Enhanced extended depth of focusing on biological samples
A system and method for constructing a digital composite image of a three-dimensional biological sample. The system includes an optical system that captures images of cells and tissue presented on a specimen slide. The system systematically acquires a stack of images at different segments across the specimen slide. For each segment, the system dynamically calculates an optimal focal plane. Once an optimal focal plane is determined for each of the stacks of images, the system generates a composite image by copying the sharpest objects from each of the optimal focal planes.
1. A method for generating a composite digital image of a biological sample from a plurality of images of the biological sample, each of the plurality of images taken along a single axis, the method comprising:
(a) identifying a first focal plane, wherein the first focal plane comprises a first z distance, for a first collection of image objects at a first x-y location of the biological sample, and
(b) identifying a second focal plane, wherein the second focal plane comprises a second z distance, for a second collection of image objects at a second x-y location of the biological sample, and
(c) identifying a first optimal focal plane and a second optimal focal plane based on (a) and (b) respectively, and
(d) combining image objects from a first optimal focal plane and image objects from a second optimal focal plane and generates the composite digital image,
and using a computer apparatus to calculate a sharpness value for the image objects in the first focal plane and calculate a sharpness value for the image objects in the second focal plane and generate a two-dimensional map of the location of the objects having the highest sharpness values, and
identifying an optimal first focal plane and optimal second focal plane for the first and second collection of image objects by performing one or more dilations and erosions on the two-dimensional map of the location of the image objects having highest sharpness values in the first and second focal planes wherein the number of erosions and dilations is proportional to the distance between cell objects of interest in healthy tissue, so as to thereby identify a first and a second optimal focal plane, and wherein image objects located on the first optimal focal plane are presented in-focus and image objects below the first optimal focal plane are deemphasized, and wherein image objects located on the second optimal focal plane are presented in-focus and image objects below the second optimal focal plane are deemphasized, so as to generate a composite digital image of a biological sample.
2. The method of claim 1 , wherein one or more of the images in the plurality of images are color images and the computer apparatus converts the one or more color images in the plurality of images to greyscale prior to identifying the optimal focal plane for the plurality of images.
3. The method of claim 1 , wherein the composite image includes a honeycomb structure of the biological sample.
4. The method of claim 3 , wherein the honeycomb structure is substantially in focus.
5. The method of claim 1 , wherein the location of the objects having the highest sharpness value is a z-index and wherein the first optimal focal plane and second optimal focal plane for the first and second collection of image objects are identified by performing a number of dilations followed by an equal number of erosions.
6. The method of claim 1 , wherein the cell objects of interest are cell structures.
7. The method of claim 1 , wherein the cell objects of interest are cell nuclei.
8. The method of claim 1 , wherein the cell objects of interest are cytoplasmic mucus pockets in goblet cells.