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I agree with Bob that performance specifications for a confocal
microscope would be a great idea. However, as I know Bob is fully aware,
the performance criteria for a confocal microscope are extremely
difficult to define. Individual components of the system can be readily
defined, but overall the performance of the instrument relies on
"balancing" many, often conflicting criteria (for example, changing the
pinhole setting will result in significant changes in the S/N in the
image - but perhaps at the expense of loss of resolution, or changing
the laser intensity will also alter the S/N but perhaps at the expense
of fading the sample!).

I have been involved in work that was aimed at attempting to compare the
performance between different instruments. We found that even comparing
the same instrument on different days was fraught with considerable
difficulty! The overall performance of the instrument was difficult to
assess using S/N, resolution or sensitivity. What we did find is that a
sample with a relatively low level of fluorescence that does not unduly
fade during imaging is the best way to test the instruments overall
performance. Such a sample can be readily prepared by using a few of
your own hairs and imaging using the relatively low level of
autofluorescence present in the hair (any natural hair colour is OK,
including red, blond, black or grey! But don't use hair that is dyed!).
The hair has a low level of autofluorescence across a broad spectral
range (using 488 nm light to excite) and has fine structural details
that give a good idea as to the overall performance of the system. The
good thing is the fluorescence is very stable, and the hair sample can
be kept for weeks or months without significant loss of autofluorescence
(a simple glycerol mount is quite adequate).

What could be provided by the manufacturers is a set of performance
criteria for the individual components (the laser, lens, PMTs etc), and
suitable reliable tests that can be performed in the field to determine
if the individual component is working correctly. This would be a great
help in determining whether the instrument was working as it should -
but may not be particularly useful in determining the overall
performance of the system. I suspect this is probably the reason the
manufacturers are reluctant to give specifications as one could then
make direct comparisons of the individual components between different
manufacturers - but which may be very misleading if one were to make a
comparative study of the overall performance of different instruments
using this criteria.

Any comments from the manufacturers would be most welcome.

Regards, Alan Hibbs.

BIOCON (publishers of the manual "Confocal Microscopy for Biologists",
mailto:[log in to unmask])
7 Walhalla Drive                 Phone: 61 3 9876 9822    FAX: 61 3 8660
2290
Ringwood East VIC 3135        Director: Dr. Alan R. Hibbs
Australia


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Robert Zucker
Sent: Saturday, 31 May 2003 2:42 AM
To: [log in to unmask]
Subject: Re: Confocal service issue

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Confocal Service variability
We have had a Leica TCD-4D and a Leica TCS-SP1 for the last 8 years.
Currently I feel that the Leica service organization is the best it has
been over that eight-year period.  Contrary to what Karl has said
regarding Leica service, I feel the current the service director is very
responsive and I believe is doing the best he can to operate an
effective service organization for the customer needs in USA bearing in
mind that it is a foreign owned company that controls the product
development, design, service and almost all other aspects of the
product. I have personally found him to be responsive to my concerns.
The Leica technician, John Zhang is a college graduate in bioengineering
from a respected US University. He has provided excellent service to our
institution in his three visits to our laboratory.  I would gladly have
him service our machine in the future. I would request his service for
difficult problems and have a high regard for his ability to problem
solve technical problems.

Why is there a difference of opinion on the service organization between
two knowledgeable users of similar confocal equipment?

 I feel the problem lies with the foreign manufactures and their lack of
providing consistent protocols to their service technicians. In America,
all point scanning confocal machines that are sold are built and
developed by foreign companies. These include the following:  Biorad
(England) Nikon (Japan) Olympus (Japan) Leica (Germany) and Zeiss
(Germany). These companies build the equipment and supply the necessary
protocols to their technicians to insure that their equipment is
functioning correctly. However there are NO published performance
specifications on the equipment from these companies. Therefore
technicians go into the field without performance specifications, which
may result in an inadequate knowledge on how to set up, align and fix
these machines.  Sometimes they succeed in repairing the machines to
acceptable subjective standards and sometimes they don't succeed in
solving the repair problems leaving the core operator very frustrated.
I believe this is the reason for the difference of opinion between
competent investigators about personnel in a specific service
organization.

It seems the service performed are evaluated by subjective criteria and
that is why a technician may not sometimes be able to adequately solve a
problem.  Where are the performance specifications for the machines? To
my knowledge not one of these manufacturers has a published set of
criteria or standards by which the investigator can insure his machine
is functioning properly. Thus it does not appear that service
technicians in the field have reliable published performance
specifications in their hands for the machines they are repairing.
Therefore I do not believe it is fair to hold an American service
manager or a technician responsible for the deficiencies of a foreign
company who has not provided the proper criteria to the service
organization to insure the machine is properly functioning. I think our
focus should be on the manufacturer and not the service technicians in
America who are trying to do the best they can without performance
specifications and specific protocols from the confocal manufacturers.
As a scientific community we should demand that these manufacturers
provide better criteria to insure our machines are functioning to the
level they were designed to perform at. They all are great machines when
they work properly.

We would gladly like any manufactures to comment to this confocal user
group on why there are NO performance specifications on their high
priced machines. This is not a subjective science and our data is
important to us and it should be not only be accurate but also
reproducible between different laboratories. Contrary to some opinion --
It is not only about Pretty Pictures. Some of us take measurements from
the machines and ask the machines to do critical things for our research
that require an aligned and properly functioning machine. Where are the
performance specifications on these machines?.
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]