SYSTEMS AND METHODS FOR SERIAL STAINING AND IMAGING

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.

Description

Claims

1. A system for imaging a tissue sample comprising: a sectioning system for sectioning a tissue sample in a fluid bath into a plurality of tissue sections;a staining system operative to stain an exposed region of the tissue sample in sequence with a sectioning sequence performed by the sectioning system; anda control system that controls sequential staining and sectioning of the tissue sample to provide a plurality of stained tissue sections.

2. The system of claim 1, further comprising an imaging system that 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 2, 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 is configured to sequentially cut sections from the tissue sample to sequentially expose deeper regions of the tissue sample.

5. The system of claim 4, further comprising an imaging system that 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 2, wherein the control system further comprises a memory to store images received from the imaging system.

8. The system of claim 7, 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 is delivered to the sample with a detergent.

10. The system of claim 1, wherein the stain includes a protease.

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 at least one of a fluid pump, a stirrer and a fan to provide agitation by circulating the stain in a fluid bath containing the sample.

13. (canceled)

14. The system of claim 1, wherein the staining system includes a heating element in contact with the fluid bath, the fluid bath in thermal contact with the stain.

15. The system of claim 1, wherein the staining system includes at least one of a sonicator, an electrophoresis chamber and a microwave to retrieve antigens.

16. (canceled)

17. (canceled)

18. The system of claim 1, wherein the sectioning system includes a vibrating blade, a microtome, or a cryostat.

19. The system of claim 2, further comprising a robotic device 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 configured to increase a diffusion rate of a stain into a tissue sample, the control system being connected to the diffusion device to control diffusion rate.

21. (canceled)

22. The system of claim 1 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.

23. The system of claim 1 wherein the system controls a steric property of the tissue sample.

24. The system of claim 1 wherein the control system has a memory that stores sample processing parameters including stored sectioning parameters and stored imaging parameters for a plurality of imaging modes of one or more imaging devices.

25. The system of claim 24 wherein the sample processing parameters control a selection of a stain to be delivered to a sample surface and an amount of the stain to be delivered.

26. The system of claim 24 wherein the sample processing parameters include control parameters of a diffusion device including selection of a diffusion rate adjustment mode and selected mode operating parameters including duration of mode operation.

27. (canceled)

28. The system of claim 1 further comprising a sample stage that controls movement of a plurality of tissue samples to be processed and wherein the control system is programmed to independently control processing parameters for a plurality of tissue sections.

29. (canceled)

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.

31. The system of claim 24, further comprising an imaging device, wherein the processing parameters are calibrated so that the timing sequence between sequential staining, imaging, and sectioning operations is correlated to generate images of regions of the tissue sample at selected depths within each region of the sample.

32. A method of producing sequentially stained tissue sections from a tissue sample, comprising: staining a first surface of a tissue sample with a stain;removing a section of the tissue sample to expose a second surface of the tissue sample with a sectioning device connected to a control system; andin response to the control system, iterating the staining, and removing steps to provide a plurality of stained tissue sections.

33. The method of claim 32, further comprising imaging the tissue sample after staining and before sectioning and 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.

36. The method of claim 34, wherein agitating the stain includes circulating the staining solution through a pump.

37. The method of claim 34, wherein agitating the stain includes using a stirrer or a fan to move the staining solution.

38. The method of claim 32, wherein the stain comprises a detergent or a protease.

39. The method of claim 32, further comprising changing the temperature of the stain while the stain is in contact with the tissue sample.

40. The method of claim 39, wherein changing the temperature of the stain includes 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 antibodies, nanobodies, dyes, aptamers, nucleic acid probes, fluorescent peptide probes, nanoparticles, quantum dots, or photoacoustic probes.

46. The method of claim 32, wherein the stain includes DAPI or anti-NeuN-alexafluor488.

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

48. The method of claim 47, wherein the control system is connected to the diffusion device.

49. The method of claim 48, 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.

50. The method of claim 32, further comprising controlling a steric property of the tissue sample.

51. The method of claim 32, wherein the control system has a memory that stores sample processing parameters.

52. The method of claim 51, wherein the sample processing parameters control a selection of a stain to be delivered to a sample surface and an amount of the stain to be delivered.

53. The method of claim 51 wherein the sample processing parameters include control parameters of a diffusion device including selection of a diffusion rate adjustment mode and selected mode operating parameters including duration of mode operation.

54. The method of claim 32 wherein the control system has stored 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 wherein the control system is programmed to independently control processing parameters for different tissue samples.

56. (canceled)

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 at least one of the tissue sample and a tissue section with a robotic arm.

59. The method of claim 51 wherein the processing parameters are calibrated so that the timing sequence between iterative steps of staining, imaging, and removing is correlated to generate images of regions of the tissue sample at selected depths within each region of the sample.