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Leigh
You have asked a very simple question that is very difficult to do
correctly.  Unfortunately  confocal microscopes are not stable and
reproducible and you must run a series of tests to insure the machine is
functioning correctly. the time period for doing this has not been
adequately determined.
We and others have published different approaches to address the
following parameters that effect machine reproducibility:   Laser power
stability, colocalization  , field illumination, spectral registration,
axial resolution,  laser power,  galvanometer scanning stability,
spectral reproducibility and sensitivity. .
Articles of these papers from our laboratory are available in PDF format
on request. There will be a workshop at the ISAC 2006 meeting in Quebec
City May 20-24 to discuss these important issues on confocal microscopy
QA, Thank you for addressing this question to this confocal group.
Best wishes
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]



                                                                        
             Leigh Silvester                                            
             <Leigh.Silvester                                           
             @NOTTINGHAM.AC.U                                        To 
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Search the CONFOCAL archive at
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I have a potential user who is carrying out a series experiment over
several weeks and would like the confocal results to be comparable.

Consequently I need to establish that the confocal system (Leica TCS
SP2) is responding the same for each batch of samples.
This does not need to be calibrated in the true sense of the word, but I
do need consistency.

I had considered setting it up on a convalaria sample each time but of
course there is no gurantee that I will be in the same Z plane each
time.

Some one from Leica mentioned fluorescent opaque slides that I could put
in each time and check that the values for intensity were the same each
time but I am struggling to find a supplier.

There is the possibility of using the Molecular Probes "Fluorescent
Microsphere Standards", but is this what they are intended to be used
for? As far as I can ascertain these are primarily for checking
alignments and calibration in terms of distances. For me it is the
calibration in terms of ascertaining that the instrument settings for
gain etc are the same each time.
I know I can do an analysis and load the parameter settings from this on
subsequent occasions, but that does not give me a measure of how similar
conditions are. I guess I am looking for somwthing to act as a quality
control.

Any ideas?



Regards

Leigh Silvester
Division of Animal Physiology
University of Nottingham
School of Biosciences
Sutton Bonington Campus
Loughborough
Leicestershire
LE12 5RD, UK
tel: +44 (0)115 9515151 x18736
fax +44 (0)115 9516302
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