http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Transmission-Images-tp7393699p7463631.html
Thanks for the paper Guy, it is amusing and informative. I always wondered how to get that .3 of a pixel!
Back to the original question; perhaps you could explain to the users not to zoom more than 1000x NA. This is a more straight forward conversation than explaining the Nyquist/Shannon theorem to nascent Confocal users. I often see users applying too much zoom and collecting empty magnification.
Numbers: using a 20x/0.8NA lens and lambda of 500nm AU=1, Nyquist is ~ 165nm. If you use 4x optical zoom, you have 200x X 4 = 800x (1000 X NA of 0.8 = 800x) which at 1024x1024 gives you a pixel size of X,Y = 110nm which is comfortably under Nyquist sampling.
] On Behalf Of Renato A. Mortara
Renato A. Mortara
R. Botucatu, 862 6o andar
Citando "Joel B. Sheffield" <
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Excellent points. That paper is a joy to read.
> Joel
>
>
> On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <
[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>> Well, to put this in more easily understood terms, Nyquist (at 2B) defines
>> the limit - ie the point where you cease to be able to reconstruct the
>> wave. So, as Mark says, you need to get beyond this to actually be able
>> to get information. The often-quoted 2.3B more or less corresponds to the
>> Rayleigh resolution criterion, ie the point at which you can reconstruct
>> the wave at usable contrast. However, the other problem we face is that we
>> do NOT reconstruct the sine wave, we just look at a map of little squares.
>> This is stupid.
>>
>> Required reading should be:
>>
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square!
>> (And a Voxel is Not a Little Cube)
>> Microsoft Technical Memo 6
>> Alvy Ray Smith
>> July 17, 1995
>>
>> (Yes, that really is the title)
>>
>> It's on the Microsoft web site, or I can mail a copy to anyone who is
>> interested.
>>
>>
>> Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:
[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 April 2012 5:53 PM
>> To:
[hidden email]
>> Subject: Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
>> times the bandlimit. The Nyquist rate is defined by the sufficient
>> condition for exact reconstructability: Fs > 2B. 2B _is_ the Nyquist rate
>> as David said, it does not mean Fs = 2B is sufficient!
>>
>> Cheers
>>
>> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> > *****
>> >
>> > Hi everyone
>> >
>> > "strict Nyquist is a factor of 2."
>> >
>> > My understanding is that the Nyquist theorem is not arbitrary and that
>> the factor is actually >2. So 2.1 would do as well as 2.3. If i understood
>> well the >2 comes from this: if you want to describe a periodic signal
>> (which is what we do when we acquire an image: we describe a sum of
>> periodic signals), you need more than 2 points within 1 full period to
>> collect enough information to reconstruct the periodic signal accurately.
>> If you only give 2 points per period (e.g. only the crests and troughs),
>> you can draw the periodic signal is several ways (e.g. double the frequency
>> of the original signal). When we acquire an image we should thus sample
>> more than twice the shortest period (the edges) to acquire enough
>> information for the computer to properly reconstruct the image. This is why
>> the Nyquist criterion is 'more than 2'. Am I right?
>> >
>> > Sylvie
>> >
>> > @@@@@@@@@@@@@@@@@@@@@@@@
>> > Sylvie Le Guyader
>> > Live Cell Imaging Unit
>> > Dept of Biosciences and Nutrition
>> > Karolinska Institutet
>> > Novum
>> > 14183 Huddinge
>> > Sweden
>> > office: +46 (0) 8 5248 1107
>> > LCI room: +46 (0) 8 5248 1172
>> > mobile: +46 (0) 73 733 5008
>> >
>> >>
>> >> On 11 Apr 2012, at 22:45, "David Baddeley"
>> >> <
[hidden email]>
>> >> wrote:
>> >>
>> >>> *****
>> >>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> >>> *****
>> >>>
>> >>>
>> >>> The diagonal in z will be much 'straighter' (due to the fact that
>> >>> the voxels are
>> >> elongated in z rather than being square), making the factor much
>> >> closer to 1 (probably something like 1.1) so it can safely be
>> >> ignored. When talking about slightly oversampling, 2.3 is already
>> >> doing this - strict Nyquist is a factor of 2. It's also worth noting
>> >> that you should probably use the theoretical resolution values (ie
>> >> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and
>> >> not the observed PSF width, as these reflect the bandwidth of the
>> >> system. I this tend to reccommend a blanket 70x70x200nm pixel size
>> >> when using a high NA objective on fixed cells. In live cells, or
>> >> other delicate samples you need to exercise a little more discretion
>> >> - the artefacts introduced by slight undersampling are likely to be
>> outweighed by other considerations.
>> >>>
>> >>> My 2c,
>> >>> David
>> >>>
>> >>>
>> >>> ------------------------------
>> >>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>> >>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> >>>> *****
>> >>>>
>> >>>> Hi John,
>> >>>>
>> >>>> Indirectly, do you suggest the same for Z sampling if we are
>> >>>> interested in 3D measurements? Thanks
>> >>>>
>> >>>> Monique Vasseur
>> >>>>
>> >>>> -----Message d'origine-----
>> >>>> De : Confocal Microscopy List
>> >> [mailto:
[hidden email]] De la part de Lemasters,
>> >> John J.
>> >>>> Envoyé : 11 avril 2012 09:34
>> >>>> À :
[hidden email] Objet : Re: Nyquist and Image
>> >>>> size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> >>>> *****
>> >>>>
>> >>>> Please remember that pixel spacing on the diagonal is 1.4 that in
>> >>>> the horizontal
>> >> and vertical directions. Accordingly to meet the Nyquist criterion
>> >> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>> >> Nyquist criterion is an arbitrary threshold, and image quality will
>> >> improve somewhat with sampling greater that proposed by Nyquist.
>> >> Considering diagonal sampling, I suggest using a pixel size that is one
>> fourth of the resolving limit for the most critical work.
>> >>>>
>> >>>> John
>> >>>>
>> >>>> --
>> >>>> John J. Lemasters, MD, PhD
>> >>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
>> >>>> Center for Cell Death, Injury & Regeneration Departments of
>> >>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
>> >>>> Biology Medical University of South Carolina
>> >>>> DD504 Drug Discovery Building
>> >>>> 70 President Street, MSC 140
>> >>>> Charleston, SC 29425
>> >>>>
>> >>>> Office: 843-876-2360
>> >>>> Lab: 843-876-2354
>> >>>> Fax: 843-876-2353
>> >>>> Email:
[hidden email]
>> >>>>
http://academicdepartments.musc.edu/ccdir>> >>>>
>> >>>>
>> >>>> -----Original Message-----
>> >>>> From: Confocal Microscopy List
>> >>>> [mailto:
[hidden email]] On Behalf Of John
>> >>>> Oreopoulos
>> >>>> Sent: Wednesday, April 11, 2012 8:29 AM
>> >>>> To:
[hidden email]
>> >>>> Subject: Re: Nyquist and Image size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> >>>> *****
>> >>>>
>> >>>> Renato,
>> >>>>
>> >>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>> >>>> Nyquist
>> >> sampling rate (ie: pixel dimensions) does not change since your
>> >> objective lens did not change. The quoted pixel size at 2Kx2K you
>> >> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>> >> (and not gaining anything). Your image may look smoother but it
>> >> contains no more information than the 512x512 image with 90x90 nm
>> >> pixel sizes. Presumably the scan speed is the same between
>> >> 512x512 and 2Kx2K.
>> >>>>
>> >>>> You should decrease the galvometric mirror scan zoom setting to get
>> >>>> back to
>> >> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>> >> Effectively, you will be imaging (and properly sampling) a larger
>> >> field of view then. I'm not familiar with the Leica laser scanning
>> >> confocals so I'm not sure if it will allow you to do this. On other
>> >> systems, like the Olympus FV300 for example, you can set your image
>> >> pixel dimensions (256x256, 512x512, etc.) and your scan zoom
>> independently.
>> >>>>
>> >>>> Just out of curiosity, why image 2K x 2K when you can't easily
>> >>>> display that on
>> >> a standard computer screen or present it in a published paper without
>> downsizing?
>> >> I rarely departed from 512x512 in my laser scanning days, except when
>> >> I wanted to see a larger field of view.
>> >>>>
>> >>>> Cheers,
>> >>>>
>> >>>>
>> >>>> John Oreopoulos
>> >>>> Research Assistant
>> >>>> Spectral Applied Research
>> >>>> Richmond Hill, Ontario
>> >>>> Canada
>> >>>> www.spectral.ca
>> >>>>
>> >>>>
>> >>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>> >>>>
>> >>>>> *****
>> >>>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> >>>>> *****
>> >>>>>
>> >>>>> Dear all,
>> >>>>>
>> >>>>> Having attended the first Pawley course in Vancouver I feel highly
>> >>>>> embarassed to ask this, but I would really appreciate a
>> clarification:
>> >>>>>
>> >>>>> When estimating the highest zoom users should apply to their
>> >>>>> sample in order to accommodate for the Nyquist theorem, I
>> >>>>> estimated the optimum pixel size value by dividing the lateral
>> >>>>> resolution (eg: 0.2
>> >>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>> >>>>>
>> >>>>> The doubt: if the image size is increased from 512x512 (having
>> >>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>> >>>>> resulting pixel size (displayed by the system - Leica) the pixel
>> >>>>> size decreases
>> >>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
>> >>>>> but only the image size, what happens to Nyquist and the optimum
>> >>>>> pixel size
>> >> at 2Kx2K ?
>> >>>>>
>> >>>>> Many thanks !
>> >>>>>
>> >>>>> Renato
>> >>>>>
>> >>>>> Renato A. Mortara
>> >>>>> Parasitology Division
>> >>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
>> >>>>> São Paulo, SP
>> >>>>> 04023-062
>> >>>>> Brazil
>> >>>>> Phone: 55 11 5579-8306
>> >>>>> Fax: 55 11 5571-1095
>> >>>>> email:
[hidden email]
>> >>>>> home page:
>> www.ecb.epm.br/~ramortara<
http://www.ecb.epm.br/%7Eramortara>
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail:
[hidden email]
> URL:
http://astro.temple.edu/~jbs>
>
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