Posted by
Lutz Schaefer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-40X-N-A-1-4-Plan-APo-as-replacement-for-63X-tp7582324p7582345.html
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Hi George,
sorry, as non-biologist I have to (at least partially) disagree on your last
comment that read:
>
> 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.
>
According to Fourier synthesis any signal, whether it being originating from
the fluorescence of a cell, other material or just as a 1D signal can be
represented by sinusoidal functions. For the "survival" of their highest
frequency components after discretization it is common to use the
Nyquist-Shannon sampling theorem. The factor of two thereby is only the
critical period from which on aliasing begins to recede. Sampling at (even)
higher than Nyquist rates is always favorable but mostly impractical,
because restoring these higher frequencies is subject to a low SNR, set
aside the storage needs. Additionally, for 2D or 3D the theorem is
separable. In other words an exemplary diagonal, 1D signal inside a 2D image
can be represented by the two orthogonal XY sampling directions.
Having said that, of course it is always practical to know the shape of the
objects you are looking at, even though their emissions or signals are by
nature composed of sine waves...
Regards
Lutz
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L u t z S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy
Advanced Imaging Methodology Consultation
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-----Original Message-----
From: George McNamara
Sent: Monday, July 14, 2014 9:39 PM
To:
[hidden email]
Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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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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>>
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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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>>>
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>>> posting.
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>>>
>>> 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
http://works.bepress.com/gmcnamara/42>>
>
--
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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