Systems and methods for volumetric tissue scanning microscopy

• US 7,372,985 B2
• Filed: 08/15/2003
• Issued: 05/13/2008
• Est. Priority Date: 08/15/2003
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Engelward, Bevin, Ragan, Timothy, Kim, Ki Hean, So, Peter, Huang, Hayden, Hsu, Lily, Bahlmann, Karsten
• Primary Examiner(s): Johns; Andrew W.
• Application Number:
  US10/642,447
• Publication Number:
  US 20050036667A1
• Time in Patent Office: 1,733 Days
• Field of Search: 382/128, 382/133, 382/134, 382/154, 356/39
• US Class Current: 382/133
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 2001/282 with mapping; Identification of areas; Spatial correlated pattern
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts

Abstract

In accordance with preferred embodiments of the present invention, a method for imaging tissue, for example, includes the steps of mounting the tissue on a computer controlled stage of a microscope, determining volumetric imaging parameters, directing at least two photons into a region of interest, scanning the region of interest across a portion of the tissue, imaging a plurality of layers of the tissue in a plurality of volumes of the tissue in the region of interest, sectioning the portion of the tissue and imaging a second plurality of layers of the tissue in a second plurality of volumes of the tissue in the region of interest, detecting a fluorescence image of the tissue due to said excitation light; and processing three-dimensional data that is collected to create a three-dimensional image of the region of interest.

Claims

1. A method for imaging tissue, comprising the steps of:

mounting the tissue on a computer controlled stage of a microscope;

determining volumetric imaging parameters;

directing at least two photon excitation light onto a region of interest;

scanning the excitation light across a first portion of the tissue;

imaging a plurality of layers of the tissue in a first volume of the tissue in the region of interest to provide first image data;

sectioning the first portion of the tissue;

scanning the excitation light across a second portion of the tissue;

imaging a second plurality of layers of the tissue in a second volume of the tissue to provide second image data; and

processing the first image data and the second image data to form a three-dimensional image of the tissue.

2. The method of claim 1, wherein the microscope comprises a multi-photon microscope.

3. The method of claim 2, wherein the excitation light has a penetration depth in the tissue of the range of approximately 200-500 μ

m.

4. The method of claim 1, further comprising detecting a fluorescent image.

5. The method of claim 1, wherein the image is a confocal reflectance image.

6. The method of claim 1, wherein the step of sectioning further comprises operating a microtome system that is integral with the microscope.

7. The method of claim 1, wherein the step of imaging a plurality of layers further comprises detecting at least 5 image frames per second.

8. The method of claim 1, wherein the step of scanning further comprises video rate scanning.

9. The method of claim 1, further comprising providing a depth resolution of approximately 0.1 to 2 μ

m.

10. The method of claim 1, wherein the step of scanning further comprises a low resolution mode and a high resolution mode.

11. The method of claim 1 wherein the sectioning step comprises moving the stage from an imaging position to a sectioning position, removing a layer of tissue with a sectioning tool, and moving the stage to the imaging position.

12. The method of claim 11 wherein the removing step further comprises cutting the layer of tissue with a blade.

13. The method of claim 11 wherein the moving step comprises translating the stage in an X-Y plane and elevating the stage to position the tissue relative to the sectioning tool.

14. The method of claim 1 further comprising performing a plurality of sectioning steps to remove successive layers of tissue.

15. The method of claim 1 further comprising programming a computer to control an imaging sequence and a stage translation sequence.

16. The method of claim 1 further comprising scanning the tissue using a moving mirror.

17. The method of claim 16 further comprising rotating a mirror relative to a light beam emitted by a laser.

18. The method of claim 16 further comprising scanning the tissue using a second mirror.

19. The method of claim 1 further comprising detecting images with an image sensor.

20. The method of claim 1 further comprising detecting images with a charge coupled device or CMOS imaging device.

21. The method of claim 1 further comprising detecting light with a photomuliplier tube detector.

22. A method of imaging tissue in-vivo, comprising the steps of:

mounting the tissue in a multi-photon microscope;

directing at least two photon excitation light onto a region of interest;

scanning a plurality of layers of the tissue in the region of interest;

imaging a plurality of layers in the tissue in the region of interest;

detecting a fluorescence image of the region of interest in response to said excitation light; and

processing the detected fluorescence image comprising the steps of;

sequentially storing a plurality of three-dimensional image data sets;

enhancing the image data sets;

registering the plurality of three-dimensional data sets to generate a large three-dimensional data set; and

displaying the three-dimensional data set of the region of interest.

23. The method of claim 22, wherein the step of processing further comprises compressing the three-dimensional data set.

24. The method of claim 22, wherein the step of processing further comprises identifying and quantifying features of the region of interest.

25. The method of claim 22, wherein the step of processing further comprises analyzing the three-dimensional data set.

26. The method of claim 22, wherein the step of imaging further comprises imaging mitotic recombination in the tissue.

27. The method of claim 22, wherein the step of scanning further comprises a low resolution mode and a high resolution mode.