Microscopy of a tissue sample using structured illumination

• US 10,042,150 B2
• Filed: 05/15/2014
• Issued: 08/07/2018
• Est. Priority Date: 05/15/2013
• Status: Active Grant

• Current Assignee: Administrators Of The Tulane Educational Fund (ADMINISTRATORS|DAVID H. COY)
• Original Assignee: Administrators Of The Tulane Educational Fund (ADMINISTRATORS|DAVID H. COY)
• Inventors: Brown, Jonathon Quincy
• Primary Examiner(s): Tran, Thai
• Assistant Examiner(s): BRANIFF, CHRISTOPHER
• Application Number:
  US14/888,257
• Publication Number:
  US 20160062098A1
• Time in Patent Office: 1,545 Days
• CPC Class Codes: A61B 10/02 Instruments for taking cell samples or for biopsy A61B10/0038 and A61B10/0045 take precedence; needle locating or guiding means A61B17/3403; samplers for enzymology or microbiology C12M1/26; sampling or preparing biological specimens G01N33/48
• CPC Class Codes: A61B 2090/366 using projection of images directly onto the body
• CPC Class Codes: A61B 2090/3941 Photoluminescent markers
• CPC Class Codes: A61B 5/0071 by measuring fluorescence emission
• 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 2001/302 Stain compositions
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/0635 Structured illumination, e.g. with grating
• CPC Class Codes: G01N 2201/0675 SLM
• CPC Class Codes: G01N 2201/129 Using chemometrical methods
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• 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
• CPC Class Codes: G06F 18/24 Classification techniques
• CPC Class Codes: H04N 23/56 provided with illuminating means

Abstract

Systems and methods are provided for evaluating a fresh tissue sample, prepared as to fluoresce under illumination, during a medical procedure. A structured light source is configured to project a spatially patterned light beam onto the fresh tissue sample. An imaging system is configured to produce an image from fluorescence emitted from the illuminated fresh tissue sample. A system control is configured to provide a human-comprehensible clinically useful output associated with the medical procedure.

Claims

1. A system comprising:

a structured light system comprising;

a source configured to project a light beam onto a fresh tissue sample;

a spatial light modulator to modulate the light beam,

wherein the spatial light modulator is arranged in a plurality of orientations to form a plurality of illumination patterns onto a focal plane of the fresh tissue sample,

an imaging system configured to produce a plurality of microscopy images from fluorescence emitted from the illuminated fresh tissue sample from each of the plurality of illumination patterns; and

a system control configured to provide a human-comprehensible clinically useful output associated with the medical procedure based on the plurality of microscopy images, wherein the system control rejects out of focus light in the plurality of microscopy images to improve contrast of the output.

2. The system of claim 1, wherein the system control comprises an automated image classification algorithm.

3. The system of claim 1, wherein the system control is further configured to apply a high pass filter to the image from the fluorescence emitted from the illuminated fresh tissue sample.

4. The system of claim 1, wherein the source comprises:

a light emitting diode; and

a condensing lens positioned to collect light from the light emitting diode and provides the collected light to the spatial light modulator.

5. The system of claim 1, wherein the source comprises:

a laser; and

a beam expander positioned in an optical path of a beam from the laser.

6. The system of claim 1, wherein the spatial light modulator comprising one of a ferroelectric liquid crystal on silicon device, a digital micromirror device, a twisted nematic liquid crystal on silicon device, a mechanical grating device and a physical mask device.

7. The system of claim 1, the system control comprising:

a non-transitory computer readable medium storing executable instructions for providing the human-comprehensible clinically useful output associated with the medical procedure; and

a processor operatively connected to the non-transitory computer readable medium.

8. A method comprising:

preparing a tissue sample such that the tissue will fluoresce under illumination, wherein preparing the tissue sample comprises applying a fluorescent stain to the tissue sample selected from the group comprising acriflavine, acridine orange, alum-tetracycline, eosin, picric acid, and acid fuchsine;

projecting light comprising a first spatial pattern and a second spatial pattern onto the tissue sample;

imaging the fluorescence emitted from the tissue sample due to the first spatial pattern of light and the second spatial pattern of light during a medical procedure;

producing a microscopy image based on the fluorescence; and

providing a human-comprehensible, clinically useful output based on the microscopy image.

9. The method of claim 8, wherein applying the fluorescent stain comprises applying the strain to the sample by at least one of spraying, dripping, dipping and pouring.

10. The method of claim 8, wherein applying the fluorescent stain to the tissue sample comprises applying at least two stains selected from the group as part of a multi-color, multi-stain process.

11. The method of claim 8, wherein the light has a common spatial frequency and the second spatial pattern causes a phase shift as a function of 2.

12. The method of claim 11, wherein an intensity of the microscopy image is based on intensities of the set of images.

13. The method of claim 11, wherein imaging the fluorescence emitted from the sample to provide a given image in the set of images comprises:

arranging the sample and at least a portion of an imaging system in a first relative position;

capturing a first component image representing the fluorescence emitted from a first location on the sample;

arranging the sample and at least a portion of an imaging system in a second relative position;

capturing a second component image representing the fluorescence emitted from a second location on the tissue sample; and

combining at least the first component image and the second component image to provide the given image.

14. The method of claim 13, wherein each of capturing the first component image and capturing the second component image comprises applying an autofocus routine at the imaging system.

15. The method of claim 8, wherein the light is produced by one of a laser and a light emitting diode.

16. A method comprising:

preparing a tissue sample such that the tissue will fluoresce under illumination, comprising applying a fluorescent stain to the tissue sample selected from the group comprising acriflavine, acridine orange, alum-tetracycline, eosin, picric acid, and acid fuchsine;

projecting light through a spatial light modulator in a plurality of orientations to provide a plurality of spatial light patterns sequentially illuminating a focal plane within the tissue sample;

imaging the fluorescence emitted from the sample as each of the plurality of spatial light patterns are projected on the tissue sample to form a plurality of microscopy images; and

combining the plurality of images to provide a clinical microscopy image during the medical procedure, wherein the clinical microscopy image has an improved contrast due to rejecting out of focus light in the plurality of microscopy images.

17. The method of claim 16, wherein preparing the tissue sample comprises a administering a non-toxic, tissue-enhancing fluorescent contrast agent to a patient.

18. The method of claim 17, wherein imaging the fluorescence emitted from the sample comprises imaging the tissue in situ.

19. The method of claim 16, wherein preparing the tissue sample comprises removing the tissue sample from a patient and applying the fluorescent stain to the tissue sample via at least one of spraying, dripping, dipping and pouring.

20. The method of claim 16, further comprising providing the clinical image to an automated image classification algorithm.