Importance

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Abstract

Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields. Autofocus could be performed directly with fluorescence images, but due to photobleaching and destructive fluorescence by-products, it is best to minimize fluorescence exposure for photosensitive specimens and live cells. This exposure problem could be completely avoided by using phase-contrast microscopy, implemented through the same optics as fluorescence microscopy. Functions for both phase-contrast and fluorescence autofocus were evaluated using the present invention and the suitability of phase-contrast autofocus for fluorescence microscopy was determined. The present autofocus system for scanning microscopy can be performed at least as fast as 0.25 s/field without loss of precision. The speed of autofocus can be further increased by a volume image which is obtained by observing an image object at each image plane of a plurality of image planes, where each image plane is vertically displaced with respect to each other image plane. An electronic image representation is obtained at each image plane. The image planes are scanned over the image object and the images arm aligned timewise in a buffer. The buffer holds a volume image comprising images at the image planes, aligned by the buffer. The image plane having the best focus is selected and a microscope objective is automatically positioned at the selected plane.

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First Claim