SYSTEMS AND METHODS FOR SERIAL STAINING AND IMAGING

• US 20180045623A1
• Filed: 03/11/2016
• Published: 02/15/2018
• Est. Priority Date: 03/11/2015
• Status: Active Grant

• Current Assignee: Tissuevision, Inc.
• Original Assignee: Tissuevision, Inc.
• Inventors: Ragan, Timothy, Dimant, Chiam Moshe
• Granted Patent: US 10,788,403 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 1/06 providing a thin slice, e.g. microtome
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 1/42 Low-temperature sample treatment, e.g. cryofixation
• CPC Class Codes: G01N 2001/302 Stain compositions
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/24 Base structure
• CPC Class Codes: G02B 21/26 Stages; Adjusting means therefor
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Abstract

The present invention relates to systems and methods for sequential operation of staining, imaging and sectioning of tissue samples by a processing system. After each layer of the sample is removed by the sectioning system, the system automatically stains the exposed surface of a sample to a depth to enable imaging of the remaining tissue. The system then repeats the sectioning, staining and imaging steps in sequence to image the sample.

Claims

1. A system for imaging a tissue sample comprising:

an imaging system;

a sectioning system;

a staining system operative to stain an exposed region of the tissue sample in sequence with a sectioning sequence performed by the sectioning system; and

a control system that controls sequential staining, imaging and sectioning of the tissue sample.

2. The system of claim 1, wherein the imaging system is configured to use one or more of multiphoton, confocal, optical coherence tomography, second harmonic generation, third harmonic generation, coherent anti-Stokes Raman, photoacoustic, or Raman imaging techniques.

3. The system of claim 1, wherein the imaging system images the exposed region of the tissue sample and/or a subsurface region of the tissue sample.

4. The system of claim 1, wherein the sectioning system includes a vibrating blade, microtome, or cryostat configured to sequentially cut sections from the tissue sample to sequentially expose deeper regions of the tissue sample.

5. The system of claim 4, wherein the imaging system images the sequentially cut sections from the tissue sample.

6. The system of claim 4, wherein the sequentially cut sections have a thickness of about 50 microns or less.

7. The system of claim 1, wherein the control system further comprises a memory to store images received from the imaging system and wherein the control system is configured to generate a three-dimensional representation of the tissue sample using the images stored in the memory.

9. The system of claim 1, wherein the stain includes a protease or is delivered to the sample with a detergent.

11. The system of claim 1, wherein the staining system includes a motorized stage to move the tissue sample, agitate the stain, or both.

12. The system of claim 1, wherein the staining system includes one or more of a fluid pump, a stirrer, or a fan to provide agitation by circulating the stain in a fluid bath containing the sample.

14. The system of claim 1, wherein the staining system includes one or more of a sonicator, an electrophoresis chamber, or a heating element in contact with a water bath, the water bath in thermal contact with the stain.

17. The system of claim 1, wherein the staining system includes a microwave to enable antigen retrieval.

19. The system of claim 1, further comprising a robotic arm to move the tissue sample among the staining system, imaging system, and sectioning system.

20. The system of claim 1 further comprising a diffusion device connected to the control system and configured to increase a diffusion rate of a stain into a tissue sample.

22. The system of claim 20 wherein the control system is programmed with a software module to select one or more diffusion devices to control a diffusion rate of a stain into a sample.

24. The system of claim 7 wherein the memory of the control system stores sample processing parameters that control a selection of the stain to be delivered to a sample surface and an amount of the stain to be delivered, the sample processing parameters including at least one of:

control parameters of a diffusion device including selection of a diffusion rate adjustment mode,

selected mode operating parameters including duration of mode operation, and

sectioning parameters and stored imaging parameters for a plurality of imaging modes.

28. The system of claim 1 further comprising a sample stage that controls movement of a plurality of tissue samples to be processed.

29. The system of claim 28 wherein the control system is programmed to independently control processing parameters for each of the tissue samples on the sample stage.

30. The system of claim 28 wherein the sample stage is operative to move the plurality of samples between one or more imaging stations, one or more sectioning stations, one or more staining stations and one or more diffusion rate control stations.

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32. A method of producing sequential images of a tissue sample, comprising:

staining a first surface of a tissue sample with a stain;

imaging the tissue sample with an imaging device to generate an image;

removing a section of the tissue sample to expose a second surface of the tissue sample; and

iterating the staining, imaging, and removing steps in response to a control system.

33. The method of claim 32, further comprising saving the images in a memory or storage device.

34. The method of claim 32, further comprising agitating the stain while it is in contact with the tissue sample.

35. The method of claim 34, wherein agitating the stain includes translating a motorized stage, circulating the staining solution through a pump, or using a stirrer or a fan to move the staining solution.

39. The method of claim 32, further comprising changing the temperature of the stain while the stain is in contact with the tissue sample by placing the stain in thermal contact with a heated water bath.

41. The method of claim 32, further comprising applying electrophoresis to at least a portion of the tissue sample and the stain.

42. The method of claim 32, further comprising sonicating at least a portion of the tissue sample and the stain.

43. The method of claim 32, further comprising performing antigen retrieval to improve staining of the tissue sample.

44. The method of claim 32, further comprising staining the second surface of the tissue sample with a different stain than was used on the first surface of the tissue sample.

45. The method of claim 32, wherein the stain includes one or more of a detergent, a protease, DAPI, anti-NeuN-alexafluor488, antibodies, nanobodies, dyes, aptamers, nucleic acid probes, fluorescent peptide probes, nanoparticles, quantum dots, or photoacoustic probes.

47. The method of claim 32, further comprising configuring a diffusion device connected to a control system to increase a diffusion rate of the stain into the tissue sample.

49. The method of claim 47, wherein the control system is programmed with a software module to select one or more diffusion devices to control a diffusion rate of a stain into a sample.

51. The method of claim 32, wherein the control system has a memory that stores sample processing parameters that control a selection of the stain to be delivered to a sample surface and an amount of the stain to delivered, the sample processing parameters including at least one of:

control parameters of a diffusion device including selection of a diffusion rate adjustment mode,

selected mode operating parameters including duration of mode operation, and

sectioning parameters and stored imaging parameters for a plurality of imaging modes.

55. The method of claim 32 further comprising controlling movement of a plurality of tissue samples to be processed using a sample stage.

56. The method of claim 55 wherein the control system is programmed to independently control processing parameters for each of the tissue samples on the sample stage.

57. The method of claim 55 wherein the sample stage is operative to move the plurality of samples between one or more imaging stations, one or more sectioning stations, one or more staining stations and one or more diffusion rate control stations.

58. The method of claim 32, further comprising moving the tissue sample with a robotic arm.

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