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Hello Bob,
I generally run a high-res lambda scan across all six of our (visible)
laser lines (458,476,488,514,543,633) using a partially mirrored
coverslip to provide a reflecitve surface. I do this for each PMT, and
use a standardized PMT voltage and AOTF settings at 20%. This allows me
to monitor the relative differences in detection efficiency in different
parts of the spectrum between the PMT's as well as check the slider
calibration. I export the graph data as text and make my own graphs
with the data for every PMT on the same plot--this makes it much easier
to see the big picture than with the Leica graphing function.
An example of a plot taken when we had a stuck PMT motor combined
with mis-calibration is posted at
http://www.itg.uiuc.edu/ms/equipment/microscopes/CFM_performance/Benchmarks/LineScans_6_3_03.pdf
In this case, the PMT 1 mirrors are stuck in a closed position, and the
spectral reading for PMT's 2 and 3 are out of register. Also the slit
width on PMT 2 is set to a larger value than it should be.
An example of a functional and calibrated machine is posted at
http://www.itg.uiuc.edu/ms/equipment/microscopes/CFM_performance/Benchmarks/LineScans_6_11_03.pdf
In this case, spectra for all of the PMT's are in register, they are
accurate, and the spectral resolution is in the nieghborhood of 5-15nm.
The spectral resolution in the red range always seems to be lower than
in the blue range. Note that the relative heights between peaks for PMT
1 is different than for PMT's 2 and 3. This is because they are
different models of PMT; for a large bandwidth spectral scan, the
spectral fingerprint obtained with PMT 1 will differ from the
fingerprint for PMT's 2 and 3.
As a general rule, you should be able to figure out if one or both
of the mirrors on a particular PMT have failed or are seriously out of
calibration by comparing the spectra obtained for each PMT. For
instance, if the "blue" mirror on PMT 2 is stuck open, then PMT 1 will
not get any light, and PMT 2 will have huge background problems. If the
sliders are stuck open on PMT 3, then that PMT will have serious
background issues from reflected light, but PMT 1 and 2 may function
normally. If PMT 2 is miscalibrated, then the detection ranges for
PMT's 1 and 3 may be cut short or extended.
If the slit size is larger than it is supposed to be, then the
detection range set on the software interface will not reflect the
actual detection range, and people start to run into problems with laser
lines getting reflected in along with fluorescence. Anyone who notices
high background from reflected light should run a quick spectral scan
across a laser line--this can be done off of the coverslip/slide
interface and only takes a moment. In many cases it turns out that a
particular detector is stuck wide open. This will happen eventually on
well used systems; we've had PMT sliders stick/fail a couple of times in
the past year, and I have little doubt that it will happen again.
That's why I check it. Fluorescence peaks are broad and biological
sample preparation is complex, so it is easy to chalk things up to poor
labeling when in fact the detector is not detecting the fluorescence
peak, or the effective detection range is truncated by an open slit on
another PMT.
Cheers,
Karl
Robert Zucker wrote:
>
>
>Hi Karl
>Thanks for your comments on sliders
>How do you test that your sliders are working correctly? We use a
>Lightform lamp that shows defined spectra in the region between 400nm
>-650nm. Broader peaks indicate problems with sliders or the machine
>alignment. We are currently experiencing a major problem with PMT 2.
>Peaks are broad and shifted 10nm shifted lower values. How would you
>detect problems with the reflected light going into PMT 1 and PMT 3?
>when PMT 2 is defective. Would you just see more background with all the
>PMT's. What is the standard. It seems very subjective. Do you have a
>test slide that you use to access this spectral problem.
>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]
>
>
>
>|---------+------------------------------->
>| | Karl Garsha |
>| | <[log in to unmask]
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>| | 08/17/2004 01:08 PM |
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> | Subject: Re: Confocal Instability-shocking observation |
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>
>
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>The mechanical slits used to select spectral regions for detection can
>and do fail or change response on a periodic basis. They are
>electromechanical, and with heavy use they wear out and need to be
>replaced and properly recalibrated. It is not always obvious that there
>are problems when imaging conventional biological specimens, the biggest
>clue is an increase in background signal due to reflected light. It is
>wise to habitually check to make sure they are functioning properly, one
>bad slider motor can mess up all the detectors, because they are
>interdependant when bandwidth selections are made.
>-Karl
>
>Robert Zucker wrote:
>
>
>
>>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]
>>
>>
>>
>>
>
>--
>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
>
>
>
--
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
|
