Re: Confocal spectral calibration standards

Calibration and Validation on Laser based instruments are very important
if one is to acquire accurate and reproducible data that is to be
beleived
The following commucnication 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 poblems also
exsits 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

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 (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