Three‐dimensional optical microscopy using tilted views

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x-y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the 'missing data' and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0.25 micron in x and y, and 0.4 micron in z.