3D volumetric images of a cell, (widefield, 488nm laser
> excitation, GFP-tagged).
> In both cases, I take 10 slices over 1 second.
> In case 1, I use 100 ms exposures, with a laser intensity of 1 unit.
> In case 2, I use 50 ms exposures, with a laser intensity of 2 units. I turn
> the laser off for 50 ms between exposures.
>
> Which case bleaches the cell more? Which case is more phototoxic? Which
> case gives the most signal? Surely someone has studied this, and the answer
> is well known. I assume there's some linear range where total dose is all
> that matters, and some non-linear range where this behavior breaks down.
>
> Motivation: my users always want to turn down the laser and crank up the
> exposure time, hoping this will be gentler to the cells or give lower
> background or higher signal. I usually give my opinion that it won't make
> much difference, but I don't have a solid reference to point them to for a
> solid answer.