http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-40X-N-A-1-4-Plan-APo-as-replacement-for-63X-tp7582324p7582346.html
thanks a lot for interesting information. I was interested in
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>
> Hi Miroslav,
>
>
> "highest resolution of 2k x 2k."
>
> is number of pixels in an image, resolution is set by that and field of view
> (zoom).
>
> With the LSM 700 -- or other point scanning confocals -- you can choose the
> zoom and number of pixels to get whatever pixel size you want.
>
> Data acquired on a confocal microscope can be used to obtain
> "superresolution", as for the ~10% increase in resolution (ex. from ~214 nm
> to <200 nm for 500 nm light, Airy disk 1.0) by deconvolving at 50 nm pixel
> size. A pixel size of 130 nm is not going to give you a resolution
> improvement (and the optical resolution of visible light confocal is ~200nm,
> not 130 nm).
> There are also other ways to process images - confocal or widefield - to get
> a lot more out of the source data. Two examples:
> PiMP
http://jcs.biologists.org/content/125/9/2257.long ... which I
> preferred using 30 nm pixel size acquisition on the confocals I managed in
> Miami (LSM710, SP5).
> 3B ...
http://www.coxphysics.com/3b/ and
http://www.superresolved.com/> (later is an online forum hosted by Susan cox and Ed Rosten for all
> superduperres).
> SOFI / bSOFI etc (which I've not used) see
>
http://www.ncbi.nlm.nih.gov/pubmed/22711840 for Peter Dedecker et al's entry
> point.
>
> Finally, not required by law (or Guy Cox or Jim Pawley or Alby Diaspro) to
> set the confocal pinhole at 1.0 Airy units (for one thing, the physical size
> for that varies with wavelength). With some specimens it is useful to use a
> smaller pinhole. Zeiss has a nice PDF online on different pinhole settings -
> if you cannot find it on an internet search, ask your Zeiss confocal rep to
> find and send it.
>
> As for Nyquist - he had a thing for sine waves. anyone imaging sine waves
> perfectly aligned in X or Y (or rotate scan view to perfectly align) can use
> the Nyquist sampling value (around 2.3). So, to sample correctly for 2D
> Nyquist, need to sample to account for the worse case sine wave at 45
> degrees. Few biological specimens viewed on a confocal or widefield
> fluorescence microscope are sine waves (pattern A at
>
http://argolight.com/product-micro/ comes close ... with modest NA objective
> lens probably close enough, but then that is a calibration slide, not a cell
> ... muscle fibers can come close).So, stop thinking Nyquist, and start
> thinking cells.
>
> Enjoy,
>
> George
> p.s. a heavily used objective will generally not perform as well as a brand
> new objective lens.
>
>
> On 7/14/2014 2:54 AM, Miroslav Varecha wrote:
>>
>> Hello,
>> I would like to react to George. In my humble opinion, best lateral
>> optical resolution for confocal microscope is around 130 nm, so going
>> to resolution 60x60 nm is overshoot and it is just wasting your drive
>> space as you are not collecting any new real information. Resolutions
>> of 50x50nm and such are area of superresolution microscopy. Our
>> confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k.
>> High quality CCDs have even less, but sCMOS can get higher than 2k x
>> 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you
>> that it is the most used objective (usually we observe stem cells)
>> even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger
>> field of view of 40x. You still get great detail and many cells in one
>> image. But if you want to observe really up close organels and such I
>> would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible
>> objective for vast majority of observations.
>> Miroslav
>>
>>
>> 2014-07-14 1:31 GMT+02:00 George McNamara<
[hidden email]>:
>>
>>>
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>>> To join, leave or search the confocal microscopy listserv, go to:
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>>> *****
>>>
>>> Hi Michael,
>>> why 2kx2k?
>>> If the 40x lens has a 250x250 um field of view, this would be
>>> undersampling,
>>> pixel size 125x125 nm. If even larger field of view, undersampling even
>>> more.
>>> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step
>>> 200
>>> nm, maybe closer),
>>> George
>>>
>>> On 7/11/2014 8:53 AM, Cammer, Michael wrote:
>>>
>>>>
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>>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>>> Post images on
http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo?
>>>> This
>>>> is something I've wanted for a long time, the ability to take large
>>>> fields
>>>> of view (2k X 2k pixels) at high resolution instead of having to do
>>>> tiling.
>>>> Also, with the new cameras that have oodles of small pixels...
>>>>
>>>> I'm considering replacing our 63X with this new 40X. Any experience
>>>> with
>>>> this, other than the battle of having to explain to other scope users
>>>> why
>>>> this is not really lower magnification?
>>>>
>>>> Regards,
>>>>
>>>> Michael
>>>>
>>>>
>>>>
>>>> ===========================================================================
>>>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU
>>>> Langone Medical Center
>>>> Cell: 914-309-3270 Lab: 212-263-3208
>>>>
http://ocs.med.nyu.edu/microscopy&>>>>
http://www.med.nyu.edu/skirball-lab/dustinlab/>>>>
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>>>
>>>
>>>
>>> --
>>>
>>>
>>>
>>> George McNamara, Ph.D.
>>> Single Cells Analyst
>>> L.J.N. Cooper Lab
>>> University of Texas M.D. Anderson Cancer Center
>>> Houston, TX 77054
>>> Tattletales
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>>
>>
>
>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales
http://works.bepress.com/gmcnamara/42