Autofocus system for scanning microscopy
First Claim
1. A sequential method of autofocus for a microscope having a focus mechanism, the method comprising:
- scanning a specimen on a microscope stage;
sequentially receiving digital images of the specimen at a plurality of vertical positions along a Z axis;
for each vertical position of the plurality of vertical positions;
applying a digital filter to a digital image acquired at the vertical position to obtain a filtered image; and
applying a measurement function to the filtered image to obtain a value representing a degree of focus;
applying a weight to the value representing a degree of focus to obtain a weighted value;
applying a non-interative function to a plurality of weighted values of focus to obtain a focus position; and
causing the microscope stage to move to a vertical position corresponding to the focus position.
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Accused Products
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.
331 Citations
20 Claims
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1. A sequential method of autofocus for a microscope having a focus mechanism, the method comprising:
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scanning a specimen on a microscope stage; sequentially receiving digital images of the specimen at a plurality of vertical positions along a Z axis; for each vertical position of the plurality of vertical positions; applying a digital filter to a digital image acquired at the vertical position to obtain a filtered image; and applying a measurement function to the filtered image to obtain a value representing a degree of focus; applying a weight to the value representing a degree of focus to obtain a weighted value; applying a non-interative function to a plurality of weighted values of focus to obtain a focus position; and causing the microscope stage to move to a vertical position corresponding to the focus position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. In an autofocus apparatus for a microscope having a focus method, an improvement comprising:
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means for scanning a specimen on a microscope stage; means for acquiring a digital image of the specimen at a plurality of vertical positions along a Z axis; means for applying a digital filter to a digital image acquired at each vertical position of the plurality of vertical positions to obtain a filtered image; means for applying a measurement function and a weight to a filtered image to sequentially obtain a plurality of weighted values, each weighted value representing a degree of focus; means for applying a non-iterative function to the plurality of weighted values to obtain a focus position; and means for causing the microscope stage to move to a vertical position corresponding to the focus position. - View Dependent Claims (15, 16, 17, 18, 19, 20)
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Specification