3-Dimensional Microscopy Using Tilted Views

Peter J. Shaw

doi:10.1117/12.962682

22 December 1989 3-Dimensional Microscopy Using Tilted Views

Peter J. Shaw

Proceedings Volume 1161, New Methods in Microscopy and Low Light Imaging; (1989) https://doi.org/10.1117/12.962682
Event: 33rd Annual Technical Symposium, 1989, San Diego, United States

Abstract

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.

Citation Download Citation

Peter J. Shaw "3-Dimensional Microscopy Using Tilted Views", Proc. SPIE 1161, New Methods in Microscopy and Low Light Imaging, (22 December 1989); https://doi.org/10.1117/12.962682

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available