Absolute intensity determination for a fluorescent light source in low level light imaging systems

• US 7,663,664 B2
• Filed: 09/18/2006
• Issued: 02/16/2010
• Est. Priority Date: 06/20/2001
• Status: Active Grant

• Current Assignee: Xenogen Corporation (Revvity, Inc.)
• Original Assignee: Xenogen Corporation (Revvity, Inc.)
• Inventors: Rice, Bradley, Cable, Michael D.
• Primary Examiner(s): Natnael; Paulos M
• Application Number:
  US11/523,480
• Publication Number:
  US 20070013780A1
• Time in Patent Office: 1,247 Days
• Field of Search: 348187-194, 348/180, 348/181, 348/184, 348/185, 382/162, 382/167
• US Class Current: 348/187
• CPC Class Codes: G01J 1/08 Arrangements of light sources specially adapted for photometry standard sources, also using luminescent or radioactive material
• CPC Class Codes: G01N 21/278 Constitution of standards
• CPC Class Codes: G01N 21/763 Bioluminescence
• CPC Class Codes: H04N 23/81 for suppressing or minimising disturbance in the image signal generation

Abstract

The invention describes systems and methods to obtain and present imaging data in absolute units. The systems and methods convert relative image data produced by a camera to absolute light intensity data using a compensation factor. The compensation factor accommodates for hardware and specific imaging conditions in the imaging system that variably affect camera output. The present invention determines the compensation factor based on assessing the output of the camera against a known light source for a specific set of imaging conditions in the imaging system. The compensation factor is then stored in memory corresponding to the specific set of imaging conditions. Upon subsequent imaging with the set of imaging conditions, the corresponding compensation factor is called from memory and applied to the camera output. A compensation factor may be determined and stored for each hardware state and imaging condition available to the imaging system.

Claims

1. A method for generating absolute fluorescent light intensity data for a fluorescent light source being imaged at low intensity light levels, the method comprising:

determining a compensation factor for converting data output by a camera into absolute fluorescent intensity light data, the compensation factor corresponding to a set of imaging conditions;

storing the compensation factor;

calling the compensation factor based on the set of imaging conditions when the set of imaging conditions are used in imaging the fluorescent light source; and

modifying image data, output by the camera, by the compensation factor to obtain the absolute fluorescent light intensity data for the fluorescent light source, wherein the fluorescent light source emits light with an intensity in the range of about 10³ to about 10¹⁰ photons/second/centimeter squared/steradian, and wherein the fluorescent light source includes fluorescing molecules.

2. The method of claim 1 wherein the fluorescing molecules are included in a mammal.

3. The method of claim 2 wherein the mammal is a mouse.

4. The method of claim 1 wherein the fluorescing molecules are included in one of:

a tissue culture plate, a multi-well plate, and a plant containing light-emitting fluorescent molecules.

5. The method of claim 1 further comprising capturing an image of the fluorescent light source using the camera.

6. The method of claim 1 further comprising placing a portable light calibration device in the imaging box, the light calibration device including an array of low intensity light supplies.

7. The method of claim 6 further comprising receiving the light from the one or more of the low intensity light supplies using the camera.

8. The method of claim 7 wherein determining the compensation factor comprises comparing the light received by the camera with a known light emission for the one or more of the low intensity light supplies.

9. The method of claim 1 wherein the compensation factor is stored in a look up table.

10. The method of claim 9 wherein the compensation factor is stored in the look up table according to the set of imaging conditions.

11. The method of claim 1 wherein the absolute fluorescent light data is expressed in physical units.

12. The method of claim 11 wherein the absolute fluorescent light data is expressed in radiance.

13. The method of claim 1 wherein the compensation factor accommodates for a hardware structure that produces a difference between the amount of light emitted by the fluorescent light source and data output by the camera.

14. The method of claim 13 wherein the compensation factor accommodates for a difference resulting from one of:

a lens disposed between the source and the camera, an f-stop associated with the camera, an iris associated with the camera, and a window disposed between the source and the camera.

15. A method for imaging a light source at low intensity light levels in an imaging system including a camera and an imaging box, the method comprising:

placing a portable light calibration device in the imaging box, said portable light calibration device having been calibrated against a known absolute intensity light source;

setting one or more image capture conditions in the imaging system;

determining a compensation factor that accommodates for differences between light emitted from the portable light calibration device and data output by the camera based on said one or more image capture conditions;

removing said portable light calibration device from the imaging box;

placing a light source which is to be imaged in the imaging box;

capturing image data from the light source using the camera; and

converting image data produced by the camera to absolute light intensity data for the light source using said compensation factor.

16. The method of claim 15 wherein the absolute intensity data includes an intensity in the range of about 10³ to about 10¹⁰ photons/second/centimeter squared/steradian.

17. The method of claim 15 further comprising:

storing the compensation factor.

18. The method of claim 17 wherein the compensation factor accommodates for a hardware structure that produces a difference between the amount of light emitted by the light source and data output by the camera.

19. A computer readable medium including instructions for generating absolute light intensity data for a fluorescent light source being imaged at low intensity light levels in an imaging box, the instructions comprising:

instructions for determining a compensation factor for convening relative intensity data output by a camera into absolute light data, the compensation factor corresponding to differences between light emitted from a portable light calibration device and data output by the camera for a set of imaging conditions within the imaging box;

instructions for storing the compensation factor;

instructions for calling the compensation factor based on the set of imaging conditions when the set of imaging conditions are used in imaging the light source within the imaging box; and

instructions for modifying image data, output by the camera, by the compensation factor to obtain the absolute light intensity data for the light source.