Posted by
Karl Garsha-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Precisely-driving-several-devices-from-IgorPro-through-National-Instruments-board-possible-tp4907273p4934891.html
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