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

[Stanislav Vitha]

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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

[Guy Cox-2]

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As I understand it, in principle there should be no difference.  But in
practice there could be a difference depending on the bit depths the
system could handle.  For example, early Bio-Rad systems (I'm showing my
age here) could only collect a single image at 8-bit depth, but averaged
into a 16-bit buffer, so you would do much better by averaging (so long
as you saved the final result as 16-bit).

                              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 Stanislav Vitha
Sent: Friday, 17 June 2011 2:26 AM
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

-----
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Checked by AVG - www.avg.com
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[Mark Cannell]

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It also depends on how the readout  bandwidth is controlled for different scan speeds...

Cheers
On 16/06/2011, at 5:26 PM, Stanislav Vitha wrote:

> *****
> 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

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Mark,

    By my reckoning it's after 4 in the morning in Auckland - what are
you doing replying at this hour?  (By the way, I'm in Taiwan, not Oz).

                                   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 Mark Cannell
Sent: Friday, 17 June 2011 2:41 AM
To: [hidden email]
Subject: Re: 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
*****

It also depends on how the readout  bandwidth is controlled for
different scan speeds...

Cheers
On 16/06/2011, at 5:26 PM, Stanislav Vitha wrote:

> *****
> 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

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1382 / Virus Database: 1513/3706 - Release Date: 06/15/11

[Lloyd Donaldson]

*****
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*****

Stan

From my practical experience longer dwell times can reduce noise. The relative effects of averaging vs accumulation depend on the detector gain so averaging at high gain is slightly more noisy than accumulation at lower gain but I think the difference is small. Combining averaging and accumulation does seem to be better for spectral imaging at narrow bandwidth for example. If your sample can stand it, increasing laser power can make much more difference, likewise making your sample brighter. I have noticed some difference in noise behaviour on the 2 systems I have used suggesting there is a significant effect of all the electronics behind the microscope.
I would be interested in your results if you do some experiments.

Lloyd


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Next Generation Biomaterials
Private Bag 3020, Rotorua
New Zealand 3010

Ph: 64 7 343 5581



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, 17 June 2011 4:46 a.m.
To: [hidden email]
Subject: Re: 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
*****

Mark,

    By my reckoning it's after 4 in the morning in Auckland - what are
you doing replying at this hour?  (By the way, I'm in Taiwan, not Oz).

                                   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 Mark Cannell
Sent: Friday, 17 June 2011 2:41 AM
To: [hidden email]
Subject: Re: 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
*****

It also depends on how the readout  bandwidth is controlled for
different scan speeds...

Cheers
On 16/06/2011, at 5:26 PM, Stanislav Vitha wrote:

> *****
> 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

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1382 / Virus Database: 1513/3706 - Release Date: 06/15/11



This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Moninger, Thomas]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Stan,

I've been told by Carl Z. engineers that in general averaging (I usually use line, not frame) tends to yield better S/N then does increasing dwell time. As Lloyd commented this may be model specific. I have not done any analysis to confirm this however....

Tom

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lloyd Donaldson
Sent: Thursday, June 16, 2011 3:14 PM
To: [hidden email]
Subject: Re: 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
*****

Stan

From my practical experience longer dwell times can reduce noise. The relative effects of averaging vs accumulation depend on the detector gain so averaging at high gain is slightly more noisy than accumulation at lower gain but I think the difference is small. Combining averaging and accumulation does seem to be better for spectral imaging at narrow bandwidth for example. If your sample can stand it, increasing laser power can make much more difference, likewise making your sample brighter. I have noticed some difference in noise behaviour on the 2 systems I have used suggesting there is a significant effect of all the electronics behind the microscope.
I would be interested in your results if you do some experiments.

Lloyd


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Next Generation Biomaterials
Private Bag 3020, Rotorua
New Zealand 3010

Ph: 64 7 343 5581



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Friday, 17 June 2011 4:46 a.m.
To: [hidden email]
Subject: Re: 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
*****

Mark,

    By my reckoning it's after 4 in the morning in Auckland - what are
you doing replying at this hour?  (By the way, I'm in Taiwan, not Oz).

                                   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 Mark Cannell
Sent: Friday, 17 June 2011 2:41 AM
To: [hidden email]
Subject: Re: 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
*****

It also depends on how the readout  bandwidth is controlled for
different scan speeds...

Cheers
On 16/06/2011, at 5:26 PM, Stanislav Vitha wrote:

> *****
> 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

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1382 / Virus Database: 1513/3706 - Release Date: 06/15/11



This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
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________________________________
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________________________________

[Julio Vazquez]

*****
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*****

This is what I noticed empirically on our Zeiss LSM 510, where averaging tends to give somewhat better noise reduction than increasing dwell time. Under "normal" imaging conditions, we typically use a dwell time of 1.6-3.2 microseconds. Increasing the dwell time to greater than  3.2 microseconds tends to result in more bleaching and somewhat reduced signal. Typically, we use 1.6 microseconds dwell time, and 2-4 averages, depending on the sample.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org/


On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:

> Stan,
>
> I've been told by Carl Z. engineers that in general averaging (I usually use line, not frame) tends to yield better S/N then does increasing dwell time. As Lloyd commented this may be model specific. I have not done any analysis to confirm this however....
>
> Tom

[David Baddeley]

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*****

One of the possible explanations for favoring averaging over dwell times is that
scanning fast and averaging lets the triplet relax between scans, thus reducing
the rate of photobleaching (see the T-REX papers from S. Hells group for
example). I also seem to remember there being some (percieved - although not
strictly quantified) benefit from doing frame averaging rather than line
averaging on an old Leica TCS-NT.

cheers,
David


----- Original Message ----
From: Julio Vazquez <[hidden email]>
To: [hidden email]
Sent: Fri, 17 June, 2011 9:20:36 AM
Subject: Re: 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
*****

This is what I noticed empirically on our Zeiss LSM 510, where averaging tends
to give somewhat better noise reduction than increasing dwell time. Under
"normal" imaging conditions, we typically use a dwell time of 1.6-3.2
microseconds. Increasing the dwell time to greater than  3.2 microseconds tends
to result in more bleaching and somewhat reduced signal. Typically, we use 1.6
microseconds dwell time, and 2-4 averages, depending on the sample.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org/


On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:

> Stan,
>
> I've been told by Carl Z. engineers that in general averaging (I usually use
>line, not frame) tends to yield better S/N then does increasing dwell time. As
>Lloyd commented this may be model specific. I have not done any analysis to
>confirm this however....
>
> Tom

[Armstrong, Brian]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have not done a careful analysis of this either, and I am not quite sure how you would, however I share the viewpoint of Julio exactly, 1.6usec/pix and 2-4 ave.



Brian Armstrong PhD
Light Microscopy Core
Beckman Research Institute
1450 East Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
 
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Thursday, June 16, 2011 2:21 PM
To: [hidden email]
Subject: Re: 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
*****

This is what I noticed empirically on our Zeiss LSM 510, where averaging tends to give somewhat better noise reduction than increasing dwell time. Under "normal" imaging conditions, we typically use a dwell time of 1.6-3.2 microseconds. Increasing the dwell time to greater than  3.2 microseconds tends to result in more bleaching and somewhat reduced signal. Typically, we use 1.6 microseconds dwell time, and 2-4 averages, depending on the sample.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org/


On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:

> Stan,
>
> I've been told by Carl Z. engineers that in general averaging (I usually use line, not frame) tends to yield better S/N then does increasing dwell time. As Lloyd commented this may be model specific. I have not done any analysis to confirm this however....
>
> Tom


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[G. Esteban Fernandez]

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I agree with Julio and Brian about the Zeiss 510 - averaged images were
always less noisy than "dwelled" images.  I always taught that point in
training sessions and showed side by side comparisons; newbies could clearly
see the benefit of averaging over dwelling.  However this isn't the case on
my 710 or 700, I see no difference (just by eye!) between averaging and
dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x slower
than whatever max. is [1~3 us]).

-Esteban


On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian <[hidden email]>
wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have not done a careful analysis of this either, and I am not quite sure
how you would, however I share the viewpoint of Julio exactly, 1.6usec/pix
and 2-4 ave.

>
>
>
> Brian Armstrong PhD
> Light Microscopy Core
> Beckman Research Institute
> 1450 East Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Julio Vazquez
> Sent: Thursday, June 16, 2011 2:21 PM
> To: [hidden email]
> Subject: Re: 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
> *****
>
> This is what I noticed empirically on our Zeiss LSM 510, where averaging
tends to give somewhat better noise reduction than increasing dwell time.
Under "normal" imaging conditions, we typically use a dwell time of 1.6-3.2
microseconds. Increasing the dwell time to greater than  3.2 microseconds
tends to result in more bleaching and somewhat reduced signal. Typically, we
use 1.6 microseconds dwell time, and 2-4 averages, depending on the sample.

> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org/
>
>
> On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>
>> Stan,
>>
>> I've been told by Carl Z. engineers that in general averaging (I usually

use line, not frame) tends to yield better S/N then does increasing dwell
time. As Lloyd commented this may be model specific. I have not done any
analysis to confirm this however....
>>
>> Tom
>
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or
entity to which they are addressed. This communication may contain
information that is privileged, confidential, or exempt from disclosure
under applicable law (e.g., personal health information, research data,
financial information). Because this e-mail has been sent without
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view the information, forward it to others or tamper with the information
without the knowledge or consent of the sender. If you are not the intended
recipient, or the employee or person responsible for delivering the message
to the intended recipient, any dissemination, distribution or copying of the
communication is strictly prohibited. If you received the communication in
error, please notify the sender immediately by replying to this message and
deleting the message and any accompanying files from your system. If, due to
the security risks, you do not wish to receive further communications via
e-mail, please reply to this message and inform the sender that you do not
wish to receive further e-mail from the sender.
>
> ---------------------------------------------------------------------
>

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I'm probably risking the wrath of the manufacturers here, but I'd like
to suggest that the difference in these cases could be caused by slight
inaccuracy in the scan.  In other words, when you do an averaged image
you are (to a small extent) averaging each pixel with its neighbour, and
hence smoothing the image.  Increasing the dwell time in a single scan
will not have this effect. The implication here is that the 710 has a
more precise scan than the 510.

                                    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 G. Esteban Fernandez
Sent: Friday, 17 June 2011 9:48 AM
To: [hidden email]
Subject: Re: 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
*****

I agree with Julio and Brian about the Zeiss 510 - averaged images were
always less noisy than "dwelled" images.  I always taught that point in
training sessions and showed side by side comparisons; newbies could
clearly
see the benefit of averaging over dwelling.  However this isn't the case
on
my 710 or 700, I see no difference (just by eye!) between averaging and
dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x
slower
than whatever max. is [1~3 us]).

-Esteban


On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian <[hidden email]>
wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have not done a careful analysis of this either, and I am not quite
sure
how you would, however I share the viewpoint of Julio exactly,
1.6usec/pix
and 2-4 ave.

>
>
>
> Brian Armstrong PhD
> Light Microscopy Core
> Beckman Research Institute
> 1450 East Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome
.htm
>
>
> -----Original Message-----
> From: Confocal Microscopy List
[mailto:[hidden email]]
On Behalf Of Julio Vazquez
> Sent: Thursday, June 16, 2011 2:21 PM
> To: [hidden email]
> Subject: Re: 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
> *****
>
> This is what I noticed empirically on our Zeiss LSM 510, where
averaging
tends to give somewhat better noise reduction than increasing dwell
time.
Under "normal" imaging conditions, we typically use a dwell time of
1.6-3.2
microseconds. Increasing the dwell time to greater than  3.2
microseconds
tends to result in more bleaching and somewhat reduced signal.
Typically, we
use 1.6 microseconds dwell time, and 2-4 averages, depending on the
sample.

> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org/
>
>
> On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>
>> Stan,
>>
>> I've been told by Carl Z. engineers that in general averaging (I

usually
use line, not frame) tends to yield better S/N then does increasing
dwell
time. As Lloyd commented this may be model specific. I have not done any
analysis to confirm this however....
>>
>> Tom
>
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the
individual or
entity to which they are addressed. This communication may contain
information that is privileged, confidential, or exempt from disclosure
under applicable law (e.g., personal health information, research data,
financial information). Because this e-mail has been sent without
encryption, individuals other than the intended recipient may be able to
view the information, forward it to others or tamper with the
information
without the knowledge or consent of the sender. If you are not the
intended
recipient, or the employee or person responsible for delivering the
message
to the intended recipient, any dissemination, distribution or copying of
the
communication is strictly prohibited. If you received the communication
in
error, please notify the sender immediately by replying to this message
and
deleting the message and any accompanying files from your system. If,
due to
the security risks, you do not wish to receive further communications
via
e-mail, please reply to this message and inform the sender that you do
not
wish to receive further e-mail from the sender.
>
> ---------------------------------------------------------------------
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi Stan,

Also depends on the fluorophore(s), probably with respect to both
photobleaching (destruction) and driving to triplet state(s) - re T-Rex
mentioned in previous reply.

On our Leica SP5, Jonathan Boyd of Leica showed me a comparison of:

* resonant scanner (8000 Hz), 10 frame average
vs
* standard scanner, 800 Hz, single scan

PMT gain (and offset), field of view, number of pixels in image, were
the same. Sorry, I forget what specimen (could have been Convallaria stem).

Resonant scanner mode was clearly brighter signal.

I suppose it is possible that Leica's digitizer was doing things
differently in the two modes.

On our Zeiss LSM510, different scan speeds have some impact on
intensity. The Zeiss field service engineer told me they try to
calibrate all speeds to produce the same intensity. Our 510 produces
brightest signal - but also noisiest - at fastest scan speed (i.e. on
Chroma fluorescent slides where I assume items in line one above are not
an issue).

Upshot, your signal may vary for reasons beyond your expectations.

Enjoy,

George


On 6/16/2011 12:26 PM, Stanislav Vitha wrote:

> *****
> 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
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[James Pawley]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi all,

I haven't seen these images but the "dwell" image (slower scan?)
image "should" be better for a given total acquisition time, because
a smaller fraction of the total exposure time is spent in retrace
(about 30% of the time is usually used for retrace in fast scan.
Ergo, the fraction of the frame time spent actually collecting signal
is about 70% at 'fast scan" and more like 90% at a 4x slower scan
rate. However, you seem to know the actual pixel dwell time, so this
accurate (rather than calculated from other data) then retrace should
not be a factor.

I haven't read this whole thread but has anyone mentioned the
electronic bandwidth of the amplifier leading up to the digitizer?
This should be set to a time constant that is at least 4x slower
(maybe 5x if we consider the lower proportion of retrace time)? If it
is not, then any benefit of longer dwell time will be lost (because
the value of the signal at the instant it is digitized will only be
characteristic of the shorter, fast scan pixel). To my knowledge only
BioRad did true box-car averaging where they integrated all the
current presented during the pixel, no matter how long it took.

As far as vibration or drift(of the stage or the scanning coil
currents) is concerned, fast scan will cause blur to the whole image,
while slower scanning will more likely cause distortion. Although
changing the scan speed by only a factor of 4 (rather than maybe 100
or even 1,000, as in a SEM) should not show much of this effect.

As far as analog vs, photon counting: In photon counting, the
bandwidth argument has no validity (assuming that the system merely
counts the pulses for a longer time as slow scan.) because what is
important then is the bandwidth up until the counter.

But it is a rare system that can do photon counting at "normal"
signal levels without losing significant signal to pulse pileup.
Pileup will tend to clip bright parts of the image, perhaps making
them look "smoother" .

Finally, what did you all decide you meant by "averaging"? Kalman
averaging will give similar results (apart from the factors above. A
running (or exponential) average, will give a factor of at least 2
less efficient use of the signal (maybe worse if you get into weak
signals).

You talk about a "camera expert" and "noise suppression", is he
talking about the tricks used to make pictures made with digital
cameras look better? That is a whole 'nuther can of worms and needs a
longer discussion. It should have little bearing on microscopical
imaging unless you are taking images off your Yokogawa using a Nikon
D3.

As far as the effect of scan rate on bleaching is concerned, there
has long been a debate over whether spreading the damage around fast
perhaps stops the buildup of, say singlet oxygen, to dangerous levels
in one location. Those scanning at video rate claimed that they could
watch their specimens longer because of this effect (I guess that the
assumption was that natural mechanisms for detoxification were
overwhelmed if the beam sat in one place too long.).

I once tried to  prove this by spending some time at the Noran
factory, but never got anything definitive. But it could be true,
because the disk-scanner folk make the same claim with some support.

Jim Pawley
At the 16th UBC Course.

>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I agree with Julio and Brian about the Zeiss 510 - averaged images were
>always less noisy than "dwelled" images.  I always taught that point in
>training sessions and showed side by side comparisons; newbies could clearly
>see the benefit of averaging over dwelling.  However this isn't the case on
>my 710 or 700, I see no difference (just by eye!) between averaging and
>dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x slower
>than whatever max. is [1~3 us]).
>
>-Esteban
>
>
>On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian <[hidden email]>
>wrote:
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  I have not done a careful analysis of this either, and I am not quite sure
>how you would, however I share the viewpoint of Julio exactly, 1.6usec/pix
>and 2-4 ave.
>>
>>
>>
>>  Brian Armstrong PhD
>>  Light Microscopy Core
>>  Beckman Research Institute
>>  1450 East Duarte Rd
>>  Duarte, CA 91010
>>  626-256-4673 x62872
>>
>http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
>>
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Julio Vazquez
>>  Sent: Thursday, June 16, 2011 2:21 PM
>>  To: [hidden email]
>>  Subject: Re: 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
>>  *****
>>
>>  This is what I noticed empirically on our Zeiss LSM 510, where averaging
>tends to give somewhat better noise reduction than increasing dwell time.
>Under "normal" imaging conditions, we typically use a dwell time of 1.6-3.2
>microseconds. Increasing the dwell time to greater than  3.2 microseconds
>tends to result in more bleaching and somewhat reduced signal. Typically, we
>use 1.6 microseconds dwell time, and 2-4 averages, depending on the sample.
>>  --
>>  Julio Vazquez
>>  Fred Hutchinson Cancer Research Center
>>  Seattle, WA
>>
>>  http://www.fhcrc.org/
>>
>>
>>  On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>>
>>>  Stan,
>>>
>>>  I've been told by Carl Z. engineers that in general averaging (I usually
>use line, not frame) tends to yield better S/N then does increasing dwell
>time. As Lloyd commented this may be model specific. I have not done any
>analysis to confirm this however....
>>>
>>>  Tom
>>
>>
>>  ---------------------------------------------------------------------
>>  SECURITY/CONFIDENTIALITY WARNING:
>>  This message and any attachments are intended solely for the individual or
>entity to which they are addressed. This communication may contain
>information that is privileged, confidential, or exempt from disclosure
>under applicable law (e.g., personal health information, research data,
>financial information). Because this e-mail has been sent without
>encryption, individuals other than the intended recipient may be able to
>view the information, forward it to others or tamper with the information
>without the knowledge or consent of the sender. If you are not the intended
>recipient, or the employee or person responsible for delivering the message
>to the intended recipient, any dissemination, distribution or copying of the
>communication is strictly prohibited. If you received the communication in
>error, please notify the sender immediately by replying to this message and
>deleting the message and any accompanying files from your system. If, due to
>the security risks, you do not wish to receive further communications via
>e-mail, please reply to this message and inform the sender that you do not
>wish to receive further e-mail from the sender.
>>
>>  ---------------------------------------------------------------------
>>

--
                    ****************************************
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.

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I was talking about sub-pixel scan inaccuracy, which would not be
sufficient to cause visible blurring but could give some smoothing.  

Image 'brightness', mentioned by another contributor to this thread, is
not a meaningful figure (particularly without any figures quoted).  For
example, resonant scans might be digitized at 8-bit for the sake of
speed, and non-resonant ones at 12 bit.  Brightness is then just a
function of the display algorithm.

                   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 James Pawley
Sent: Friday, 17 June 2011 3:36 PM
To: [hidden email]
Subject: Re: 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
*****

Hi all,

I haven't seen these images but the "dwell" image (slower scan?)
image "should" be better for a given total acquisition time, because
a smaller fraction of the total exposure time is spent in retrace
(about 30% of the time is usually used for retrace in fast scan.
Ergo, the fraction of the frame time spent actually collecting signal
is about 70% at 'fast scan" and more like 90% at a 4x slower scan
rate. However, you seem to know the actual pixel dwell time, so this
accurate (rather than calculated from other data) then retrace should
not be a factor.

I haven't read this whole thread but has anyone mentioned the
electronic bandwidth of the amplifier leading up to the digitizer?
This should be set to a time constant that is at least 4x slower
(maybe 5x if we consider the lower proportion of retrace time)? If it
is not, then any benefit of longer dwell time will be lost (because
the value of the signal at the instant it is digitized will only be
characteristic of the shorter, fast scan pixel). To my knowledge only
BioRad did true box-car averaging where they integrated all the
current presented during the pixel, no matter how long it took.

As far as vibration or drift(of the stage or the scanning coil
currents) is concerned, fast scan will cause blur to the whole image,
while slower scanning will more likely cause distortion. Although
changing the scan speed by only a factor of 4 (rather than maybe 100
or even 1,000, as in a SEM) should not show much of this effect.

As far as analog vs, photon counting: In photon counting, the
bandwidth argument has no validity (assuming that the system merely
counts the pulses for a longer time as slow scan.) because what is
important then is the bandwidth up until the counter.

But it is a rare system that can do photon counting at "normal"
signal levels without losing significant signal to pulse pileup.
Pileup will tend to clip bright parts of the image, perhaps making
them look "smoother" .

Finally, what did you all decide you meant by "averaging"? Kalman
averaging will give similar results (apart from the factors above. A
running (or exponential) average, will give a factor of at least 2
less efficient use of the signal (maybe worse if you get into weak
signals).

You talk about a "camera expert" and "noise suppression", is he
talking about the tricks used to make pictures made with digital
cameras look better? That is a whole 'nuther can of worms and needs a
longer discussion. It should have little bearing on microscopical
imaging unless you are taking images off your Yokogawa using a Nikon
D3.

As far as the effect of scan rate on bleaching is concerned, there
has long been a debate over whether spreading the damage around fast
perhaps stops the buildup of, say singlet oxygen, to dangerous levels
in one location. Those scanning at video rate claimed that they could
watch their specimens longer because of this effect (I guess that the
assumption was that natural mechanisms for detoxification were
overwhelmed if the beam sat in one place too long.).

I once tried to  prove this by spending some time at the Noran
factory, but never got anything definitive. But it could be true,
because the disk-scanner folk make the same claim with some support.

Jim Pawley
At the 16th UBC Course.

>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I agree with Julio and Brian about the Zeiss 510 - averaged images were
>always less noisy than "dwelled" images.  I always taught that point in
>training sessions and showed side by side comparisons; newbies could
clearly
>see the benefit of averaging over dwelling.  However this isn't the
case on
>my 710 or 700, I see no difference (just by eye!) between averaging and
>dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x
slower

>than whatever max. is [1~3 us]).
>
>-Esteban
>
>
>On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian <[hidden email]>
>wrote:
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  I have not done a careful analysis of this either, and I am not

quite sure
>how you would, however I share the viewpoint of Julio exactly,
1.6usec/pix

>and 2-4 ave.
>>
>>
>>
>>  Brian Armstrong PhD
>>  Light Microscopy Core
>>  Beckman Research Institute
>>  1450 East Duarte Rd
>>  Duarte, CA 91010
>>  626-256-4673 x62872
>>
>http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHom

e.htm
>>
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List
[mailto:[hidden email]]
>On Behalf Of Julio Vazquez
>>  Sent: Thursday, June 16, 2011 2:21 PM
>>  To: [hidden email]
>>  Subject: Re: 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
>>  *****
>>
>>  This is what I noticed empirically on our Zeiss LSM 510, where
averaging
>tends to give somewhat better noise reduction than increasing dwell
time.
>Under "normal" imaging conditions, we typically use a dwell time of
1.6-3.2
>microseconds. Increasing the dwell time to greater than  3.2
microseconds
>tends to result in more bleaching and somewhat reduced signal.
Typically, we
>use 1.6 microseconds dwell time, and 2-4 averages, depending on the
sample.

>>  --
>>  Julio Vazquez
>>  Fred Hutchinson Cancer Research Center
>>  Seattle, WA
>>
>>  http://www.fhcrc.org/
>>
>>
>>  On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>>
>>>  Stan,
>>>
>>>  I've been told by Carl Z. engineers that in general averaging (I

usually
>use line, not frame) tends to yield better S/N then does increasing
dwell
>time. As Lloyd commented this may be model specific. I have not done
any
>analysis to confirm this however....
>>>
>>>  Tom
>>
>>
>>
---------------------------------------------------------------------
>>  SECURITY/CONFIDENTIALITY WARNING:
>>  This message and any attachments are intended solely for the
individual or
>entity to which they are addressed. This communication may contain
>information that is privileged, confidential, or exempt from disclosure
>under applicable law (e.g., personal health information, research data,
>financial information). Because this e-mail has been sent without
>encryption, individuals other than the intended recipient may be able
to
>view the information, forward it to others or tamper with the
information
>without the knowledge or consent of the sender. If you are not the
intended
>recipient, or the employee or person responsible for delivering the
message
>to the intended recipient, any dissemination, distribution or copying
of the
>communication is strictly prohibited. If you received the communication
in
>error, please notify the sender immediately by replying to this message
and
>deleting the message and any accompanying files from your system. If,
due to
>the security risks, you do not wish to receive further communications
via
>e-mail, please reply to this message and inform the sender that you do
not
>wish to receive further e-mail from the sender.
>>
>>
---------------------------------------------------------------------
>>


--
                    ****************************************
Prof. James B. Pawley,                                     Phone:
604-822-7801
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.

[Straatman, Kees (Dr.)]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy,

The Leica SP5 uses a 12-bit digitizer in either resonant (8000 Hz) or
standard (hi-res, wide variety of scan speeds, standard is 400 Hz, can
go 1 Hz to 1400 Hz) scan mode. Resonant scan mode output can be 8-bit or
12-bit, standard scan mode can be 8, 12, or 16-bit. Same whether
standard PMT mode or HyD photon counting mode.

Jim - the new HyD detector is pretty fast, pulse pile up did not seem to
be a problem at the Yale Microscopy Workshop last week (thanks again to
Ann, Derek, Joerg, the other Yalies helping wit the workshop, vendors
and attendees).

The interaction of the fluorophore(s) with the mounting media components
can trump the digitizer details - see, for example, the streaking in the
non-ROXS figure panels of:

Fluorophores: Single-Molecule STED Microscopy with Photostable Organic
Fluorophores (Small 13/2010). </pubmed/20589865> Kasper R, Harke B,
Forthmann C, Tinnefeld P, Hell SW, Sauer M., Small. 2010 Jun 29;6(13):
1379-1384. PMID: 20521266.



George

On 6/17/2011 2:47 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was talking about sub-pixel scan inaccuracy, which would not be
> sufficient to cause visible blurring but could give some smoothing.
>
> Image 'brightness', mentioned by another contributor to this thread, is
> not a meaningful figure (particularly without any figures quoted).  For
> example, resonant scans might be digitized at 8-bit for the sake of
> speed, and non-resonant ones at 12 bit.  Brightness is then just a
> function of the display algorithm.
>
>                     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 James Pawley
> Sent: Friday, 17 June 2011 3:36 PM
> To: [hidden email]
> Subject: Re: 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
> *****
>
> Hi all,
>
> I haven't seen these images but the "dwell" image (slower scan?)
> image "should" be better for a given total acquisition time, because
> a smaller fraction of the total exposure time is spent in retrace
> (about 30% of the time is usually used for retrace in fast scan.
> Ergo, the fraction of the frame time spent actually collecting signal
> is about 70% at 'fast scan" and more like 90% at a 4x slower scan
> rate. However, you seem to know the actual pixel dwell time, so this
> accurate (rather than calculated from other data) then retrace should
> not be a factor.
>
> I haven't read this whole thread but has anyone mentioned the
> electronic bandwidth of the amplifier leading up to the digitizer?
> This should be set to a time constant that is at least 4x slower
> (maybe 5x if we consider the lower proportion of retrace time)? If it
> is not, then any benefit of longer dwell time will be lost (because
> the value of the signal at the instant it is digitized will only be
> characteristic of the shorter, fast scan pixel). To my knowledge only
> BioRad did true box-car averaging where they integrated all the
> current presented during the pixel, no matter how long it took.
>
> As far as vibration or drift(of the stage or the scanning coil
> currents) is concerned, fast scan will cause blur to the whole image,
> while slower scanning will more likely cause distortion. Although
> changing the scan speed by only a factor of 4 (rather than maybe 100
> or even 1,000, as in a SEM) should not show much of this effect.
>
> As far as analog vs, photon counting: In photon counting, the
> bandwidth argument has no validity (assuming that the system merely
> counts the pulses for a longer time as slow scan.) because what is
> important then is the bandwidth up until the counter.
>
> But it is a rare system that can do photon counting at "normal"
> signal levels without losing significant signal to pulse pileup.
> Pileup will tend to clip bright parts of the image, perhaps making
> them look "smoother" .
>
> Finally, what did you all decide you meant by "averaging"? Kalman
> averaging will give similar results (apart from the factors above. A
> running (or exponential) average, will give a factor of at least 2
> less efficient use of the signal (maybe worse if you get into weak
> signals).
>
> You talk about a "camera expert" and "noise suppression", is he
> talking about the tricks used to make pictures made with digital
> cameras look better? That is a whole 'nuther can of worms and needs a
> longer discussion. It should have little bearing on microscopical
> imaging unless you are taking images off your Yokogawa using a Nikon
> D3.
>
> As far as the effect of scan rate on bleaching is concerned, there
> has long been a debate over whether spreading the damage around fast
> perhaps stops the buildup of, say singlet oxygen, to dangerous levels
> in one location. Those scanning at video rate claimed that they could
> watch their specimens longer because of this effect (I guess that the
> assumption was that natural mechanisms for detoxification were
> overwhelmed if the beam sat in one place too long.).
>
> I once tried to  prove this by spending some time at the Noran
> factory, but never got anything definitive. But it could be true,
> because the disk-scanner folk make the same claim with some support.
>
> Jim Pawley
> At the 16th UBC Course.
>
>    
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I agree with Julio and Brian about the Zeiss 510 - averaged images were
>> always less noisy than "dwelled" images.  I always taught that point in
>> training sessions and showed side by side comparisons; newbies could
>>      
> clearly
>    
>> see the benefit of averaging over dwelling.  However this isn't the
>>      
> case on
>    
>> my 710 or 700, I see no difference (just by eye!) between averaging and
>> dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x
>>      
> slower
>    
>> than whatever max. is [1~3 us]).
>>
>> -Esteban
>>
>>
>> On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian<[hidden email]>
>> wrote:
>>      
>>>   *****
>>>   To join, leave or search the confocal microscopy listserv, go to:
>>>   http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>   *****
>>>
>>>   I have not done a careful analysis of this either, and I am not
>>>        
> quite sure
>    
>> how you would, however I share the viewpoint of Julio exactly,
>>      
> 1.6usec/pix
>    
>> and 2-4 ave.
>>      
>>>
>>>
>>>   Brian Armstrong PhD
>>>   Light Microscopy Core
>>>   Beckman Research Institute
>>>   1450 East Duarte Rd
>>>   Duarte, CA 91010
>>>   626-256-4673 x62872
>>>
>>>        
>> http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHom
>>      
> e.htm
>    
>>>
>>>   -----Original Message-----
>>>   From: Confocal Microscopy List
>>>        
> [mailto:[hidden email]]
>    
>> On Behalf Of Julio Vazquez
>>      
>>>   Sent: Thursday, June 16, 2011 2:21 PM
>>>   To: [hidden email]
>>>   Subject: Re: 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
>>>   *****
>>>
>>>   This is what I noticed empirically on our Zeiss LSM 510, where
>>>        
> averaging
>    
>> tends to give somewhat better noise reduction than increasing dwell
>>      
> time.
>    
>> Under "normal" imaging conditions, we typically use a dwell time of
>>      
> 1.6-3.2
>    
>> microseconds. Increasing the dwell time to greater than  3.2
>>      
> microseconds
>    
>> tends to result in more bleaching and somewhat reduced signal.
>>      
> Typically, we
>    
>> use 1.6 microseconds dwell time, and 2-4 averages, depending on the
>>      
> sample.
>    
>>>   --
>>>   Julio Vazquez
>>>   Fred Hutchinson Cancer Research Center
>>>   Seattle, WA
>>>
>>>   http://www.fhcrc.org/
>>>
>>>
>>>   On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>>>
>>>        
>>>>   Stan,
>>>>
>>>>   I've been told by Carl Z. engineers that in general averaging (I
>>>>          
> usually
>    
>> use line, not frame) tends to yield better S/N then does increasing
>>      
> dwell
>    
>> time. As Lloyd commented this may be model specific. I have not done
>>      
> any
>    
>> analysis to confirm this however....
>>      
>>>>   Tom
>>>>          
>>>
>>>
>>>        
> ---------------------------------------------------------------------
>    
>>>   SECURITY/CONFIDENTIALITY WARNING:
>>>   This message and any attachments are intended solely for the
>>>        
> individual or
>    
>> entity to which they are addressed. This communication may contain
>> information that is privileged, confidential, or exempt from disclosure
>> under applicable law (e.g., personal health information, research data,
>> financial information). Because this e-mail has been sent without
>> encryption, individuals other than the intended recipient may be able
>>      
> to
>    
>> view the information, forward it to others or tamper with the
>>      
> information
>    
>> without the knowledge or consent of the sender. If you are not the
>>      
> intended
>    
>> recipient, or the employee or person responsible for delivering the
>>      
> message
>    
>> to the intended recipient, any dissemination, distribution or copying
>>      
> of the
>    
>> communication is strictly prohibited. If you received the communication
>>      
> in
>    
>> error, please notify the sender immediately by replying to this message
>>      
> and
>    
>> deleting the message and any accompanying files from your system. If,
>>      
> due to
>    
>> the security risks, you do not wish to receive further communications
>>      
> via
>    
>> e-mail, please reply to this message and inform the sender that you do
>>      
> not
>    
>> wish to receive further e-mail from the sender.
>>      
>>>
>>>        
> ---------------------------------------------------------------------
>    
>>>        
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[George McNamara]

*****
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

[simon walker (BI)]

*****
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

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

George,

      Thanks for the clarification - I'm not in Australia so I couldn't
check our own SP5.  But my point remains that 'brightness' means
nothing.  Handling the output of a high-speed scanner is still
computationally challenging, and may use different algorithms compared
to slow scan acquisition.  The only measure of image quality is signal
to noise.

                                        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 George McNamara
Sent: Friday, 17 June 2011 8:58 PM
To: [hidden email]
Subject: Re: 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
*****

Hi Guy,

The Leica SP5 uses a 12-bit digitizer in either resonant (8000 Hz) or
standard (hi-res, wide variety of scan speeds, standard is 400 Hz, can
go 1 Hz to 1400 Hz) scan mode. Resonant scan mode output can be 8-bit or

12-bit, standard scan mode can be 8, 12, or 16-bit. Same whether
standard PMT mode or HyD photon counting mode.

Jim - the new HyD detector is pretty fast, pulse pile up did not seem to

be a problem at the Yale Microscopy Workshop last week (thanks again to
Ann, Derek, Joerg, the other Yalies helping wit the workshop, vendors
and attendees).

The interaction of the fluorophore(s) with the mounting media components

can trump the digitizer details - see, for example, the streaking in the

non-ROXS figure panels of:

Fluorophores: Single-Molecule STED Microscopy with Photostable Organic
Fluorophores (Small 13/2010). </pubmed/20589865> Kasper R, Harke B,
Forthmann C, Tinnefeld P, Hell SW, Sauer M., Small. 2010 Jun 29;6(13):
1379-1384. PMID: 20521266.



George

On 6/17/2011 2:47 AM, Guy Cox wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I was talking about sub-pixel scan inaccuracy, which would not be
> sufficient to cause visible blurring but could give some smoothing.
>
> Image 'brightness', mentioned by another contributor to this thread,
is
> not a meaningful figure (particularly without any figures quoted).
For

> example, resonant scans might be digitized at 8-bit for the sake of
> speed, and non-resonant ones at 12 bit.  Brightness is then just a
> function of the display algorithm.
>
>                     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 James Pawley
> Sent: Friday, 17 June 2011 3:36 PM
> To: [hidden email]
> Subject: Re: 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
> *****
>
> Hi all,
>
> I haven't seen these images but the "dwell" image (slower scan?)
> image "should" be better for a given total acquisition time, because
> a smaller fraction of the total exposure time is spent in retrace
> (about 30% of the time is usually used for retrace in fast scan.
> Ergo, the fraction of the frame time spent actually collecting signal
> is about 70% at 'fast scan" and more like 90% at a 4x slower scan
> rate. However, you seem to know the actual pixel dwell time, so this
> accurate (rather than calculated from other data) then retrace should
> not be a factor.
>
> I haven't read this whole thread but has anyone mentioned the
> electronic bandwidth of the amplifier leading up to the digitizer?
> This should be set to a time constant that is at least 4x slower
> (maybe 5x if we consider the lower proportion of retrace time)? If it
> is not, then any benefit of longer dwell time will be lost (because
> the value of the signal at the instant it is digitized will only be
> characteristic of the shorter, fast scan pixel). To my knowledge only
> BioRad did true box-car averaging where they integrated all the
> current presented during the pixel, no matter how long it took.
>
> As far as vibration or drift(of the stage or the scanning coil
> currents) is concerned, fast scan will cause blur to the whole image,
> while slower scanning will more likely cause distortion. Although
> changing the scan speed by only a factor of 4 (rather than maybe 100
> or even 1,000, as in a SEM) should not show much of this effect.
>
> As far as analog vs, photon counting: In photon counting, the
> bandwidth argument has no validity (assuming that the system merely
> counts the pulses for a longer time as slow scan.) because what is
> important then is the bandwidth up until the counter.
>
> But it is a rare system that can do photon counting at "normal"
> signal levels without losing significant signal to pulse pileup.
> Pileup will tend to clip bright parts of the image, perhaps making
> them look "smoother" .
>
> Finally, what did you all decide you meant by "averaging"? Kalman
> averaging will give similar results (apart from the factors above. A
> running (or exponential) average, will give a factor of at least 2
> less efficient use of the signal (maybe worse if you get into weak
> signals).
>
> You talk about a "camera expert" and "noise suppression", is he
> talking about the tricks used to make pictures made with digital
> cameras look better? That is a whole 'nuther can of worms and needs a
> longer discussion. It should have little bearing on microscopical
> imaging unless you are taking images off your Yokogawa using a Nikon
> D3.
>
> As far as the effect of scan rate on bleaching is concerned, there
> has long been a debate over whether spreading the damage around fast
> perhaps stops the buildup of, say singlet oxygen, to dangerous levels
> in one location. Those scanning at video rate claimed that they could
> watch their specimens longer because of this effect (I guess that the
> assumption was that natural mechanisms for detoxification were
> overwhelmed if the beam sat in one place too long.).
>
> I once tried to  prove this by spending some time at the Noran
> factory, but never got anything definitive. But it could be true,
> because the disk-scanner folk make the same claim with some support.
>
> Jim Pawley
> At the 16th UBC Course.
>
>    
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I agree with Julio and Brian about the Zeiss 510 - averaged images

were
>> always less noisy than "dwelled" images.  I always taught that point
in
>> training sessions and showed side by side comparisons; newbies could
>>      
> clearly
>    
>> see the benefit of averaging over dwelling.  However this isn't the
>>      
> case on
>    
>> my 710 or 700, I see no difference (just by eye!) between averaging
and

>> dwelling (~800 volt gain comparing 4-8 averages to dwell times 4-8x
>>      
> slower
>    
>> than whatever max. is [1~3 us]).
>>
>> -Esteban
>>
>>
>> On Thu, Jun 16, 2011 at 2:38 PM, Armstrong, Brian<[hidden email]>
>> wrote:
>>      
>>>   *****
>>>   To join, leave or search the confocal microscopy listserv, go to:
>>>   http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>   *****
>>>
>>>   I have not done a careful analysis of this either, and I am not
>>>        
> quite sure
>    
>> how you would, however I share the viewpoint of Julio exactly,
>>      
> 1.6usec/pix
>    
>> and 2-4 ave.
>>      
>>>
>>>
>>>   Brian Armstrong PhD
>>>   Light Microscopy Core
>>>   Beckman Research Institute
>>>   1450 East Duarte Rd
>>>   Duarte, CA 91010
>>>   626-256-4673 x62872
>>>
>>>        
>>

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHom

>>      
> e.htm
>    
>>>
>>>   -----Original Message-----
>>>   From: Confocal Microscopy List
>>>        
> [mailto:[hidden email]]
>    
>> On Behalf Of Julio Vazquez
>>      
>>>   Sent: Thursday, June 16, 2011 2:21 PM
>>>   To: [hidden email]
>>>   Subject: Re: 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
>>>   *****
>>>
>>>   This is what I noticed empirically on our Zeiss LSM 510, where
>>>        
> averaging
>    
>> tends to give somewhat better noise reduction than increasing dwell
>>      
> time.
>    
>> Under "normal" imaging conditions, we typically use a dwell time of
>>      
> 1.6-3.2
>    
>> microseconds. Increasing the dwell time to greater than  3.2
>>      
> microseconds
>    
>> tends to result in more bleaching and somewhat reduced signal.
>>      
> Typically, we
>    
>> use 1.6 microseconds dwell time, and 2-4 averages, depending on the
>>      
> sample.
>    
>>>   --
>>>   Julio Vazquez
>>>   Fred Hutchinson Cancer Research Center
>>>   Seattle, WA
>>>
>>>   http://www.fhcrc.org/
>>>
>>>
>>>   On Jun 16, 2011, at 2:08 PM, Moninger, Thomas wrote:
>>>
>>>        
>>>>   Stan,
>>>>
>>>>   I've been told by Carl Z. engineers that in general averaging (I
>>>>          
> usually
>    
>> use line, not frame) tends to yield better S/N then does increasing
>>      
> dwell
>    
>> time. As Lloyd commented this may be model specific. I have not done
>>      
> any
>    
>> analysis to confirm this however....
>>      
>>>>   Tom
>>>>          
>>>
>>>
>>>        
> ---------------------------------------------------------------------
>    
>>>   SECURITY/CONFIDENTIALITY WARNING:
>>>   This message and any attachments are intended solely for the
>>>        
> individual or
>    
>> entity to which they are addressed. This communication may contain
>> information that is privileged, confidential, or exempt from

disclosure
>> under applicable law (e.g., personal health information, research
data,

>> financial information). Because this e-mail has been sent without
>> encryption, individuals other than the intended recipient may be able
>>      
> to
>    
>> view the information, forward it to others or tamper with the
>>      
> information
>    
>> without the knowledge or consent of the sender. If you are not the
>>      
> intended
>    
>> recipient, or the employee or person responsible for delivering the
>>      
> message
>    
>> to the intended recipient, any dissemination, distribution or copying
>>      
> of the
>    
>> communication is strictly prohibited. If you received the

communication
>>      
> in
>    
>> error, please notify the sender immediately by replying to this
message

>>      
> and
>    
>> deleting the message and any accompanying files from your system. If,
>>      
> due to
>    
>> the security risks, you do not wish to receive further communications
>>      
> via
>    
>> e-mail, please reply to this message and inform the sender that you

do

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George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

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[James Pawley]

*****
To join, leave or search the confocal microscopy listserv, go to:
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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.