# US 10,871,640 B2 · Methods and systems for automated imaging of three-dimensional objects

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- Number: US 10,871,640 B2
- Document ID: US10871640B2
- Status: Granted
- Kind: B2
- Application number: 16/458,147
- Filed: 2019-06-30
- Published: 2020-08-20
- Granted: 2020-12-22
- Earliest claimed: 2019-02-15
- Assignees: PerkinElmer Cellular Technologies Germany GmbH
- Applicants: S Chris Hinnah; Kaupo Palo; Ollavi Ollikainen; Thomas Steinkamp; Hartwig Preckel
- Inventors: S Chris Hinnah; Kaupo Palo; Ollavi Ollikainen; Thomas Steinkamp; Hartwig Preckel
- Web page: https://patentgrep.com/patent/US10871640B2

## CPC

- G02B 21/002: {Scanning microscopes (scanning near field optical microscopes G01Q60/18)}
- G02B 21/008: {Details of detection or image processing, including general computer control}
- G02B 21/0032: {Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06)}
- G02B 21/0036: {Scanning details, e.g. scanning stages}
- G02B 21/02: Objectives
- G02B 21/244: {using image analysis techniques}
- 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}
- G02B 30/52: the three-dimensional [3D] volume being constructed from a stack or sequence of two-dimensional [2D] planes, e.g. depth sampling systems
- G06T 7/571: from focus
- H04N 13/395: with depth sampling, i.e. the volume being constructed from a stack or sequence of two-dimensional [2D] image planes
- G06T 2207/10056: Microscopic image
- G06T 2207/10148: Varying focus
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06V 20/69: Microscopic objects, e.g. biological cells or cellular parts
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION
- G06V: IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING

## Abstract

Certain configurations are described of methods and systems that can be used to image three-dimensional objects such as biological cells, biological tissues or biological organisms. The methods and systems can image the three-dimensional objects at reduced imaging times and with reduced data volumes.

## Claims

### Claim 1

A method of imaging a three-dimensional object present in a matrix using a microscope, the method comprising:
pre-scanning the matrix using a first magnification objective to identify an x-y location of the three-dimensional object in the matrix and to identify a z-height for the three-dimensional object in the matrix; and
scanning the identified three-dimensional object using a second magnification objective in a z-direction using the identified z-height of the identified three-dimensional object to provide a three-dimensional image of at least some portion of the three-dimensional object present in the matrix, wherein the second magnification objective is greater than or equal to the first magnification objective.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pre-scanning comprises obtaining a plurality of individual z-plane images using the first magnification objective of the microscope to provide a first image set of discrete z-plane images used to identify the z-height.

### Claim 3 (depends on 2)

The method of claim 2, wherein the scanning comprises obtaining a plurality of discrete z-plane images of the identified three-dimensional object starting at the identified z-height of the three-dimensional object and using the second magnification objective of the microscope to provide a second image set.

### Claim 4 (depends on 3)

The method of claim 3, wherein the plurality of discrete z-plane images of the second image set are used to provide an entire three-dimensional image of the three-dimensional object in the matrix.

### Claim 5 (depends on 4)

The method of claim 4, wherein image correction is applied to the first image set to provide a corrected, first image set prior to scanning the identified three-dimensional objects using the second magnification objective.

### Claim 6 (depends on 5)

The method of claim 5, wherein corrected x-y locations of the three-dimensional object and a corrected z-height for the three-dimensional object are obtained from the corrected, first image set and used in the scanning of the three-dimensional object using the second magnification objective to provide the second image set.

### Claim 7 (depends on 6)

The method of claim 6, wherein image correction is applied to the second image set to provide a corrected, second image set that is used to provide the three-dimensional image of the three-dimensional object.

### Claim 8 (depends on 1)

The method of claim 1, wherein the pre-scanning using the first magnification objective is performed using a 10× air objective of the microscope, and the scanning using the second magnification objective is performed using a 40× water immersion objective of the microscope.

### Claim 9 (depends on 1)

The method of claim 1, wherein a laser confocal scanning microscope is used to perform both the pre-scanning and the scanning.

### Claim 10 (depends on 9)

The method of claim 9, wherein the three-dimensional object is a biological organism, a biological organ, a biological tissue, a biological cell or a component or organelle thereof.

### Claim 11 (depends on 9)

The method of claim 9, wherein the pre-scanning and scanning of the biological organism, the biological organ, the biological tissue, the biological cell or the component or the organelle thereof is performed in a matrix comprising a hydrogel or in a matrix comprising a three-dimensional scaffold.

### Claim 12 (depends on 9)

The method of claim 9, further comprising pre-scanning the matrix using the first magnification objective of the microscope to identify an x-y location of each of a plurality of individual three-dimensional objects in the matrix and to identify a z-height for each of the identified plurality of three-dimensional objects in the matrix, and scanning each identified three-dimensional object using the second magnification objective in a z-direction using a respective identified z-height of each identified three-dimensional object to provide a three-dimensional image of at least some portion of each of the three-dimensional objects present in the matrix.

### Claim 13 (depends on 12)

The method of claim 12, wherein the pre-scanning using the first magnification objective is performed using a 10× air objective of the microscope, and the scanning using the second objective magnification is performed using a 40× water immersion objective of the microscope.

### Claim 14 (depends on 13)

The method of claim 13, wherein each of the three-dimensional objects is independently a biological organism, a biological organ, a biological tissue, a biological cell or a component or organelle thereof, and wherein the matrix comprises a hydrogel or a three-dimensional scaffold.

### Claim 15 (depends on 1)

The method of claim 1, further comprising using the pre-scanning to identify a z-width of the identified three-dimensional object in the matrix and using the identified z-width to select a scan time or to select a data volume obtained from the scanning of the three dimensional object in the matrix.

### Claim 16

A microscope system configured to image a three-dimensional object in a three-dimensional matrix, the system comprising:
a sample holder configured to receive a sample comprising the three-dimensional object in the three-dimensional matrix;
a first light source optically coupled to the sample holder and configured to illuminate at least some portion of the three-dimensional object in the three dimensional matrix that is received by the sample holder, wherein an wavelength provided by the first light source is selected to excite at least one species present in the three-dimensional object;
at least one objective optically coupled to the light source and configured to receive a light emission from the at least one excited species present in the three-dimensional object in the three-dimensional matrix;
a detector optically coupled to the at least one objective and configured to receive the light emission from the objective;
a processor electrically coupled to the detector,
wherein the microscope system is configured to pre-scan the three-dimensional matrix using a first magnification objective to identify an x-y location of the three-dimensional object and to identify a z-height for the three-dimensional object, and wherein the microscope system is configured use a second magnification objective to scan the identified three-dimensional object in a z-direction using the identified z-height of the identified three-dimensional object to provide a three-dimensional image of at least some portion of the three-dimensional object.

### Claim 17 (depends on 16)

The microscope system of claim 16, wherein the system comprises a first objective and a second objective, and wherein first objective is used as the first magnification objective and the second objective is used as the second magnification objective, and wherein the second objective is different than the first objective and provides a magnification greater than or equal to a magnification of the first objective.

### Claim 18 (depends on 17)

The microscope system of claim 17, wherein the first objective is an air objective and the second objective is a water immersion objective.

### Claim 19 (depends on 18)

The microscope system of claim 18, wherein the detector comprises at least one camera and the first light source comprises at least one laser, and wherein the system is configured to use the camera and the laser to perform laser scanning confocal microscopy during the pre-scan and scan of the three-dimensional object.

### Claim 20 (depends on 19)

The microscope system of claim 19, wherein the processor is configured to execute instructions to construct a three-dimensional image of the three-dimensional object in the three-dimensional matrix from an image set obtained from the scan step.