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Natural Science Forum / Physics / Optics / February 2006Confocal Microscope principle
Hi, I understand the role of a pinhole and its size in selectively imaging a layer of a thick 3D specimen. My question is, why not place an array of finitely small pinholes in a plane at the focal length of the objective lens rather than using having random or spiral arrangements of pinholes in a rotating nipkow disk. I guess there is an explanation. I would appreciate your replies. Thanks very much, Madhu. Interesting question. Let me guess. I assume when you talk about "an array of finitely small pinholes" you mean a regular/symetric array of equally sized pinholes. The pinholes are located in a conjugated aperture plane to restrict the illumination inside the specimen to a thin slice in the object plane. If you take a periodic arrangement of pinholes, you will have a regular pattern of interfering waves in the object plane. Just take a regular arrangment of white dots on black background and calculate the 2D FFT of this. (You can use ImageJ for this.) - The purpose of the pinhole(s) is to illuminate just a thin section evently. A random arrangement or a single pinhole located in the center of the Fourier plane (called Gamma point) is more suitable to accomplish this. - Other explanations? Gregor > Hi, > [quoted text clipped - 7 lines] > Thanks very much, > Madhu. > Interesting question. Let me guess. I assume when you talk about "an > array of finitely small pinholes" you mean a regular/symetric array of [quoted text clipped - 22 lines] >>Thanks very much, >>Madhu. The reason for the Nipkow disk is to get uniform illumination over the field. If you wanted to use a stationary array of pinholes, they'd have to be far enough apart that they didn't generate interference fringes--which is much farther than one pixel pitch. You probably don't want to trade a nice 10**6 pixel image for 10**4 isolated dots. Cheers, Phil Hobbs PS: Back in the day, I helped Gordon Kino and Guoxing Xiao with their first Nipkow microscope--Guoxing built it in the lab next to mine. Thanks for the information. Could you explain, how a rotating pinhole won't get interference from the reflected light from the surrounding region? Sincerely, Madhu. > Thanks for the information. Could you explain, how a rotating pinhole > won't get interference from the reflected light from the surrounding > region? > > Sincerely, > Madhu. It isn't the rotation, it's the wide separation that avoids fringes. The rotation just allows a Nipkow wheel with 1% clear aperture to cover the whole field uniformly. The Nipkow design (logarithmic spirals) provides constant average brightness over the whole diameter. Cheers, Phil Hobbs To reiterate, point-scanning (with appropriate pinhole size) is the basic principle behind confocal; simultaneous measurements using more pinholes will collect light from planes adjacent to the focal plane from the specimen. A Nipkow disc is one of the optimal ways (?) to scan the imaging area using optimally positioned pinholes (logarithmic spirals as you mentioned) for real-time full-image formation. Consider I have only one pinhole. With appropriately small pinhole size, most of the light that passes through the pinhole will be from the on-focus plane. Now, if I add another pinhole adjacent to the first pinhole, I would expect the second pinhole (even though closer to the first) to allow most of the light from a point on the on-focus plane (this point on the on-focus plane will be inline with the second pinhole). Can you explain where exactly does the confocal principle compromised here? I appreciate your explanations. Madhu. Is it about the specimen illumination? I am not sure if Nipkow disc illuminates only the point(s) in the specimen that are being imaged. If the argument is that: simultaneous illumination of all the points in the specimen (which should happen for imaging according to my initial question of using a array of pinholes) will cause light interaction from all the planes and result in a blurred image. I apologize if this sounds stupid, but I appreciate any help to clarrify. Madhu. It is infact true that the confocal principle relies on both the controlled illumination of specimen (a very small volume) and controlled reception of light coming from the specimen (could be the light that pass through the specimen) as in original Marvin Minsky's design or the light reflected from the specimen as in most of the modern confocal system. If more than one point in the object plane is illuminated, it will cause light interaction from adjacent point and result in a blurred image; so it is necessary to illuminate the specimen point wise; for eg., tandem scanning system uses nipkow disk pinholes to illuminate points in the specimen. Reflected light out-of-focus volumes are reduced by the pinholes. So as I understand, the confocal principle relies on: 1. Illuminate small volumes (or multiple infinitesimal volumes that are sufficient apart) that do not cause interaction of light from adjacent points 2. Reduce light from out-of-focus planes by proper position of the pinholes with appropriately small pinhole sizes. Reading Prof. Marvin Minsky's memoir helped me gain a good understanding. Here is a link to his memoir on inventing confocal, if there is any interest: http://web.media.mit.edu/~minsky/papers/ConfocalMemoir.html Thanks all. Sincerely, Madhu. > It is infact true that the confocal principle relies on both the > controlled illumination of specimen (a very small volume) and [quoted text clipped - 18 lines] > there is any interest: > http://web.media.mit.edu/~minsky/papers/ConfocalMemoir.html Thanks. Your previous Feb 14 idea was quite right, and the Yokogawa company in Tokyo has made a disk with thousands of microlenses--instead of pinholes! This scheme transmits so much light that the instrument shows movies at 30 Hertz. (My prototype took about 30 seconds per frame.) Right now they have an exhibit in Tokyo, which has my prototype on the same table with their latest instrument. Also, you were right about the size of those 'pinholes'. When I used a 100x objective lens, with a focal plane about 8 inches away from lens, the pinholes were really rather big. I just used a small safety pin to punch a hole in a thin piece of brass foil. I guess this is a reply from Prof. Minsky himself. Its wonderful to hear the views from the inventor himself. I have read about the disk with microlenses for improving the light throughput. Also I read about a patent-pending disk scanning unit (DSU) with Olympus that has a pattern slits (instead of pinholes) but creates a virtual pinholes when spun at 3000rpm (Ref: http://www.olympusamerica.com/seg_section/seg_product.asp?product=963) that has the advantages of Nipkow scanning speed with much better light throughput. I cannot wait to see how a confocal with slits really work. Thanks for writing. Madhu. I found this very readable web site about this topic http://www.olympusfluoview.com/theory/confocalscanningsystems.html Gregor |
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