Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
The sliders for PMT 1, 2 and 3 are controlled by the board that I
mentioned in my last post (board needs a giant heatsink). So are the
x,y and z galvo's, and various other motors (scan rotation, etc..). It
sounds like your board is overheating and giving bad signals to the
stepper motors. The thermal paste thing helps (of course a qualified
service guy should slap on the paste, and have a (working) replacement
board ready if that doesn't solve the problem).
-Karl
Carl Boswell wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi Bob,
>While I don't know anything practical about the Leica SP, your problem with
>warm vs. cold systems suggests a bad connection in the electronics of the
>PMT2 board. Classically, the condition of warming causing an intermittent
>or inconsistent connection because of expansion, either at contacts or
>within a circuit, that doesn't exist when the hardware is cold. You might
>try getting a loaner replacement PMT and see if that makes a difference.
>
>I've appreciated the discussion of testing the spectral system if for no
>other reason that I hope one day to have one and would need to know how to
>validate it.
>
>Good luck,
>Carl
>
>
>Carl Boswell, Ph.D.
>Dept. of Molecular and Cellular Biology
>Univ. of Arizona
>520-626-8469
>520-621-3709 FAX
>----- Original Message -----
>From: "Robert Zucker" <[log in to unmask]>
>To: <[log in to unmask]>
>Sent: Wednesday, August 18, 2004 3:51 PM
>Subject: Re: Confocal Instability-#2
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Confocal Instability update. Is there a relationship between ,machine
>temperature, hours of operation and stability?
>
>Additional data regarding temperature effects on our Leica SP confocal
>systems.
>Room temperature is very stable at 20C ( 69F)
>We have checked our system with the LightForm lamp for proper spectral
>calibration. Result:: Lamp is very stable --it is a standard-- however
>machine is NOT Stable!!!!
>.
>Data
>8-6-04 Friday morning --- STABLE ( cold system)--good spectral data in
>all PMTs
>8-6-04 Friday afternoon --UNSTABLE ( warm system) 4-6 hours of operation
>-- PMT 1 PMT 3 show good spectral data--PMT 2 shows bad spectral
>data--10nm shift with bad very resolution. multiple reflections.
>8-9-04 Monday morning --STABLE (cold system )--good spectral data with
>all PMTs
>8-17-04 Tuesday afternoon --UNSTABLE-(warm system) 2 hours running --bad
>spectral resolution with PMT 2 -- PMT 1,3 OK
>8-18- 04 evening (6PM) STABLE (cold system) PMT 2 is NOW GOOD --
>proper spectral registration
>
>How many hours of operation exists before the system goes into the
>unstable mode producing some really BAD data!!!!
>
>Does anyone has an idea on what is happening in this confocal system?
>More importantly what should be done about this problem?. Has anyone
>else seen this problem on their machine? Lightform lamp data seems to
>be correlated with increased refections in the system making the
>bandpass useable variable spectral range very narrow. Is the solution
>the following: Replace the slider in front of the PMT or just do a
>re-calibration of the system. Any ideas?
>Best wishes and thanks for the discussion.
>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]
>
>
>
>|---------+------------------------------->
>| | Stefan Terjung |
>| | <[log in to unmask]> |
>| | Sent by: Confocal |
>| | Microscopy List |
>| | <[log in to unmask]
>| | UFFALO.EDU> |
>| | |
>| | |
>| | 08/18/2004 11:32 AM |
>| | Please respond to |
>| | Confocal Microscopy |
>| | List |
>| | |
>|---------+------------------------------->
>
>
>
>>---------------------------------------------------------------------------
>>
>>
>-----------------------------------|
> |
>|
> | To: [log in to unmask]
>|
> | cc:
>|
> | Subject: Re: Confocal Instability-shocking observation
>|
>
>
>
>>---------------------------------------------------------------------------
>>
>>
>-----------------------------------|
>
>
>
>
>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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