Derek,
Bleaching is a handy reference. I too recommend it, and if you have a
standard sample (e.g., 1 um fluorescent bead or smaller) that has
been bleached on a "reference" scope which has been laser line power
calibrated, then you can actually use that to estimate the power
delivered on a different scope. The 1/e curves follow a single
exponential pretty well, though this will depend on the bead fluor.
This effect will be completely independent of the PMT gain or any
other electronic component. You just want to have enough signal to
generate a good curve. (Derek, I think you mean a signal just short
of saturation as opposed to minimal saturation which would be zero
signal.) I would recommend doing this on ten beads or more to get
some decent statistics, but once done, you can do this on any test
system and compute the power delivered, and determine the S/N for
power delivered.
This all assumes the same lens NA and beads and bead environment are
used. With enough scans one can bleach the sample to the point where
instrument noise is comparable to the signal providing the useful
dynamic range of the confocal. But one cannot escape the need for a
careful and reliable power calibration for this approach to work
well. If done correctly it becomes more than just a crude comparison.
Mario
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1Ļocal
>
>A crude method I use for looking at sensitivity is to look at
>photobleaching. If your specimen is zoomed to the same size between
>systems (fill the screen) then you should be able to record a series
>of images over time and plot the degree to which the specimen
>bleaches. Ideally you would put a light meter at the objective to
>make sure the laser powers employed were similar, but I have just
>put the PMT voltage to around 800 and adjusted the power to the
>point where the specimen has minimal saturation (use a color LUT
>that highlights saturated pixels). A more sensitive system will
>require less laser power to image and will subsequently photobleach
>less. Plot the intensity of the image over time and you will see if
>there is a significant difference. You will have to decide on what
>system to use, but beads on a slide should work. The assumptions
>taken here are that a pinhole of 1airy disk is in fact the said
>amount between systems. Take a slide along with you next time and
>give this a try.
>
>Essentially, in real life we want the best signal to noise without
>much photobleaching.
>
>At 01:08 PM 8/14/02 -0400, you wrote:
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1Ļocal
>>
>>--- You wrote:
>>It would be good if NIST or some other prestigious and neutral body came up
>>with a good physical standard for dim fluorescence,
>>--- end of quote ---
>>Coincidently, we have been grappling with this issue over the past
>>few weeks (because we are in the market for a new confocal system).
>>We have been using beads as well as solutions of fluorochrome as
>>low fluorescence standards. But they are unstable and annoying.
>>Our current idea is that we would like to have plastic fluorescent
>>slides of varying (including particularly low) fluorescence
>>intensities. Who makes those slides? Can't we get them to make
>>slides of low intensities so that we can use them to look at
>>instrument sensitivity over background (a blank slide)? What I
>>would really like is one slide with segments of zero, very very
>>low, very low,low, medium, and high fluorescence levels. Any
>>ideas of who could make something like this?
>>
>>Alice
>>
>>Alice L. Givan
>>Englert Cell Analysis Laboratory
>>of the Norris Cotton Cancer Center
>>Dartmouth Medical School
>>Lebanon, New Hampshire NH 03756
>>tel 603-650-7661
>>fax 603-650-6130
>>[log in to unmask]
>
>Derek Schulze
>Flow Cytometry and Confocal Microscopy Facility Manager
>Cancer Research Labs ext.36635
>Queen's University
>Kingston, ON
>Canada
>http://meds.queensu.ca/medicine/crl/flow/
--
_________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies
FAX (510) 528-8076
1561 Posen Ave
Berkeley, CA
94706
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