# US 10,768,402 B2 · Microscopy of a tissue sample using structured illumination

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- Number: US 10,768,402 B2
- Document ID: US10768402B2
- Status: Granted
- Kind: B2
- Application number: 16/054,235
- Filed: 2018-08-03
- Published: 2018-12-06
- Granted: 2020-09-08
- Earliest claimed: 2013-05-15
- Assignees: The Administrators of the Tulane Educational Fund
- Applicants: The Administrators of the Tulane Educational Fund
- Inventors: Jonathon Quincy Brown
- Web page: https://patentgrep.com/patent/US10768402B2

## CPC

- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- A61B 5/0071: {by measuring fluorescence emission}
- 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)}
- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)}
- 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}
- G06F 18/24: Classification techniques
- H04N 23/56: provided with illuminating means
- A61B 2090/366: {using projection of images directly onto the body}
- A61B 2090/3941: {Photoluminescent markers}
- G01N 2001/302: {Stain compositions}
- G01N 2021/6419: {Excitation at two or more wavelengths}
- G01N 2021/6421: {Measuring at two or more wavelengths}
- G01N 2021/6441: {with two or more labels}
- G01N 2201/0635: Structured illumination, e.g. with grating
- G01N 2201/0675: SLM
- G01N 2201/129: Using chemometrical methods
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G06F: ELECTRIC DIGITAL DATA PROCESSING (computer systems based on specific computational models G06N)
- H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION

## 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

### Claim 1

A method comprising:
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 an excised tissue sample, wherein the tissue sample is pre-stained with a fluorescent contrast agent;
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 microscopy images to form an image of an optical section of the excised tissue sample, wherein the image exhibits an improved contrast due to rejecting out of focus light in the plurality of microscopy images during the combining.

### Claim 2 (depends on 1)

The method of claim 1, further comprising preparing the excised tissue sample such that the tissue will fluoresce under illumination, wherein preparing the excised tissue sample comprises applying a solution of the fluorescent contrast agent to the excised tissue sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the fluorescent contrast agent comprises at least one of an anionic dye solution or a cationic dye solution.

### Claim 4 (depends on 1)

The method of claim 1, wherein the fluorescent contrast agent comprises an anthraquinone dye, Eosin, or Acridine orange.

### Claim 5 (depends on 1)

The method of claim 1, wherein the fluorescent contrast agent comprises Acriflavine, Proflavine, Acid fuchsine, Methylene blue, Picric acid, Cresyl violet, Tetracycline, Alum tetracycline, Fluorescein, Aniline blue Fast green, boron-dipyrromethene, Rhodamine, Sulforhodamine, 4′,6-diamidino-2-phenylindole, Propidium iodide, or Alcian blue.

### Claim 6 (depends on 1)

The method of claim 1, wherein the light is produced by a laser and/or a light emitting diode.

### Claim 7

A system comprising:
a light source to project a light beam onto an excised 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 excised tissue sample; and
an imaging system to produce a plurality of microscopy images from fluorescence emitted from the tissue sample due to a plurality of illumination patterns, wherein the plurality of microscopy images are combined to provide an image of an optical section of the excised tissue sample with an improved contrast, wherein the improved contrast is provided by rejecting out of focus light in the plurality of microscopy images.

### Claim 8 (depends on 7)

The system of claim 7, wherein the light source comprises:
a light emitting diode; and
a condensing lens positioned to collect light from the light emitting diode and provide the collected light to the spatial light modulator.

### Claim 9 (depends on 7)

The system of claim 7, wherein the light source comprises:
a laser; and
a beam expander positioned in an optical path of a beam from the laser.

### Claim 10 (depends on 7)

The system of claim 7, wherein the spatial light modulator comprises: ferroelectric liquid crystal on silicon device, a digital micromirror device, a twisted nematic liquid crystal on silicon device, a mechanical grating device or a physical mask device.

### Claim 11 (depends on 7)

The system of claim 7, wherein the excised tissue sample is stained with a fluorescent contrast agent.

### Claim 12 (depends on 11)

The system of claim 11, wherein the fluorescent contrast agent comprises an anionic dye solution or a cationic dye solution.

### Claim 13 (depends on 11)

The system of claim 11, wherein the fluorescent contrast agent comprises an anthraquinone dye.

### Claim 14 (depends on 11)

The system of claim 11, wherein the fluorescent contrast agent comprises Eosin, or Acridine orange.

### Claim 15 (depends on 11)

The system of claim 11, wherein the fluorescent contrast agent comprises Acriflavine, Proflavine, Acid fuchsine, Methylene blue, Picric acid, Cresyl violet, Tetracycline, Alum tetracycline, Fluorescein, Aniline blue Fast green, boron-dipyrromethene, Rhodamine, Sulforhodamine, 4′,6-diamidino-2-phenylindole, Propidium iodide, or Alcian blue.