Hi All,
By « Flat images » what do you mean compare to dark
images? And what is the right procedure to acquire them? Thanks a lot,
Monique Vasseur
tél. (514) 343-6111 poste 5148
De : Confocal
Microscopy List [mailto:[hidden email]] De la part de Karl
Garsha
Envoyé : 20 avril 2010 22:51
À : [hidden email]
Objet : Re: photons vs. photoelectrons?
Hello All,
The photo-electron measurement can be considered to be the electrons which are
registered by the camera pixel, the conversion to photons is a calculation that
takes the quantum efficiency into account. The conversion to photons makes some
assumptions about the wavelength and bandwidth of the photon population that
delivered the photoelectron count (consider fluorescence emission and objective
transmission curves and filters convolved with the quantum efficiency of the
camera chip).
In my experience the evolve calibration technology is defensible from an
analytical standpoint; it is also valuable in a practical context. I have no
commercial interest in making this statement. I concur that it’s advisable to
understand what such tools do, and I don’t think there is any reason to believe
that technology obfuscates the theory behind it. Most of us probably don’t
contemplate how our mass air flow sensors affect spark timing in our
automobiles on our way to work, yet the information is available, and it can be
empowering under the right circumstances.
Because my cameras have to be calibrated, and I work with several cameras, I
submit that rigorous gain calibrations aren’t all that painless. The situation
with even the most advanced EMCCD technology can be substantially less trivial.
The type of automated gain calibration under discussion can take a number of
noise factors into account and make a non-trivial situation much more
manageable, accurate and precise.
With the evolve tool, the calibration is handled responsibly. I’ve made the
effort to convince myself of this. The automated calibration produces
more precise calibration than I’m likely to produce manually in the absence of
such automated calibration tools, but the big advantage is convenience. The
calibration is handled at every gain level (in multiple replicates) using a
uniform field illumination built into the camera. There is indeed quite a bit
more to it (mean-variance / photon transfer curve calculations using bias’s and
flats, bias stability management, etc.. as well as sophisticated voltage
management of the EM gain register), but my point is that this is done in
minutes. It would be prohibitive for me, or many other busy scientists, to be
doing this routinely. This technology makes it straightforward to have a
summer undergraduate intern, junior research associate or senior scientist all
collaborating to gather advanced quantitative data in the context of the ‘big
picture’ (no pun intended) without us worrying about whether someone calibrated
the camera at a given gain state correctly.
If others have opinion that departs from my experience, then it’s worth
discussion; it can be healthy to challenge new tools and pose questions. But we
should do so based on evidence. Data I gathered using an evolve clearly
indicates the calibration performed by the camera is accurate –when I
tested the linearity of the EM Gain on a calibrated unit the actual least
squares fit I recorded had an R squared value of 0.9995. The gain reported is
the measurable gain, to the best of my ability to verify. This isn’t an
exercise I would repeat for fun, but I can speak to the results. The technology
does work, quite well. Quantitative work with EM cameras raises responsibility
for considerations beyond those typical of interline cameras. There are
different sources of error, noise etc.
I can put on a slide prepared a year ago on one of my instruments and tell if
it changed and by how much. I require this level of instrument
characterization. This brings up an important point however: analytical imaging
technology is a system level calibration. Fluorescence is a real-time
photochemical phenomenon, and variability can arise from both the instrument
and sample. If you want to truly resolve sample differences, both the
illumination and the camera need to be well characterized (assuming
standardized optics). I’ve witnessed 30% discrepancy between instruments
because of light guide aging (all other things being equal, new arc lamps etc).
Technologies like closed loop illumination and sample plan calibration can be
enormously helpful in helping to efficiently assure data integrity. The recent
introduction of practical quantitative illumination and calibration tools is an
important advance that makes quantitative work more accessible, reliable and
convenient.
So, in the spirit of informative discussion, I've added my input as well.
Best Regards,
Karl Garsha
On Mon, Apr 19, 2010 at 9:27 PM, Mark Cannell <[hidden email]> wrote:
Hi Steve
As EM gain calibration is so trivial -I couldn't help but be unimpressed :-P
To calibrate in terms of average photoelectrons across the image is also
trivial when you reduce the signal to << 1 photon per pixel and take
plenty of frames. But that is not still not accounting for the pixel to pixel
sensor variation. My point is that is that you can't calibrate an _image_
by assuming that the gain and offset of every pixel is the same -you need darks
and flats to do this and only then can you provide an image calibrated in
'photons' captured. I may be getting old but I don't like seeing quite
complicated ideas being distilled by "turn key" solutions to the
point where a user thinks they have something accurately calibrated -but never
know what the calibration means or its assumptions.
I know that many folks these days don't seem to want to know anything about the
limitations of the methods they use because they think results are more
important (than actually understanding what their machine actually does). But
as you know, my view is that unless you "understand the process of imaging
you risk imagination" (c).
another 2c.
Cheers Mark
Stephen Cody wrote:
Dear List,
I just checked the Evolve web page again, it is not a "Dark
Calibration" but a light calibration. A shutter is closed, and an
internal light source in the camera activated to calibrate the EMCCD.
Below I've extracted the relevant text..... I have no commercial
affiliation with Photometrics.
From Photometrics Web page (This is from a commercial company, I have
no affiliation ad I have no personal experience of this product).
"EMCCD cameras are subject to aging of the EMCCD register as a result
of its usage. The Evolve has a simple calibration feature that
performs the industry’s most accurate EM calibration within 3 minutes.
A simple turn of the camera’s nose-piece closes a shutter and
activates a light source which the detector uses to calibrate its EM
gain. This ensures that users will receive the most accurate EM gain
and EM gain applied matches what the user requests.
Simple software control will allow the user to use this feature as a
manual shutter in order to block all light from the sensor in order to
take dark reference frames if necessary."
On 20 April 2010 09:37, Stephen Cody <[hidden email]> wrote:
Dear Mark et al,
As I understand from the promotional material for this camera, there
is a dark calibration procedure built into the camera. The eVolve web
site while very glittzy is quite informative (if you can stand the
hype)
Stephen Cody
On Saturday, April 17, 2010, Mark Cannell <[hidden email]>
wrote:
Hi All
I must admit to being unimpressed by this 'improvement'. It removes (from the
researcher) the need to understand what a camera really does and I doubt that
it is accurate. Before someone howls at this, I would point out that
astronomers who routinely produce calibrated images use a dark and a flat frame
to achieve this. Without a dark, you cannot calibrate the camera image -even if
you assume it is flat (which it isn't). The problem is that the camera changes
it's properties (especially the EM register) so no single calibration is going
to be accurate. Since it is easy to actually use darks and flats to calculate
actual photon numbers, why rely on a manufacturer calibration? I suggest it's a
bit like assuming your Gilson/Eppendorf is still correct and everyone knows
that's not GLP -right? But let's be clear, most people don't give a damn about
how many photoelectrons there are -they just want a pretty image. For the few
cases where photo-electron numbers are needed, the time taken to take darks and
flats are trivial compared to the time taken in precise experiments.
my 2c
Mark Cannell
*Van:* Confocal Microscopy List [mailto:[hidden email]]
*Namens *John Oreopoulos
*Verzonden:* vrijdag 16 april 2010 16:04
*Aan:* [hidden email]
*Onderwerp:* photons vs. photoelectrons?
The recent release of the Photometrics EMCCD "eVolve" camera which
has the ability to output images with pixel values that correspond to
photoelectron counts......
--
Stephen H. Cody
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