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

Posted by Claudiu Brumaru on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Signal-bleaching-and-toxicity-vs-exposure-time-for-constant-total-light-dosage-tp7580528p7580532.html

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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 <
[hidden email]> wrote:

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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.
>