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Thanks Jeremy,
    Your statement is well put and I couldn't agree more.  Ours is a
multi-disciplinary institution and about 70% of our applications for LSM
are heavily influenced by materials scientists and physical chemists,
and as the boundaries between biology, bio-engineering and more strictly
quantitative sciences blur, I believe the emphasis on data integrity for
laser scanning microscopy on the part of end users will increase.   Such
groups joke about marketing claims such as having "the best optics in
the world" when the specifics for various performance parameters are
kept under wraps.  Faith based acceptance of marketing claims is
contradictory to the training and methods employed by many scientists.
    Biology has a history of using qualitative evidence to support
claims, and measurements tend to be relative rather than absolute.  Many
biologists are familiar with widefield based systems with few
electro-mechanical and electro-optical components that run for years and
years requiring only light bulb centration and occasional computer
reboots.  Even electron microscopes are more robust in terms of
performance over time than laser scanning instruments.  Biological
specimens are amorphous, and instrument calibration and testing is
sometimes only addressed on the first day of undergraduate physics for
non-majors laboratory (an experience much loathed by many biology
majors).   Feeding into the faith in instrumentation held by many
biologists are service demonstrations that don't really measure
anything; such "look, it's working great" type tests are usually
designed to give positive results under all but the most dire of
circumstances, and end users are generally (perhaps purposely) not
trained how to perform meaningful diagnostics by LSM companies.
    I've had clients try to use operational LSM instrumentation in other
departments on campus (while we are having service issues resolved) only
to find that the equipment was so out of calibration that it was
unusable in the context of their application.  The regular end users of
these instruments had no idea there was anything wrong.
    As the market matures and new sophisticated applications are
developed which employ laser scanning instrumentation, it is hoped that
some of the MBA training which influences the relative importance
between marketing and service/support will be tempered by closer
attention to qualitative measures of instrument performance documented
by administrators of such equipment.  It's not always the sample prep
that has gone south, and it is important that administrators of LSM
instrumentation know how to confirm this quickly and efficiently.
Cheers,
Karl

Jeremy Lerner wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> This message is a manufacturer's view of instrument calibration and
> validation, and is prompted by postings over the last month.  I am the
> President of LightForm, Inc., a company that designs and manufacturers
> spectroscopic instruments, non-confocal analytical spectral imaging
> systems,
> and is also the distributor of a multi-ion discharge lamp (MIDL).
> Readers
> are probably aware that Bob Zucker suggested that an MIDL could be
> used as a
> light source for standardizing, calibrating and validating the
> performance
> of confocal spectral systems (CSI).
>
> Most of my professional experience has been in the physics and chemistry
> communities each of which is well served by the National Institute of
> Standards and Technology (NIST) (www.nist.gov).  As you are probably
> aware,
> this institute develops, or recommends, certified standards and protocols
> with the full participation of the user, academic and industrial
> communities.    As a relative newcomer to the confocal community, I have
> been amazed that there is any debate about the merits of calibrating and
> validating CSI systems.  If we start from the premise that the
> integrity of
> a spectrally dependent image is a function of the quality of the spectral
> data on which it is based, then all CSI systems, under the same operating
> conditions, should obtain identical data.  If this is a reasonable
> expectation, then every CSI operator has a right to demonstrably,
> absolutely, and unambiguously prove that a CSI system is capable of
> acquiring high quality spectral data, at the time the data is acquired.
>
> To achieve this means that an absolute-standard spectrum acquired on one
> instrument should be identical to that acquired on every instrument of
> the
> same model and/or operating characteristics.   As you may have guessed by
> now, instrument operators in the analytical, physics, chemistry (as
> well as
> the image intensive remote Earth resource) communities, expect and demand
> the ability to prove that their instruments are in optimal condition.
> CSI
> operators have a right (perhaps obligation) to make no less than the same
> demand.
>
> As this does not appear to happen too often, I can only speculate that
> there
> are members of the CSI community that do not recognize the need.
> Perhaps a
> tradition of imaging through bandpass filters has made very expensive CSI
> systems into nothing more than "super filters".  I note however, that
> even
> the least expensive filter comes with a certificate showing a wavelength
> scan of its profile.  I have heard the claim that CSI systems are
> "relative"
> devices and are not "spectroscopic" or "analytical"; therefore
> standardization or user performed calibration and validation is
> unnecessary
> or too expensive. Does arguing against instrumental standardization,
> calibration and validation ever make sense in the sciences? One or
> other of
> us is either missing the point, is in denial, or is trying to postpone
> the
> inevitable.
>
> The hard fact is that very nice looking images can be acquired with an
> instrument that is both out of alignment and spectrally inaccurate.
> Perhaps
> a large part of the problem is the human reluctance to discard or
> undermine
> a "good-looking" image, especially when that image is self-serving.
> If you
> go for an MRI or blood test the instrument operator has your interest in
> mind and is, therefore obligated to confirm that it meets a minimal
> standard.  If all CSI operators were performing work for others, and
> careers
> or lives were at stake, there would be no issue whether or not to
> standardize and calibrate.  Having said this I would have thought that
> core
> imaging facility directors would demand that CSI manufactures provide
> calibration and validation tools, given that they too have
> responsibilities
> to their clients.  If an instrument is bought without user demonstrable
> standards or specifications, let both the buyer and operator beware!
>
> CSI operators should be aware that confirming that a CSI system is
> performing optimally is a trivial task that takes no more than a
> couple of
> minutes.  There is no mystery to a spectrometer - it may be covered by a
> black box, but its operation is transparent, described in the literature,
> and very well understood.  Even though all spectrometers distort or
> change
> the profile of a natural emission, these distortions can be accurately
> emulated and mathematically predicted.  Consequently, we know how to
> determine the theoretical appearance of any natural spectrum when
> characterized by any CSI system, or spectroscopic device.  All
> well-designed
> spectrometers, in good alignment and focus, will produce predictable
> theoretical spectral profiles every time.  It follows that if we use a
> universally accepted absolute-standard spectral calibration light
> source, we
> have a tool to assess whether an instrument is performing optimally or
> not.
>
> Fortunately, these tools have been available off-the-shelf for over 50
> years. The most common is a multi-ion discharge lamp, and is an
> absolute-standard light source that emits a series of spectral
> features that
> are stable, and known with very great accuracy and precision.  In
> fact, NIST
> publishes a list of emission lines for many elements, including those
> found
> in standard Hg/Ar wavelength calibration lamps. (Visit
> http://physics.nist.gov/PhysRefData/Handbook/ for a full listing of the
> elements covered).  A wavelength scan of the lamp's emission enables
> you to
> prove wavelength accuracy, focus, contrast, and relative
> wavelength-to-wavelength ratios without doubt or compromise.
>
> As an instrument operator you have rights. You have a right to be able to
> differentiate between expected spectral performance and degradation in
> optical integrity due to opto-mechanical instability, misalignment, or
> temperature.  You, or your colleagues, have a right to be able to
> reproduce
> your data on a similar system elsewhere. You have a right to be able to
> communicate objectively with colleagues, a service technician or a
> manufacturer using a universally accepted standard as a reference
> point.  It
> is the logical and scientific way to solve problems.  Have no doubt,
> it is
> emphatically in your interest to be able to differentiate between
> normal and
> abnormal instrumental behavior.
>
> The take home message is that performing QA on an instrument is in
> everybody's interest, there is no good excuse not to.  And no, it
> should not
> add to the cost of the instrument! I recommend that a committee be set
> up,
> made up of CSI operators and manufacturers, to jointly develop and
> formalize
> testing protocols. It would be best if the committee were to operate
> through
> NIST, ISAC or some other professional organization.
>
> (As a side note, for those interested in the details, I co-authored a
> paper
> that includes a tutorial on the theory and practice of spectral
> instrumental
> optical functions and their contributions to CSI system performance.
> It has
> been accepted for publication in Cytometry, if you would like a copy
> contact
> Bob Zucker ([log in to unmask]), or me.)
>
> Best wishes
> Jeremy
> Jeremy Lerner
> LightForm, Inc.,
> Tel: (908) 281-9098
> Cell: (908) 963-4262
> eMail: [log in to unmask]
> Web: www.lightforminc.com
> "There is nothing worse than a sharp image of a fuzzy concept."
> Ansel Adams
>


--
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