22 December 1989 3-Dimensional Microscopy Using Tilted Views
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The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x-y plane, typically by a factor of 5 or more. This is true of conventional or confocal microscopes. In order to alleviate this problem we have used multiple tilted views to supply the 'missing data' and thus increase the resolution in z. A special tilting microscope stage has been constructed, which allows specimens mounted on thin glass capillary tubes to be rotated through large angles. Through-focal data sets can then be collected from several mutually tilted viewing directions. The relative orientation, translation and z-sampling parameters for the data sets can then be determined by use of a novel phase/wiener cross-correlation function. Finally, the data sets, once brought to a common coordinate system, can be combined using amplitude and phase combination techniques in Fourier space borrowed from X-ray crystallography. We have applied this technique to metaphase chromosomes in intact embryos of Drosophila melanogaster. Images were collected using a liquid nitrogen cooled CCD array camera, and processed using a VAX computer. As judged from the maximum extent of significant power in the Fourier transform, the resolution of the final reconstruction was about 0.25,urn in x and y and better than 0.4,um in z. Although we have shown an application to data collected from a conventional fluorescence microscope, the technique is equally applicable to any other imaging mode or to confocal images.
© (1989) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Peter J. Shaw, Peter J. Shaw, } "3-Dimensional Microscopy Using Tilted Views", Proc. SPIE 1161, New Methods in Microscopy and Low Light Imaging, (22 December 1989); doi: 10.1117/12.962682; https://doi.org/10.1117/12.962682


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