Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One important point: the side of the scanhead on the SP-2 is a giant
heatsink for the control board. The control board gets very hot; in
order for the heat to be pulled off of the board effectively, the
junction between the aluminum plates that hold the board and the side of
the scan head (the part with black fins) should be coated with thermal
paste--this enhances the heat transfer by an order of magnitude or
more. A warm room is a bad idea; if the board gets too hot, the
confocal may begin to behave erratically or the board may start to fail.
-Karl
Stefan Terjung wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear confocal stability interested,
>
>referring to the comment by Robert Atkinson that aluminium alloys (and other
>alloys) expand with temperature, I would like to stress that it is very
>important to keep the imaging conditions as constant as possible for
>reliable measurements.
>
>Regarding (spectral) confocal performance I think we should consider the
>following issues:
>
>- The room temperature should be kept as constant as possible (air
>conditioning often creates fluctuating temperature curves, sometimes
>resulting in different performance of confocals).
>
>- Keeping the confocal system (scan head with spectral detectors)
>temperature as constant as possible. If the room temperature fluctuates
>(e.g. due to the air conditioning system) an additional climate chamber
>around the microscope is often used to stabilize the focus over longer
>periods. If spectral reliability in fluorescence detection is important, it
>could be considered to build a climate chamber around the scanning unit. But
>of course the temperature of this box should be at the 'optimum working
>temperature' of the scanning unit (typically around 21°C). If the specimen
>should be kept at 37°C perhaps two climate chambers would be neccessary (Iīm
>not sure if this is applicable, any opinions on that?).
>
>- The calibration of the confocal (especially the spectral detection) should
>be done after the system warmed up sufficiently (also service technicians
>are proposing to warm up the system before starting to work for reliable
>results). Maybe Bob Zuckerīs system has been spectrally calibrated before it
>reached a stabilized temperature? Possibly this could explain why he
>describes PMT1 and PMT2 as excellent in the morning but having strange
>effects in the late afternoon ?
>
>Regards,
>
>Stefan
>
>
>
>Stefan Terjung, PhD
>EMBL, Meyerhofstr.1
>69117 Heidelberg, Germany
>Cell Biology/Biophysics Program
>Advanced Light Microscopy Facility
>Phone ++49-6221-3878-467 FAX-242
>www.embl.de/almf/
>www.embl.de/eamnet/
>
>
>
>
>>Hi Bob,
>>Aluminium alloys expand at around 23 microns per degree C for
>>every meter of material. This makes it important that the
>>equipment is aligned and used at the same stabilised
>>temperature. This is not always easy to achieve. It can be
>>especially difficult during long automated processes.
>>
>>Regards,
>>Robert.
>>
>>
>>Confocal Users
>>How stable is the alignment in your confocal microscopy
>>system? Does the beam wander or are the mechanical components
>>affected by heat?
>>
>>We Tested our Leica SP1 spectral system with a LightForm lamp
>>as described our MM 2004 abstract and in a tutorial review on
>>spectroscopic imaging to be published in Cytometry (in press)
>>" Calibration and Validation of Confocal Imaging System." The
>>test measures spectral registration of defined peaks between
>>400-650 using the LightForm Spectral Lamp.
>>
>>Morning: The system showed that PMT 1and PMT 2 were
>>excellent, with sharp narrow wavelength reference spectra
>>peaks. PMT 3 had less resolution than the other two for an
>>unknown reason. Late Afternoon: Strange image data recorded
>>in PMT 2 below the 647-excitation line while measuring
>>TOPRO-3 (PMT3) and Alexa 568 (PMT 2) -Possible reflections
>>were occurring in the detecting region assigned to PMT 2.
>>
>>We next tested the three PMTs in system with LightForm lamp.
>>It was observed that PMT 1 and PMT 3 were identical to the
>>morning values. PMT 2 shifted 5nm to lower wavelength values
>>and the FWHM of the peaks were now very broad. This suggests
>>that resolution was lost using this PMT 2 (our best PMT in
>>the morning). We put sliders over the laser line and found
>>the unusable range below the laser lines to be 8nm (488)
>>16nm(568) and 32nm (647). They supposedly should be 7nm or less.
>>
>>How is this occurring? Why is the system going out of
>>calibration? Must we test at multiple times in the day to
>>insure a calibrated system? How would you test for laser or
>>machine drift on your confocal machine? It is a shocking
>>observation that other confocal users need to be aware of, as
>>the ramifications are very serious for good reproducible
>>data. Let's discuss this confocal instability on this user group.
>>
>>PS-Same effect observed about 5 different times on our
>>machine over a 2-year period and at least twice on another
>>machine located in a different geographical area. We do not
>>check for this problem all the time. Do You?
>>
>>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 72 Research Triangle
>>Park, North Carolina, 27711
>>Tel: 919-541-1585; fax 919-541-4017
>>e-mail: [log in to unmask]
>>
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--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu
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