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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 +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of James Pawley
> Sent: Friday, 17 June 2011 3:36 PM
> To: [log in to unmask]
> 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<[log in to unmask]>
>> 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:[log in to unmask]]
>
>> On Behalf Of Julio Vazquez
>>
>>> Sent: Thursday, June 16, 2011 2:21 PM
>>> To: [log in to unmask]
>>> 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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--
George McNamara, PhD
Analytical Imaging Core Facility
University of Miami
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