# US 2019/0170646 A1 · Light-sheet microscope with parallelized 3d image acquisition

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- Number: US 2019/0170646 A1
- Document ID: US20190170646A1
- Status: Published application
- Kind: A1
- Application number: 16/093,561
- Filed: 2017-04-12
- Published: 2019-06-06
- Earliest claimed: 2016-04-12
- Assignees: Board of Regents, The University of Texas System
- Applicants: The Board of Regents of the University Of Texas System
- Inventors: Reto P. Fiolka
- Web page: https://patentgrep.com/patent/US20190170646A1

## CPC

- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- 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 21/0076: {arrangements using fluorescence or luminescence}
- G02B 21/008: {Details of detection or image processing, including general computer control}
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes 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}
- G01N 2021/6463: {Optics}
- G01N 2201/061: Sources
- G02B 27/1066: {for enhancing image performance, like resolution, pixel numbers, dual magnifications or dynamic range, by tiling, slicing or overlapping fields of view}
- G02B 27/144: {using partially transparent surfaces without spectral selectivity (G02B27/147 takes precedence)}
- 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

This disclosure includes an imaging system that is configured to image in parallel multiple focal planes in a sample uniquely onto its corresponding detector while simultaneously reducing blur on adjacent image planes. For example, the focal planes can be staggered such that fluorescence detected by a detector for one of the focal planes is not detected, or is detected with significantly reduced intensity, by a detector for another focal plane. This enables the imaging system to increase the volumetric image acquisition rate without requiring a stronger fluorescence signal. Additionally or alternatively, the imaging system may be operated at a slower volumetric image acquisition rate (e.g., that of a conventional microscope) while providing longer exposure times with lower excitation power. This may reduce or delay photo-bleaching (e.g., a photochemical alteration of the dye that causes it to no longer be able to fluoresce), thereby extending the useful life of the sample.

## Claims

### Claim 1

A method for performing parallelized image acquisition with reduced photo-bleaching and blurring, the method comprising:
generating, by a light source, a beam of light;
splitting the beam of light to produce a plurality of beams of light of equal power, where the power of each of the plurality of beams of light is less than the power of the beam of light generated by the light source;
illuminating a sample using the plurality of beams of light, where the sample is disposed on a coverslip, and the coverslip is oriented such that different portions of the sample are illuminated in parallel by a different one of the plurality of beams of light, and beams of light that illuminate adjacent portions of the sample are spatially separated with respect to each other;
capturing, by a plurality of cameras, image data representative of fluorescence emitted by the illuminated portions of the sample, where each of the plurality of cameras captures image data representative of fluorescence emitted by a respective one of the different portions of the sample in parallel; and
generating one or more images representative of the sample based on the captured image data.

### Claim 2 (depends on 1)

The method of claim 1, where the plurality of beams of light comprises at least a first beam of light and a second beam of light, and the plurality of cameras comprises at least a first camera and a second camera.

### Claim 3 (depends on 2)

The method of claim 2, where the first camera captures first image data representative of fluorescence emitted by a first portion of the sample that has been illuminated by the first beam of light, and the second camera captures second image data representative of fluorescence emitted by a second portion of the sample that has been illuminated by the second beam of light.

### Claim 4 (depends on 1)

The method of claim 1, where, during the illumination, each of the plurality of beams of light is separated from adjacent ones of the plurality of beams of light by a threshold distance.

### Claim 5 (depends on 1)

The method of claim 1, where the cover slip is oriented at an angle with respect to an illumination objective and a detection objective, the illumination objective is configured to illuminate the different portions of the sample using the plurality of beams of light, and the detection objective is configured to detect the fluorescence emissions emitted by the illuminated sample.

### Claim 6 (depends on 1)

The method of claim 1, further comprising:
moving the coverslip to a new location, where movement of the coverslip causes additional portions of the sample to be illuminated by the plurality of beams of light, different ones of the additional portions of the sample are illuminated in parallel by different ones of the plurality of beams of light, and the beams of light that illuminate adjacent ones of the additional portions of the sample are spatially separated with respect to each other;
capturing additional image data representative of fluorescence emitted by the additional portions of the sample, where each of the plurality of cameras captures image data representative of fluorescence emitted by a respective one of the additional portions of the sample in parallel; and
generating one or more additional images representative of the additional image data.

### Claim 7 (depends on 6)

The method of claim 6, further comprising generating a 3D data set for each camera, where, for each camera, the 3D data set comprises a series of 2D images generated based on the image data captured while the sample is at an initial position and additional image data captured after the sample has been moved to one or more additional locations.

### Claim 8 (depends on 7)

The method of claim 7, further comprising generating a 3D image of the entire sample by combining the 3D data sets generated for each of the cameras.

### Claim at position 9

-15. (canceled)

### Claim 16

An imaging system for performing parallelized image acquisition with reduced photo-bleaching and blurring, the imaging system comprising:
a light source to generate a beam of light;
one or more beam splitters configured to produce a plurality of beams of light of equal power, where the power of each of the plurality of beams of light is less than the power of the beam of light generated by the light source;
an illumination objective to illuminate a sample using the plurality of beams of light, where the sample is disposed on a coverslip;
a detection objective to detect fluorescence emitted by the sample, where the coverslip is oriented such that different portions of the sample are illuminated in parallel by a different one of the plurality of beams of light in a staggered configuration such that beams of light that illuminate adjacent portions of the sample are spatially separated with respect to each other along a detection direction;
a plurality of cameras to capture image data representative of fluorescence emitted by the illuminated portions of the sample as detected by the detection objective, where each of the plurality of cameras captures image data representative of fluorescence emitted by a respective one of the different portions of the sample in parallel;
at least one processor to generate one or more images representative of the sample based on the captured image data; and
a memory coupled to the at least on processor.

### Claim 17 (depends on 16)

The imaging system of claim 16, where the plurality of beams of light comprises at least a first beam of light and a second beam of light, the plurality of cameras comprises at least a first camera and a second camera, the first camera captures first image data representative of fluorescence emitted by a first portion of the sample that has been illuminated by the first beam of light, and the second camera captures second image data representative of fluorescence emitted by a second portion of the sample that has been illuminated by the second beam of light, and the first portion and the second portion of the sample are staggered with respect to each other.

### Claim 18 (depends on 16)

The imaging system of claim 16, where the cover slip is oriented at a forty five degree (45°) angle with respect to the illumination objective and the detection objective, and the angle of the cover slip provides the staggered configuration.

### Claim 19 (depends on 16)

The imaging system of claim 16, further comprising:
means for moving the coverslip to a new location, where movement of the coverslip causes additional portions of the sample to be illuminated by the plurality of beams of light, different ones of the additional portions of the sample are illuminated in parallel by different ones of the plurality of beams of light, and the beams of light that illuminate adjacent ones of the additional portions of the sample are spatially separated with respect to each other, and the plurality of cameras capture additional image data representative of fluorescence emitted by the additional portions of the sample.

### Claim 20 (depends on 19)

The imaging system of claim 19, where the image data and the additional image data are stored in the memory, and the at least one processor is configured to:
generate a 3D data set for each camera, where, for each camera, the 3D data set comprises a series of 2D images generated based on the image data captured while the sample is at an initial position and additional image data captured after the sample has been moved to one or more additional locations; and
generate a 3D image of the entire sample by combining the 3D data sets generated for each of the cameras.

### Claim 21

A method for performing parallelized image acquisition with reduced photo-bleaching and blurring, the method comprising:
generating, by a light source, a beam of light;
process the beam of light to produce a plurality of pencils of light;
illuminating a sample using the plurality of pencils of light, where the sample is disposed within a volume, and where the plurality of pencils of light are staggered such that different portions of the sample are illuminated in parallel by a different one of the plurality of pencils of light, and where pencils of light that illuminate adjacent portions of the sample are spatially separated with respect to each other;
capturing, by one or more cameras, image data representative of fluorescence emitted by the illuminated portions of the sample, where each of the plurality of cameras captures image data representative of fluorescence emitted by a respective one of the different portions of the sample in parallel;
modifying the illumination of the sample within the volume using the plurality of pencils of light, where additional image data is captured by the one or more cameras subsequent to modifying the illumination of the sample; and
generating one or more images representative of the sample based on the captured image data.

### Claim 22 (depends on 21)

The method of claim 21, where modifying the illumination of the sample within the volume comprises:
sweeping the plurality of pencils of light across the volume in a first direction; and
controlling one or more galvanometers to facilitate the sweeping of the plurality of pencils of light in the first direction.

### Claim 23 (depends on 22)

The method of claim 22, where modifying the illumination of the sample within the volume comprises:
modifying a location of the plurality of pencils of light in a second direction to illuminate additional portions of the sample within the volume; and
controlling the one or more galvanometers to facilitate the modifying of the location of the plurality of pencils of light in the second direction.

### Claim 24 (depends on 21)

The method of claim 21, where the one or more cameras comprises a single camera, the method further comprising:
capturing, by the single camera, the image data representative of fluorescence emitted by the illuminated portions of the sample, where, for each of the plurality of the plurality of pencils of light, the single camera is configured to capture image data representative of fluorescence emitted by the sample in response to illumination by the plurality of pencils of light in parallel, where fluorescence emitted by the sample in response to illumination by one of the plurality of pencils of light is mapped to first active pixels of the camera, and where fluorescence emitted by the sample in response to illumination by another one of the plurality of pencils of light is mapped to second active pixels of the camera;
correlating image data corresponding to a particular one of the plurality of pencils of light to generate one or more images representative of the sample based on the captured image data, each of the one or more images corresponding to fluorescence emitted by the sample in response to illumination by a respective one of the plurality of pencils of light; and
combining the one or more images to generate a three dimensional image.

### Claim 25 (depends on 21)

The method of claim 21, where the one or more cameras comprises two or more cameras, the method further comprising:
capturing, by a first camera of the two or more cameras, first image data representative of fluorescence emitted by a first portion of the sample in response to illumination by a first one of the plurality of the plurality of pencils of light; and
capturing, by a second camera of the two or more cameras, second image data representative of fluorescence emitted by a second portion of the sample in response to illumination by a second portion of the sample by a second pencil of light of the plurality of the plurality of pencils of light, where at least a portion of the first image data and the second image data is captured in parallel; and
generating the one or more images based on image data captured by each of the two or more cameras, where a first image of the one or more images is generated based on image data captured by the first camera, and where a second image of the one or more images is generated based on image data captured by the second camera; and
combining the images generated based on the image data captured by the first camera and the second camera to generate a three dimensional image.