# US 11,079,719 B2 · Lens-free holographic optical system for high sensitivity label-free microbial growth detection and quantification for screening, identification, and susceptibility testing

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- Number: US 11,079,719 B2
- Document ID: US11079719B2
- Status: Granted
- Kind: B2
- Application number: 16/028,287
- Filed: 2018-07-05
- Published: 2019-01-10
- Granted: 2021-08-03
- Earliest claimed: 2017-07-05
- Assignees: Accelerate Diagnostics, Inc.
- Applicants: Accelerate Diagnostics, Inc.
- Inventors: Oleg Gusyatin
- Web page: https://patentgrep.com/patent/US11079719B2

## CPC

- G03H 1/10: using modulated reference beam
- C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)}
- G01N 15/0227: using imaging; using holography
- G01N 15/1433: using image recognition
- G01N 15/1434: Optical arrangements
- G01N 23/20: by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G02B 21/0008: {Microscopes having a simple construction, e.g. portable microscopes}
- G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)}
- G03H 1/0443: {Digital holography, i.e. recording holograms with digital recording means (holobject computation G03H1/0866)}
- G01N 2015/0233: {using holography}
- G01N 2015/1006: {for cytology}
- G01N 2015/1454: {using phase shift or interference, e.g. for improving contrast}
- G01N 2015/1493: {Particle size}
- G01N 2015/1497: {Particle shape}
- G03H 1/0866: {Digital holographic imaging, i.e. synthesizing holobjects from holograms}
- G03H 2001/0033: {in hologrammetry for measuring or analysing}
- G03H 2001/0447: {In-line recording arrangement}
- G03H 2001/2655: {Time multiplexing, i.e. consecutive records wherein the period between records is pertinent per se}
- G03H 2222/13: Multi-wavelengths wave with discontinuous wavelength ranges (G03H2222/18 takes precedence)
- G03H 2222/52: Divergent beam
- G03H 2226/13: Multiple recording means
- G03H 2227/03: Means for moving one component (G03H1/0476, G03H2001/2695 take precedence)
- 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})
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- 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

## Abstract

Disclosed are optical interrogation apparatus that can produce lens-free images using an optoelectronic sensor array to generate a holographic image of sample objects, such as microorganisms in a sample. Also disclosed are methods of detecting and/or identifying microorganisms in a biological sample, such as microorganisms present in low levels. Also disclosed are methods of using systems to detect microorganisms in a biological sample, such as microorganisms present in low levels. In addition or as an alternative, the methods of using systems may identify microorganisms present in a sample and/or determine antimicrobial susceptibility of such microorganisms.

## Claims

### Claim 1

An automated system, comprising:
an automated holographic optical apparatus situated to determine at least antimicrobial susceptibility of a microorganism corresponding to an object in a sample volume based on a detected variation over time of a hologram of the sample volume, an output of at least one deeply supervised convolutional neural network, and a phenotypical behavior of the microorganism, wherein the phenotypical behavior of the microorganism is classified based on the detected variation and the output of the at least one deeply supervised convolutional neural network,
wherein the holographic optical apparatus is an in-line holographic apparatus and the hologram is an in-line hologram; and
wherein the in-line holographic optical apparatus includes a reference beam source situated to direct a reference beam to the sample volume, a sample receptacle situated to hold the sample volume in view of the reference beam, an optical sensor situated to detect the in-line hologram formed by the reference beam and the sample volume, and a controller coupled to the optical sensor and that includes at least one processor and one or more computer-readable storage media including stored instructions that, responsive to execution by the at least one processor, cause the controller to determine the variation over time of the in-line hologram.

### Claim 2 (depends on 1)

The system of claim 1, wherein the controller is configured to reconstruct the spatial characteristics of the sample volume based on the detected in-line hologram, diffraction propagation approximation, and a phase retrieval algorithm.

### Claim 3 (depends on 2)

The system of claim 2, wherein the controller is configured to determine a focal plane of the microorganism in the sample volume based on the reconstructed spatial characteristics.

### Claim 4 (depends on 1)

The system of claim 1, wherein the at least one deeply supervised convolutional neural network includes a spatial reconstruction deeply supervised convolutional neural network configured to produce an output corresponding to a reconstruction of the spatial characteristics of the sample volume based on a trained set of network layers, and wherein the controller is configured to reconstruct the spatial characteristics of the sample volume using the reconstruction deeply supervised convolutional neural network.

### Claim 5 (depends on 1)

The system of claim 1, wherein the at least one deeply supervised convolutional neural network includes a microorganism identification deeply supervised convolutional neural network configured to produce an output corresponding to a microorganism identification, microorganism morphology identification, microorganism movement identification, and/or microorganism phenotypic classification for the microorganism in the sample volume based on a trained set of network layers, and wherein the controller is configured to identify the microorganism, microorganism morphology, microorganism movement, and/or classify the microorganism phenotypical behavior using the microorganism identification deeply supervised convolutional neural network.

### Claim 6 (depends on 1)

The system of claim 1, wherein the controller is configured to determine a 3D position and/or morphological characteristics of the microorganism based on the in-line hologram.

### Claim 7 (depends on 1)

The system of claim 1, wherein the controller is configured to associate the object detected in a later hologram with the object detected in an earlier hologram, based on proximity or morphological characteristics of the objects detected from the variation over time of the in-line hologram.

### Claim 8 (depends on 1)

The system of claim 1, wherein the controller is configured to form an object track for the object in the sample volume based on the detected variation over time of the in-line hologram.

### Claim 9 (depends on 1)

The system of claim 1, wherein the controller is configured to identify the object as the microorganism in the sample volume based on the detected variation over time of the in-line hologram.

### Claim 10 (depends on 1)

The system of claim 1, wherein the controller is configured to classify a phenotypical behavior of the microorganism in the sample volume based on the detected in-line hologram.

### Claim 11 (depends on 10)

The system of claim 10, wherein the controller is configured to determine a correspondence between the phenotypic behavior of the microorganism and presence, concentration, and taxon of the microorganism in the sample volume.

### Claim 12 (depends on 11)

The system of claim 11, wherein the sample volume includes a plurality of sample volume portions situated in a respective at least one growth control, at least one selective media, and at least one antimicrobial flow cell that are held by the sample receptacle, and the controller is configured to determine the presence, taxon, and an antibiogram of the microorganism or multiple microorganisms based on the at least one growth control, the at least one selective media, and the at least one antimicrobial flow cell.

### Claim 13 (depends on 1)

The system of claim 1, wherein the optical sensor is a complementary metal oxide semiconductor (CMOS) sensor having a pixel pitch of 1.5 μm or smaller.

### Claim 14 (depends on 1)

The system of claim 1, wherein the optical sensor has a pixel pitch of 1 μm/pixel or smaller and the controller is configured to determine, based on the detected in-line hologram, morphological characteristics of the microorganism.

### Claim 15 (depends on 1)

The system of claim 1, wherein the reference beam source includes a plurality of pinhole apertures spaced apart from each other by 1 mm or less with each of the pinhole apertures configured to emit respective reference subbeams at different respective wavelengths.

### Claim 16 (depends on 1)

The system of claim 1, wherein the reference beam source includes a pinhole aperture situated to receive illumination from an illumination source and the reference beam source is configured to direct the reference beam lens-free from the pinhole aperture to the sample volume and optical sensor.

### Claim 17 (depends on 16)

The system of claim 16, wherein the illumination source is configured to generate illumination at multiple wavelengths.

### Claim 18 (depends on 16)

The system of claim 16, wherein the illumination received from the illumination source by the pinhole aperture is incoherent and the reference beam comprises incoherent illumination.

### Claim 19 (depends on 1)

The system of claim 1, wherein the reference beam source is situated to direct a plurality of reference beams to the sample volume and to adjacent portions of the optical sensor so as to mosaic the field of view of the in-line holographic apparatus.

### Claim 20 (depends on 19)

The system of claim 19, wherein the adjacent portions of the optical sensor correspond to separate CMOS sensors.

### Claim 21 (depends on 19)

The system of claim 19, wherein the sample volume includes a plurality of sample volume portions, including a first sample volume portion situated in a first sample reaction chamber that is held by the sample receptacle, wherein the first sample volume portion is situated as a growth control volume by having an absence of an antimicrobial agent, and including a second sample volume portion situated in a second sample reaction chamber, wherein the second volume portion is situated as an antimicrobial susceptibility test volume in the presence of a predetermined antimicrobial agent.

### Claim 22 (depends on 21)

The system of claim 21, wherein the sample reaction chambers include a plurality of growth channels having selective media.

### Claim 23 (depends on 1)

The system of claim 1, wherein the holographic apparatus is situated to determine a presence of the microorganism based on the detected variation with the sample volume having a microorganism concentration of 10 cfu/mL or less.

### Claim 24 (depends on 1)

The system of claim 1, wherein the holographic apparatus is situated to display a time-lapse image associated with the sample volume at a time-resolution that is faster than a microorganism division rate.

### Claim 25 (depends on 24)

The system of claim 24, wherein the time-lapse image corresponds to one or more of the hologram and one or more planes of the sample volume.

### Claim 26 (depends on 1)

The system of claim 1, wherein a time period of the detected variation corresponds to four or fewer microorganism doubling events.

### Claim 27 (depends on 1)

The system of claim 1, wherein a time period of the detected variation corresponds to three or fewer microorganism doubling events.

### Claim 28 (depends on 1)

The system of claim 1, wherein the microorganism is immobilized in the sample volume.