> Search the CONFOCAL archive at
> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> One way is to increase the magnification of the objective. Olympus now
> sells 150x/1.45 TIRF objective that uses standard immersion oil and
> coverslips.
>
> Julian Borejdo
>
>>>> John Oreopoulos <
[hidden email]> 08/01/07 11:06 AM
>>>>
> Search the CONFOCAL archive at
> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list server,
>
> This topic has come up before, but I need a little more details on
> the subject. I have a 1.45 NA 60x Olympus objective mounted on an
> IX70 inverted microscope to which a Roper Cascade 512B EMCCD camera
> is attached on the trinocular. The camera has 16x16 um pixels.
> According to the Rayleigh criterion, my maximum possible resolution
> is (assuming 500 nm light):
>
> R = 0.61*lambda/NA = 0.61*500/1.45 = 210 nm
>
> According to the Nyquist sampling theorem, to properly reconstruct
> the image, I should sample the image every 210/2.3 = 91 nm in the x
> and y direction.
>
> With the 512B EMCCD camera, the back projected size of the pixel
> length using the 60x 1.45 NA objective will be:
>
> Back projected size of pixel length = real pixel length/objective
> magnification = 16/60 = 0.267 um = 267 nm
>
> Therefore, I am currently under-sampling the images. I need my back
> projected pixel length to be an additional 267 / 91 = 3x smaller.
>
> If I am wrong with any of my calculations at this point, please
> correct me. If I'm not wrong, then the only way to correct this
> problem is to magnify the image further so that features in the image
>
> can be more finely sampled by the 16x16 um pixels of the camera. For
> my application, I am not "light starved", and so I think I can afford
>
> the signal loss that will accompany the additional magnification as a
>
> result of spreading the light in the image over a larger area.
> Resolution is more critical and hence the need for better sampling.
>
> Here is my question: What kind of lens (focal length, diameter,
> apochromat, etc.) should I use to magnify the image further for
> proper Nyquist sampling, and how and where should I fit this to my
> microscope? Unfortunately my knowledge of geometric optics is very
> basic and I don't want to ask my boss to buy the wrong lens.
> Because the objective is infinity corrected, I imagine I can insert
> the lens anywhere in infinity space. The logical position is between
> the camera mount and the microscope trinocular, correct? But if I do
> that, I may have to change the vertical position of the camera so
> that the image on the camera is parfocal with the eypieces. Is there
> a suitable lens that can be used without having the extend the
> distance of the camera from the scope too much like seen in this
> youtube video:
>
> <a href="http://www.youtube.com/watch?v=9ncrx6NkXmQ&mode=related&search=" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">
http://www.youtube.com/watch?v=9ncrx6NkXmQ&mode=related&search=
>
> Is there a standard modular part from Olympus that can be inserted
> into the microscope that can do the magnification easily (the
> "optovar")? Would any special microscope and camera adaptors be
> needed as well? Note that the microscope that I'm using has dual use
> as a epifluorescence and TIRF microscope which both use the same
> objective. All previous posts on this topic on the list server were
> talking about spinning disk confocals I think.
>
> Once again, thanks to everyone on the list server for being such a
> great resource for microscope information. I don't know how I'd
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