Precisely driving several devices from IgorPro through National Instruments board possible?
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Ponti, Aaron |
Precisely driving several devices from IgorPro through National Instruments board possible?
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Dear list
we would like to set up a system for behavioral experiments (conditioning) with mice where several pieces of hardware should be driven and, most importantly, precisely synchronized (at the few-ms scale). We currently are thinking of using WaveMetrics Igor Pro the Igor NIDAQ Tools MX to program the experiment protocols and then drive all the hardware through a National Instruments NI PCI-6229 board. In our setup, we would need to control (1) a laser through analog and digital outputs (->aotf and ->ttl) to modulate the laser power and/or to switch it on and off; (2) a sound source (digital), (3) a camera (digital); and possibly other recordings. Does anybody have any experience with using Igor Pro for this kind of things? Is it possible to trigger all this hardware precisely? The laser pulses will have down to 5ms period, is this time resolution possible at all? Is it conceivable to be able to react to measurements, i.e. modify the generated output signals on the fly in response to measured inputs? Can you otherwise suggest other approaches? Thanks a lot for any suggestions ---------------------------------------------------------- | Dr. Aaron C. Ponti | Friedrich Miescher Institute for Biomedical Research | Facility for Advanced Microscopy and Imaging | Image processing and analysis / software development | Maulbeerstrasse 66 CH-4058, Basel | WRO-1066.0.16 | Tel: +41 61 696 3513 | Fax: +41 61 697 3976 | http://www.fmi.ch/faim ---------------------------------------------------------- |
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Craig Brideau |
Re: Precisely driving several devices from IgorPro through National Instruments board possible?
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You will probably want to use the onboard high-speed counters to
handle the laser modulation. Rather than doing analog control, just use pulse-width modulation with the counters. If the overall repetition rate is very high the subjects should not be able to perceive the flashing. If you try to use the digital I/O lines and trigger them in software you will be at the mercy of the computer's task-handling capabilities. Since PCs are not designed to be real-time devices this could lead to some irregularities in the software triggering. Best of luck! Craig On Thu, Apr 15, 2010 at 6:48 AM, Ponti, Aaron <[hidden email]> wrote: > Dear list > > we would like to set up a system for behavioral experiments > (conditioning) with mice where several pieces of hardware should be > driven and, most importantly, precisely synchronized (at the few-ms > scale). We currently are thinking of using WaveMetrics Igor Pro the Igor > NIDAQ Tools MX to program the experiment protocols and then drive all > the hardware through a National Instruments NI PCI-6229 board. > In our setup, we would need to control (1) a laser through analog and > digital outputs (->aotf and ->ttl) to modulate the laser power and/or to > switch it on and off; (2) a sound source (digital), (3) a camera > (digital); and possibly other recordings. > Does anybody have any experience with using Igor Pro for this kind of > things? Is it possible to trigger all this hardware precisely? The laser > pulses will have down to 5ms period, is this time resolution possible at > all? Is it conceivable to be able to react to measurements, i.e. modify > the generated output signals on the fly in response to measured inputs? > Can you otherwise suggest other approaches? > > Thanks a lot for any suggestions > > ---------------------------------------------------------- > | Dr. Aaron C. Ponti > | Friedrich Miescher Institute for Biomedical Research > | Facility for Advanced Microscopy and Imaging > | Image processing and analysis / software development > | Maulbeerstrasse 66 CH-4058, Basel > | WRO-1066.0.16 > | Tel: +41 61 696 3513 > | Fax: +41 61 697 3976 > | http://www.fmi.ch/faim > ---------------------------------------------------------- > |
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Mark Cannell |
Re: Precisely driving several devices from IgorPro through National Instruments board possible?
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In reply to this post by Ponti, Aaron
Using DMA will certainly give good timing but at the expense of limited
overall cycle time. Without knowing precisely your timing protocol/rep rate regime you've asked a question that is hard to answer. You might want to experiment with a cheaper board -do you really need so many I/O lines? Hope this helps. Mark Ponti, Aaron wrote: > Dear list > > we would like to set up a system for behavioral experiments > (conditioning) with mice where several pieces of hardware should be > driven and, most importantly, precisely synchronized (at the few-ms > scale). We currently are thinking of using WaveMetrics Igor Pro the Igor > NIDAQ Tools MX to program the experiment protocols and then drive all > the hardware through a National Instruments NI PCI-6229 board. > In our setup, we would need to control (1) a laser through analog and > digital outputs (->aotf and ->ttl) to modulate the laser power and/or to > switch it on and off; (2) a sound source (digital), (3) a camera > (digital); and possibly other recordings. > Does anybody have any experience with using Igor Pro for this kind of > things? Is it possible to trigger all this hardware precisely? The laser > pulses will have down to 5ms period, is this time resolution possible at > all? Is it conceivable to be able to react to measurements, i.e. modify > the generated output signals on the fly in response to measured inputs? > Can you otherwise suggest other approaches? > > Thanks a lot for any suggestions > > ---------------------------------------------------------- > | Dr. Aaron C. Ponti > | Friedrich Miescher Institute for Biomedical Research > | Facility for Advanced Microscopy and Imaging > | Image processing and analysis / software development > | Maulbeerstrasse 66 CH-4058, Basel > | WRO-1066.0.16 > | Tel: +41 61 696 3513 > | Fax: +41 61 697 3976 > | http://www.fmi.ch/faim > ---------------------------------------------------------- > |
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The recent release of the Photometrics EMCCD "eVolve" camera which has the ability to output images with pixel values that correspond to photoelectron counts (instead of arbitrary digital count units) has me wondering a bit something. The idea behind this camera, as I understand it, is that having images reported in terms of photoelectrons instead of counts (ie: an absolute scale vs a relative scale) will allow a better comparison of image data between labs around the world, and even for single user comparing images acquired with a particular microscope from day to day. Seems reasonable to me and sounds like a pretty good idea. What I'm confused about is that I've seen some papers in the literature, mainly those that deal with single-molecule studies, that report image data in terms of actual photons detected. Is there a difference, and more importantly, can someone explain to me the advantage of using photon counts vs. photoelectron counts?
(No commercial interest for Photometrics) John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging |
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Dear John,
When people measure single molecule they tend to report in terms
of detected photons, probably because the amount of detected photons is a
parameter that (co)-defines the quality of the fluorescent probe they
use.
And if one
uses an APD detector you obtain a photon/s signal and then there is of course
also the method of single photon counting as a method to measure fluorescence
lifetimes.
Of course
that what is being measured are indeed photo-electrons, but some people tend to
correct the number of actually detected photons for detection
effiency.
Kind
regards,
Peter
________________________________ Van: Confocal Microscopy List [mailto:[hidden email]] Namens John Oreopoulos Verzonden: vrijdag 16 april 2010 16:04 Aan: [hidden email] Onderwerp: photons vs. photoelectrons? (No commercial interest for Photometrics) John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Dit bericht kan informatie bevatten die niet voor u is bestemd. Indien u niet de geadresseerde bent of dit bericht abusievelijk aan u is toegezonden, wordt u verzocht dat aan de afzender te melden en het bericht te verwijderen. De Staat aanvaardt geen aansprakelijkheid voor schade, van welke aard ook, die verband houdt met risico's verbonden aan het elektronisch verzenden van berichten. This message may contain information that is not intended for you. If you are not the addressee or if this message was sent to you by mistake, you are requested to inform the sender and delete the message. The State accepts no liability for damage of any kind resulting from the risks inherent in the electronic transmission of messages. Nederlands Forensisch Instituut Ministerie van Justitie |
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In reply to this post by John Oreopoulos
The recent release of the Photometrics EMCCD "eVolve" camera which has the ability to output images with pixel values that correspond to photoelectron counts (instead of arbitrary digital count units) has me wondering a bit something. The idea behind this camera, as I understand it, is that having images reported in terms of photoelectrons instead of counts (ie: an absolute scale vs a relative scale) will allow a better comparison of image data between labs around the world, and even for single user comparing images acquired with a particular microscope from day to day. Seems reasonable to me and sounds like a pretty good idea. What I'm confused about is that I've seen some papers in the literature, mainly those that deal with single-molecule studies, that report image data in terms of actual photons detected. Is there a difference, and more importantly, can someone explain to me the advantage of using photon counts vs. photoelectron counts? (No commercial interest for Photometrics) John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Hi John,
First of all, congratulations to Photometrics! (I think that
other companies may also offer this feature?)
Those of us who have been following developments in
phototdetectors have long recommended this approach although the
EM-CCD really made it essential.
Photoelectron counts are the best you can do. You can't actually
count photons until they have been turned into photoelectrons. The
quantum efficiency is the fraction of photons that are converted into
photoelectrons (and subsequently detected). It is often a strong
function of wavelength.
There are many advantages of this sort of calibration. Although,
as others suggest, it facilitates inter-lab calibrations etc, I think
that the main advantage is that is allows the operator to make a good
estimate of Poisson Noise and without this, any flirtation with
quantitative fluorescence is impossible.
Poisson noise defines the uncertainty of the photon signal
intensity as the sqrt of the number of quantum events
(photoelectrons), not the sqrt of some number stored in the computer
that is only proportional to the signal intensity.
Finally, it allows you to greatly compress the data without loss.
In the early days, NASA used to convert their CCD signal into
electrons and then take the sqrt of this number before sending it back
home (through a very slow, noisy signal channel). It halves the number
of bits needed.
Now, if we could just get the confocal manufacturers to do the
same. Admittedly determining the proper calibration is much harder to
do with PMTs than CCDs, but it is not at all impossible.
Cheers,
JIm Pawley
-- *********************************************************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ |
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In reply to this post by John Oreopoulos
In general, one photon detected by the camera will produce one
photoelectron. This is true for photons in the visible - CCDs are also
sensitive to X-rays, where a single photon will produce multiple
photoelectrons.
For more info on CCD physics, see James Janesick's excellent books. Kurt On 4/16/2010 7:04 AM, John Oreopoulos wrote: The recent release of the Photometrics EMCCD "eVolve" camera which has the ability to output images with pixel values that correspond to photoelectron counts (instead of arbitrary digital count units) has me wondering a bit something. The idea behind this camera, as I understand it, is that having images reported in terms of photoelectrons instead of counts (ie: an absolute scale vs a relative scale) will allow a better comparison of image data between labs around the world, and even for single user comparing images acquired with a particular microscope from day to day. Seems reasonable to me and sounds like a pretty good idea. What I'm confused about is that I've seen some papers in the literature, mainly those that deal with single-molecule studies, that report image data in terms of actual photons detected. Is there a difference, and more importantly, can someone explain to me the advantage of using photon counts vs. photoelectron counts? |
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In reply to this post by Zoon, Peter
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 (instead of arbitrary digital count units) has me > wondering a bit something. The idea behind this camera, as I > understand it, is that having images reported in terms of > photoelectrons instead of counts (ie: an absolute scale vs a relative > scale) will allow a better comparison of image data between labs > around the world, and even for single user comparing images acquired > with a particular microscope from day to day. Seems reasonable to me > and sounds like a pretty good idea. What I'm confused about is that > I've seen some papers in the literature, mainly those that deal with > single-molecule studies, that report image data in terms of actual > photons detected. Is there a difference, and more importantly, can > someone explain to me the advantage of using photon counts vs. > photoelectron counts? > > (No commercial interest for Photometrics) > > > John Oreopoulos, BSc, > > PhD Candidate > > University of Toronto > > Institute For Biomaterials and Biomedical Engineering > > Centre For Studies in Molecular Imaging > > > > Dit bericht kan informatie bevatten die niet voor u is bestemd. Indien > u niet de geadresseerde bent of dit bericht abusievelijk aan u is > toegezonden, wordt u verzocht dat aan de afzender te melden en het > bericht te verwijderen. > De Staat aanvaardt geen aansprakelijkheid voor schade, van welke aard > ook, die verband houdt met risico's verbonden aan het elektronisch > verzenden van berichten. > > This message may contain information that is not intended for you. If > you are not the addressee or if this message was sent to you by > mistake, you are requested to inform the sender and delete the > message. The State accepts no liability for damage of any kind > resulting from the risks inherent in the electronic transmission of > messages. > > Nederlands Forensisch Instituut > Ministerie van Justitie > |
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Stephen Cody-2 |
Re: photons vs. photoelectrons?
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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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Stephen Cody-2 |
Re: photons vs. photoelectrons?
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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 > -- Stephen H. Cody |
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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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Karl Garsha-2 |
Re: photons vs. photoelectrons?
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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 |
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Monique Vasseur |
Re: photons vs. photoelectrons?
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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 Hello All, On Mon, Apr 19, 2010 at 9:27 PM, Mark Cannell <[hidden email]> wrote: Hi Steve
Dear List, Dear Mark et al, Hi All --
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In reply to this post by Karl Garsha-2
Well I could not let this dumbing down analogy go by. My point is that
if you are a scientist doing research (even publishing a paper) on the air intake of your car you should know how the air mass sensor works and if its accurate!!! But this still misses the point that knowing what the EM gain is (and its very easy to measure) doesn't tell you how many photons arrived at the camera face. You need to know the A/D conversion factor AND some idea of camera flatness. Karl Garsha wrote: > Hello All, > <snipped> > > 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. Cheers Mark |
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In reply to this post by Monique Vasseur
A flat is the image obtained with a uniformly illuminated field. Uniform
illumination is not always easy to achieve, but if you could take images of a thin uniform dye layer as a reasonable measure. Then when you average many such frames you should have captured the non-uniformities in your optical system that affect the flatness of your fluorescence image. This link may help you: http://www.aavso.org/observing/programs/ccd/manual/3.shtml Regards, Mark > 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 > > > > |
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deepak sharma-2-3 |
Re: photons vs. photoelectrons?
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In reply to this post by John Oreopoulos
******Commercial Interest ******Reply from Photometrics******
I would first like to thank the contributors to this subject matter for their input. Photometrics would like to provide information and actual data which should help answer the points raised and discussed. This can be found at: http://www.photomet.com/useroutreach/042210.php We discuss measuring analog gain i.e. the cameras e/ADU conversion factor using a mean-variance analysis, how to calculate electron multiplication gain and the flatness or photo-response non-uniformity. We also explain that Rapid-CAL and Quant-View features of the evolve camera utilize a combination of these measurements to provide accurate data integrity. We hope this data and information helps clarify some of the questions being asked and we are always happy to answer any questions you may have. Deepak Sharma, Ph.D. Senior Product Manager Photometrics 3440 East Britannia Drive Tucson, AZ 85706-5006 |
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Hi All,
Hate to say I told you so... but you do need to do the calibration yourself . By the way, the method they propose has an important and fundamental assumption .... I suggest that if Photometrics would like to add/explain the conditions under which their calibration method is justified (and they assume _only_ Poisson noise :-O) on the web page that would be a great help to users. My tenet (that you need to understand/check what is being done) would seem justified :-) my last 2c Mark Deepak Sharma wrote: > ******Commercial Interest ******Reply from Photometrics****** > > I would first like to thank the contributors to this subject matter for > their input. > > Photometrics would like to provide information and actual data which should > help answer the points raised and discussed. This can be found at: > > http://www.photomet.com/useroutreach/042210.php > > We discuss measuring analog gain i.e. the cameras e/ADU conversion factor > using a mean-variance analysis, how to calculate electron multiplication > gain and the ‘flatness’ or photo-response non-uniformity. We also explain > that Rapid-CAL and Quant-View features of the evolve camera utilize a > combination of these measurements to provide accurate data integrity. > > We hope this data and information helps clarify some of the questions being > asked and we are always happy to answer any questions you may have. > > > Deepak Sharma, Ph.D. > Senior Product Manager > Photometrics > 3440 East Britannia Drive > Tucson, AZ 85706-5006 > |
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Andreas Bruckbauer |
Re: photons vs. photoelectrons?
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I have a few questions regarding
this:
- What is the point in knowing how many photoelectrons have been detected when photons get lost all the way through the microscope and the number of photons depends on other parameters like illumination intensity and environment of the dye? - Mark, you seem to be so confident about your way to calibrate the camera, how do you do it? - The method with dark frames and flats is described by Gosh and Webb in Biophysical Journal Volume 66 May 1994 1301-1318, they write: "this provides a lower boundary for the actual number of photons detected, because other noise contributions with similar square-root dependencies may exist." - Has anyone actually compared the results of these calibration with a result of an illumination of a known number of photons? best wishes Andreas -----Original Message-----
From: Mark Cannell <[hidden email]> To: [hidden email] Sent: Fri, 23 Apr 2010 4:07 Subject: Re: photons vs. photoelectrons?
Hi All,
Hate to say I told you so... but you do need to do the calibration yourself . By the way, the method they propose has an important and fundamental assumption .... I suggest that if Photometrics would like to add/explain the conditions under which their calibration method is justified (and they assume _only_ Poisson noise :-O) on the web page that would be a great help to users. My tenet (that you need to understand/check what is being done) would seem justified :-) my last 2c Mark Deepak Sharma wrote: > ******Commercial Interest ******Reply from Photometrics****** > > I would first like to thank the contributors to this subject matter for > their input. > > Photometrics would like to provide information and actual data which should > help answer the points raised and discussed. This can be found at: > > http://www.photomet.com/useroutreach/042210.php > > We discuss measuring analog gain i.e. the cameras e/ADU conversion factor > using a mean-variance analysis, how to calculate electron multiplication > gain and the ‘flatness’ or photo-response non-uniformity. We also explain > that Rapid-CAL and Quant-View features of the evolve camera utilize a > combination of these measurements to provide accurate data integrity. > > We hope this data and information helps clarify some of the questions being > asked and we are always happy to answer any questions you may have. > > > Deepak Sharma, Ph.D. > Senior Product Manager > Photometrics > 3440 East Britannia Drive > Tucson, AZ 85706-5006 > |
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Hi Andreas
As I said at the beginning, there are very few cases where actual photon numbers are needed, but it adds a veneer of precision/expertise to put out an image "calibrated" in photoelectrons. Now I don't mind that, but if it's to be done that way I would like it to be correct/honest. I hope you can see my point. As in other areas, the purpose of calibration is to allow reference to others. But in my experience it is hard to do a good calibration of most complex measurements so it's better if a result can be expressed in terms of a change... The only cases I can think of where actual quantum numbers are needed are for some statistical tests or fitting to theory. The trouble with EMCCD is that the multiplicative noise reduces the S/N so it's as if you actually got about half the number of photons. (So, if you are in a regime where your signal for the exposure time is much greater than the read noise you should not use an EMCCD. While most EMCCDs also allow you not to use the EM register, the read out amplifier for the CCD shift register is also very noisy by good CCD standards. ) But With an EMCCD, 'accurate' calibration is actually easier when you can detect a signal with mean signal per pixel <<1 phot. Now when you _count_ (by thresholding) events you have removed the problem of multiplicative noise so when you take the average signal intensity (minus dark frames of course) you know how many photoelectron events are associated with it. As far as I know, no software/camera does this -but you can. Cheers Andreas Bruckbauer wrote: > I have a few questions regarding this: > > - What is the point in knowing how many photoelectrons have been > detected when photons get lost all the way through the microscope and > the number of photons depends on other parameters like illumination > intensity and environment of the dye? > > - Mark, you seem to be so confident about your way to calibrate the > camera, how do you do it? > > - The method with dark frames and flats is described by Gosh and Webb > in Biophysical Journal Volume 66 May 1994 1301-1318, they write: "this > provides a lower > boundary for the actual number of photons detected, because other > noise contributions with similar square-root dependencies may exist." > > - Has anyone actually compared the results of these calibration with a > result of an illumination of a known number of photons? > > best wishes > > Andreas > > > |
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Andreas Bruckbauer |
Re: photons vs. photoelectrons?
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Hi Mark,
sorry if anyone is bored by this topic now, but i think counting single photon events after thresholding is a bad idea because the result depends very much on the threshold settings, the signal for one photon and read out noise is not well enough separated, i would rather trust the established methods. best wishes Andreas -----Original Message-----
From: Mark Cannell <[hidden email]> To: [hidden email] Sent: Fri, 23 Apr 2010 22:37 Subject: Re: photons vs. photoelectrons?
Hi Andreas
As I said at the beginning, there are very few cases where actual photon numbers are needed, but it adds a veneer of precision/expertise to put out an image "calibrated" in photoelectrons. Now I don't mind that, but if it's to be done that way I would like it to be correct/honest. I hope you can see my point. As in other areas, the purpose of calibration is to allow reference to others. But in my experience it is hard to do a good calibration of most complex measurements so it's better if a result can be expressed in terms of a change... The only cases I can think of where actual quantum numbers are needed are for some statistical tests or fitting to theory. The trouble with EMCCD is that the multiplicative noise reduces the S/N so it's as if you actually got about half the number of photons. (So, if you are in a regime where your signal for the exposure time is much greater than the read noise you should not use an EMCCD. While most EMCCDs also allow you not to use the EM register, the read out amplifier for the CCD shift register is also very noisy by good CCD standards. ) But With an EMCCD, 'accurate' calibration is actually easier when you can detect a signal with mean signal per pixel <<1 phot. Now when you _count_ (by thresholding) events you have removed the problem of multiplicative noise so when you take the average signal intensity (minus dark frames of course) you know how many photoelectron events are associated with it. As far as I know, no software/camera does this -but you can. Cheers Andreas Bruckbauer wrote: > I have a few questions regarding this: > > - What is the point in knowing how many photoelectrons have been > detected when photons get lost all the way through the microscope and > the number of photons depends on other parameters like illumination > intensity and environment of the dye? > > - Mark, you seem to be so confident about your way to calibrate the > camera, how do you do it? > > - The method with dark frames and flats is described by Gosh and Webb > in Biophysical Journal Volume 66 May 1994 1301-1318, they write: "this > provides a lower > boundary for the actual number of photons detected, because other > noise contributions with similar square-root dependencies may exist." > > - Has anyone actually compared the results of these calibration with a > result of an illumination of a known number of photons? > > best wishes > > Andreas > > > |
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