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Dear Martin
I think it is a step in the right direction that a major confocal
microscopy company is making measurements that insure their system
(Leica TCS SP2 AOBS) is optically efficient and the PMTs are absorbing
all of the photons that are possible so the sensitivity is improved and
cellular damage is reduced. This is the aim of all confocal microscopes
and we congratulate you on the technical improvements in your new Leica
TCS SP2 AOBS.
It is important for the confocal user to be able to test this
performance level in their own laboratories to insure that these
fantastically designed machines are working at the levels they were
designed to work at and at the levels they were tested in the factory
prior to shipment. Occasionally, the machines in the field do not
always perform to the levels that the manufactures have designed them to
operate at. I said occasionally. This is a problem that all confocal
core laboratories have to deal with at different times. How do we test
the machines to insure that they are working correctly in our
laboratories? It does us little good if we have the best designed
machine that does not function at those levels in our laboratory. We
need to test the whole system not just individual components. Sometimes
individual components may all have fantastic performance specifications
but when you put them together in one system you may get junk as they
are not aligned correctly or they may be just incompatible. What is the
increase in sensitivity of the Leica TCS SP2 AOBS relative to the TCS
SP1 or SP2 filter based system? I find it hard to believe that there
would be a 25-45% increase in sensitivity by removing just one dichroic
element.How many other elements were removed in this filterless system?
Perhaps the other components in the new system have different
specifications that increase the sensitivity. I believe, it is
important to test the whole system not just individual components to
conclude that the machine is 25-45% more sensitive.
We have published a few papers on the subject of Confocal QA that allows
the user to test some aspects of their equipment.This is a starting
point and not the end of the testing procedure. Although we sometimes
try to test individual components in a confocal microscope to learn the
limitations of the machine, our focus is primarily on testing the whole
system performance to optimize the machine operation. We, as scientists
that use confocal microscopes , could use the help of all manufacturers
to release some testing guidelines and procedures that will help
confocal users to test their machines in the field. Without direct
input from the manufacturers papers and books have been written by
Pauley, Paddock, Hibbs, Frohlich, Carter, Zucker, and others that will
serve as the reference procedures to test this equipment. I am sure you
may not agree with all of the testing protocols in these publications,
but in my opinion they are better than subjective measurements that are
made on a histological slide that are sometimes used to measure
performance and sensitivity of a confocal microscope.
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]
Martin Hoppe
<[log in to unmask]> To: [log in to unmask]
Sent by: Confocal cc:
Microscopy List Subject: Re: Sensitivity
<[log in to unmask]
UFFALO.EDU>
08/18/02 12:46 PM
Please respond to
Confocal Microscopy List
Robert,
Numbers, no marketing hype: I have been comparing the Acousto-Optical
Beam Splitter (AOBS) transmission to conventional beam splitters, which
are replaced by this element in the Leica TCS SP2 AOBS systems. The
spectral transmission characteristics of the individual elements are
measured e.g. by placing them in a conventional spectrometer. The
measurement results then can be overlayed to the emission curves of the
fluorochromes, giving you quantitative results of the improvement in
collection efficiency for a certain fluorochrome (or fluorochrome
combination).
Leica has such spectral transmission and sensitivity data for all of the
elements of their confocal systems. These data are generally not
distributed to the public.
However, like you we clearly see the need for better customer
information about how collection efficiency is influenced quantitatively
in a confocal microscope. Therefore we have developed a simulation
program, which shows the effects of important elements in a confocal
microscope to the final "live" image. Besides beam splitters, AOBS, and
detector types, the program also includes live separation of overlapping
emissions by linear unmixing (which is far better, if your system has
good primary collection efficiency). You can simulate different laser
excitation intensities and see the effects on dye separation and
signal-to-noise ratio in the final images. Dye combinations can be
picked from a database of fluorochromes. The simulation has been
"reality-checked".
The program can be demonstrated by the local Leica Microsystems Confocal
Representatives. You may also contact me directly for further
information or if you would like to get it.
Best regards
Martin
-------------------------
Martin Hoppe, Ph.D.
Leica Microsystems Heidelberg GmbH
Am Friedensplatz 3
D 68165 Mannheim, Germany
E-Mail: [log in to unmask]
Phone: +49-621-7028-1100
Faqx: +49-621-7028-1180
Search the CONFOCAL archive at
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We just returned from the Microscopy meetings in Quebec MM2002. Great
area of the world to visit in the summer and perhaps in the winter. On
our return we found that there was an interesting discussion on
sensitivity of confocal microscopes being discussed on the confocal
list. How exactly does one measure that a system without filters is
more sensitive and 25-45 % brighter than another system with filters.
Is
this a marketing statement or is there a detailed approach that one can
use to measure sensitivity??
To test sensitivity, last year, we presented a method in Cytometry in
which we first measure the power on the stage of a specific laser line.
Then we put a reference particle ( i.e. 10u bead or fluorescent plastic
) on the stage Next we take a CV of the pixel distribution in the
image
and compare the CV value to that obtained from different CLSM systems.
The CV number will be related to the sensitivity of the system and
the
ability of the system to collect light with low PMT settings. Lower
PMT
settings in general will translate into better sensitivity but the PMT
number is not a valid indicator of sensitivity as PMT voltages are only
crude estimates of system performance.
It is very difficult to compare two different manufacturers machines by
this technique as all acquisition parameters have to be equivalent.
However, it is possible and if done correctly one may make a statement
that one configuration is 25-45% more sensitive than another,
especially
if both systems are form the same manufacturer.
Our procedure to measure sensitivity has been describe in two Cytometry
publications last year that are available on request in PDF file
format.
The two papers have been published in the August 2001 issue of
Cytometry.
Zucker, R.M. Price OT Evaluation of confocal system performance.
Cytometry 44:273-294 2001.
Zucker, R.M. Price OT Statistical evaluation of confocal microscopy
images.
Cytometry 44:295-308 2001
How does Dr Martin Hoppe of Leica make the statement that their unit is
the most sensitive confocal microscope and is 25-45% brighter than a
filter based system? Are they using subjective criteria or are they
using a method similar to what we have outlined in these two
publications? Could this actually be the start of confocal
manufacturers
issuing performance specifications on their equipment? It would be a
step in the right direction if this is indeed the case.
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]
Martin Hoppe
<[log in to unmask]> To:
[log in to unmask]
Sent by: Confocal cc:
Microscopy List Subject: Re:
Simultaneous four channel imaging
<[log in to unmask]
UFFALO.EDU>
08/07/02 03:36 PM
Please respond to
Confocal Microscopy List
Robert,
to answer your question: the AOBS - I like to re-name it "Golden Eye",
our project code name, although not really in line with our corporate
ID
:-) - improves optical collection efficiency by 25-40% over
conventional
high-performance dichroics, depending on the fluorochrome combination.
This means you can lower the excitation intensity significantly, reduce
bleaching and improve cell viability. In addition, excitation light is
virtually completely eliminated from the emission spectrum, you can
even
record spectra across the excitation lines.
Best regards
Martin
-------------------------------------------------
Martin Hoppe, Ph.D.
VP Marketing & Sales
Leica Microsystems Heidelberg GmbH
Am Friedensplatz 3
D 68165 Mannheim/Germany
Email: [log in to unmask]
Phone: +49-621-7028-1100
Fax: +49-621-7028-1180
In a message dated 07.08.2002 14:43:10 Westeuropäische Sommerzeit,
[log in to unmask] writes:
There is no big gap as far as I am concerned. We are using a Leica SP
with
four detectors (UV/Ar/GreNe/HeNe lasers) and the AcoustoOptical Beam
Splitter (AOBS - I've told them they need a different acronym...).
Empirically, light thoughput (or perhaps I should say useful spectral
windows) are very nice - samples are quite "bright" compared with our
old
dichroic-based system. I think someone really ought to do the
quantitative
work on this and I think Leica has some "relative" numbers (relative to
their own filter-based system).
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hello Confocalists
>
>We are beginning to think about new microscopes and I'd appreciate any
>info on the following question:
>
>Is there a system out there which will allow simultaneous detection /
>imaging of four fluorophores emitting from blue to far red (eg AMCA /
>Cy2 / Cy3 / Cy5)? We do this sort of imaging routinely in wide-field
and
>increasingly we need to it at confocal level. (Indeed if five channels
>were available we'd use them!)
>
>I know that the lasers are available (eg blue diode plus Kr/Ar or Ar
>plus red diode or whatever... I know that Leica and Zeiss have their
>spectral-detection systems (discussed here not long ago). From what I
>can figure out, it looks like BioRad has the lasers but only three
>detection channels; Zeiss seems to have the detection system but do
they
>have the lasers? I got lost in Leica's website and gave up...(Sorry
>Leica!)
>
>Since the discussion a couple of months ago, has anyone come up with
new
>info / ideas about the real value of the Leica and Zeiss approaches?
>From what has appeared on the list so far, I suspect there is a big
gap
>between theory and practice here...
>
>Feel free to reply off list if appropriate.
>
>Thanks
>
>IAN
>
>
>
>--
>Professor Ian Gibbins
>Anatomy &Histology
>Flinders University of South Australia
>GPO Box 2100, Adelaide, SA 5001
>Australia
>
>Phone: +61-8-8204 5271
>FAX: +61-8-8277 0085
>Email: [log in to unmask]
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 308
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396
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