Differential scan imaging systems and methods

• US 9,134,241 B2
• Filed: 04/11/2011
• Issued: 09/15/2015
• Est. Priority Date: 04/11/2011
• Status: Active Grant

• Current Assignee: Li-Cor Biotech LLC
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Bouzid, Ahmed
• Primary Examiner(s): Kelley, Christopher S
• Assistant Examiner(s): Zhou, Zhihan
• Application Number:
  US13/084,371
• Publication Number:
  US 20120257087A1
• Time in Patent Office: 1,618 Days
• Field of Search: 348/241, 382/128, 382/129, 356/311, 250/208.1, 250/208.2
• US Class Current: 1/1
• CPC Class Codes: G01N 2021/1785 Three dimensional
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

Systems and methods for producing background-reduced fluorescence imaging signals include an illumination system that provides illumination light from an illumination source to a targeted area on the sample platform, a sensor adapted to detect light and having an array of sensing locations, and collection optics arranged and configured to project light emanating from the sample platform onto the sensor. In typical operation, light from the targeted area is projected onto a first portion of the sensor comprising a first plurality of the sensing locations and light from proximal to the targeted area on the platform is projected onto a second portion of the sensor comprising a second plurality of the sensing locations, and a second signal detected by the second portion of the sensor is subtracted from a first signal detected by the first portion of the sensor to produce a background-reduced signal, e.g., a signal with reduced background related noise.

Claims

1. A fluorescence imaging system, comprising:

a sample platform; and

an illumination and detection system, including;

an illumination system that provides a beam of excitation light from a source to a targeted area on the sample platform, wherein the targeted area includes a fluorescent material, and Wherein the excitation light includes light having an excitation wavelength of the fluorescent material;

a sensor adapted to detect light and having an array of sensing locations; and

imaging optics arranged and configured to image light emanating from the sample platform onto the sensor,

wherein fluorescent light emitted from the fluorescent material in the targeted area is imaged onto a first portion of the sensor comprising a first plurality of the sensing locations and background light from a re ion proximal to the targeted area on the platform is simultaneously imaged onto a second portion of the sensor comprising a second plurality of the sensing locations different from the first plurality of the sensing locations, wherein the region proximal to the targeted area. on the platform is not directly illuminated by the beam of excitation light, and wherein the background light includes scattered excitation light, and

wherein a second signal detected by the second portion of the sensor is subtracted from a first signal detected by the first portion of the sensor to produce a. background-reduced signal, wherein the first signal and the second signal are simultaneously detected.

2. The system of claim 1, further including an intelligence module communicably coupled with the sensor and adapted to process signals received from the sensor.

3. The system of claim 2, wherein the first and second signals are processed in the intelligence module to produce the background-reduced signal.

4. The system of claim 2, further including readout circuitry coupled with the sensor, and Wherein the first and second signals are processed during readout from the sensor in the readout circuitry to produce the background-reduced signal.

5. The system of claim 1, wherein the sensor includes a plurality of sensor elements, each element defining one or more of the sensing locations.

6. The system of claim 1, wherein the sensor includes a single sensor element having a plurality of sensing locations arranged in an array.

7. The system of claim 1, wherein the sensor includes a sensor element selected from the group consisting of a CCD array chip, a CMOS array chip, a plurality of CCD array chips, a plurality of CMOS array chips, one or a plurality of photodiodes, a focal plane array, and an array of photomultiplier devices.

8. The system of claim 1, wherein the sensor includes one or more of a CCD array chip, a CMOS array chip, a photomultiplier device, a photodiode, a plurality of photodiodes, and a focal plane array.

9. The system of claim 1, the system further including a mechanism to move one or more of the sample platform, the illumination, and detection system such that the targeted area on the sample platform is scanned in one or both directions defining a plane of the sample platform.

10. The system of claim 9, wherein the system is one of a point scanning system Wherein the targeted area includes one or more focused spots onto one or a plurality of sensing locations, or a line scanning system wherein the targeted area includes an extended linear array of the sensing locations.

11. The system of claim 1, wherein the illumination light and the light from the targeted area imaged onto the first portion of the sensor are coaxial at a plane defined by the sample platform.

12. The system of claim 1, wherein the illumination light and the light from the targeted area imaged onto the first portion of the sensor are not coaxial at a plane defined by the sample platform.

13. The system of claim 1, wherein the illumination source includes a source selected from the group consisting of a laser, an LED and a broadband lamp.

26. The system of claim 1, wherein the illumination system includes a scanning mechanism that moves the source such that the targeted area on the sample platform is scanned in one or both directions defining a plane of sample platform.

14. A fluorescence imaging method, comprising:

illuminating a targeted area of a sample platform with a beam of excitation light, wherein the targeted area includes a fluorescent material, and wherein the excitation light includes light having an excitation wavelength of the fluorescent material;

imaging light emanating from the sample platform onto a sensor having an array of sensing locations, wherein fluorescent light emitted from the fluorescent material in the targeted area is imaged onto a first portion of the sensor comprising a first plurality of the sensing locations and background light from a region proximal to the targeted area on the platform is simultaneously imaged onto a second portion of the sensor comprising a second plurality of the sensing locations different from the first plurality of the sensing locations, and wherein the region proximal to the targeted area on the platform is not directly illuminated by the beam of excitation light, and wherein the background light includes scattered excitation light; and

subtracting a second signal detected by the second portion of the sensor from a first signal detected by the first portion of the sensor to produce a background-reduced signal, wherein the first signal and the second signal are simultaneously detected.

15. The method of claim 14, wherein subtracting is performed in an intelligence module communicably coupled with the sensor.

16. The method of claim 14, wherein subtracting is performed during readout from the sensor readout circuitry communicably coupled with the sensor.

17. The method of claim 14, further including scanning the targeted area over the sample platform so as to build up an image of a sample on the sample platform over time.

18. The method of claim 17, wherein scanning includes moving the sample platform relative to a fixed illumination and detection system.

19. The method of claim 17, wherein scanning includes moving an illumination and detection. system relative to a fixed sample platform.

20. The method of claim 17, wherein the sample platform and detection system are fixed, and wherein scanning includes scanning the illumination across the sample platform.

21. The method of claim 14, wherein illuminating includes directing the beam of excitation light from a source onto the targeted area of the sample platform so that the beam of excitation light is substantially coaxial, at the sample platform, with the light being imaged onto the sensor.

22. The method of claim 14, further comprising displaying a representation of the noise reduced signal on an output or display device.

23. The method of claim 14, wherein the sensor includes one of a single sensor element having a plurality of sensing locations arranged in an array, or a plurality of sensor elements, each element defining one or more of the sensing locations.

24. The method of claim 14, wherein illuminating includes directing the beam of excitation light from a source onto the targeted area of the sample platform at an angle relative to a normal to a plane defined by the sample platform.

25. The method of claim 14, further including;

seaming the targeted area over the sample platform so as to build up a two dimensional image of a sample on the sample platform at a first depth of focus; and

one or more times;

i) adjusting the depth of focus; and

ii) scanning image of a sample on the sample platform so as to build up a two dimensional image of a sample on the sample platform at a different depth of focus; and

thereafter;

combining the two dimensional images to produce a three-dimensional image.

27. A fluorescence imaging scanner, comprising:

a sample platform; and

an illumination and detection system, including;

an illumination system that provides a beam of excitation light from a source to a targeted area on the sample platform, wherein the targeted area includes a fluorescent material, and wherein the excitation light includes light having an excitation wavelength of the fluorescent material;

sensor adapted to detect light and having an array of sensing locations; and

imaging optics arranged and configured to image light emanating from the sample platform onto the sensor,

wherein fluorescent light, emitted from the fluorescent material in the targeted area is imaged onto a first portion of the sensor comprising a first plurality of the sensing locations and background light from .a region proximal to the targeted area on the platform is simultaneously imaged onto a second portion of the sensor comprising a second plurality of the sensing locations different from the first plurality of the sensing locations, wherein the region proximal to the targeted area on the platform is not directly illuminated by the beam of excitation light, and wherein the background light includes scattered excitation light, and

a scanning mechanism configured to move the sample platform or the illumination and detection system such that the targeted area is scanned along the sample platform over a plurality of scan positions;

wherein, iteratively over the plurality of scan positions, a second signal is detected by the second portion of the sensor and a first signal is detected by the first portion of the sensor, wherein the first signal and the second signal are simultaneously detected at each scan position, and wherein the second signals are subtracted from the first signals to produce a background-reduced image of the plurality of scan positions of the sample platform.