Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/averaging-vs-accumulation-for-noise-reduction-is-there-a-difference-tp6483751p6485963.html
*****
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 +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
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
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.