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Hi Julio,

Confocal microscope output is not simple. For one thing, the instrument 
manufacturers do things "under the hood" with the data between the PMT 
and the user interface you see. For example, on the Zeiss LSM510, the 
service engineers were supposed to tweak some setting(s) that resulted 
in approximately the same output value regardless of scan speed (of 
course you could just acquire one pixel, but then it would not be a 
scanner ... and you'd probably photobleach the spot before too long). 
Try scanning at the same speed (pixel dwell time) with say 16x16 pixels 
[which I nominate we call "Jim's box" in honor of Jim Pawley ... oops, 
maybe that's Guy's box and Jim's should be 25x25 pixels, assuming you 
have your bead perfectly centered in the middle] vs max # pixels (ex. 
8192x8192 pixels - may take a while ... of course if your Z or XY drift 
in that time, too bad).

Besides photobleaching, if your laser power is high enough, your 
fluorophores will go triplet and stuck in the triplet state(s) until 
either (a) the laser spot moves on, (b) you hit the off switch, or (c) 
they die. Amount of oxygen (which can quench excited state triplets but 
also can kill the fluorophore) or other triplet state quenchers. Of 
course trivial other things like too low or too high a PMT voltage (Cho 
and Lockett favor relatively low PMT gain with sensible choice of A/D 
converter), wrong pinhole size, scattering by the specimen (including 
refractive index differences between specimen, coverglass, immersion 
medium, lens). Not that you should expect any of your lasers to have 
constant power output to the specimen (try scanning to the transmitted 
light detector overnight for this, though the photodetector in the T 
path is not a PMT).

A good paper on confocal microscope calibration is:

Calibration and standardization of the emission light path of confocal 
microscopes. <http://www.ncbi.nlm.nih.gov/pubmed/16872427>

*Cho* EH, *Lockett* SJ.

J Microsc. 2006 Jul;223(Pt 1):15-25.

PMID:
    16872427


Ted Young, Yuval Garini et al, published a similar paper,
http://repository.tudelft.nl/view/ir/uuid%3A2cdbdf0c-45be-4ae0-8daa-a48c8058e756/
Reading Ted and Yuval et al's abstract reminded me to mention to the 
entire listserv readership: please do not use "arbitrary units" in your 
manuscripts (or in your minds for that matter).

George
p.s. monitors and room lighting, LUT choice (compare blue to gray), and 
contrast adjustment of the image display (and adjacent white/gray/black 
space on the GUI) all have a visual impact on what you see: quantify the 
data by the numbers and graphs.


On 9/30/2013 4:39 PM, Julio Vazquez wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear confocalists,
>
> I have been thinking about the relative brightness of different objectives on a point laser scanning confocal microscope. The formulas one finds in books, etc, report brightness as proportional to NA^2 / M^2 for transmitted light, and NA^4 / M^2 for epifluorescence. However, I have never seen a specific discussion of single point scanners vs widefield. It is my impression that on a point scanner, the laser beam is focussed to a spot whose radius depends on the NA; on the collection side, light emitted by the spot is collected back onto a PMT, with a collection efficiency also related to the NA. At neither stage does the magnification seem to play a role. This leads me to think that the brightness of an objective on a point scanner is proportional to the fourth power of the NA, and independent of the magnification. I did some quick and dirty measurements on 200 nm beads which seemed to support this (although complicated by various factors such as the size of the back aperture, which made it difficult to get precise measurements of excitation power at the sample with high NA lenses, for example, and other things). However, I am puzzled I have never seen mention of this anywhere. Is it correct that magnification is irrelevant for image brightness on a point scanner, or am I way off the mark?
>
>
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> 1100 Fairview Ave N., DE-512
> Seattle, WA 98109
>
> http://www.fhcrc.org/en.html
>
>    


-- 



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
http://works.bepress.com/gmcnamara/26/