Methods for scaling images to differing exposure times

1. A method for scaling an image taken at a given exposure time to a selected exposure time, comprising,

determining a dark pixel intensity of an imaging device, wherein determining the dark pixel intensity comprises,

setting an exposure time to zero, wherein the exposure time comprises a time interval over which pixel intensity signals are read from the imaging device;

acquiring an intensity image; and

calculating a mean intensity for the entire intensity image;

acquiring a first image at a given exposure time, wherein the first image comprises a fluorescence microscopy image of a biological material comprising a first biomarker;

acquiring a second image at a different exposure time, wherein the second image comprises a fluorescence microscopy image of a biological material comprising a second biomarker;

adjusting a pixel intensity of one or more pixels in the first image, based at least in part on the dark pixel intensity, for a second exposure time that is different from the given exposure time; and

adjusting a pixel intensity of one or more pixels in the second image, based at least in part on the dark pixel intensity, for a second exposure time that is different from the given exposure time,

wherein adjusting the pixel intensity at least in part uses the following equation:

I

t

2

=

t

2

t

1

⁢

I

t

1

-

(

t

2

t

1

-

1

)

⁢

DarkpixelIntensity

wherein t 1 is the given exposure time, t 2 is the second exposure time, I t1 is a signal intensity of a given pixel for exposure time t 1 , I t2 is an estimated signal intensity for exposure time t 2 and Darkpixellntensity is the dark pixel intensity for the imaging device.

2. The method of claim 1 , wherein the given exposure time is based at least in part on one or more settings for the imaging device.

3. The method of claim 1 , wherein the biomarker is a fluorescent biomarker.

4. The method of claim 1 , wherein the first image of the biological material exhibits a signal in a fluorescent channel.

5. The method of claim 1 , further comprising registering the first and second image to form a composite image.

6. The method of claim 1 , further comprising acquiring an autofluorescence image of the biological material.

7. The method of claim 6 , further comprising subtracting the autofluorescence image of the biological material from the first image.

8. The method of claim 7 , further comprising applying a biomarker to the biological material, wherein the first image is acquired after the biomarker is applied to the biological material.

9. The method of claim 8 , further comprising registering the autofluorescence image and the first image.

10. The method of claim 7 , further comprising applying another biomarker to the biological material, and acquiring a second image of the biological material.

11. The method of claim 10 , further comprising registering the first image and the second image.

12. The method of claim 10 , further comprising applying one or more additional biomarkers, each corresponding to a channel, to the biological material, and acquiring one or more additional images for each of the biomarker channels.

13. The method of claim 12 , further comprising registering the first, second and additional images to form a composite image of the channels.