EMCCD gain, photon scissors, bogeyman avoidance

[Karl Garsha-2]

Hi All,

If I am to recant my worldly ways and seek enlightenment in the manner of this 'one true religion' of EMCCD calibration, I'd like to know who we can credit. Can someone please help me with a citation or reference for this straight-forward <<1 photon approach?

Perhaps it is my over-simple yet pragmatic approach to problems that limits me, but I'm having trouble getting my head around the practical application of the simple darks and flats translating into subphoton imaging that we are being prescribed here. Exposure to Hulkamania, Bay Watch and monster truck rallys here in the US have likely so rotted my intellect that I fear I may have trouble grasping the subtle nuances of The Way. In all the excitement I must have misplaced my photon scissors and I don't know where to begin. What happens if the light source fluctuates while I'm madly clipping away at the feeble illumination with my photon scissors? Does this impact my calibration?

I can appreciate we should isolate the camera at least, so that we can measure within the constraints of camera noise and not total imaging system noise. We can begin with this. We should have our favorite bonafide even-field illumination methods ready.

I would like to point out that we should keep in mind that we shouldn't just calibrate our EMCCD once, this should be done at regular intervals. EM gain registers change they're properties as the camera is used, in other words, EMCCD cameras 'age'. Some cameras have built in calibration features that offset this change in the gain mapping through camera electronics, but that is technology and a primary tenent of our new lack of faith is that leveraging technology will make us dumber. So we'll try to do this the smart way. Depending on how heavily we use our cameras we may want to re-map gain on a weekly basis. After a while it should become routine, removing the camera, mounting on the even field illumination station, making sure the light source output is stable and calibrated. After a while it may seem tedious. To be completely throrough, I suggest playing 'Thriller' on an audio loop and working on a bogeyman dance while you do the weekly calibration. After just 52 weeks you will have a well characterized camera as well as a very impressive dance. It won't hurt anything unless you drop the camera moonwalking back to the microscope, and this measure will protect calibrations from any residual influence of evil spirits or bad juju.

To simplify and look at the big picture, let us contemplate, for the sake of discussion. What if, we first calculate our gain under non-EM conditions, using the non-EM read port. (by the way, the read noise here on a good EMCCD should be around 5-6 e- which is well in the range of a respectable fluorescence grade sony 285 interline). We can figure out the gain (full well capacity/bit depth) using the well-established mean variance method. We can do this gain calculation at a short exposure time, and over again at incrementally increased the exposure times. Calculate the gain at each exposure time, it should be consistent. But as the exposure time gets longer, both the mean pixel value and variance should increase in a proportional manner. This demonstrates linearity and implies a Poisson distribution of intensity values in accordance with what we would expect from quantum uncertainty. If I'm wrong, please help me gain a better understanding, I'm here to learn too, and I'm not infallible. The slope of this plot should reflect the constant 'gain' in electrons per grey level. In simple terms, as the amount of light captured increases, so does the pixel value. This happens in a predictable manner. If you look at a brightness level, you have some idea of the charge collected in that pixel, but only at a single gain level. This is a simple experiment, we hold the gain constant and the light constant and changed the exposure time, and saw how that translates into pixel values.

Now, what if, we start using the gain register. We can hold the light constant, hold the exposure constant, and take an image with zero gain applied. Subtract the zero-exposure 'bias' image. See what the modal brightness value is. Does it correspond to a place on the linear plot we created earlier? Raise the gain some convenient increment, does the (bias-subtracted modal) pixel value fall on our non-em gain plot of pixel values somewhere? How much exposure time difference corresponds to the two points at different EM gain settings? What does this relationship imply? What if you compare the mean value instead of the modal value?  You can start to look at other things like the distribution of pixel values and coefficient of variation at various EM gain settings as well. Are we measuring anything here or is this another grand illusion perpetrated by the Dark Prince of sub-photon imaging? Did the number of photons coming out of the light and hitting the chip change to half as many per unit time because we're using EM gain?

A natural extension of this is to now do this simple heretical experiment from zero gain to 1000x EM gain at regular increases in gain. At a constant light level, does the increase in modal pixel intensities follow a linear trend with linear incremental increase on the gain scale? Because this is easy, check this many times for each gain level...how precise are the values? If you do this many times at the same gain setting, does the value keep changing? This could be temporal gain drift, a source of imprecision. Does your camera have a specification for gain drift? Or is it the light source? Let us hope not...you better have a stable light to measure the camera, in accordance to the isolation principle.

Check the bias...collect a series of images at zero exposure time. Plot the modal values as a function of time in the series...did the bias drift? Does the gain drift and the bias drift? Now what. How can you subtract bias from a series of images if the bias is fluctuating, and what if the gain fluctuates as a function of time too? High quality EMCCD cameras have provisions to minimize this and provide a specification for both bias stability and gain stability, but it's technology. You can choose to leverage it, or not. Linearizing that gain curve isn't trivial either, so if you have a situation with different people using 'almost' the same gain they may be working with samples that are very different in quantum yeild, especially towards the higher side of gain amplification. 

What this all boils down to is ability to report differences within and between samples in some form of responsible unit. Arbitrary units are subjective, mabye even passe'. Yes you can have an internal control and use ratios to normalize, but not if you change that gain between channels. Reporting in units that actually mean something allows results acquired at different gain states to be compared in a meaningful manner, it's more than just veneer. It's a means of ensuring consistent results and comparing data, and the point of this level of technology is to transcend what can simply be percieved through the eyepieces. This equipment isn't for making pretty pictures, we may as well go back to drawing our results with pen and ink for that. To extract information we would not otherwise be able to get...that is the key...but to get there we need some sort of tool. Our species is widely renown for the development and clever use of tools. We accept some risk of being wrong any time we perform a measurement. We all have our limitations. 

Hopefully this will stimulate some thought, if I'm wrong we'll at least be able to measure how wrong at some point.

Best Regards,

Karl

On Fri, Apr 23, 2010 at 2:37 PM, Mark Cannell <[hidden email]> wrote:

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

[Mark Cannell]

Karl Garsha wrote:
>
> To simplify and look at the big picture, let us contemplate, for the
> sake of discussion. What if, we first calculate our gain under non-EM
> conditions, using the non-EM read port. (by the way, the read noise
> here on a good EMCCD should be around 5-6 e- which is well in the
> range of a respectable fluorescence grade sony 285 interline).
Which EM chip has this read noise? I'd be interested in getting it for
our next EMCCD. FYI the charge amplifier should age much more slowly
than the EM register so you only need to calibrate when you need to
calibrate (which for most imaging is never, as I said).

Cheers

[Andreas Bruckbauer]

So i guess the question is if the mean variance method is working wel when using the  EMCCD chip witout the non-EM port, the problem might be aditional noise. I guess Karl at photometrics has tested this extensively. If you want to varify you probably need a calibrated light source.

best wishes

Andreas

-----Original Message-----
From: Mark Cannell <[hidden email]>
To: [hidden email]
Sent: Wed, 28 Apr 2010 2:47
Subject: Re: EMCCD gain, photon scissors, bogeyman avoidance

Karl Garsha wrote: 
> 
> To simplify and look at the big picture, let us contemplate, for the > sake of discussion. What if, we first calculate our gain under non-EM > conditions, using the non-EM read port. (by the way, the read noise > here on a good EMCCD should be around 5-6 e- which is well in the > range of a respectable fluorescence grade sony 285 interline). Which EM chip has this read noise? I'd be interested in getting it for our next EMCCD. FYI the charge amplifier should age much more slowly than the EM register so you only need to calibrate when you need to calibrate (which for most imaging is never, as I said). 
 
Cheers 

[Karl Garsha-2]

Karl is not at or with Photometrics and hasn't been for years. 

I am a Senior Scientist affiliated with a large organization headquartered in Europe, and I serve as lead investigator on a number of projects concerned with developing a new generation of tissue-based molecular cancer diagnostics. The technology program I'm accountable for is hoped to contribute to more informed treatment decisions and improve the lives of cancer patients on a global scale. I define stringent performance specifications, and require the equipment I select to meet them. 

This also means I'm accountable to be able to explain the synergy of technologies that allow us to extract this medically relevant information, to some of the mightiest regulatory agencies in the world. I need to know what my cameras are doing and how to qualify and verify them, and how to communicate, transparently and clearly. There is nothing to be gained by muddying of the waters or baffling with arcane theory in this arena.   

My opinions are my own and do not necessarily reflect those of my current or former employers. I'm free to state my mind and say whatever I wish, although I hope most of it is of redeeming value. I'm footloose and fancy free as cannons go. This is precisely why I chose not to broadcast my affiliation, lest my opinions or advice be misconstrued as representing those of any particular organization.

For matters pertaining to legitimate scienctific discussion I can be contacted directly at my professional address: [hidden email]

My interest here is primarily to help foster responsible exchange of useful practical information and tested theory and help the group to avoid misinformation. Yes, I have in-depth experience with cameras as well as characterization of  numerous other analytical imaging technologies, it is my profession to develop and apply such technologies to relevant practice. I work in an environment where tight-tolerances and high-performance results are expected and there are no good excuses for anything below expectations. Technologies that I feel I can trust and refer to have earned it.

The slope of mean-variance plot will indeed be different at 0 gain on the EM port, this is due to addtional noise factors and reflected by increased variance, but the response should remain linear at different exposure times. 

Sincerely,
Karl

On Thu, Apr 29, 2010 at 11:30 PM, Andreas Bruckbauer <[hidden email]> wrote:

So i guess the question is if the mean variance method is working wel when using the  EMCCD chip witout the non-EM port, the problem might be aditional noise. I guess Karl at photometrics has tested this extensively. If you want to varify you probably need a calibrated light source.

best wishes

Andreas

-----Original Message-----
From: Mark Cannell <[hidden email]>
To: [hidden email]

Sent: Wed, 28 Apr 2010 2:47
Subject: Re: EMCCD gain, photon scissors, bogeyman avoidance

Karl Garsha wrote: 
> 
> To simplify and look at the big picture, let us contemplate, for the > sake of discussion. What if, we first calculate our gain under non-EM > conditions, using the non-EM read port. (by the way, the read noise > here on a good EMCCD should be around 5-6 e- which is well in the > range of a respectable fluorescence grade sony 285 interline). Which EM chip has this read noise? I'd be interested in getting it for our next EMCCD. FYI the charge amplifier should age much more slowly than the EM register so you only need to calibrate when you need to calibrate (which for most imaging is never, as I said). 
 
Cheers 

[Mancini, Michael A]

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[hidden email]

 

[Andreas Bruckbauer]

My apologies for placing Karl with Photometrics, must have been a quite old link which came up in google. I enjoyed your comment.
best wishes

Andreas

-----Original Message-----
From: Karl Garsha <[hidden email]>
To: [hidden email]
Sent: Fri, 30 Apr 2010 23:22
Subject: Re: EMCCD gain, photon scissors, bogeyman avoidance

Karl is not at or with Photometrics and hasn't been for years. 

I am a Senior Scientist affiliated with a large organization headquartered in Europe, and I serve as lead investigator on a number of projects concerned with developing a new generation of tissue-based molecular cancer diagnostics. The technology program I'm accountable for is hoped to contribute to more informed treatment decisions and improve the lives of cancer patients on a global scale. I define stringent performance specifications, and require the equipment I select to meet them. 

This also means I'm accountable to be able to explain the synergy of technologies that allow us to extract this medically relevant information, to some of the mightiest regulatory agencies in the world. I need to know what my cameras are doing and how to qualify and verify them, and how to communicate, transparently and clearly. There is nothing to be gained by muddying of the waters or baffling with arcane theory in this arena.   

My opinions are my own and do not necessarily reflect those of my current or former employers. I'm free to state my mind and say whatever I wish, although I hope most of it is of redeeming value. I'm footloose and fancy free as cannons go. This is precisely why I chose not to broadcast my affiliation, lest my opinions or advice be misconstrued as representing those of any particular organization.

For matters pertaining to legitimate scienctific discussion I can be contacted directly at my professional address: [hidden email]

My interest here is primarily to help foster responsible exchange of useful practical information and tested theory and help the group to avoid misinformation. Yes, I have in-depth experience with cameras as well as characterization of  numerous other analytical imaging technologies, it is my profession to develop and apply such technologies to relevant practice. I work in an environment where tight-tolerances and high-performance results are expected and there are no good excuses for anything below expectations. Technologies that I feel I can trust and refer to have earned it.

The slope of mean-variance plot will indeed be different at 0 gain on the EM port, this is due to addtional noise factors and reflected by increased variance, but the response should remain linear at different exposure times. 

Sincerely,
Karl

On Thu, Apr 29, 2010 at 11:30 PM, Andreas Bruckbauer <[hidden email]> wrote:

So i guess the question is if the mean variance method is working wel when using the  EMCCD chip witout the non-EM port, the problem might be aditional noise. I guess Karl at photometrics has tested this extensively. If you want to varify you probably need a calibrated light source.

best wishes

Andreas

-----Original Message-----
From: Mark Cannell <[hidden email]>
To: [hidden email]

Sent: Wed, 28 Apr 2010 2:47
Subject: Re: EMCCD gain, photon scissors, bogeyman avoidance

Karl Garsha wrote: 
> 
> To simplify and look at the big picture, let us contemplate, for the > sake of discussion. What if, we first calculate our gain under non-EM > conditions, using the non-EM read port. (by the way, the read noise > here on a good EMCCD should be around 5-6 e- which is well in the > range of a respectable fluorescence grade sony 285 interline). Which EM chip has this read noise? I'd be interested in getting it for our next EMCCD. FYI the charge amplifier should age much more slowly than the EM register so you only need to calibrate when you need to calibrate (which for most imaging is never, as I said). 
 
Cheers