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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
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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]
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| | Stefan Terjung |
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| | 08/18/2004 11:32 AM |
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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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