System and method for time-resolved fluorescence imaging and pulse shaping
A time-resolved fluorescence imaging (TRFI) system that images a target medium without lifetime fitting. Instead of extracting the lifetime precisely, the system images the fluorophore distribution to allow for a simple and accurate method to obtain the fluorescence image without lifetime-extraction for time-resolved fluorescence imaging. An illumination source circuit for TRFI is also disclosed that shapes the excitation pulse. In one embodiment, the illumination source comprises an LED and stub line configured for generating a linear decay profile.
1. A method for imaging a sample medium, the method comprising;
exciting the sample medium with a square excitation light pulse;
generating a first image of the medium, said first image comprising data relating to at least a first fluorophore corresponding to a first component of the medium and a second fluorophore corresponding to a second component of the medium, the first fluorophore having a longer fluorescence lifetime than the second fluorophore;
generating a second image of the medium at a specified time subsequent to said first image while the first fluorophore and second fluorophore still have decaying fluorescence values, said second image comprising data relating to the first fluorophore and the second fluorophore; and
generating a third image as a function of first image and the second image to identify a lifetime-based contrast between the first component and the second component within the medium.
2. A method as recited in claim 1 :
wherein the second image is generated after decay of the second fluorophore such that data relating to the second fluorophore is absent from the second image; and
wherein generating the third image comprises subtracting the first image from the second image such that data relating to the first fluorophore is absent from the second image.
3. A method as recited in claim 2 , wherein the second image is normalized to the intensity of the first image prior to subtracting the first image from the second image.
4. A method as recited in claim 1 :
wherein generating the third image comprises dividing the second image by the first image.
5. A method as recited in claim 4 , further comprising:
multiplying the third image by a constant.
6. A method as recited in claim 1 , wherein the sample medium comprises human tissue.
7. A system for imaging a sample medium, the system comprising:
(a) a processor; and
(b) programming executable on said processor and configured for:
(i) exciting the sample medium with a square excitation light pulse;
(ii) generating a first image of the medium, said first image comprising data relating to at least a first fluorophore corresponding to a first component of the medium and a second fluorophore corresponding to a second component of the medium, the first fluorophore having a longer fluorescence lifetime than the second fluorophore;
(iii) generating a second image of the medium at a specified time subsequent to said first image while the first fluorophore and second fluorophore still have decaying fluorescence values, said second image comprising data relating to the first fluorophore and the second fluorophore; and
(iv) generating a third image as a function of first image and the second image to identify a lifetime-based contrast between the first component and the second component within the medium.
8. A system as recited in claim 7 :
wherein the second image is generated after decay of the second fluorophore such that data relating to the second fluorophore is absent from the second image; and
wherein generating the third image comprises subtracting the first image from the second image such that data relating to the first fluorophore is absent from the second image.
9. A system as recited in claim 8 , wherein the second image is normalized to an intensity of the first image prior to subtracting the first image from the second image.
10. A system as recited in claim 7 :
wherein generating the third image comprises dividing the second image by the first image.
11. A system as recited in claim 10 , wherein the programming is further configured for:
multiplying the third image by a constant.
12. A system as recited in claim 7 , wherein the sample medium comprises human tissue.