Hello all,

I commend Bob's list of variables.

As an exercise, each June the participants in the Live-Cell course compile
a list of the factors that can effect the one type of a data the confocal
microscope produces: a number in a computer.

The first year we got 39 variables for living cells. Last year it was even
more:

Illumination:
Laser power setting, Stability, Dye saturation, Wavelength, Pinhole
setting, ND filter, Barrier filter, Dichroic filter, Alignment,
Pre-exposure; Dye: Quantum yield, Bleaching, Orientation, Concentration,
Metabolism, Sequestration, Quenching, FRET, Ion- concentration; Immersion
medium: Reflection at optical surfaces, Spherical aberration; Objective:
NA, "finger prints," Transmission, Coverslip thickness, Coverslip
correction;

Detector:
Dark current, Sensor Reflectance, Q.E., Aquisition time, Averaging, PMT
voltage, PMT Temperature, Electronic gain, Gamma, DC offset, Clipping;
Specimen: Thickness, Structure, Physiology, Opacity, Timing, Medium IR,
Medium Temperature, "Gasses," Glucose, pH, Osmolarity, Autofluorescence;
Digitizing: Stray currents, Bandwidth, Aliasing, Post-processing of data
including Deconvolution.

In the Handbook, there are some suggestions for reducing this array to just
those in the detection pathway by using the PMT to measure a "signal"
generated by the normal transmission light source.  It is a start but don't
expect great accuracy for a while.

It also helps if you can look at a data set in which the numbers stored are
equal to the photons detected (photon counting) rather than just looking at
some number having an arbitrary and unknown scaling factor.

Jim P.

>Don
>I have also asked the fundamental question on instrument performance.
>How do you know that your confocal is working correctly? Unfortunately
>there is not one particle or test that can accomplish this task easily. We
>have tried many particles and many tests. Our best test particle is a 10
>micron bead slide made by Jeff Wang of Spereotech.
>
>To evaluate system performance you must consider field illumination,
>laser power spectral resolution PMT performance, dichroic filters and
>axial resolution, and especially Noise.  One particle can not accomplish all
>of these tests. We are continuing to devise tests that will access the
>functioning of our confocal machine.  Our initial attempt to describe the
>tests procedures are discussed in an article that will be published this
>spring.
>
>Zucker, R.M. Price,OT  --Practical Confocal Microscopy and the
>Evaluation of System Performance  METHODS of Enzymology Anda
>Cornea editor
>
>I hope it brings a suitable discussion among confocal users so
>procedures can be adopted that are similar to flow cytometer methods in
>evaluating system performance . This is essential if we want to quantify
>fluorescence which is one of the aims of our laboratory.
>
>Bob
>
>
>Robert M Zucker. PhD
>United States Environmental Protection Agency
>National Health and Environmental Effects Research Laboratory
>Reproductive Toxicology Division  MD-72
>Research Triangle Park North Carolina 27711
>tel-- 919-541-1585   fax--919-541-4017
>
>
>
>Robert M Zucker. PhD
>United States Environmental Protection Agency
>National Health and Environmental Effects Research Laboratory
>Reproductive Toxicology Division  MD-72
>Research Triangle Park North Carolina 27711
>tel-- 919-541-1585   fax--919-541-4017
              ****************************************
Prof. James B. Pawley,                             Ph.  608-263-3147
Room 223, Zoology Research Building,               FAX  608-265-5315
1117 Johnson Ave., Madison, WI, 53706  [log in to unmask]
"A scientist is not one who can answer questions
but one who can question answers."
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