We have published a few articles on measurements of power which may be
useful for this discussion I have listed a few references below that
have been published and have added some paragraphs from some of those
papers that may be helpful. .Many factors influence these power
measurements between different machines. Some of these factors were
discussed in these papers.
I would like to mention that there is "instability" in the power
measurement due to heat dissipation, fiber polarization or AOTF
problems. This data on power fluctuations is discussed in the July 2006
issue of Cytometry A and will impact on power measured. PDF files of the
July 2006 papers will be available shortly and will be sent out on
request.
Bob
1. Zucker RM Confocal Slide Based System :Performance Cytometry In
press July 2006.
2. Zucker RM Confocal Microscopy Slide Based Systems: Instability
Cytometry In Press July 2006
3. Zucker, R.M.. Confocal Microscopy System Performance. Laser Power
. Microscopy Today. 10: 20-22 Nov/Dec 2002
4. Zucker, R.M. Evaluation of Confocal Microscopy System Performance.
Cell Imaging Techniques. Douglas Taajets editor Humana Press Chapter 5
77-135 2005
Laser power
The laser power test appears to be one of the most useful tests to
evaluate if a system is misaligned or functioning suboptimal by
recording insufficient laser power readings. The laser power test can
indicate if the system is aligned properly up to the plane of excitation
on the stage or if the machine has a defective component (i.e. a dying
laser, defective AOTF or a broken fiber optic). In our experience,
without sufficient power throughput in the system, the PMT voltages will
have to be increased to high values to visualize fluorescence derived
from specimens. This will result in reduced image quality.
Visible Laser power test
Connect the suitable UV or visible probe (Coherent probe detectors
(UV-L818, Vis-LN36) a power meter (Lasermate/Q, or FieldMaster,
Coherent, Auburn California) and place it directly on top of the dry 10x
objective. The power meter is adjusted to the specific wavelength (365,
488, 568, or 647 nm). A lens holder can also be fabricated in the
machine shop and placed on the stage. Laser power will be affected by
the objective (magnification and NA
The CLSM zoom factor is set from 8-32 to reduce the beam scan and to
focus it into the “sweet spot” of the detector. The scanner is set at
bi-directional-slow speed to reduce the time period that the power meter
is reading “0”. The maximum digital reading from the power meter is then
recorded. The power derived from this measurement is dependent on the
type magnification and NA of the lens used. Each lens will have a
unique set of values, which is dependent on objective’s NA and other
transmission factors. The power meter diode was not reliable in this
system and could only be used as a crude estimate of the functioning of
the laser. This may change in the future with better-designed systems.
In Zeiss LSM 510 confocal systems the power measurement obtained must be
multiplied by a factor 2 to get the correct reading as there is
blanking( system shut off during return sweep) that occurs in the
measurement.
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 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]
Paul Rigby
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Hi All,
I am attempting to make direct measurements of the laser power at the
sample plane on a Leica TCS SP2 confocal. This will be done for each of
the visible laser lines available on our instrument (458, 488, 514, 561,
594 and 633nm). Determining absolute power as well as relative power
between lines is the aim.
The sensor is mounted in the specimen plane and a low NA objective is
used in an attempt to collect as much of the laser beam as possible
(thereby hopefully minimising the effects due to focus position on the
sensor). The zoom is set to maximum (x32) in an attempt to get the
scanned region as small as possible and unidirectional scanning is
performed at 200Hz (slowest setting available).
When I observe the readout power on the meter, the values fluctuate
widely, although a maximum value can be seen. I do not see this
variation when I do the same type of measurements on my Biorad
MRC1000/1024 UV system. Can anyone explain what might be happening here?
Could it be related to the function of the AOBS (or similar optical
components) in the system? Perhaps my meter does not respond quickly
enough for the blanking performed during laser beam flyback? How do
others do this type of direct measurement of laser power in the sample
plane on their Leica (or other) instruments?
All comments, suggestions and criticisms welcome.
Cheers
Paul
---------------
Dr Paul J Rigby
Director and Senior Lecturer
Biomedical Imaging & Analysis Facility (M510)
The University of Western Australia
35 Stirling Highway, Crawley, WA, 6009
Australia
Ph (61-8) 9346 2819
Fx (61-8) 9346 3469
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