# US 2021/0279876 A1 · Automated holographic video microscopy assay

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- Number: US 2021/0279876 A1
- Document ID: US20210279876A1
- Status: Published application
- Kind: A1
- Application number: 17/196,895
- Filed: 2021-03-09
- Published: 2021-09-09
- Earliest claimed: 2020-03-09
- Assignees: New York University; Spheryx, Inc.
- Applicants: New York University
- Inventors: David G. GRIER; Fook Chiong CHEONG; Kaitlynn SNYDER; Rushna QUADDUS; Lauren E. ALTMAN; Kent KIRSHENBAUM
- Web page: https://patentgrep.com/patent/US20210279876A1

## CPC

- G06T 7/0014: {using an image reference approach}
- G01N 15/0227: using imaging; using holography
- G01N 15/1429: Signal processing
- G01N 15/1433: using image recognition
- G01N 15/1434: Optical arrangements
- G01N 15/1459: {the analysis being performed on a sample stream}
- G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)}
- G03H 1/0005: {Adaptation of holography to specific applications (holographic optical element G02B5/32; holographic scanner G02B26/106; recognition using holographic mask G06V10/88; holographic memories G11B7/0065, G11C13/042)}
- G03H 1/0443: {Digital holography, i.e. recording holograms with digital recording means (holobject computation G03H1/0866)}
- G03H 1/0465: {Particular recording light; Beam shape or geometry (G03H1/06 takes precedence)}
- G06T 7/62: of area, perimeter, diameter or volume
- G01N 15/01: specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05)
- G01N 2015/0233: {using holography}
- G01N 2015/1006: {for cytology}
- G01N 2015/1027: {Determining speed or velocity of a particle}
- G01N 2015/1445: {Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography}
- G01N 2015/1454: {using phase shift or interference, e.g. for improving contrast}
- G03H 2001/0033: {in hologrammetry for measuring or analysing}
- G03H 2001/005: {in microscopy, e.g. digital holographic microscope [DHM]}
- G03H 2001/0447: {In-line recording arrangement}
- G03H 2001/0471: {Object light being transmitted through the object, e.g. illumination through living cells}
- G03H 2001/266: {Wavelength multiplexing}
- G03H 2210/55: Having particular size, e.g. irresolvable by the eye
- G03H 2210/62: Moving object
- G03H 2222/13: Multi-wavelengths wave with discontinuous wavelength ranges (G03H2222/18 takes precedence)
- G03H 2222/34: Multiple light sources
- G06T 2207/10056: Microscopic image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- 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)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G03H: HOLOGRAPHIC PROCESSES OR APPARATUS (holograms, e.g. point holograms, used as ordinary optical elements G02B5/32; producing stereoscopic or other three-dimensional effects G02B30/00; diffraction-grating systems G02B27/44; systems using moiré fringes G02B27/60; optical logic elements G02F3/00; stereo-photography G03B35/00; photosensitive materials or processes for photographic purposes G03C; {stereo-photographic or similar processes G03C9/00}; apparatus for processing exposed photographic materials G03D; analogue computers performing mathematical operations with the aid of optical elements G06E3/00; authentication by radiation, of concealed information carried by holograms or diffraction gratings G06K19/16; holographic storage G11B7/0065, G11C13/04; {stereoscopic or other three dimensional effects in television systems H04N13/00})

## Abstract

An in-line holographic microscope can be used to analyze a video stream to track individual colloidal particles' three-dimensional motions. The system and method can provide real time nanometer resolution, and simultaneously measure particle sizes and refractive indexes. An assay using the holographic microscope for holographic particle characterization directly detect viruses, antibodies and related targets binding to the surfaces of specifically functionalized micrometer-scale colloidal probe beads. The system detects binding of targets by directly measuring associated changes in the bead's diameter without the need for downstream labeling and analysis.

## Claims

### Claim 1

A method of characterizing a parameter of a viral protein by holographic microscopy, comprising the steps of:
receiving holographic image data having an a set of concentric bright and dark rings associated with a functionalized probe bead;
determining by Lorenz-Mie analysis an effective sphere estimate of each of the functionalized probe bead's effective sphere diameter, and effective sphere refractive index; and
determining a diameter difference between the effective sphere diameter with a predetermined diameter for unbound functionalized probe beads, with diameter difference representing the result of material bound to the bead and medium within the effective sphere estimate, wherein the effective sphere diameter (dp) is equal to
dp=d0+2δf
wherein dp is the effective sphere diameter, d0 is the predetermined diameter for unbound functionalized probe beads, δ is effective optical thickness of a complete layer of the viral protein, and f is the fraction of binding sites on the functionalized probe bead.

### Claim 2 (depends on 1)

The method of claim 1, wherein the material is a protein associated with SARS-CoV-2.

### Claim 3 (depends on 1)

The method of claim 1, determining by Maxwell Garnet effective-medium theory the effective diameter of the probe bead, and effective refractive index;

### Claim 4 (depends on 1)

The method as defined in claim 1 wherein an associated plurality of holograms are formed by interaction between the sample and the plurality of wavelengths of coherent light, thereby enabling determination of different responses of the sample to the different wavelengths of coherent light and analyzing the different response to identify the parameters of the sample.

### Claim 5 (depends on 1)

The method as defined in claim 1 wherein determining the centroid further includes determining the Lorenz-Mie functionality scattering function fs(kr) along a line segment R=|r−rp| and interpolating to obtain a function fs(k(r−rp)) thereby reducing processing time and providing real time analysis of the sample.

### Claim 6 (depends on 1)

The method as defined in claim 1 further including the steps of performing the Lorenz-Mie analysis and obtaining comparisons between of the image data a particle being in an untreated state and another particle having undergone a treatment, thereby enabling real time characterization of molecular layers present on the treated particle versus the untreated particle.

### Claim 7 (depends on 6)

The method as defined in claim 6 wherein the real time characteristics are selected from the group of index of refraction and particle radius.

### Claim 8 (depends on 1)

The method as defined in claim 1 further including the step of estimating in-plane coordinates of the particle by the Lorenz-Mie analysis and then estimating axial coordinate of the particle by back-propagating the measured light field applying a Rayleigh-Sommerfeld propagator.

### Claim 9 (depends on 1)

The method as defined in claim 1 wherein the analysis step includes applying a Levenburg-Marquardt fitting routine to identify the in-plane co-ordinate within 3 nm.

### Claim 10 (depends on 1)

The method as defined in claim 1 further including the steps of determining velocity of the particle in a flowing form.

### Claim 11 (depends on 10)

The method as defined in claim 10 further including the step of mapping a three-dimensional flow field of the particle.

### Claim 12 (depends on 10)

The method as defined in claim 10 wherein the step of analyzing the information comprises simultaneously tracking and characterizing individual ones of the particles.

### Claim 13 (depends on 1)

The method as defined in claim 1 wherein the step of measuring includes identifying molecular-scale coatings on functionalized forms of the particle by detecting variations in apparent increase in radius.

### Claim 14

A system for characterizing a parameter of a sample by holographic microscopy, comprising:
a holographic microscope including a laser beam source and an objective lens, the laser beam positioned to interact with a fluidic sample channel having media, probe beads, and target objects, scattering from the sample comprising probe beads having target objects bound to a surface and interacting with an unscattered portion of the laser beam to provide a holographic scattering pattern,
an image collection device for collecting image data characteristic of the scattering pattern from the holographic microscope; and,
a computer system including a processor and memory, the memory having stored thereon computer readable instructions, the computer readable instructions configured to:
receive holographic image data of the sample;
determine a first estimate of the number of coated probe beads in the holographic image data, each object associated with a set of concentric bright and dark rings;
determine, for each set of concentric bright and dark rings a centroid defined by an approximate x, y position in a plane, with each pixel of the image data voting for pixels in a transformed image that may be centroids;
determine an estimate of the axial position (z) of each of the coated probe beads;
determine by Lorenz-Mie analysis an estimate of each of the coated probe beads effective sphere radius and effective sphere refractive index, where each coated probe bead is treated as an effective sphere for Lorenz-Mie analysis;
determine by Maxwell Garneet effective-medium theory the effective coating diameter, and effective coating refractive index, an effective coating comprising bound target objects and media within an associated effective sphere;
compare one of the radius and refractive index for each of the objects the same of radius or refractive index of a control sample; and
determine for each one of the objects if that one object is bound to a material relative to the control sample.

### Claim 15 (depends on 14)

The system of claim 14, wherein the holographic microscope includes a plurality of laser beams corresponding to multiple wavelengths and wherein the computer readable memory is configured to receive multi-color holographic image data.

### Claim 16 (depends on 15)

The system as described in claim 15 wherein each of the plurality of laser beams are configured to engage a microfluidic channel having the plurality of particles.

### Claim 17

A method of characterizing parameters for holographic microscopy of objects comprising the steps of:
receiving, by a processor, image data from a storage medium;
transforming, by a processor, the image data using a Hough circular transform to determine by a voting algorithm a first estimate of the number of objects in the image data and the objects approximate x, y position in a plane;
applying, by a processor, Lorenz-Mie analysis of the image data for each of the objects to determine an estimate of the radius and refractive index of each of the objects;
applying, by a processor, Maxwell Garnett effective-medium theory analysis of the image data for each of the objects to determine the effective diameter of a coating associated with the object;
identifying, by a processor, from one or both of estimated radii and estimated refractive indices those particles having a coating; and
generating a signal to the user indicating completed process.

### Claim 18 (depends on 17)

The method of claim 17, wherein the determination of a centroid is by application of a circular Hough transformation wherein each pixel in an original image votes for the pixels in a transformed image.

### Claim 19 (depends on 13)

The method as defined in claim 13 wherein the coatings are associated with the bind proteins of SARS-CoV-2.

### Claim 20 (depends on 13)

The method as defined in claim 13 wherein partial coatings are filled with a fluid medium.