Systems and methods for volumetric tissue scanning microscopy

• US 8,718,351 B2
• Filed: 12/26/2012
• Issued: 05/06/2014
• Est. Priority Date: 08/15/2003
• Status: Expired due to Fees

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Engelward, Bevin, Ragan, Timothy, Bahlman, Karsten, Kim, Ki-Hean, Laiho, Lily Hsu, Huang, Hayden, So, Peter T. c.
• Primary Examiner(s): Johns, Andrew W
• Application Number:
  US13/726,931
• Publication Number:
  US 20130142413A1
• Time in Patent Office: 496 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 system for imaging a tissue sample comprising:

a multiphoton light source that emits excitation light to illuminate a region of interest;

a microscope optically coupled to the light source such that light is coupled through a lens of the microscope onto the region of interest within a biological sample at a plurality of focal locations, the microscope being operated in response to microscope scan parameters;

a controlled stage to support the biological sample to be sectioned into a plurality of layers from the biological sample after imaging of each layer in sequence, the controlled stage being operated in response to selected stage scan parameters;

a scanner to scan a plurality of focal locations within the region of interest;

an imaging device that receives light from the plurality of focal locations within the biological sample to detect fluorescence images of the region of interest within each of the plurality of layers; and

a data processor that processes the plurality of images to produce stored electronic image data of the region of interest.

2. The system of claim 1 further comprising a sectioning device positioned to remove a layer of the sample after imaging to expose a further layer of the sample for imaging.

3. The system of claim 2 wherein the sectioning device is one of a microtome, a vibrating blade microtome and a rotating blade.

4. The system of claim 1 wherein imaging device comprises a plurality of photomultiplier tubes.

5. The system of claim 4 wherein each of the plurality of photomultiplier tubes images the sample at a different wavelength range.

6. The system of claim 1 wherein the microscope comprises a confocal microscope.

7. The system of claim 1 wherein the light source is a laser.

8. The system of claim 1 wherein the light source is one of a picosecond laser and a femtosecond laser.

9. The system of claim 1 further comprising a rotating polygonal minor that provides a fast scanning axis.

10. The system of claim 1 further comprising a galvanometer driven mirror that provides a slow scanning axis.

11. The system of claim 1 further comprising a piezoelectric-driven lens translator that provides a depth axis.

12. The system of claim 1 further comprising at least one diode to generate a reference signal.

13. The system of claim 1 wherein the excitation light is in the range of approximately 650-1200 nm.

14. The system of claim 1 further comprising a low resolution scanning mode and a high resolution mode for focusing at a depth within a region of interest.

15. The system of claim 1 further comprising a processor that controls the scanner to provide a tissue scanning rate of at least 100 cells per second.

16. The system of claim 1 wherein the imaging device comprises a multi anode photomultiplier tube.

17. A method for mulitphoton imaging of a sample comprising:

illuminating a first region of a biological sample with excitation light to deliver at least two photons onto the first region of the sample at a plurality of focal locations;

a microscope assembly that optically couples the illuminating light onto the sample through a microscope lens;

imaging the first region of the sample with a plurality of detector elements that receive light from the sample to detect light from the plurality of focal locations within the first region of interest;

subsequently sectioning the first region of the sample to expose a second region of the sample; and

imaging the second region of the sample with the plurality of detector elements to provide a fluorescence image of the second region of the sample.

18. The method of claim 17 further comprising using a microlens array to illuminate the first region of the sample.

19. The method of claim 17 further comprising using a galvanometer driven mirror that provides a slow scanning axis.

20. The method of claim 17 further comprising using a piezoelectric-driven lens translator that provides an adjustable depth axis.

21. The method of claim 17 further comprising using at least one diode to generate a reference signal.

22. The method of claim 17 further comprising using the plurality of photomultiplier tube arrays to image at different wavelengths.

23. The method of claim 17 further comprising using a sectioning device that comprises one of a microtome, a vibrating blade microtome and a rotating blade.

24. The method of claim 17 further comprising using a low resolution scanning mode and a high resolution mode for focusing on a region of interest.

25. The method of claim 17 further comprising using a data processor to control a scanner that translates illuminating light through the first region of the sample to provide a scanning rate of at least 100 cells per second.

26. The method of claim 17 further comprising illuminating the plurality of focal locations at a selected depth within each layer.

27. The method of claim 17 further comprising a dichroic mirror to separate the light received from the sample into a first channel that includes a first array photomultiplier tubes and into a second channel that includes a second array photomultiplier tubes.

28. The method of claim 27 further comprising detecting an image with a multi anode photomultiplier tube.

29. The method of claim 17 further comprising detecting a plurality of images of adjacent regions in each layer of tissue.