http://confocal-microscopy-list.275.s1.nabble.com/averaging-vs-accumulation-for-noise-reduction-is-there-a-difference-tp6483751p6486977.html
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
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.
> *****
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>
> 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
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> ______________________________________________
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>
> -----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:
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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
>>>>
>>>
>>>
>>>
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