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I agree with John Lemasters on XY:

    John: 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.
       

I recommend to my users to take the Airy XY resolution (conventional 
optics) and divide by ~3.5. So, for Lambda = 500 nm and 1.4 NA objective 
lens:

distance (Airy XY) = 0.6 * lambda / NA = 0.6 * 500 / 1.4 = 214 nm /// 
divide by 3.5 for pixel size gives 61.2 nm, which I round to 60 nm (or 
whatever Leica LAS AF or Zeiss ZEN rounds to).

For Z, while I would like to acquire with Zeiss or Leica's "optimized" 
setting, photobleaching of most specimens leads me to usually recommend 
"half overlap" (0.5 of optical slice thickness) or "layer cake" (Z-step 
size equal to manufacturer recommended optical slice thickness). If I 
open the pinhole, then I use layer cake (yummy).

//

Leica LAS AF owners: check out "STED/confocal deconvolution" in Process 
tab. Tedious to use (I've complained about the tedious workflow - I 
encourage every Leica owner to complain too). I recommend:
1. acquire as above - I use 12-bit acquisition mode [digitzer output is 
12-bit on the SP5] (welcome to go for Leica recommended Z step size if 
thin and/or non-bleachable specimen)
2. Generate PSF image ... FWHM = Airy distance calculated as above (214 
nm for confocal, I used 80 nm for the CW-STED demo). ... I admist to not 
checking the online help to see if there is a better value (of course 
this whole step is part of the tedious workflow: the software should not 
need to generate a stupid "dot" image at all).
2a. I use Lorentz. Why? Sounds cooler than Gauss.
3. in the STED/confocal deconv command, select the correct pair of 
images (PSF and image to deconvolve), I use the default numeric value 
(0.001?), and like "Signal Energy" (cooler sounding than the other 
options, though unfortunately not able to make the default).
** Result image (or Z-series) is pretty quick on a cropped image. I did 
a 4 channel 4kx4k Z-series (single tile) recently, did not take too long 
(don't believe "95% done" on the progress meter).
    One (of many) issues: the command autoscales to the full dynamic 
range brightness (i.e. 4095 for 12-bit). This makes negative controls as 
well as images with bright junk look "hmmm". Best wasy to deal with it 
(until Leica fixes this ... and no, it is not just "signal energy"): 
Learn to contrast adjust! I hope you used modest gain (600 to 800 for 
the internal SP5 PMTs, offset 0 works well for our SP5s, just above zero 
intensity without any illumination).
4. Contrast adjust both the original and the STED/confocal deconv images.

Memo to Leica and other confocal manufacturers: With GPU card(s) 
deconvolution (and other image processing) commands should be 
instantaneous. GPU card(s) are a tiny fraction of the price of a 
confocal microscope. If three people from NIST (maybe most of the heavy 
lifting by one student?) can speed up 3D-SIM by 90x, the confocal and 
nanoscope manufacturers ought to be able to figure out GPU programming:

    Lefman J </pubmed?term=%22Lefman%20J%22%5BAuthor%5D>, Scott K
    </pubmed?term=%22Scott%20K%22%5BAuthor%5D>, Stranick S (2011)
    </pubmed?term=%22Stranick%20S%22%5BAuthor%5D>Live, video-rate
    super-resolution microscopy using structured illumination and rapid
    GPU-based parallel processing. Microsc Microanal. <#> 2011
    Apr;17(2):191-6.

    Structured illumination fluorescence microscopy is a powerful
    super-resolution method that is capable of achieving a resolution
    below 100 nm. Each super-resolution image is computationally
    constructed from a set of differentially illuminated images.
    However, real-time application of structured illumination microscopy
    (SIM) has generally been limited due to the computational overhead
    needed to generate super-resolution images. Here, we have developed
    a real-time SIM system that incorporates graphic processing unit
    (GPU) based in-line parallel processing of raw/differentially
    illuminated images. By using GPU processing, the system has achieved
    a 90-fold increase in processing speed compared to performing
    equivalent operations on a multiprocessor computer--the total
    throughput of the system is limited by data acquisition speed, but
    not by image processing. Overall, more than 350 raw images (16-bit
    depth, 512 × 512 pixels) can be processed per second, resulting in a
    maximum frame rate of 39 super-resolution images per second. This
    ultrafast processing capability is used to provide immediate
    feedback of super-resolution images for real-time display. These
    developments are increasing the potential for sophisticated
    super-resolution imaging applications. PMID:21385522.

For another example:

    A distributed multi-GPU system for high speed electron microscopic
    tomographic reconstruction. </pubmed/21741915> Zheng SQ, Branlund E,
    Kesthelyi B, Braunfeld MB, Cheng Y, *Sedat* JW, *Agard* DA.
    Ultramicroscopy. 2011 Jul;111(8):1137-43. PMID: 21741915.




George



On 4/11/2012 8:29 AM, John Oreopoulos wrote:
> *****
> 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: [log in to unmask]
>> home page: www.ecb.epm.br/~ramortara
>>      
>