http://confocal-microscopy-list.275.s1.nabble.com/Precisely-driving-several-devices-from-IgorPro-through-National-Instruments-board-possible-tp4907273p4939825.html
A flat is the image obtained with a uniformly illuminated field. Uniform
of a thin uniform dye layer as a reasonable measure. Then when you
image. This link may help you:
> 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] <mailto:
[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]
> <mailto:
[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]
> <mailto:
[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]
> <mailto:
[hidden email]>] *Namens *John Oreopoulos
> *Verzonden:* vrijdag 16 april 2010 16:04
> *Aan:*
[hidden email]
> <mailto:
[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
>
>
>
>