Re: Signal, bleaching and toxicity vs. exposure time for constant total light dosage

Posted by Jurek Dobrucki on Jun 19, 2013; 11:43pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Signal-bleaching-and-toxicity-vs-exposure-time-for-constant-total-light-dosage-tp7580528p7580531.html

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Andrew,

longer acquisition at a lower intensity of exciting light will often
results in far less bleaching (and less photodamage in live cells).

You are right - this issue was studied. An example is provided by our paper:

Bernas T, Zarebski M, Cook PR, Dobrucki JW .Minimizing photobleaching
during confocal microscopy of fluorescent probes bound to chromatin:
role of anoxia and photon flux.
J Microsc. 2004 Sep;215(Pt 3):281-96

Figure 4I demonstrates that it is possible to (almost) eliminate
photobleaching of eGFP if the photon flux is significanly reduced. The
same dose of light delivered with a laser
beam of much higher intensity results in a rapid loss of signal.

Hope this helps,

Jurek

Jerzy Dobrucki, Ph.D., D.Sc.
Professor of Biophysics
Head, Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
    Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone]; fax. [phone]
[hidden email]
http://www.wbbib.uj.edu.pl/helios




On Wed, Jun 19, 2013 at 8:42 PM, Andrew York
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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.