Methods and systems for removing autofluorescence from images

1. A method for removing any inherent autofluorescence associated with a biological material, comprising the steps of:

a) acquiring a first reference image of the biological material;

b) acquiring a first set of one or more images of the biological material using one or more filters corresponding to one or more information dyes;

c) applying the one or more additional dyes to the biological material and then acquiring a second set of one or more images comprising a separate image of the biological material with each of the filters corresponding to the information dyes and a second reference image of the biological material;

d) coregistering the first and second reference images;

e) and then removing any inherent autofluorescence exhibited in the images acquired in steps c).

2. The method of claim 1 , wherein the first and second reference images are acquired using a filter corresponding to a cyan fluorescent protein.

3. The method of claim 1 , wherein step a) further comprises the step of, applying a reference dye, having a high signal to autofluorescence ratio, to the biological material and wherein the first and second reference images are acquired using a filter corresponding to the reference dye.

4. The method of claim 3 , wherein the dye applied in step a) comprises a dye that corresponds to the UV spectrum.

5. The method of claim 4 , wherein the dye applied in step a) corresponding to the UV spectrum is DAPI.

6. The method of claim 3 , wherein the dye applied in step a) comprises a dye that corresponds to the IR spectrum.

7. The method of claim 1 ,

wherein the first and second reference images of the biological material are acquired using a filter corresponding to a cyan fluorescent protein; and

wherein the first reference image is a fixed image having a coordinate system and second reference image is a moving image having a coordinate system, and wherein the reference images are coregistered at least in part, to form a composite having a coordinate system, by estimating one or more underlying transformation parameters to map the moving image onto the fixed image coordinate system.

8. The method of claim 1 ,

wherein step a) further comprises the step of, applying a reference dye, having a high signal to autofluorescence ratio, to the biological material;

wherein the first and second reference images are acquired using a filter corresponding to the reference dye; and

wherein the first reference image is a fixed image having a coordinate system and second reference image is a moving image having a coordinate system, and wherein the reference images are coregistered at least in part, to form a composite having a coordinate system, by estimating one or more underlying transformation parameters to map the moving image onto the fixed image coordinate system.

9. The method of claim 1 , wherein the reference images are coregistered, at least in part, using a similarity transform that uses one or more transformation parameters selected from a group consisting of: translation, rotation and scaling.

10. The method of claim 1 , wherein the reference images are coregistered by estimating one or more underlying transformation parameters to map a moving image onto a fixed image coordinate system, where;

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11. A system for removing inherent autofluorescence associated with a biological material, comprising:

a) a processing device adapted to analyze a reference image of a biological material that exhibits the presence of one or more reference dyes, having a high signal to autofluorescence ratio;

b) one or more filters corresponding to the reference dye and one or more filters corresponding to one or more information dyes;

c) a digital imaging device adapted to acquire a first set of one or more images of the biological material, in conjunction with the filters corresponding to the reference dye and the one or more information dyes; and further adapted to acquire a second set of one or more images of the biological material, wherein the biological material further exhibits the presence of one or more of the information dyes, in conjunction with the filters corresponding to the reference dye and the one or more information dyes;

wherein the second set of images comprises a separate image of the biological material with each of the filters corresponding to the reference dye and the one or more information dyes; and

wherein the processing device is further adapted to coregister the reference images; and then remove the inherent autofluorescence, from the images exhibiting the presence of one or more of the information dyes.

12. The system of claim 11 , wherein at least one of the reference dyes corresponds to the UV spectrum.

13. The system of claim 12 , wherein at least one of the dyes that corresponds to the UV spectrum is DAPI.

14. The system of claim 11 , wherein at least one of the reference dyes corresponds to the IR spectrum.

15. The system of claim 11 ,

wherein the digital imaging device is adapted to acquire a reference image of the biological material using a reference filter corresponding to a cyan fluorescent protein; and

wherein the processor is adapted to coregister the reference images by estimating one or more underlying transformation parameters to map the image coordinate system.

16. The system of claim 11 , wherein the reference images are coregistered using an intensity-based or a featured based parameters.

17. The system of claim 11 , wherein the reference images are coregistered, at least in part, using a similarity transform that incorporates translation, rotation, and scaling.

18. The system of claim 11 wherein the reference images are coregistered by estimating one or more underlying transformation parameters to map a moving image onto a fixed image coordinate system, where;

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19. A system for removing inherent autofluorescence associated with a biological material, comprising:

a) a processing device adapted to analyze a reference image of a biological material taken using a reference filter corresponding to a cyan fluorescent protein, having a high signal to autofluorescence ratio;

b) one or more filters corresponding to one or more information dyes;

c) a digital imaging device adapted to acquire a first set of one or more images of the biological material, in conjunction with the reference filter and the filters corresponding to the one or more information dyes; and further adapted to acquire a second set of one or more images of the biological material, wherein the biological material further exhibits the presence of one or more of the information dyes, in conjunction with the reference filters and the filters corresponding to the one or more information dyes;

wherein the second set of images comprises a separate image of the biological material with the reference filter and with each of the filters corresponding to the one or more information dyes; and

wherein the processing device is further adapted to coregister the reference images; and then remove the inherent autofluorescence, from the images exhibiting the presence of one or more of the information dyes.

20. The system of claim 19 , wherein the reference images are coregistered using an intensity-based or a featured based parameters.

21. The system of claim 19 , wherein the reference images are coregistered, at least in part, using a similarity transform that incorporates translation, rotation, and scaling.

22. The system of claim 19 wherein the reference images are coregistered by estimating one or more underlying transformation parameters to map a moving image onto a fixed image coordinate system, where;

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