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Hi Claudiu,

you are right, but there is one important note to add: in *both* cases 
(doubling exposure time vs doubling intensity, linear regime) you collect *
twice* the number of photons which translates to sqrt(2) improvement of S/N,
provided you are limited by photon noise. Anyway, both cases are equivalent,
you get the same result with the same photon dose.

To all:

Generally, of course, there may be other (nonlinear) effects or other 
sources of noise (e.g. dark current) that may make a difference. But this is
unlikely in widefield with good camera.

It is strange that (as noted by others) microscopists try to reduce 
phototoxicity and photobleaching by decreasing the peak intensity in a point
scanning confocal (faster scanning with averaging) on one hand, while trying
to increase peak intensity in widefield by pulsed illumination... I have not
seen a really convincing paper stating that pulsed illumination reduces 
photobleaching.

Best,

zdenek svindrych



---------- Původní zpráva ----------
Od: Claudiu Brumaru <[log in to unmask]>
Datum: 20. 6. 2013
Předmět: Re: Signal, bleaching and toxicity vs. exposure time for constant 
total light dosage

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Hi Andrew,

Regarding the signal intensity, one can double the emitted fluorescence by
doubling the laser intensity (considering that this intensity is low enough
to stay within the linear dependence between the emission and excitation
light) while doubling the integration time leads to only a square root of 2
increase in S/N.

Claudiu.


On Wed, Jun 19, 2013 at 9:42 PM, Andrew York <
[log in to unmask]> 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.
>"