Multiple-label fluorescence imaging using excitation-emission matrices

• US 7,532,326 B2
• Filed: 07/06/2005
• Issued: 05/12/2009
• Est. Priority Date: 07/07/2004
• Status: Expired due to Fees

• Current Assignee: Timothy C. Corcoran
• Original Assignee: Timothy C. Corcoran
• Inventors: Corcoran, Timothy C.
• Primary Examiner(s): Punnoose; Roy M
• Assistant Examiner(s): PAJOOHI GOMEZ, TARA S
• Application Number:
  US11/175,955
• Publication Number:
  US 20060007439A1
• Time in Patent Office: 1,406 Days
• Field of Search: 356317-318
• US Class Current: 356/332
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/021 using plane or convex mirrors, parallel phase plates, or particular reflectors
• CPC Class Codes: G01J 3/14 using refracting elements, e.g. prisms G01J3/18, G01J3/26 take precedence prisms per se G02B5/04
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6423 Spectral mapping, video display
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/10 Scanning
• CPC Class Codes: G01N 2201/1042 X, Y scan, i.e. object moving in X, beam in Y

Abstract

Methods and devices are disclosed which apply an excitation-emission matrix (EEM) to a heterogeneous, two-dimensional sample, allowing a considerably larger number of emitting, e.g. fluorescent, labels to be used simultaneously. This may be accomplished by employing a spectroscopic method of excitation-emission matrices which allows discrimination of species with similar emission spectra, and also allows positive identification of energy transfer between emitting species. The methods and devices may employ a novel excitation-light scanning technique which allows imaging of the emission from the heterogeneous sample both in two spatial dimensions (length and width) and in two spectral dimensions (excitation and emission wavelength). This light scanning technique maximizes the throughput of excitation light, increasing the sensitivity and hence the reading speed of the instrument.

Claims

1. An apparatus, comprising:

a light source for producing spectral excitation light having wavelength variation along a first dimension across a spectral range;

a scanning device for scanning the spectral excitation light across a sample substantially parallel to the first dimension such that the wavelength variation across the spectral range of the spectral excitation light interacts with a spatial position along the sample to emit a secondary light; and

an imaging device for capturing the secondary light as a spectral emission light having wavelength variation along a second dimension.

2. The apparatus of claim 1, wherein the sample comprises a width and the spectral excitation light comprises a repetition of the spectral range along the first dimension such that a single scan of the spectral excitation light across the sample exhibits the spectral range to each point across the width.

3. The apparatus of claim 1, wherein the sample comprises a two-dimensional array.

4. The apparatus of claim 1, wherein the light source comprises a lamp and a wavelength dispersion device.

5. The apparatus of claim 1, wherein the imaging device comprises an imaging spectrograph equipped with a two-dimensional detector.

6. The apparatus of claim 1, wherein the scanning device comprises a scanning mirror for reflecting the spectral excitation light and moving the reflected spectral excitation light across the sample substantially parallel to the first dimension.

7. The apparatus of claim 1, wherein the scanning device comprises a movable platform supporting the sample for moving the sample substantially parallel to the first dimension.

8. The apparatus of claim 1, further comprising a movable platform supporting the sample for moving the sample in a second dimension distinct from the first dimension following each scan of the spectral excitation light across the sample to repetitiously scan and capture the secondary light to produce an excitation-emission matrix for each spatial position on the sample.

9. The apparatus of claim 8, further comprising a data processor for identifying sample emission due to energy transfer between at least two emitting species from data of the excitation-emission matrix.

10. The apparatus of claim 9, wherein the data processor identifies a signature in the data of the excitation-emission matrix of a donor species and an acceptor species having the properties of the donor species along an excitation wavelength axis and the properties of the acceptor species along an emission wavelength axis.

11. The apparatus of claim 9, wherein the data processor corrects error in quantification of amount of the at least two emitting species due to the energy transfer between the at least two emitting species from the data of the excitation-emission matrix.

12. A method, comprising:

producing spectral excitation light having wavelength variation along a first dimension across a spectral range;

scanning the spectral excitation light across a sample substantially parallel to the first dimension such that the wavelength variation across the spectral range of the spectral excitation light interacts with a spatial position along the sample to emit a secondary light; and

capturing the secondary light as a spectral emission light having wavelength variation along a second dimension.

13. The method of claim 12, wherein the sample comprises a width and producing the spectral excitation light comprises repeating the spectral range along the first dimension such that scanning the spectral excitation light across the sample exhibits the spectral range to each point across the width.

14. The method of claim 12, wherein the sample comprises a two-dimensional array.

15. The method of claim 12, wherein producing the spectral excitation light comprises producing and wavelength dispersing a broadband light.

16. The method of claim 12, wherein capturing the secondary light comprises wavelength dispersing the secondary light along the second dimension.

17. The method of claim 12, wherein scanning comprises reflecting the spectral excitation light and moving the reflected spectral excitation light across the sample substantially parallel to the first dimension.

18. The method of claim 12, wherein scanning comprises moving the sample substantially parallel to the first dimension.

19. The method of claim 12, further comprising repeating the scanning of the spectral excitation light across the sample and supporting and moving the sample in a second dimension distinct from the first dimension following each scan of the spectral excitation light across the sample and repeating the capturing of the secondary light to produce an excitation-emission matrix for each spatial position on the sample.

20. The method of claim 19, further comprising identifying sample emission due to energy transfer between at least two emitting species from data of the excitation-emission matrix.

21. The method of claim 20, further comprising identifying a signature in the data of the excitation-emission matrix of a donor species and an acceptor species having the properties of the donor species along an excitation wavelength axis and the properties of the acceptor species along an emission wavelength axis.

22. The method of claim 20, further comprising correcting error in quantification of amount of the at least two emitting species due to the energy transfer between the at least two emitting species from the data of the excitation-emission matrix.

23. An apparatus, comprising:

means for producing spectral excitation light having wavelength variation along a first dimension across a spectral range;

means for scanning the spectral excitation light across a sample substantially parallel to the first dimension such that the wavelength variation across the spectral range of the spectral excitation light interacts with a spatial position along the sample to emit a secondary light; and

means for capturing the secondary light as a spectral emission light having wavelength variation along a second dimension.