Systems and methods for volumetric tissue scanning microscopy
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.
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 μm.
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 μm.
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.