averaging vs. accumulation for noise reduction - is there a difference?

[Guy Cox-2]

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Kees,

         The major source of noise in confocal images is shot noise,
that is the random fluctuation in photon number arriving at a pixel.
There are other sources of noise, of course, but shot noise predominates
(see a certain book edited by one J. Pawley).  In any case, in most of
the examples given here, other 'obvious' factors, such as PMT voltage,
were controlled.  Prima facie, therefore, the images should be
equivalent.  We therefore have to look to other factors, such as the way
the microscope talks to its hardware, or excitation into triplet states.


         On a simplistic basis, we might expect that there would be more
overhead - ie dead time - when acquiring an image at high speed rather
than slow, so that a slow scan should win out over averaging.  However,
the reported claims go in the opposite direction.  I have already
suggested one possible explanation.  But the other explanation, that
things are over-excited, seems to be running around (sorry!) and there
is a simple experiment we can do to control for this.  Do your averaged
scans, then turn your laser power to exactly 50% of the original value
and average for twice the time.  If the results are not identical then
you are saturating your fluorophore and that is the source of the
problem.

         It must be at least 20 years since I first pointed out that you
should check for saturation, yet I've never met anyone who does it.  In
any experiment you should try reducing the laser power, and check that
the fluorescence decreases proportionally.  If it doesn't, you are
saturating and, in simple terms, making a rod for your own back.  

                                       Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Straatman, Kees R. (Dr.)
Sent: Friday, 17 June 2011 5:53 PM
To: [hidden email]
Subject: Re: averaging vs. accumulation for noise reduction - is there a
difference?

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Very interesting discussion.

I was just wondering. We talk about S/N. If you acquire a single frame
for longer I would expect you also accumulate more noise in the image,
if you average your signal in each image your sample should be more or
less the same while the noise (random) is averaged. So would you not
expect a better S/N with averaging when all else is equal?

Kees

Senior Experimental Officer
Centre for Core Biotechnology Services
University of Leicester, UK

http://www.le.ac.uk/biochem/microscopy/home.html


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Stanislav Vitha
Sent: 16 June 2011 17:26
To: [hidden email]
Subject: averaging vs. accumulation for noise reduction - is there a
difference?

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Hallo,
this is a very basic question, but I cannot figure this out from what I
have
been reading, so a simple explanation for a non-physicist would be much
appreciated:

Is there a real difference in the improvement of the signal to noise
ratio
between frame averaging (or accumulation) and longer dwell times (slower

scan) for a point-scanning confocal witrh a PMT detector?

For instance, using single point scanning confocal, 12-bit acquisition.

a) averaging (or accumulating) 5 frames, 4 microseconds per pixel
b) acquiring a single frame, 20 microseconds per pixel

Assumptions:
no saturation of the detector;
stable environmental conditions, no focus drift, etc

Would it matter (for the dfference between the two scenarios) if it was
analog
detection or photon counting detection?

I will run this little test later, but I am curious what you think.

I thought that at least for the photon counting mode, the two important
factors would be the dark counts and the total number of counts
detected, so
whether it is acquired in one scan or in 5 scans, it should be the same.
My
camera expert here insists that the averaging scheme will give better
noise
suppression.

Thanks!


Stan Vitha  
 
Microscopy and Imaging Center
Texas A&M University

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[Straatman, Kees (Dr.)]

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Dear Jim and Guy,

Thanks for the answers. Should have thought before I wrote.

Kees




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: 17 June 2011 15:18
To: [hidden email]
Subject: Re: averaging vs. accumulation for noise reduction - is there a difference?

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Dear Kees,

In the confocal (or 2photon) microscope, their is very little
read-noise (noise signal generated by and within the photodetector,
in this case usually a PMT.). The main source of noise is Poisson
Noise which is a statistical noise, namely the fact that the
uncertainty of any measurement of quantum events is the square root
of the number of these events actually detected.

Suppose that you have a "constant signal" which, after being measured
many times, would average out to say, 100 photons/pixel. Any
particular measurement of this signal will be in the range of 90 to
110 photons about 67% of the time while 37% of the measurements are
even farther from the "real" intensity.  We say that such a signal
has 10% Poisson noise.

On the other hand, confocal signals from the stained areas of the
specimen tend to be much less than 100 photons/pixel: often closer to
9 photon/pixel, giving +/-30% noise. Dark areas are likely to be 1's
and zeroes.

Multiplicative noise in the PMT (also related to Poisson statistics)
means that each photoelectron is not amplified by the same amount,
smearing out the intensity actually recorded. This means that even
though the recorded image of a weakly fluorescent object may seem to
have values everywhere from 0 to 256, you may in fact be only
recording a "true" signal of 1s and zeros with a few 2's thrown in by
the statistics. i.e., Unless you do photon counting ,you have little
idea of the actual number of photons represented by the number
recorded in the memory.

People commonly say that the signal gets "noisy" if they turn up the
PMT too far. While it is possible that their PMT is breaking down in
some way when used with higher voltages on the dynodes, the more
likely explanation is that they turn up the PMT because the signal is
so weak and weak signals have very poor Poisson statistics.

Counting longer or Kalman averaging allows more photons/pixel to be
counted. Although counting more events increases the absolute
uncertainty, (the sqrt of a larger number is bigger than the sqrt of
a smaller number), the ratio of the signal to this noise improves
with the sqrt of the total signal detected.

To bring another variable into the discussion, deconvolving confocal
or 2-photon data effectively averages the signal over all the voxels
needed to define the PSF. If you assume 2 voxels are needed for each
"resel" (the resolution element, from the center of the Airy Disk to
the first dark ring on one side), then you need at least 4x4x4 = 64
voxels to define the PSF, and deconvolving such a signal in 3D will
effectively average the signal of a point object over these 64
nearest neighbors, drastically improving the S/N (In this case the
S/N would improve 8x, the same as averaging for 64 scans. The
contrast of the image would also appear to decrease but this is an
artefact of averaging and can be increased using a look-up table.)

Hope that helps.

Jim Pawley
at the 2011 UBC Course.

>
>Very interesting discussion.
>
>I was just wondering. We talk about S/N. If you acquire a single
>frame for longer I would expect you also accumulate more noise in
>the image, if you average your signal in each image your sample
>should be more or less the same while the noise (random) is
>averaged. So would you not expect a better S/N with averaging when
>all else is equal?
>
>Kees
>
>Senior Experimental Officer
>Centre for Core Biotechnology Services
>University of Leicester, UK
>
>http://www.le.ac.uk/biochem/microscopy/home.html
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Stanislav
>Vitha
>Sent: 16 June 2011 17:26
>To: [hidden email]
>Subject: averaging vs. accumulation for noise reduction - is there a
>difference?
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hallo,
>this is a very basic question, but I cannot figure this out from what I have
>been reading, so a simple explanation for a non-physicist would be much
>appreciated:
>
>Is there a real difference in the improvement of the signal to noise ratio
>between frame averaging (or accumulation) and longer dwell times (slower
>scan) for a point-scanning confocal witrh a PMT detector?
>
>For instance, using single point scanning confocal, 12-bit acquisition.
>
>a) averaging (or accumulating) 5 frames, 4 microseconds per pixel
>b) acquiring a single frame, 20 microseconds per pixel
>
>Assumptions:
>no saturation of the detector;
>stable environmental conditions, no focus drift, etc
>
>Would it matter (for the dfference between the two scenarios) if it was analog
>detection or photon counting detection?
>
>I will run this little test later, but I am curious what you think.
>
>I thought that at least for the photon counting mode, the two important
>factors would be the dark counts and the total number of counts detected, so
>whether it is acquired in one scan or in 5 scans, it should be the same. My
>camera expert here insists that the averaging scheme will give better noise
>suppression.
>
>Thanks!
>
>
>Stan Vitha
>
>Microscopy and Imaging Center
>Texas A&M University

--
                    ****************************************
Prof. James B. Pawley,                                     Phone: [phone]
3D Microscopy of Living Cells: Summer Course   CELL: 778-919-3176
Info at: http://www.3dcourse.ubc.ca
"If it isn't diffraction, it is statistics":Microscopist's complaint, Anon.

[George McNamara]

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Hi Simon,

In addition to our old LSM510, I manage two Leica SP5 confocal
microscopes (one with multiphoton, FLIM and FCS) that are open access
(UM, South Florida, worldwide) and a Zeiss LSM710 (closed access, for
two institutes). All three of these have spare capacity, even once I
move all LSM510 users to the Leica's.  I know of another 15 confocal
microscopes at UM. We have plenty of confocal microscope capacity. In
our OMX (3D-SIM) demo we have already seen amazing new details not
resolvable by confocal (NIH shared instrumentation grant submitted for
OMX in March 2011 - 3D-SIM, TIRF and Applied Precision's Monet precision
localization). We are also considering submitting for another NIH
instrumentation program a complementary nanoscope that uses a different
technology and has confocal capability by just leaving off the depletion
laser - very different technology that will not have the same potential
artifacts as 3D-SIM (and vice versa) and that give us additional nano
imaging capabilities.

Sincerely,

George

On 6/17/2011 9:13 AM, simon walker (BI) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> "My core is moving to nanoscope(s)"
> Surely these technologies are complementary, not mutually exclusive.  We're getting super res in the not-too-distant future, but these systems certainly won't be replacing our confocals.  I guess it all depends on the application, but I can't see many core facilities losing their confocal imaging capabilities in preference to super res.
> Simon
>
>
>
> To: [hidden email]
> Subject: selling our Zeiss LSM510
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocal listserv,
>
> I now have approval to sell our Zeiss LSM510 confocal microscope.
> Briefly, 4 PMT system (not META), Axiovert 200M motorized stand, Argon
> ion visible laser, HeNe543, HeNe633, several objective lenses. UV laser
> ready. You can optionally purchase from us the Coherent Enterprise I UV
> laser, power supply, LP5 chiller - UV laser is not currently operation
> so needs refurbishing ($8500 from Evergreen Laser, similar price from
> Laser Innovations, no including shipping). System was under service
> contract until Feb 1 of this year. Three PMT's were replaced last year,
> so in pretty good shape.
>
> Zeiss currently has a promotion to trade in an LSM510 or Bio-Rad
> confocal for $75K credit toward a LMS710 or 780. My core is moving to
> nanoscope(s), so I am not going to use the promotion (and Zeiss is not
> offering it for their PAL-m or SIM or even TIRF systems). Even if you
> don't get in on the promotion, you could negotiate for that amount with
> your Zeiss rep.
>
> I have a potential buyer so don't wait around long. We can arrange with
> Zeiss field service to find shipping crates and they can pack and
> unpack. You can visit Miami to try it out (sorry - rainy season just
> started).
>
> Sincerely,
>
> George
> [hidden email]
>
>
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Axel Kurt Preuss]

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Yup, we definitely won't loose focus
axel

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of simon walker (BI)
Sent: Friday, 17 June, 2011 9:14 PM
To: [hidden email]
Subject: Re: selling our Zeiss LSM510

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"My core is moving to nanoscope(s)"
Surely these technologies are complementary, not mutually exclusive.  We're getting super res in the not-too-distant future, but these systems certainly won't be replacing our confocals.  I guess it all depends on the application, but I can't see many core facilities losing their confocal imaging capabilities in preference to super res.
Simon



To: [hidden email]
Subject: selling our Zeiss LSM510

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Dear Confocal listserv,

I now have approval to sell our Zeiss LSM510 confocal microscope.
Briefly, 4 PMT system (not META), Axiovert 200M motorized stand, Argon ion visible laser, HeNe543, HeNe633, several objective lenses. UV laser ready. You can optionally purchase from us the Coherent Enterprise I UV laser, power supply, LP5 chiller - UV laser is not currently operation so needs refurbishing ($8500 from Evergreen Laser, similar price from Laser Innovations, no including shipping). System was under service contract until Feb 1 of this year. Three PMT's were replaced last year, so in pretty good shape.

Zeiss currently has a promotion to trade in an LSM510 or Bio-Rad confocal for $75K credit toward a LMS710 or 780. My core is moving to nanoscope(s), so I am not going to use the promotion (and Zeiss is not offering it for their PAL-m or SIM or even TIRF systems). Even if you don't get in on the promotion, you could negotiate for that amount with your Zeiss rep.

I have a potential buyer so don't wait around long. We can arrange with Zeiss field service to find shipping crates and they can pack and unpack. You can visit Miami to try it out (sorry - rainy season just started).

Sincerely,

George
[hidden email]



--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

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