Methods, software, and apparatus for focusing an optical system using computer image analysis

Claim 3

The method of claim 1 , wherein the focusing element comprises a focus stage.

Claim 4

The method of claim 1 wherein the focusing element comprises at least one lens associated with the imaging instrument.

Claim 5

The method of claim 1 , wherein the focusing element comprises an image detector associated with the imaging instrument.

Claim 6

The method of claim 1 , wherein the plurality of focus positions comprises positions above and positions below a predicted optimal focal position.

Claim 7

The method of claim 1 , wherein the high-density microarray comprises densities of 4 binding sites per square millimeter to 10 4 binding sites per square millimeter.

Claim 10

The method of claim 7 , wherein the container is a reaction plate comprising between 96 and 1536 wells.

Claim 11

The method of claim 7 , wherein the container is a custom multi-well reaction plate.

Claim 12

The method of claim 7 , wherein the container is a plurality of capillaries in an array.

Claim 13

The method of claim 7 , wherein the container is a plurality of individual sample tubes arranged in an array configuration.

Claim 14

The method of claim 7 , wherein the container is a plurality of individual sample locations in a microfluidic card.

Claim 15

The method of claim 7 , wherein the plurality of subregions numbers between four and one-hundred.

Claim 16

The method of claim 7 , wherein the sample image is substantially rectangular in configuration and wherein the plurality of subregions comprises substantially rectangular images, which are formed by separating the sample image into rows and columns of subimages.

Claim 17

The method of claim 7 wherein the sample image is captured by an image detector comprising a plurality of pixels and calculating the image intensity statistical dispersion values comprises calculating standard deviations of pixel intensity values associated with the plurality of pixels.

Claim 18

The method of claim 7 , wherein determining the optimal focus position further comprises determining whether a predetermined percentage of the subregion focus positions fit within a focal window associated with the imaging instrument.

Claim 19

The method of claim 7 further comprising: identifying extreme foci within the subregion focus positions; and determining the optimal focus position based on the extreme foci.

Claim 20

The method of claim 17 , wherein the optimal focus position is determined by calculating a midpoint between the extreme foci.

Claim 21

The method of claim 17 , wherein the optimal focus position is determined by using an average of the subregion focus positions within a focal window associated with the imaging instrument.

Claim 22

The method of claim 17 , further comprising: ignoring outlier positions in the subregion focus positions.

Claim 23

The method of claim 7 , wherein determining the optimal focus position comprises determining the optimal focus position for received electromagnetic energy of a particular wavelength.

Claim 24

The method of claim 7 , wherein determining the optimal focus position comprises determining the optimal focus position for received electromagnetic energy of different wavelengths.

Claim 27

The method of claim 23 , wherein the target sample comprises a microarray.

Claim 28

The method of claim 24 , wherein the scattered light from fiducials associated with the microarray is used to determine the optimal focus position.

Claim 29

An instrument for analyzing biological samples, the instrument comprising: an electromechanical movement coupled to and operable to move a focusing element to a plurality of focus positions within a focusing element movement range; a digital camera coupled to the imaging instrument operable to capture a sample image of a target sample at the plurality of focus positions; an image processor operable to resolve the sample image into a plurality of subregions; and a digital processor operable to execute instructions to calculate image intensity statistical dispersion values within the plurality of subregions and to select a focus window delineating a desired degree of focus and determined on the basis of image intensity statistical dispersion versus focus element position and further operable to execute instructions to evaluate image intensity statistical dispersion values versus focus element position for the plurality of subregions based on the calculated image intensity statistical dispersion values and to determine an optimal focus position for the focusing element which maximizes the number of subregions residing within the focus window, wherein the target sample is a high-density microarray or high-density biological sample container.

Claim 30

An instrument for analyzing biological samples, the instrument comprising: an electromechanical movement coupled to and operable to move a focusing element to a plurality of focus positions within a focusing element movement range; a digital camera coupled to the imaging instrument operable to capture a sample image of a target sample at the plurality of focus positions; an image processor operable to resolve the sample image into a plurality of subregions; and a digital processor operable to execute instructions to calculate image intensity statistical dispersion values within the plurality of subregions and to select a focus window delineating a desired degree of focus and determined on the basis of image intensity statistical dispersion versus focus element position and further operable to execute instructions to evaluate image intensity statistical dispersion values versus focus element position for the plurality of subregions based on the calculated image intensity statistical dispersion values and to determine an optimal focus position for the focusing element which maximizes the number of subregions residing within the focus window, wherein the target sample is a high-density microarray or high-density biological sample container.