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These are real world comments in a core facility like the one I work. We
have come across with almost all situations described by Geoff. 
I would add to couple of points to this as one should consider the room
temperature, a fluctuation of 5 degrees Celsius can significantly drift
the pinholes as well as the alignment of lasers.
In addition, Leigh can obtain a reflected mirror slide (I think Zeiss
can sell one-it is used for 2Photon excitation finger printing) to check
the excitation intensities at identical power, dichroics and path might
help (by viewing the histogram for the both intensity distribution and
peaks). 
A control measurement not only at the start and end but also in the
middle might help too.
Even with all these precautions, and if one still questions the
stability of the system on intensity based measurements then one could
provide the data from Time domain (FLIM) to validate the claims.
Shiv
 

Mayandi Sivaguru, PhD
Associate Director
Molecular Cytology Core
Biomolecular Research Facilities
120 Christopher S. Bond Life Sciences Center
University of Missouri
Columbia, MO 65211
 
Voice: 573-882-4895
Fax: 573-884-9676
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www.biotech.missouri.edu/mcc
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Williams, Geoffrey
Sent: Monday, May 08, 2006 3:20 PM
To: [log in to unmask]
Subject: Re: standardisation/calibration

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Great comments to this point.

I will refrain from commenting about how stable our system or systems
I've used in the past are as they bear no aid for Leigh's problem (okay
well I will refrain from mentioning manufacture names).

However, there is one potential solution that could help.  Two ways to
deal with this and one is to set the laser using beads or what not.
Another is to approach it in the manner that one would to set a
calibrated intensity using those metal halide liquid guided systems as
discussed on the list.  Specifically collect a reference fluorescent
image prior to starting and make a correction based on the relative
intensities.  It does involve a standard, but that is something every
confocal facility should have. 

On the more controllable side, depending on the budget, purchase a laser
power meter that would fit over the objective, that or find an
enterprising Physics student to make you one (extremely simple to
measure intensity).

Good luck, Oh I should add, I've had one service engineer tell me that
the Argon laser lines take 15 minutes to reach about 50% power and then
slowly increase to 100% over first hour, and another service engineer
say that they are good to go from the get go.  I've known some lasers to
be absolutely rock solid and others to fluctuate very significantly.
Some users report to me that if they come in to use the system at
different times of the day (say 4pm vs 8am) only to find that a 4pm
intensity to be much lower than if they use the system when they turned
it on at 8am to be brighter.  

Do not forget about variance of Pinholes or light-path components over
the day (heat soak or fields).  Knowing the laser output is still only
1/2 the battle.  The detector has to see exactly the same intensity with
the same laser power time point to time point (simple PMT variance -
500V one day may be 550V the next - theoretically).  That's why the
collection of a reference image first and last (per session) can be the
only way to go even with all the fancy measurement gadgets you can buy.


Forgive the longwinded elaboration, as always please feel free to poke
holes in my comments in public or private...  the holes let the light
through ;-)

Geoff Williams
Leduc Bioimaging Facility Manager
Brown University
 
http://www.brown.edu/Facilities/Leduc_Bioimaging_Facility/
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Leigh Silvester
Sent: Monday, May 08, 2006 9:31 AM
To: [log in to unmask]
Subject: standardisation/calibration

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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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