Re: A manufacturer's view of the instrumental calibration controversy

Steve and Paul	--can you post this with a new subject heading

Calibration and Validation on Laser based instruments are very important
if one is to acquire accurate and reproducible data that is to be
believed
The following communication was posted by a colleague on the Confocal
listserver. I am reposting on the flow cytometry listserver as the QA
issues are relevant to the Cytometry community. Do these problems also
exists with flow cytometers?
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: zucker.robert@epa.gov


Re: A manufacturer's view of the instrumental calibration controversy

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 a shadow of 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 manufactures, 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 (Zucker.Robert@epamail.epa.gov) or
me.)

Best wishes
Jeremy

Jeremy Lerner
LightForm, Inc.,
Tel: (908) 281-9098
Cell: (908) 963-4262
eMail: jlerner@lightforminc.com
Web: www.lightforminc.com
"There is nothing worse than a sharp image of a fuzzy concept."
Ansel Adams