http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-40X-N-A-1-4-Plan-APo-as-replacement-for-63X-tp7582324p7582341.html
Oh, thanks for explanation. I work with scanning confocal only for short
time. I was using spinning disc microscope with EMCCD before and resolution
was pretty much set by camera. It is very interesting to use deconvolution
to reach even higher resolution with confocal. Unfortunately noone here
wanted very high resolutions for their data so far. I will do some test
I apologize. This approach is new to me.
Dne 14. 7. 2014 16:28 "Feinstein, Timothy" <
> *****
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> *****
>
> Hi Miroslav,
>
> You are correct that even in the best case a traditional optical
> microscope can only separate point sources > 100 nm apart. However the
> theoretical resolution limit is far from the same thing as the ideal
> resolution for sampling. As George mentioned if you want to get the most
> out of an optical image then you should deconvolve out the point spread
> function. In that case the optimal sampling for proper deconvolution
> depends on seeing enough of the PSF from a given point source to guess at
> its center, in which case you need to sample at roughly twice as fine as
> the theoretical limit of a given NA/wavelength/refractive index. This
> sampling theorem was worked out by a fellow named Harry Nuqiust during his
> work with radio signals. In practice I recommend that pretty much
> everyone who works near the resolution limit of their system take the time
> to deconvolve their data.
>
> Cheers,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
> Phone: 616-234-5819 | Email:
[hidden email]
>
>
>
>
>
>
>
> On 7/14/14, 3:54 AM, "Miroslav Varecha" <
[hidden email]> wrote:
>
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >
>
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> >roscopy
> >Post images on
> >
>
http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiEz> >AT3VrQA&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your
> >posting.
> >*****
> >
> >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]>:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >>
> >>
>
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> >>icroscopy
> >> Post images on
> >>
>
http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4DfB> >>tS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your
> >>posting.
> >> *****
> >>
> >> 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:
> >>>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>>
> >>>
>
http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df> >>>E5ThZ2a_w&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
> >>>lmicroscopy
> >>> Post images on
> >>>
>
http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df> >>>BtS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom 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
> >>>
> >>>
>
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> >>>
> >>>
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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
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