Multifunction Autofocus System and Method for Automated Microscopy

Abstract

Multifunction autofocus for automated microscopy includes automatic coarse focusing of an automated microscope by reflective positioning, followed by automatic image-based autofocusing of the automated microscope performed in reference to a coarse focus position. In some aspects, the image-based autofocusing utilizes astigmatism in microscope optics for multi-planar image acquisition along a Z axis direction of a microscope. In some other aspects, the image-based autofocusing utilizes astigmatism in microscope optics in combination with chromatic aberration for multi-planar image acquisition along the Z axis direction.

We claim: 
     
         1 . An automated microscopy system for simultaneously measuring image characteristics of a plurality of focal planes in a magnified image with an image-based autofocus system, in which the image-based autofocus system includes at least one optical pathway to transfer light from which the magnified image is formed and the optical pathway includes an astigmatic optical device that introduces astigmatism into the light in the at least one optical pathway. 
     
     
         2 . The automated microscopy system of  claim 1 , in which the astigmatic optical device includes a cylindrical lens. 
     
     
         3 . The automated microscopy system of  claim 1 , in which the astigmatic optical device includes two half cylindrical lenses. 
     
     
         4 . The automated microscopy system of  claim 1 , in which the image-based autofocus system further includes means for calculating a best focus based on the image characteristics. 
     
     
         5 . The automated microscopy system of  claim 1 , in which the optical pathway introduces chromatic aberration into the light that causes different colors of light to be focused at different focal planes. 
     
     
         6 . The automated microscopy system of  claim 5 , in which the astigmatic optical device is a cylindrical lens. 
     
     
         7 . The automated microscopy system of  claim 5 , in which the astigmatic optical device includes two half cylindrical lenses. 
     
     
         8 . The automated microscopy system of  claim 1 , in which the image-based autofocus system acquires a sequence of magnified images simultaneously and each magnified image is acquired from a respective focal plane of the plurality of focal planes, and a controller calculates image characteristics for each magnified image and determines a best focus from the image characteristics. 
     
     
         9 . The automated microscopy system of  claim 1 , in which the image-based autofocus system acquires a sequence of magnified images simultaneously and each magnified image is acquired from a respective focal plane of the plurality of focal planes, and a controller calculates a focus index for autofocus. 
     
     
         10 . The automated microscopy system of  claim 1 , in which the image-based autofocus system acquires a sequence of magnified images simultaneously and each magnified image is of a particular color of light, each color of light provides an image focused at a different focal plane due to chromatic aberration, and a controller calculates image characteristics from more than one focal plane for each image. 
     
     
         11 . The automated microscopy system of  claim 1 , in which the optical pathway transfers the light to respective multi-planar optical subsystems, each including a camera, the cameras are positioned at different focal planes, and a beam splitter divides the light between the multi-planar optical subsystems. 
     
     
         12 . The automated microscopy system of  claim 11 , in which each camera generates a sequence of image characteristics for the plurality of focal planes and a controller calculates a focus index for each of the plurality of focal planes using the image characteristics. 
     
     
         13 . The automated microscopy system of  claim 1 , in which the plurality of focal planes are distributed along an optical axis, and the image-based autofocus system simultaneously acquires a plurality of magnified images at the plurality of focal planes. 
     
     
         14 . The automated microscopy system of  claim 1 , including a means for estimating a coarse focus corresponding to a predetermined distance from an object surface, a means for estimating a fine focus position corresponding to a focal plane of the plurality of focal planes, and a programmable controller for combining the two estimates to provide a best focus position and adjusting focus to the best focus position. 
     
     
         15 . The automated microscopy system of  claim 14 , including means for scanning a specimen and multifunction autofocus means for updating the best focus position with respect to the specimen at each scan location of a plurality of scan locations. 
     
     
         16 . The automated microscopy system of  claim 15 , including means for making measurements of the specimen. 
     
     
         17 . A method of focusing an automated microscopy system including an objective, a stage, and an optical axis passing through the objective and the stage, by:
 obtaining a coarse focus value with a reflective positioning system;   obtaining a coarse focus corresponding to the coarse focus value;   obtaining a fine focus value with an image-based autofocus system;   updating the coarse focus value with the fine focus value; and,   causing relative movement between the objective and the stage to obtain a fine focus corresponding to the updated coarse focus value.   
     
     
         18 . The method of  claim 17  in which obtaining a fine focus value with an image-based autofocus system includes directing light from a specimen to the Image-based autofocus system, introducing astigmatism into the light when the light is transferred to the image-based autofocus system, simultaneously generating a stack of focal plane images of the specimen from the light and obtaining a best focus value from the stack, wherein the best focus value is the fine focus value. 
     
     
         19 . The method of  claim 18 , in which simultaneously generating a stack of focal plane images of the specimen from the light includes introducing chromatic aberration into the light along with the astigmatism. 
     
     
         20 . The method of  claim 18 , in which directing light from a specimen to the Image-based autofocus system includes directing oblique or dark field reflected illumination through the objective to the specimen, directing the oblique or dark field illumination scattered by the specimen through the objective to the image-based autofocus system. 
     
     
         21 . The method of  claim 20 , in which simultaneously generating a stack of focal plane images of the specimen from the light includes introducing chromatic aberration into the dark field illumination scattered by the specimen. 
     
     
         22 . The method of  claim 21 , in which obtaining a coarse focus value with a reflective positioning system includes sensing a reflected image through one of the objective and a sensor beside the objective. 
     
     
         23 . A method of operating an automated microscopy system including an objective, a stage, and an optical axis passing through the objective and the stage, by the automated-microscopy-system-executed steps of:
 obtaining a coarse focus position in response to an image reflected from an object plane;   and then, adjusting the coarse focus position by a best focus value associated with a focal plane of a stack of focal planes extending along the optical axis simultaneously in an image by a means that introduces astigmatism into an optical system; followed by,   positioning one of the objective, the stage, and an external fine focus mechanism to a focus position corresponding to the best focus value.   
     
     
         24 . The method of  claim 23  performed during the automated-microscopy-system-executed X-Y scanning of the stage. 
     
     
         25 . The method of  claim 24  in which the X-Y scanning is one of incremental and continuous scanning. 
     
     
         26 . The method of  claim 25 , a respective image is obtained from each focal plane of the stack of focal planes, further including the automated-microscopy-system-executed steps of:
 obtaining a focus index for each respective image;   calibrate each focus index;   determining presence of image content for each calibrated focus index; and,   calculating the best focus if sufficient image content is present for at least one focus index.