System, device and method for three-dimensional imaging of moving particles

Abstract

Disclosed are methods, devices, systems, and applications for camera-free, high-throughput three-dimensional imaging of particles in motion. In some aspects, the system includes: a particle moving device to allow the particle to move along a travel path; an optical illumination system to generate an asymmetric illumination area of ​​light in the range of the particle's travel path, the optical illumination system scanning multiple portions of the particle at multiple time points while the particle moves; an optical detection system optically connected to the particle moving device to obtain light signal data associated with different portions of the particle corresponding to the particle volume during movement on the travel path; and a data processing unit to process the light signal data obtained by the optical detection system and generate data including information indicating the 3D characteristics of the particle.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent document claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 710,576, entitled “SYSTEMS, DEVICES AND METHODS FOR THREE-DIMENSIONAL IMAGING FLOWCYTOMETRY,” filed on February 16, 2018. The entire contents of the above patent application are incorporated herein by reference as part of the disclosure of this patent document.

[0003] Federally Sponsored Research or Development

[0004] This invention was made with government support from the National Institutes of Health (NIH) under Grant No. DA045460. The government has certain rights in this invention. Technical Field

[0005] This patent document relates to techniques and systems for flow cytometry. Background Art

[0006] Flow cytometry is a technique for detecting and analyzing particles such as living cells when they flow through a fluid. For example, a flow cytometer can be used to characterize the physical and biochemical properties of cells and / or biochemical molecules or molecular clusters based on the optical, electrical, acoustic and / or magnetic responses of cells and / or biochemical molecules or molecular clusters when they are interrogated in a serial manner. Typically, flow cytometry uses an external light source to interrogate particles, from which light signals caused by one or more interactions (e.g., forward scattering, side scattering and fluorescence) between the input light and the particles can be detected. The properties measured by flow cytometry include the relative size, granularity and / or fluorescence intensity of the particles. Summary of the invention

[0007] Disclosed are methods, devices, systems and applications related to three-dimensional imaging flow cytometry (3D-IFC).

[0008] In some aspects, a system for three-dimensional (3D) imaging of moving particles includes: a particle moving device including a substrate for allowing the particle to move along a travel path in a first direction; an optical illumination system for generating an asymmetric illumination region of light within the range of the travel path of the particle, the optical illumination system scanning multiple portions of the particle at multiple time points while the particle moves, the asymmetric illumination region of light including an illumination dimension that is thinner than other illumination dimensions and forms a shape similar to an illumination plane, the optical illumination system including a light source that generates a light beam, the light source being optically coupled to a light redirecting device to modify the light beam to a different angle by redirecting the light beam so that the asymmetric illumination region of light is directed toward the travel path of the particle; an optical detection system, optically connected to a particle moving device and operable to obtain light signal data associated with different portions of a particle corresponding to a volume of the particle during movement of the particle along a travel path, wherein the optical detection system comprises: one or more photodetectors and a spatial filter located between the particle moving device and the one or more photodetectors, the spatial filter comprising a plurality of apertures to selectively allow a portion of an asymmetric illumination area of ​​light on a scanned portion of the particle to pass through the spatial filter and be detected by the one or more photodetectors; and a data processing unit in communication with the optical detection system, the data processing unit comprising a processor, wherein the processor is configured to process the light signal data obtained by the optical detection system and generate data comprising information indicative of 3D features of the particle.

[0009] In some aspects, a method for three-dimensional (3D) imaging of a moving particle includes: moving the particle along a first direction; scanning multiple portions of the particle portion by portion by directing separate asymmetric illumination areas of light to corresponding ranges while the particle is moving; spatially filtering portions of the asymmetric illumination areas of light on the scanned portions of the moving particle to allow detection of certain light signals corresponding to the particle volume passing through the spatial filter; and detecting the spatially filtered light signals to obtain separate voxels in three dimensions mapped to the detection time point.

[0010] The subject matter described in this patent document and the attached appendix can be implemented in a specific way providing one or more of the following features. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A A block diagram of an exemplary embodiment of a three-dimensional imaging flow cytometry (3D-IFC) system in accordance with the present technology is shown.

[0012] Figure 1B Shows Figure 1A FIG. 1 is a block diagram of an exemplary embodiment of a data processing system for a 3D-IFC system.

[0013] Figure 1CA diagram illustrating an exemplary implementation of a 3D-IFC system in accordance with the present technology.

[0014] Figure 1D Shown in Figure 1C The cells flowing at the object plane and the Figure 1C Illustration of the spatial filter at the image plane.

[0015] FIG. 2 shows a diagram illustrating the principle of the relationship between a time domain signal and spatial information including spatial light intensity.

[0016] Figure 3A and Figure 3B A data graph of a time domain signal from an exemplary 3D-IFC system is shown, according to some exemplary embodiments.

[0017] FIG. 4A to FIG. 4I Data graphs showing 2D and 3D images captured by an exemplary 3D-IFC system, according to some exemplary embodiments.

[0018] Figure 5A A diagram illustrating an exemplary implementation of a 3D-IFC system in accordance with the present technology.

[0019] Figure 5B Shown is depicted along Figure 5A The object plane flows through the cells and is located Figure 5A Illustration of the spatial filter at the image plane.

[0020] Figure 5C An example of a 3D reconstruction space generated by a data processing system of an exemplary 3D IFC system is shown.

[0021] FIG. 5D to FIG. 5F An example output of an example 3D-IFC system demonstrating time to 3D space mapping is shown in accordance with some example embodiments.

[0022] FIG. 6A to FIG. 6C Shown are images and data graphs of an exemplary 3D-IFC system using CFSE-stained HEK-293 cells bound to 1 μm fluorescent beads, according to some exemplary embodiments.

[0023] Fig. 7A and Figure 7B Shown are images and data graphs of fluorescent γH2AX foci imaged by an exemplary 3D-IFC system, according to some exemplary embodiments.

[0024] FIG. 8A to FIG. 8D Shown are images and data graphs depicting leukocyte cell morphology determined by an exemplary 3D-IFC system, according to some exemplary embodiments.

[0025] Fig. 9 An example of a microfluidic system for use in an exemplary 3D-IFC system is shown, according to some exemplary embodiments.

[0026] Fig.10 A diagram showing an example for spatial filter design, according to some exemplary embodiments.

[0027] Fig.11 A diagram illustrating an exemplary implementation of a 3D-IFC system in accordance with the present technology. DETAILED DESCRIPTION

[0028] A central challenge in biology is to relate the phenotypes of heterogeneous individuals in a population to their genotypes to understand the extent to which they conform to observed group behavior or stand out as exceptions that drive disease or as threats to health. While optical microscopy has been the foundational method for studying the morphology and molecular composition of biological specimens, flow cytometry has become the gold standard for quantitative high-throughput single-cell characterization in many biomedical applications. Conventional imaging flow cytometry (IFC) is a technique that combines some aspects of optical imaging with flow cytometry. For example, IFC can simultaneously generate ensemble-averaged measurements and high-content spatial measures from individual cells in a large population of cells without interference caused by changes in experimental conditions. However, an important limitation of existing IFC systems is that only 2D cell images can be obtained, regardless of the optical detection method and computational algorithm used. The lack of 3D tomography results in occlusion of objects, blurring due to depth of focus, loss of z-axis spatial resolution, and artifacts caused by projecting 3D cells into 2D images. For example, with 2D microscopy, if a fluorescent probe is observed at the center of a cell, its location (e.g., membrane, cytosol, nucleus) is ambiguous. For some applications (e.g., internalization measurements, probe colocalization, and spot counting) that are relevant to 2D imaging (which depends on the cell being oriented to the imaging plane), 3D images can provide more complete and accurate phenotypes of cell and organelle morphology as well as nucleic acid and protein localization to support biological insights, where 2D imaging depends on the cellular orientation of the imaging plane. Therefore, rapid and continuous 3D image acquisition of single cells in flow is required.

[0029] The need for continuous, sub-millisecond temporal resolution using fluorescence microscopy remains unaddressed. Imaging cells in flow can rapidly perform high-throughput morphology, translocation, and cell signaling analysis on large populations of cells. Compared to single-point flow cytometry, three-dimensional imaging flow cytometry (3D-IFC) provides high-content information about the 3D spatial distribution of fluorescence and / or scattered light, which can be used for high-throughput rare cell detection and heterogeneous phenomenon studies.

[0030] Methods, devices, systems, and applications for camera-free, high-throughput three-dimensional imaging of particles in motion are disclosed. In certain aspects, for example, a three-dimensional imaging flow cytometry (3D-IFC) system is arranged in a light sheet fluorescence microscope structure that illuminates a specimen in a single plane while detecting signals in a perpendicular direction, for example, through a spatial filter. For example, a 3D-IFC system may include: an acousto-optic deflector that provides high-speed scanning in the z direction. For example, a 3D-IFC system may include a single-element photodetector to detect fluorescent optical signals that pass through a spatial filter in each signal channel and reconstruct a 3D image from the time-domain detector output. The 3D-IFC system integrates the scanning light sheet, the flow movement of cells, and the spatial filter to enable the detector to detect a single point in the cell at a time, which allows for space-to-time mapping on a one-to-one basis, i.e., so-called spatiotemporal conversion.

[0031] Although some disclosed embodiments of systems, methods, and apparatus for three-dimensional imaging of moving particles are described herein as being primarily used for imaging flow cytometry to facilitate understanding of the basic concepts, it should be understood that the disclosed embodiments may also include techniques and systems for moving particles in other ways or forms and may be used for other applications.

[0032] The disclosed systems, methods, and apparatus are capable of mapping 3D features of a moving object (e.g., a cell, particle, or other object) to a time-series signal at the detector output on a pixel-by-pixel basis as the cell / particle / object passes through (e.g., by flow, sliding, falling, or other mechanism) an optical interrogation area defined by an optical illumination area, wherein the optical illumination area is referred to as a "sheet of light," i.e., a rapid scan. In some embodiments, the disclosed systems, methods, and apparatus include a spatial filter / mask placed in front of the optical detector to allow only light signals generated from a specific volume (e.g., a point volume) and at a specific time to reach the detector. Signals outside the specific volume or at a specific point in time are blocked by the spatial mask.

[0033] Figure 1AA block diagram of an exemplary embodiment of a system labeled 100 for three-dimensional imaging of moving particles according to the present technology is shown. The system 100 includes a particle moving system 110, a scanning sheet light illumination system 120 connected to the particle moving system 110, an optical detection system 130 connected to the particle moving system 110, and a data processing system 140 in communication with the optical detection system 130 and / or the scanning sheet light illumination system 130. In some embodiments, the system 100 operates as a single point detector by converting optical detection information about a sample flowing between space and time to construct a three-dimensional image based on the converted information. For example, when the sample moves through the particle moving system 110, the illumination system 120 does not focus light on a single point of the sample, but focuses light along a 2D plane, while the optical detection system 130 detects individual light points from the three-dimensional space around the planar illuminated sample based on a spatial filter map having a pattern that allows only certain portions of light to pass through to limit certain point signals from entering the detector. This optomechanical technique structures light on a three-dimensional object in two dimensions (e.g., a planar "sheet of light") while scanning an optically shielded one-dimensional point of the planar sheet of light as the object moves. The system 100 is capable of producing 3D cytoscopy images of particles, cells, or other moving objects at fast flow rates (e.g., 10 cm / sec), allowing high throughput 3D imaging.

[0034] In some embodiments of the system 100, the particle moving device 100 includes a substrate that allows the particle to move along a travel path in a first direction (e.g., a moving direction). The scanning light sheet illumination system 120 is configured to generate a light sheet within the range of the particle's travel path, and the scanning light sheet illumination system 120 scans multiple parts of the particle at multiple time points while the particle moves. The generated scanning light sheet is an asymmetric illumination area of ​​light, which includes an illumination dimension that is thinner than other illumination dimensions to form a shape similar to an illumination plane. The scanning light sheet illumination system 120 includes a light source (e.g., a laser) that generates a light beam, and the light source is optically coupled to a light redirection device to modify the light beam by redirecting the light beam to different angles so that the light sheet is directed to the travel path of the particle. The optical detection system 130 is optically connected to the particle moving device 110 and is operable to obtain light signal data associated with different parts of the particle during the movement of the particle along the travel path, wherein the different parts of the particle correspond to the volume of the particle. The optical detection system 130 includes one or more photodetectors and a spatial filter located between the particle moving device 110 and the one or more photodetectors, wherein the spatial filter includes one or more holes to selectively allow a portion of the asymmetric illumination area of ​​light above the scanned portion of the particle to pass through the spatial filter and be detected by the one or more photodetectors. The data processing unit 140 communicates with the optical detection system 130 (and, in some embodiments, with the scanning sheet light illumination system 120) and is configured to process the optical signal data obtained by the optical detection system 130 and generate data including information indicating the 3D characteristics of the particle.

[0035] In some embodiments, the particle movement system 110 is sometimes referred to as a "flow cell" in such related embodiments, and includes a substrate having a fluid channel, wherein the fluid channel is used to carry a fluid sample containing a flowing sample (e.g., particles, living cells, or other objects). In some embodiments, the particle flow cell system includes a flow focusing system to provide a confined sample fluid to a thin stream in the fluid channel. In some embodiments, the fluid channel of the flow cell is configured to be optically transparent in the interrogation range, thereby allowing a clear light path. The fluid containing the flowing sample flows through the interrogation area in the fluid channel along the flow direction, wherein the imaging system 120 obtains optical data for each particle including a single particle or a single cell.

[0036] However, in some embodiments, the particle movement system 110 includes a substrate to which one or more particles, cells (or other objects for interrogation) are fixed, wherein the substrate moves relative to the scanning light illumination system 120 and the optical detection system 130 so that the particles, cells, etc. are brought into an interrogation range, where the scanning light is illuminated and from which light at and / or passing through the particles, cells, etc. can be detected. In such embodiments, the particle movement system 110 includes a device positioning system that includes a moving stage (e.g., driven by a motor or other drive device) to move the substrate so that the particles fixed thereon move along a travel path and are brought into the range over which the scanning light is illuminated.

[0037] In some embodiments, the scanning light sheet illumination system 120 includes a light source that generates light that will be configured as a sheet of light for illuminating particles flowing in a fluid channel of the particle moving system 110. In some embodiments, the light source includes a laser; and / or in some embodiments, the light source includes a high brightness LED or a superluminescent diode. The illumination system 120 includes a device for redirecting a light beam generated by the light source to different angles, which can be programmed to produce periodic angle changes at high speed (e.g., in the range of at least 100kHz to 1MHz). The beam redirection device can be optically coupled with a passive optical component (e.g., a cylindrical lens or other optical component, such as a lens, prism, or spatial filter) to shape the redirected light and produce a structured light sheet illumination that allows optical sectioning of moving particles. For example, the illumination system 120 can be used to illuminate a thin slice or thin line of particles at a time, such as a scanning light sheet on the particles.

[0038] In some embodiments, for example, the light redirection device includes an acousto-optic deflector (AOD) device. For example, an AOD can provide controllable, high-speed beam angle changes with high optical efficiency, where the AOD moves the energy of the beam without losing beam portions. While the AOD can reliably, quickly, and efficiently redirect light, it is wavelength sensitive and may not be optimal for some applications where multiple beams of different wavelengths are to be employed in an implementation of the system 100.

[0039] For example, in some embodiments, in addition to or in lieu of the AOD, the light redirection device may include a multiplexed array of scanning mirrors and / or a multiplexed array of scanning gratings that are not wavelength-restricted. For example, a single optical component such as a scanning mirror will provide a scanning speed of approximately 20 kHz; however, a multiplexed array of scanning mirrors may be configured to provide a high speed of, for example, 200 kHz.

[0040] In some embodiments, the optical detection system 130 can be placed at any angle orthogonal to the path of travel of particles, cells, etc., for example, perpendicular to a fluid channel with a clear optical path of a particle flow pool. In some embodiments, the optical detection system 130 includes a photodetector and a spatial filter located at the image plane of the photodetector to selectively allow light from a specific position of the particle to pass through for detection by the photodetector. Based on a combination of the following conditions, the optical detection system 130 can be operated to detect 1D optical data points of a 3D object in time by detecting light intensity (e.g., including fluorescence and scattering) from each position of the particle in a sequentially timed manner based on (a) the flow movement of the particle, (b) scanning sheet light illumination, and (c) a combination of spatial mapping filters. Therefore, the time domain signal recorded by the photodetector of the optical detection system 130 has a one-to-one relationship from space to time. In some implementations, the optical detection system 130 includes a filter to collect and detect fluorescence and scattered light. For example, the optical detection system 130 may include multiple photodetectors for detecting multiple channels of fluorescence and / or scattered light.

[0041] In some embodiments, the spatial filter includes a surface having a plurality of holes (openings or slits) arranged in a pattern on the surface. In some embodiments, the slits are spaced apart along a first direction in which the particles move and a second direction perpendicular to the first direction, wherein the positions of adjacent slits vary along the second direction relative to another slit of the pattern. In some examples, the holes may be configured as other geometric images and shapes, for example, including but not limited to squares, rectangles, trapezoids, triangles, etc., or other curved shapes including ellipses, circles, etc. In various embodiments, the size of the hole may be selected based on the desired resolution of the image data to be constructed by the detectable light signal. For example, the diameter or length of the hole may be determined by multiplying the magnified intensity of the light directed to the image plane by the desired resolution. In one example, if the desired resolution is 1 μm and the objective lens of the detection module 170 is 20x, the slit will be configured to have a diameter of 20 μm. Similarly, for example, if the desired resolution is 0.5 μm and the objective lens of the detection module 170 is 50x, the slit will be configured to have a diameter of 25 μm. In some implementations, for example, a relatively small aperture (relative to the size of the particle) effectively allows the detected light to be processed as a delta function.

[0042] The data processing system 140 is configured to process the detected optical signal data and generate data (e.g., a data set) indicating three-dimensional features of particles, cells, etc., from which a 3D image or density distribution of particles, cells, etc. can be constructed. For example, the data may represent an optical density distribution in a 3D space, which is very different from a 3D image obtained from a conventional 3D imaging mode (e.g., a confocal microscope). In some embodiments, the data processing system 140 includes a data stream transmission unit that communicates with the optical detection system 130. The data processing system 140 may include a processor and a memory (a data processing unit) to reconstruct a 3D image for each particle based on the detected optical signal data captured at the optical detection system 130. In some implementations, for example, an image reconstruction algorithm resides on the data processing unit, which includes specifications for the illumination and detection protocols and controls the scanning sheet light illumination system 120 and the optical detection system 130 accordingly. In some embodiments, the data processing system 140 includes an image processing unit to process the image data to determine properties associated with the particles.

[0043] Figure 1BA block diagram of an exemplary implementation of a data processing system 140 of the exemplary system 100 is shown. In various implementations, the data processing system 140 is embodied in one or more personal computing devices (e.g., including desktop or laptop computers), one or more computing devices in a computer system or communication network accessible via the Internet (referred to as the "cloud") (including servers and / or databases in the cloud), and / or one or more mobile computing devices, such as smartphones, tablet computers, or wearable computer devices (including smart watches or smart glasses). The data processing system 140 includes a processor and a memory, wherein the processor is used to process data and the memory communicates with the processor to store and / or buffer data. For example, the processor may include a central processing unit (CPU) or a microcontroller unit (MCU). In some implementations, the processor may include a field programmable gate array (FPGA) or a graphics processing unit (GPU). For example, the memory may include and store processor executable code that, when executed by the processor, configures the data processing system 140 to perform various operations, such as receiving information, commands and / or data, processing information and data such as from the system 100, and transmitting or providing the processed information / data to another device such as an actuator or an external display. To support the various functions of the data processing system 140, the memory may store information and data, such as instructions, software, values, images, and other data processed or referenced by the processor. For example, various types of random access memory (RAM) devices, read-only memory (ROM) devices, flash memory devices, and other suitable storage media may be used to implement the storage function of the memory. In some implementations, the data processing system 140 includes an input / output (I / O) unit to connect the processor and / or memory to other modules, units, or devices. In some embodiments, such as for mobile computing devices, the data processing system 140 includes a wireless communication unit, such as a transmitter (Tx) or a transmitter / receiver (Tx / Rx) unit. For example, in such an embodiment, the I / O unit may connect the processor and memory to a wireless communication unit, such as using various types of wireless interfaces compatible with typical data communication standards, which may be used in the communication of the data processing system 140 with other devices, for example, between one or more computers in the cloud or in a user device. Data communication standards include, but are not limited to, Bluetooth, Bluetooth Low Energy (BLE), Zigbee, IEEE 802.11, Wireless Local Area Network (WLAN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access (WiMAX)), 3G / 4G / LTE / 5G cellular communication methods, and parallel interfaces. In some implementations, the data processing system 140 may be connected to other devices using a wired connection via the I / O unit.The data processing system 140 may also be connected to other external interfaces, data storage sources, and / or visual or audio displays, etc., to retrieve and transmit data and information that can be processed by the processor, stored in the memory, or displayed on a display device or output unit of an external device.

[0044] Figure 1C A diagram of an exemplary embodiment of a 3D-IFC system 150 according to some embodiments of the system 100 is shown. As shown, the 3D-IFC system 150 includes a particle flow cell 151, which includes a fluid channel for carrying a fluid sample connected to a light sheet illumination module 160 and a detector module 170, wherein the fluid sample includes a flowing sample (e.g., particles, living cells, etc.). The light sheet illumination module 160 includes an acousto-optic deflector (AOD) 165 optically coupled to a cylindrical lens 161 and an objective lens (OL) 163, which are arranged in an optical illumination path with a light source 169 (e.g., a laser, such as a 488nm laser). For example, the cylindrical lens 161 is placed after the AOD 165 to focus the light (e.g., focus the laser beam) in one direction to form a scanning light sheet. For each frequency, the AOD will produce a deflected first-order beam at a different angle, thereby producing a scan over time. A detector module 170 including a spatial filter (SP) 171 is arranged in an optical detection path between an objective lens 172 and an optical photodetector 179 (e.g., a photomultiplier tube (PMT)) connected to the flow system 151 to capture optical signal data from flow objects (e.g., cells, particles, etc.). In some implementations, the detector module 170 detects one or more specific spectral ranges of the optical signal data. Similarly, in Figure 1C In some embodiments shown, for example, the detector module 170 includes: a spatial filter 171 optically coupled to a dichroic mirror (DM) 173 in a split beam path (wherein the dichroic mirror (DM) 173 is optically coupled to a bandpass filter (BP) 175) and a photodetector 179 (e.g., a PMT). For example, illumination can be performed perpendicular to the detection. The spatial filter 171 is placed at the image plane. In some embodiments, the 3D-IFC system 150 includes a lens or light guide to focus and / or guide light at a specified location of another component of the system. In some embodiments, for example, the PMT signal is collected by a digital recording device such as 100 megasamples per second (MS / s) per channel.

[0045] In some exemplary embodiments, the 3D-IFC system 150 is arranged in a light sheet fluorescence microscope configuration, which illuminates the flowing sample in a single plane while detecting signals in the perpendicular direction. The acoustic optical deflector (AOD) 165 can provide high-speed scanning in the z direction. Single-element photodetectors such as PMT 179 detect fluorescence passing through the spatial filter 171 in each channel, and a reconstructed image is output by the time domain detector. Combining the scanning light sheet, the flow movement of the cell, and the spatial filter, the detector detects only a single point in the cell at a time, thereby enabling space-to-time conversion.

[0046] Figure 1D A diagram depicting an exemplary flow sample (e.g., a cell) flowing along an object plane and a spatial filter 171 located at an image plane is shown. In some embodiments, the spatial filter 171 comprises a surface having a plurality of holes (openings) arranged in a pattern (e.g., slits), wherein each hole of the pattern is spaced along the y-axis (parallel to the direction of flow) and the position of each hole of the pattern varies along the x-axis (perpendicular to the direction of flow) relative to another hole of the pattern such that each hole along the y-axis is at a different position along the x-axis. Figure 1D In the exemplary spatial filter 171 shown, the apertures include circular (eg, elliptical or circular) slits having a diameter of approximately 20 μm.

[0047] As shown in the figure, a planar sheet of light is created in the xy plane, where the width of the light is defined along the y-axis (particle flow direction), and the x-axis (illumination direction) is the weight of the light. In the figure, the z-axis is the scanning axis controlled by AOD 165. In some implementations, the thickness of the sheet of light (z-axis) is about 1 μm. As the cell moves in the channel, an optical point scan of the cell is performed along the y-axis; and the x-axis scan is completed by the spatial filter diagram 171. In this example, scanning is performed in the z-direction, and the sheet of light generated by the sheet illumination module 160 has a waist of 0.6 μm (z-direction) and a length of 250 μm in the y-direction. The spatial filter has ten slits of 20 μm (x-direction) by 10 μm (y-direction), which are placed one by one.

[0048] FIG. 2A to FIG. 2D A diagram illustrating the relationship between a time domain signal (right) and spatial information (left) depicted as a cube in 3D space, showing spatial light intensity. Figure 2A An example of a time domain signal from a 3D-IFC representing the light intensity of a 3D object (a sphere in the example) is shown; each time point corresponds to a specific voxel in 3D space. Figure 2B An example of an envelope of a signal representing an xy plane projection of the entire 3D object is shown. Figure 2CExamples are shown where each highlighted section represents the light intensity of one yz plane section of 3D space. In these examples, the duration of each section is the time for the object to travel 20 μm in the object plane, which is equal to the time it takes for the image to pass from one slit to another along the spatial filter. Figure 2D An example is shown in which a single peak highlighted represents the light intensity of a row in the z direction, where the light sheet performs one scanning cycle.

[0049] The following equations may be used to describe some aspects of embodiments of the system 100. For example, when the frequency of the sound sent to the acoustic transducer of the acousto-optic deflector (AOD) varies sinusoidally, the position of the light sheet in the z direction, f(t), may be described as

[0050] f(t∈(0,T / 2))=v 1 t

[0051] f(t∈(T / 2,T))=2z 0 -v 1 t (1)

[0052] For example, if the audio frequency varies in a sawtooth manner, then f(t) can be described as

[0053] f(t)=v 1 t, (2)

[0054] Where T is the scanning period, v 1 is the scanning speed in the z direction, z 0 is the waist of the light in the z direction.

[0055] Sheet light I(x, y, z, t) can be written as

[0056]

[0057] The characteristic function F(x, y) of the spatial filter is designed as

[0058]

[0059] Where x=1, 2, ..., N is the number of slits on the spatial filter, and L is the slit length in the y direction.

[0060] The measured PMT signal S(t) can be expressed as

[0061] S(t)=∫ x，y，z Cell(x,y-Mv 2 t, z) I (x, y, z, t) F (x, y) dxdydz = ∫ x，y Cell(x,y-Mv 2t,f(t))F(x,y)dxdy (5)

[0062] Wherein, M is the magnification of the detection system, and Cell(x, y, z) is the 3D cell fluorescence intensity distribution map.

[0063] This exemplary method maps 3D cell images into time-domain light intensity on a one-to-one basis.

[0064] Implementation of the exemplary embodiment of the system 100 can be used for imaging and characterization of biological samples such as living cells. For example, a single cell can be imaged in a suspension flowing at a speed of 0.1 m / s in a flow cell tube. In the exemplary embodiments described herein, some of the single cell samples that have been imaged include HEK293T cells stained with CellTrace CFSE, where 1 μm fluorescent beads are bound to the cell membrane of the HEK293T cells.

[0065] Figure 3A and Figure 3B An example of a data plot of a time domain signal from an exemplary 3D-IFC system 150 is shown. The object (i.e., HEK293T cells stained with CellTrace CFSE with 1 μm fluorescent beads bound to their cell membranes) may flow at a speed of 0.1 m / s. Figure 3A , the data graph shows the time domain data output from a PMT of one channel (e.g., 520 / 40 nm bandpass), Figure 3B Time domain data from the PMT output of another channel (eg, 650 nm long pass) is shown. The detected envelope can be used to reconstruct a 2D image of the cell. Figure 3A and Figure 3B The data graphs of 150 and 151 show time domain signal cropping of a cell using the 3D-IFC system 150 as an example. In these exemplary implementations, the light sheet is scanned at 200 kHz over a range of 25 μm in the z direction.

[0066] FIG. 4A to FIG. 4I An exemplary data graph showing 2D and 3D images of HEK293T cells captured by an exemplary 3D-IFC system 150 is shown, wherein the HEK293T cells are stained with CellTrace CFSE and have 1 μm fluorescent beads bound to their cell membranes. FIG. 4A to FIG. 4C In the data graphs, 2D images showing green channel (401), red channel (402), and overlay (403) image data are shown, respectively, where the 2D images are captured and reconstructed by the exemplary 3D-IFC system 150. In these data graphs, the scale bar represents 5 μm. FIG. 4D to FIG. 4F, data plots illustrate 3D reconstructed images showing green channel (404), red channel (405), and merged (406) 3D reconstructed image data generated by an exemplary 3D-IFC system 150. In these data plots, a cubic cage represents a space of 20 μm×20 μm×20 μm. FIG. 4G to FIG. 4I Six different 2D perspectives of 3D imaged cells in the green channel (perspectives 407-411), red channel (perspectives 412-417) and overlay (perspectives 418-423) of the 3D image are shown. As shown in the 3D reconstructed images, the 2D perspectives at different viewing angles have different characteristics.

[0067] FIG. 4A to FIG. 4I The 3D image shown in Figure 2 shows four fluorescent beads on the cell membrane, but the 2D image contains unclear information about the number of beads. In a flow system, there is little control over the orientation of the object, so capturing a 2D image with only one viewing angle can produce detection errors, especially when relative position is important. Since biological samples are inherently three-dimensional, capturing 3D images provides complete information about the sample.

[0068] An exemplary method for implementing and / or manufacturing an exemplary 3D-IFC system 150 is described. In the exemplary implementation described above, a 100mW 488nm laser (iBeam-SMART, Toptica) was used, which had an elliptical beam with a Gaussian energy distribution, which was collimated, focused, and expanded to illuminate an area of ​​1.2μm (z direction) by 250μm (y direction). An AOD (Isomet) driven by a modular, swept RF power supply (Isomet) was used to generate a deflected laser beam with different output angles at different times, and thus scan the laser beam in the z direction. A cylindrical lens and a 10× / 0.25 objective lens were used to form a sheet of light. Fluorescence emission from the sample was collected by a 10× / 0.25 objective lens (Mitutoyo), and the fluorescence emission from the sample was detected by one PMT (Hamamatsu) for each channel. The PMT readings were recorded by a PCIE DAQ card (Advantech).

[0069] The manufacture of spatial filters may include the following techniques. The design of the spatial filter is drawn in AutoCAD and printed onto a transparent mask at a speed of 20,000 dots per inch (dpi). A layer of negative photoresist (NR9-1500PY, Futurrex, Inc.) is spun at a speed of 3,000 revolutions per minute (rpm) on a 6-inch glass wafer. The wafer is heated on a hot plate at 150°C for 3 minutes and then exposed to UV light (EVG620NT, EV Group) through a transparent mask. After ultraviolet irradiation, the wafer is baked at 100°C for another 3 minutes and then developed in RD6 (Futurrex, Inc.) for 12 seconds. A 300nm thick aluminum film is sputtered onto the glass wafer. After metal stripping, the pattern of the spatial filter is formed and the glass wafer is cut into 12mm by 12mm slices. To help fix the spatial filter in the flow cytometry system, a spatial filter with ten 20 μm by 10 μm slits was mounted onto a sample holder fabricated by a 3D printing method.

[0070] Preparation of cell samples may include the following techniques. HEK293T human embryonic kidney cell samples were cultured in 10 cm culture dishes with culture medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin) to 90% confluence. After 100-fold dilution of 1.0 μm fluorescent beads (Ex / Em: 488 / 645 nm, T8883, Thermo Fisher) in the stock solution, 100 μL of the diluted solution was mixed with 10 mL of fresh cell culture medium and added to the cell culture plate. After 10 hours of continuous culture, HEK293T cells were harvested and fixed with 4% paraformaldehyde, washed and resuspended in 1X phosphate-buffered saline (PBS). After fixation, the cells were stained with the CellTrace CFSE Cell Proliferation Kit (Ex / Em: 492 / 517 nm, Thermo Fisher). Before each imaging experiment, the suspension was diluted to a concentration of 200 cells / μL in PBS.

[0071] Exemplary applications of the disclosed 3D-IFC systems and methods may include the following.

[0072] DNA damage: When ionizing radiation or cytotoxic chemicals cause DNA damage and form double-strand breaks (DSBs), phosphorylated protein γ-H2AX foci are rapidly formed and represent DSBs in a 1:1 manner. Using antibodies against γ-H2AX, detection and visualization of γ-H2AX by flow cytometry and immunofluorescence microscopy are used to assess DNA damage, related DNA damage proteins, and DNA repair. 2D imaging techniques and manual quantification have been widely used by many researchers. However, counting the number of foci based on 2D images is not only labor-intensive, but also unreliable when the number of foci is large and overlaps at an angle. Compared with 2D techniques, obtaining 3D spatial resolution at high throughput has significant advantages for analyzing the number, area, and density of γ-H2AX. For example, an exemplary embodiment of the 3D-IFC system 150 described herein can provide a high-throughput cytometer suitable for quantification of γ-H2AZ foci.

[0073] FISH: Fluorescence in situ hybridization (FISH) can effectively detect and locate the presence or absence of specific DNA sequences on chromosomes, and achieve high accuracy in disease diagnosis and prognosis assessment. FISH signal analysis is usually performed on 2D microscopic images. However, detection errors may occur when two detected FISH signals overlap in the projected image plane that is translocated in the depth direction. For early or subtle diseases, detecting a small number of abnormal cells can make a difference in diagnosis, and this detection error may be crucial. The exemplary embodiment of the 3D-IFC system 150 described herein may be suitable for 3D visualization of FISH signals and may improve the overall accuracy of analysis for some clinical cases.

[0074] Internalization / endocytosis: Many cellular processes, such as (1) recognition, binding, internalization, and elimination of pathogens and cellular debris by phagocytes; (2) uptake and presentation of cellular antibodies; (3) uptake of exosomes and other nanoparticles by cells; and (4) assessment of cellular drug uptake and processing by measuring internalization. 2D wide-field microscopy images or imaging cytometry images provide only incomplete information about relative positions. For example, from a 2D microscopy image, if a particle is observed to be located exactly in the center of the cell, it is difficult to tell whether the particle is on the cell membrane (at the top of the cell) or whether the particle is in the cytoplasm or even in the nucleus. Instead, images are taken in multiple focal planes to resolve such issues. Exemplary embodiments of the 3D-IFC system 150 described herein can perform continuous, high-throughput analysis for these internalization studies.

[0075] Further embodiments and implementations of camera-free, high-throughput 3D-IFC systems, methods, and apparatus for imaging objects such as cells based on the capture of 3D fluorescence and dark-field images of single cells in flow using optical section microscopy are described. The disclosed camera-free, high-throughput 3D-IFC systems and methods provide the ability to jointly capture 3D fluorescence and label-free side scatter images of single cells in flow at a throughput of approximately 500 cells per second.

[0076] For example, in some exemplary implementations, a high-throughput 3D-IFC system utilizes sheet light scanning illumination technology and spatiotemporal conversion detection technology, so that fluorescence and label-free 3D cell image reconstruction can be achieved by single-element photodetector readings without a camera. In an exemplary implementation, taking advantage of the speed and sensitivity of photomultiplier tubes (PMTs), an exemplary 3D-IFC system uses a variety of scanning techniques to add spatial information to the flow cytometry architecture. 3D imaging can be achieved by using laser scanning on the first (z-) axis, cell transformation through the flow through the second (y-) axis, and multiple pinholes arranged along the third (x-) axis to generate fluorescence and label-free information from 60,000 voxels per cell. By accurately mapping time to space, the photodetector reading at one time point corresponds to one voxel in 3D space. The exemplary results described below show a 0.2 m·s flow rate corresponding to a throughput of approximately 500 cells per second. -1 Fluorescence and 90-degree label-free side scatter (SSC) imaging of single cells at flow rates by 3D-IFC.

[0077] Figure 5AAnother exemplary embodiment of a 3D-IFC system, labeled 550, according to some embodiments of the system 100 is shown. As shown, the 3D-IFC system 550 includes a flow system 551, which includes a fluid channel for carrying a fluid sample, wherein the fluid sample includes a flowing sample (e.g., particles, living cells, etc.) connected to a light sheet illumination module 560 and a detector module 570 of the 3D IFC system 550. The light sheet illumination module 560 includes: an optically coupled cylindrical lens (CL) 561 and an acousto-optic deflector (AOD) 565 and an illumination objective (IO) 563 (e.g., a 50X / 0.55 illumination objective), arranged on an optical illumination path with a light source 569 (e.g., a laser). The cylindrical lens 561 is placed after the AOD 565 to focus the light in one direction (e.g., to focus a laser beam) to form a scanning light sheet. For example, in some embodiments, the light sheet illumination module 560 includes a mirror element 564 between the cylindrical lens 561 and the AOD 565 to allow for various positions of the components of the illumination module 560 while preserving the optical path to deliver light on the flowing sample. In some embodiments, for each frequency, the AOD 565 produces a first order beam deflected at a different angle and thus produces a scan over time. In some implementations, the fluid sample is 2D hydrodynamically focused by a sheath before entering a square cross-section quartz flow cell, as shown in FIG. Fig. 9 Further discussion.

[0078] The detector module 570 includes a spatial filter (SP) 571 disposed in an optical detection path between a detector objective (DO) 572 (e.g., a 10X / 0.28 detection objective) and an optical photodetector 579 to capture optical signal data from a flow object (e.g., a cell, a particle, etc.), wherein the detector objective (DO) 572 is connected to the flow system 551. The spatial filter 571 is placed at an image plane of the photodetector 579. For example, the photodetector 579 may be configured to include one or more photodetectors (e.g., such as a photomultiplier tube (PMT)) so that the detector module 570 detects a specific spectral range of the optical signal data. Figure 5A As shown, in some embodiments, for example, the detector module 570 includes one or more dichroic mirrors (DM) 573 and a spatial filter 571 optically coupled, wherein the dichroic mirror (DM) 573 is optically coupled to a corresponding photodetector 579 (e.g., a PMT) in a separate optical path. In some embodiments, the system 550 is configured to optically couple the one or more dichroic mirrors (DM) 573 to a corresponding bandpass filter (BP) 175 using the photodetector 579 in a separate optical path. For example, as Figure 5AAs shown, the illumination of the flowing sample is configured perpendicular to the detection orientation. In some embodiments, the 3D-IFC system 550 includes a lens or light guide to focus and / or direct light at a specified location of another component of the system.

[0079] 3D-IFC system 550 includes an exemplary embodiment of data processing system 140, which includes a digitizer (DIG) 580 (e.g., 125MS·s -1 ) connected to a processing unit (e.g., including a processor and a memory) to digitally process the output signal of the exemplary PMT of the detector module 570.

[0080] In an exemplary implementation of the 3D-IFC system 550, suspended cells may form a single file in 2D hydrodynamic focusing in a quartz flow cell having a square cross-section. Fig. 9 It is further shown in detail in Figure 5A In the example of FIG. 5 , a light sheet (xy plane) is formed on a flow cell 551 (e.g., with a diffraction-limited beam waist and a height of 200 μm to 400 μm) by laser excitation, and scanned in the z direction at a very high rate (e.g., 200 kHz). For example, when a particle or cell moves at 0.2 m·s -1 When the velocity flows through the entire optical interrogation, the pixelated field of view is represented by a 3D space with X times Y times Z voxels, such as Figure 5C shown.

[0081] Figure 5B A diagram depicting an optical interrogation region of an exemplary flowing sample (e.g., a cell) flowing along an object plane and a spatial filter 571 located at the image plane is shown. The spatial filter 571 comprises a surface having a plurality of holes arranged in a pattern, wherein each hole of the pattern is spaced along the y-axis (parallel to the flow direction) and its position varies relative to another hole of the pattern along the x-axis (perpendicular to the flow direction). Figure 5B As shown, H represents the height of the light sheet; θ represents the tilt angle between the flow (y axis) and the vertical line. The illumination light sheet is spread and scanned horizontally on the z axis, the sample flows on the y axis, and x is the orthogonal axis.

[0082] The spatial filter at the image plane uses pinholes to generate line scans in the x-axis. For example, the array of pinholes on the spatial filter is aligned at an angle θ to the flowing vapor, so the array of pinholes also travels in the x-direction. In this way, each pinhole allows light from voxels with different x-indexes to pass to the PMT detector (e.g., details of this exemplary mask are discussed later in this disclosure). When a flowing cell appears at the first pinhole of the spatial filter, the imaging process begins. During the first light scan (e.g., 5 μs), a line scan with x is collected. 1 y 1Index of voxel z 1-Z As the cell flows downstream in y to the next position x 1 y 2 , generating the corresponding voxel z 1-Z This process is repeated until the entire 2D yz-section is at x 1 When imaging at 1, the cell passes completely through pinhole 1. As the cell moves downstream in the y direction (e.g., the direction of flow), it reaches the next pinhole and is recorded at x. 2 to x X For example, see Figure 5C .

[0083] Figure 5C An example of a 3D reconstructed space generated by the data processing system of the 3D IFC system 550 is shown. From this processing, for example, the resolution on the X-axis is determined by the number of pinholes (e.g., the pixelated field of view in the x-direction); the resolution on the Y-axis is determined by the distance between the two slits (e.g., the pixelated field of view in the y-direction); and the resolution on the Z-axis is determined by the light sheet scanning range (e.g., the pixelated field of view in the z-direction).

[0084] FIG. 5D to FIG. 5F An exemplary output of the 3D-IFC system 550 is shown. In an exemplary implementation of the system 550, the detected time-domain multi-parameter signals (e.g., multicolor fluorescence, FL1 and FL2, and side scatter, SSC, light intensity) are synchronized with a reference output of a tuning voltage of an AOD driver, wherein the reference output represents the z-position of the detected voxel. For example, at a scan rate of 200 kHz, a one-dimensional (1D) intensity distribution in the z-axis is recovered from the time-domain signals in a time period of 5 μs (e.g., see Figure 5D During the cell travel period of about 100 μs between pinholes, about 20 laser scan cycles are performed and the yz-plane 2D image array is recovered from the PMT readout (see, for example, Figure 5E ). As the cell traverses the entire interrogation region, a stack of 2D yz plane images is recovered and a final 3D image is reconstructed where the ordered pinhole numbers indicate the x-position of the voxel. Fig. 5F In the example shown, a 10-pinhole spatial filter produces ten 2D yz plane images and a signal length of 1 ms corresponding to a throughput of 500 cells per second. In this implementation, the bandwidth of the PMT and digitizer used in the exemplary system 550 (e.g., 150 MHz and 125 MHz) respectively supports a throughput of 60,000 voxels per 3D cell image. Each 20 μm × 20 μm × 20 μm 3D cell image in 3D space is resized to a size of 100 × 100 × 100 pixels for 3D image analysis and quantitative measurement.

[0085] To measure the velocity of each cell for image reconstruction by spatiotemporal transformation, pairs of slits upstream and downstream of the pinhole were added to the optical spatial filter (see Fig.10 ). The measured rate of cells passing through the detection area and the known frequency of the light sheet scanning rate ensure that each voxel in the 3D-IFC image has a different time domain value and all voxels can be captured carefully in time from the single element PMT. The 3D cell image is then reconstructed from the time domain signal.

[0086] Figure 5D Depicted is a light sheet scanning cycle that produces a one-dimensional light intensity distribution on the z-axis. The PMT voltage reading at one point in time corresponds to the light intensity of one voxel on the z-axis.

[0087] Figure 5E Depicted is the 2D profile produced in the yz plane by multiple scans within one pinhole scan cycle as the object travels along the y axis. Each section (separated by dashed lines) corresponds to the light intensity of a row in the 2D image stack.

[0088] Fig. 5F It depicts that when the object completely passes through the spatial filter coverage area, the time domain signal contains complete information of the 3D contour in xyz space. Each section corresponds to a 2D image section. Fig. 5F In the figure, AOD refers to the tuning voltage of the AOD driver; FL1 refers to the PMT reading of the fluorescence detection channel 1; FL2 refers to the PMT reading of the fluorescence detection channel 2; SSC refers to the PMT reading of the side scattered light detection channel.

[0089] For example, to demonstrate the cell imaging capability of the 3D-IFC system 550, a -1 The flow rate of the system 550 is used to image a single cell in suspension. The exemplary implementation of the system 550 produces exemplary image data of mammalian cells, including two-color fluorescence and unlabeled dark field (side scatter) 3D images. For the exemplary implementation, HEK293 cells were stained with intracellular carboxyfluorescein dye (CFSE) and bound to a random number of 1 μm fluorescent carboxylate-modified polystyrene beads.

[0090] FIG. 6A to FIG. 6C Images and data graphs of an exemplary implementation of a 3D-IFC system 550 using CFSE-stained HEK-293 cells bound to 1 μm fluorescent beads are shown. Fig. 6A Depicted are examples of recovered 2D yz plane images and 3D surface rendering views of aggregates of CFSE fluorescence, beads, and SSC (lower row). Figure 6BAn example of a 3D image and histogram detection event for a cell bound to beads is depicted. The relative positional relationship in 3D space indicates that particle counts in 3D-IFC are independent of cell orientation. In the example of a cell bound to four beads, occlusion at certain angles could be a source of error for particle counting using 2D images. Figure 6C An example of intensity-based 3D SSC image processing is depicted. Figure 6C The left column shows Fig. 7A Intensity histogram of the 3D SSC image of the cell shown in . P(x, y, z) is the position of a bead of size 1 μm determined using the 3D fluorescence image; within the ±1 μm region of each bead's position, the local intensity peak in the 3D SSC image can be found. Figure 6C In the figure, the scale bar represents 5 μm. In an exemplary implementation, the flow rate is 0.2 m / s. CFSE is an intracellular carboxyfluorescein dye with Ex / Em: 488 / 517; beads are carboxylate-modified fluorescent microspheres with Ex / Em: 488 / 645; and SSC is 90 degree side scatter.

[0091] Reference Figure 6B , noting that although the 2D bead images overlap, 3D-IFC resolves the exact number of particles from the reconstructed 3D image, which is important for some localization and co-localization features. In a flow system, cells and their internal structures are randomly oriented, and therefore, the 2D images may result from unfavorable viewing angles. However, using the disclosed 3D-IFC system, multiple viewing angles can be obtained through 3D-IFC cell tomography, which can provide improved relative position relationships and point counting results, which is beneficial for machine vision and human visualization. For example, using the side scatter dark field imaging mode, the 3D-IFC system 550 produces a 3D spatial distribution of scattered light. When an object is illuminated by visible light, the refractive index changes will scatter the light, and the size of the scattering range and the refractive index distribution determine the distribution of the scattered light. The 3D SSC image represents the spatial distribution of those refractive index (n) changes between the fluid (PBS, n~1.335), cells (n~1.3-1.6), and polystyrene beads (n~1.6). As Figure 6C As shown, the intensity-based low-pass filtered SSC image indicates the cell volume, and the high-pass filtered SSC image is correlated with the fluorescent image of the beads.

[0092] The disclosed 3D-IFC system is able to obtain information that is virtually unobtainable by conventional imaging flow systems. Take the following as an example. When ionizing radiation or cytotoxic chemicals cause DNA damage in the form of double-strand breaks (DSBs), phosphorylated protein γ-H2AX (γH2AX) forms foci at the DSBs in a 1:1 manner. Through anti-γ-H2AX immunolabeling, the foci reflect DNA damage and DNA repair capacity. Currently, 2D imaging technology and manual quantification are used, but due to viewing angle dependence, foci counting from 2D images is labor intensive and unreliable. To evaluate the lesion counting of the 3D-IFC system, immunolabeled γH2AX foci in CMK3 cells (e.g., glioblastoma multiforme cell line) can be imaged after 6 Gy of γ-ray irradiation. Representative cell images are shown in Figure 2. Fig. 7A and Figure 7B This exemplary data shows that the number of γH2AX foci is independent of fluorescence intensity, and therefore, intensity-based measurements using conventional flow cytometry metrics are poor reflections of DNA damage. In contrast, the 3D-IFC system 550 is able to accurately and rapidly analyze the number of γH2AX-positive foci.

[0093] Fig. 7A and Figure 7B Images and data graphs depicting exemplary results of fluorescent γH2AX foci imaged by the 3D-IFC system 550 are shown. Fig. 7A A representative 3D image of a radiation-damaged glioblastoma CMK3 cell stained with CFSE and γH2AX antibody conjugated with PerCP / Cy5.5 is shown; and a two-color fluorescent 2D yz plane merged image section at x=10 μm. The high quality of the 3D image shows that 3D-IFC is suitable for the study of DNA damage foci. Figure 7B An example of a scatter plot of 917 detection events in γH2AX intensity and foci counts, and images of cells within the labeled range (i)-(iv) in the scatter plots are shown. The data show that foci counts are independent of the fluorescence intensity of labeled γH2AX, so intensity-based measurements using conventional flow cytometry metrics cannot assess the extent of DNA damage. In the images, the scale bar represents 5 μm. The flow rate during the exemplary implementation was 0.2 m / s. For CFSE, intracellular carboxyfluorescein dye, Ex / Em is: 488 / 517; for PerCP-Cy5.5: DNA damage antibody conjugated dye, Ex / Em is: 490 / 677.

[0094] Peripheral blood leukocyte morphology is an important clinical diagnostic and prognostic measure for acute and longitudinal evaluation. An exemplary implementation of the 3D-IFC system 550 was performed to demonstrate such peripheral blood leukocyte morphology.

[0095] FIG. 8A to FIG. 8D Images and data graphs depicting leukocyte cell morphology determined by the 3D-IFC system 550 are shown. Fig. 8A Representative 2D transmission images (left column) and 3D images of leukocytes are shown. Figure 8B Shows Fig. 8A An example of an intensity histogram of the SSC signal of the first cell is shown, along with its 3D profile in the Orthoslice view and the Volume view. The cell volume estimate based on the 3D SSC image is matched to the fluorescence volume. Figure 8C Shown is a scatter plot of 589 detected events in CFSE(FL1) volume and SSC-based estimated cell volume. Fig.8D Scatter plots of cell diameter calculated from estimated cell volume based on SSC and SSC intensity are shown. In these figures, the scale bar represents 5 μm. For the exemplary implementation, the flow rate was 0.2 m / s.

[0096] Fig. 8A Representative 3D-IFC imaging of leukocytes in three imaging modes is shown: 2D transmission image (e.g., 3D-IFC parameters) and 3D fluorescence and side scatter images. The leukocyte SSC signal not only indicates nuclear granularity, but also provides cell volume analysis obtained from low-intensity (low refractive index) SSC imaging. Here, two or more intensity bands of the 3D SSC signal can be used to generate two or more images reflecting their corresponding refractive indices, e.g., cytoplasm and nucleus; while the conventional flow SSC intensity signal is mainly contributed by the nucleus. As shown in FIG. Figure 8B and 8C As shown, 3D SSC images can be used to generate cell volumes that match the transmission and fluorescence results.

[0097] More information regarding materials and methods used in an exemplary implementation of the 3D-IFC system 550 for obtaining and producing results is described below.

[0098] In some embodiments, the particle movement system 110 (eg, a particle flow cell embodiment) may include: Fig. 9 A microfluidic system is shown that can continuously introduce suspended cells into an optical interrogation area. Since the position of the cells in the microchannel cross section is aligned with the optical field of view in all x-, y-, and z-directions, a reproducible and stable flow rate of the cells is obtained by tightly focusing the cells at the center of the square cross section. The suspended cells in the sample injected by the syringe pump are hydrodynamically focused into a single stream. The sheath flow is used to restrict the flowing cells in the x- and z-directions. An air pressure pump is used together with a liquid flow meter to provide a stable sheath flow. At the junction of the sheath flow and the sample flow, the tube end is specially tapered to maintain symmetrical flow. The flow rate ratio between the sample and the sheath is precisely controlled to 100:1 to ensure that the particles flow at a high speed within the optical field of view.

[0099] Fig. 9 An example of a microfluidic system 900 for an exemplary particle flow cell 551 of an exemplary 3D-IFC system 550 is shown. Fig. 9 Includes diagrams and photographs of an exemplary microfluidic system 900 connected to a conical tube. As shown in the figure, suspended particles or cells in a fluid can flow through a channel to an exemplary quartz flow cell having a cross-sectional dimension of 250 μm×250 μm and a length of 20 mm.

[0100] In some exemplary embodiments of system 100, the optical system is configured as a light-sheet fluorescence microscope (similar to Figure 1C or Figure 5A ) arrangement, which illuminates the sample in a single plane while detecting signals in the vertical (z-) direction. In an exemplary implementation of the 3D-IFC system 550, a 1000mW 488nm laser (coherent) with a circular beam shape with a Gaussian energy distribution is used, which is collimated, expanded, and then focused to illuminate an area of ​​0.6μm (z-direction) by 250μm (y-direction). A cylindrical lens and a 50× / 0.55 objective lens (Mitutoyo) are used to form a light sheet. An acousto-optic deflector (AOD) is configured in the optical system to produce high-speed scanning in the z-direction (e.g., 200kHz in this implementation).

[0101] A single pixel photodetector can detect the fluorescent or scattered light that passes through the spatial filter in each channel and reconstruct an image from the time domain output. For example, for spatial filter 579, the pinholes are arranged vertically. Figure 5B As shown, the flow (y-) direction is tilted to create an angle θ, which is determined by the field of view D in the x-direction. x , field of view D in the y direction y and the number of pinholes X, θ = tan -1 (D x / (X·D y )). In addition, because the z-direction illumination sheet light scans a larger range than the cell size and has a higher scanning speed, typically more than 20 times higher than the cell travel speed in the y direction, when a cell passes through a pinhole, the light intensity of a portion of a yz plane image is recorded; when the cell passes through the next pinhole, another yz plane image stack is acquired. For example, combining the scanning sheet light, the flow movement of the cell, and the spatial filter, the detector detects the fluorescence of only a single voxel in the cell at a time, allowing a one-to-one time-to-space mapping. This concept can be mathematically modeled by the following equation. The measured PMT signal S(t) can be expressed as:

[0102] S(t)=∫∫∫{∫∫C(x',y'-v Ct,z)I(z,t)psf(x-x'),y-y')dx'dy'}·F(Mx,My)dxdydz=∫∫∫{C(x,yv C t,z)I(z,t)}·F(Mx,My)dxdydz (6)

[0104] Where C(x,y,z) is the 3D cell fluorescence or scattered light intensity distribution map, I(z,t) is the sheet light illumination, F(x,y) is the characteristic function of the spatial filter, and v C is the cell flow velocity, and M is the magnification of the detection system.

[0105] The sound frequency sent to the acoustic transducer in the AOD can be changed to deflect the beam to create illumination at different z-positions. The continuously changing tuning voltage that produces the sound frequency can be generated by various types of waveforms (e.g., sine wave, triangle wave, etc.). In order to obtain the highest laser power efficiency, the tuning voltage is set to change in a sawtooth manner, so the position of the light sheet in the z direction z0(t) can be described as:

[0106] z 0 (t∈(nT,(n+1)T))=v i (t-nT)

[0107] n=0,1,2,... (7)

[0108] Where T is the light sheet illumination scanning period, v i is the scanning speed in the z direction.

[0109] By using a cylindrical lens, the laser is diverged to form a light sheet with a height of 200μm-400μm in the y direction. The scanning light sheet illumination I(z,t) can be described as a Gaussian beam:

[0110]

[0111] Using an oversampled PMT signal readout, the spatial resolution in the z direction is diffraction limited. The Gaussian beam waist is measured to be 0.73 μm and approximated as a delta function to simplify calculations:

[0112] I(z,t)≈k·δ(zz 0 (t)) (9)

[0113] exist Fig.10 Two examples of spatial filters are shown in .

[0114] Fig.10Figure showing an example for spatial filter design. Two examples of spatial filters are placed in the image plane. The top two long slits and the bottom two long slits with dimensions of 10μm×200μm are used for velocity detection. The other pinholes on the spatial filter are 10μm×20μm (left) and 10μm×10μm (right) for capturing 3D images with voxel sizes of 2μm and 1μm in the x-direction, respectively. The arrangement of the pinholes aligns the laser beam waist.

[0115] Leaving aside the slit used for speed detection, the characteristic function F(x, y) of the spatial filter is designed as

[0116]

[0117] Where q = 1, 2, ..., N is the number of pinholes on the spatial filter. The size of the pinholes together with the numerical aperture of the detection lens, the cell flow velocity and the signal sampling rate determine the spatial resolution in the x-direction and the y-direction.

[0118] In an exemplary implementation, the spatial filter is fabricated using electron beam lithography, and the light shielding region is made of chromium with a thickness of 250 nm.

[0119] With the above approximation, when y q+1 -y q When the cell size (diameter) is larger than the cell size, and the cell projection overlaps with the jth pinhole,

[0120] S(t)=∫C(x,y-Mv C t,z 0 (t))δ(xx q )δ(yy q )dxdy=C(x j ,y j -Mv C t,z 0 (t)) (11)

[0121] A 10× / 0.28 detection objective was used and cells were assumed to be within the depth of field, and exemplary system PSFs at multiple z were not considered. The light intensity signal detected by the PMT (Hamamatsu) was first amplified by an amplifier (Hamamatsu) with a bandwidth from DC to 150 MHz and then digitized by a digitizer (ADVANTECH) with a maximum sampling rate of 125 MS / s per channel.

[0122] Therefore, this exemplary method maps 3D cell images to time-domain light intensity one-to-one. According to the above equation, a 3D image construction algorithm was written in MATLAB. CDue to slight changes in the original size of the 3D image of each cell, the original size of the 3D image may be slightly different. In the exemplary implementation, the original 3D image is then resized to 100×100×100 pixels. 3D image batch processing is performed in ImageJ.

[0123] Fig.11 Another exemplary embodiment of a 3D-IFC system, labeled 1150, is shown according to some embodiments of the 3D-IFC system 550. The 3D-IFC system 1150 includes an additional detection path, for example, to provide a 2D transmission imaging mode. As shown in the figure, the shaded portion (lower right portion of the figure) depicts components for transmission image detection; it includes a spatial filter 1171 for transmission detection with a single slit (e.g., with a size of 50 μm×2 mm) in data communication with a data processing system (e.g., DIG 580), a detection objective (DO) 572 (e.g., a 50× / 0.55 detection objective), and a photodetector 1172 (e.g., a PMT, a photomultiplier tube).

[0124] Exemplary methods for preparing cells are described below.

[0125] Cells with fluorescent beads. In some exemplary implementations, human embryonic kidney 293 (HEK-293) cells were cultured in a 10 cm culture dish to 90% confluence with complete culture medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin). After 100-fold dilution of 1.0 μm fluorescent beads (Ex / Em: 488 / 645 nm, e.g., T8883, ThermoFisher) in a stock solution (2% solid), 100 μL of the diluted solution was mixed with 10 mL of fresh cell culture medium and added to a cell culture plate. After 10 hours of continuous culture, HEK-293 cells were harvested and stained with a CellTrace CFSE cell proliferation kit (Ex / Em: 492 / 517 nm, e.g., C34554, ThermoFisher) at a working concentration of 20 μM. After the staining process, cells were fixed with 4% paraformaldehyde, washed and resuspended in 1X phosphate buffered saline (PBS).Before each imaging experiment, the cell suspension was diluted to a concentration of 1000 cells / μL in PBS.

[0126] CFSE staining. In some exemplary implementations, HEK-293 cells can be cultured in a 10 cm dish with complete medium until 98% confluence, then harvested and resuspended in 1X PBS to a concentration of 1×10 6cells / mL. CFSE cell proliferation kit (Ex / Em492 / 517nm, e.g., C34554, Thermo Fisher) was added to the cell suspension at a working concentration of 20 μM. After incubating the cells at 37°C for 30 minutes, the staining process was quenched with fresh culture medium (DMEM), the HEK-293 cells were washed with 1X PBS and fixed with 4% paraformaldehyde. The fixed cells were washed and resuspended in 1X PBS. The protocol was also applied to stain CMK3 cells and human blood leukocytes.

[0127] CMK3 cell radiation treatment and immunostaining. In some exemplary implementations, human glioblastoma CMK3 cells were cultured in a culture dish with complete medium (DMEM-F12, 2% B27 supplement, 1% penicillin-streptomycin, 1% glutamine, 100 μg / L EGF, 100 μg / L FGF, 0.24% heparin). The cells were harvested and resuspended in 1X PBS to 1x10 6 The concentration of cells / mL was then stained with CFSE. In order to induce DNA double-strand breaks (DSBs), CMK3 cells were irradiated with 6Gy using a cesium source irradiator. After irradiation for 30 minutes, the treated cells were washed once with 1X PBS and fixed with 1% paraformaldehyde. The fixed cells were washed twice with PBS. Then 70% ethanol was added to the cells, and the cells were incubated on ice for 1 hour. After ethanol treatment, the cells were washed twice with PBS and incubated in 1% TritonX-100 at room temperature for 10 minutes. Then, the cells were washed once with PBS and incubated in 5% bovine serum albumin (BSA) in PBS on a shaker at room temperature for 30 minutes. The cells were then washed once with PBS and incubated on ice for one hour on a shaker at a dilution of 1:300 in anti-phospho-histone H2A.X (Ser139) antibody, clone JBW301. After primary antibody treatment, cells were washed twice with 5% BSA and incubated in PerCP / Cy5.5 anti-mouse IgG1 antibody at a 1:100 dilution on a shaker for 1 hour on ice. Finally, stained cells were washed twice with 5% BSA and resuspended in stabilization fixation buffer diluted 1:3 in MilliQ water. Prior to each imaging experiment, cells were diluted to a concentration of 500 cells / μL in PBS.

[0128] Separation and staining of leukocytes. In some exemplary implementations, fresh human whole blood was harvested from EDTA tubes from San Diego Blood Bank. The red blood cells in the whole blood were first lysed with RBC lysis buffer (00-4300-54, Invitrogen), and the leukocyte population was harvested by a soft centrifuge after the lysis process. The harvested leukocytes were then washed and resuspended in 1X PBS to 1x106 The resuspended leukocytes were stained with CFSE. After the staining process, the cells were fixed with 4% paraformaldehyde, washed and resuspended in 1X PBS. Before each imaging experiment, the cell suspension was diluted to a concentration of 500 cells / μL in PBS.

[0129] As demonstrated by the exemplary data, the disclosed 3D-IFC system is able to provide unprecedented access to high throughput 3D image capture and analysis. An exemplary implementation of the 3D-IFC system 550 features a cell flow rate of 0.2 m·s -1 , 1 μm spatial resolution on 3 axes, 20 μm field of view on all axes, and a throughput of 500 cells per second. Some embodiments have the flexibility to directly improve spatial resolution, field of view, and 3D image capture rate by changing spatial filters and using higher flow rates. Its information-rich 3D dark field (side scatter) image detection coupled with 2D transmission images provides the possibility of label-free determination.

[0130] Exemplary applications of the disclosed systems, methods, and apparatus include, but are not limited to, asymmetric division of T cells into effector and memory cells, secretory pathways of B cells, phenotypic drug discovery, protein or receptor translocation, tracking the formation or transport of organelles, chromosome structural aberrations (where 3D orientation and polarity are important), and other applications. In some implementations, the disclosed systems, methods, and apparatus can be integrated with image-based classification capabilities.

[0131] Example

[0132] In some embodiments according to the present technology (Example A1), a three-dimensional imaging flow cytometry system includes: a particle flow system; a scanning sheet light illumination system connected to the particle flow system; an optical detection system connected to the particle flow system; and a data processing system that communicates with the optical detection system and / or the scanning sheet light illumination system. The particle flow system includes: a fluid channel with a clear optical path, which carries a fluid sample containing particles; a flow focusing system for providing a confined sample fluid to a thin stream in the fluid channel. The scanning sheet light illumination system includes: a light source for illuminating particles flowing in the fluid channel; a device for redirecting the illumination light beam to different angles, which is programmable to produce periodic angle changes at high speed; and a cylindrical lens and / or other optical components to produce structured sheet light illumination to allow an optical scan of the illuminated portion of the particle at one time, for example, a thin portion or a thin line of the particle. The optical detection system includes: one or more photodetectors and a spatial filter located at an image plane of the one or more photodetectors to selectively allow light from a specific position of the particle to pass through and be detected by the one or more photodetectors. The data processing system is configured to process the detected data and generate a three-dimensional constructed image of the particle.

[0133] Example A2 includes the system of any of Examples A1 or A3-A7, wherein the optical detection system is operable to collect and detect fluorescent and / or scattered light.

[0134] Example A3 includes the system of any of Examples A1-A2 or A4-A7, wherein the optical detection system is configured to be positioned at any angle where the fluid channel has an optically transparent path.

[0135] Example A4 includes a system of any of Examples A1-A3 or A5-A7, wherein the optical detection system is operable to detect 1D optical data points of a 3D object over time based on a combination of: (a) flow movement of particles; (b) scanning sheet light illumination; and (c) a spatial mapping filter to detect light intensity (e.g., including fluorescence and scattering) at each position of the particle in a sequentially timed manner so that the time domain signal recorded by one or more photodetectors has a one-to-one relationship from space to time.

[0136] Example A5 includes the system of any of Examples A1-A4 or A6-A7, wherein the data processing system includes a data flow unit in communication with the optical detection system.

[0137] Example A6 includes the system of any of Examples A1-A5 or A7, wherein the data processing system includes an image reconstruction algorithm that includes illumination specifications and detection protocols, and controls the scanning light illumination system and the optical detection system accordingly.

[0138] Example A7 includes the system of any of Examples A1-A6, wherein the data processing system includes an image processing unit to process the image data to determine properties associated with the particles.

[0139] In some embodiments according to the present technology (Example B1), an optical imaging system for performing three-dimensional imaging in a flow cytometry system includes: a light source for illuminating particles flowing in a flow channel; one or more lenses configured above the flow channel range; an acousto-optic deflector configured in the optical path of the optical photodetector and the light source to provide high-speed scanning in the z direction; and a single-element photodetector to detect fluorescence passing through a spatial filter in each channel, wherein the light source is configured to illuminate the particles flowing in the flow channel in a single plane while the signal is detected in the vertical direction.

[0140] Example B2 includes the optical imaging system of Example B1, wherein the system is configured to acquire optical signal data to reconstruct an image from a time domain detector output based on a combination of scanning sheet light, flow movement of particles, and spatial filters so that the photodetector can detect individual points in the particle at a time, thereby allowing spatiotemporal transformations.

[0141] Example B3 includes the optical imaging system of Example B1, wherein the light source includes a laser.

[0142] In some embodiments according to the present technology (Example B4), a three-dimensional imaging flow cytometry system includes: a particle flow device configured to include a substrate, a channel formed on the substrate, which can be used to make particles including living cells flow along the flow direction of the flow channel; an imaging system, connected to the particle flow device and operable to obtain three-dimensional image data associated with the particles during flow through the channel; and a data processing and control unit that communicates with the imaging system, the data processing and control unit including a processor, which is configured to process the image data obtained by the imaging system to determine one or more characteristics associated with the cells from the processed image data, wherein the imaging system is configured as a light-sheet fluorescence microscope configuration, which can illuminate the sample in a single plane while detecting signals in a vertical direction.

[0143] Example B5 includes the three-dimensional imaging flow cytometry system of Example B4, wherein the imaging system includes the optical imaging system of any one of Examples B1-B3.

[0144] Example B6 includes the three-dimensional imaging flow cytometry system of Example B4, wherein the imaging system includes one or more light sources to provide input light within the range of the particle flow device; and an optical photodetector to capture image data from particles illuminated by the input light.

[0145] Example B7 includes the three-dimensional imaging flow cytometry system of Example B6, wherein the optical photodetector includes an objective lens of a microscope optically coupled to a spatial filter, an emission filter, and a photomultiplier tube.

[0146] Example B8 includes the three-dimensional imaging flow cytometry system of Example B7, wherein the optical photodetector further comprises: one or more light-guiding elements to guide input light in a first range to guide light emitted or scattered by cells to optical elements of the optical photodetector, or both.

[0147] Example B9 includes the three-dimensional imaging flow cytometry system of Example B8, wherein the light guiding element includes a dichroic mirror.

[0148] Example B10 includes the three-dimensional imaging flow cytometry system of Example B8, wherein the optical photodetector includes two or more photomultiplier tubes to generate two or more corresponding signals based on two or more bands or types of light emitted or scattered by the cells.

[0149] In some embodiments according to the present technology (Example C1), a three-dimensional (3D) imaging flow cytometry system includes: a particle flow device, the device including a substrate and a channel formed on the substrate to allow particles in a fluid to flow along a flow direction of the channel; an optical illumination system to generate a two-dimensional (2D) light sheet, the light sheet scans multiple parts of the particles in the fluid, one part at a time, while the particles flow along the flow direction in the channel, the optical illumination system including a light source that generates a light beam, the light source is optically coupled to a light sheet generating unit to modify the light beam into a 2D light sheet directed to the channel, wherein the light sheet generating unit includes: a light redirecting device to redirect the light beam to different angles; and a light sheet with the channel placed after the light redirecting device. a cylindrical lens in the path; an optical detection system connected to the particle flow device and operable to obtain three-dimensional light signal data associated with the particles during flow through the channel, wherein the optical detection system includes: one or more photodetectors and a spatial filter located on an image plane of the one or more photodetectors, the spatial filter including a plurality of holes to selectively allow a portion of the 2D sheet light on a scanned portion of the particle to pass through and be detected by the one or more photodetectors; and a data processing unit in communication with the optical detection system and the optical illumination system, the data processing unit including a processor configured to process the light signal data obtained by the optical detection system and generate image data, from which a 3D image of the particle can be constructed.

[0150] Example C2 includes a system of any one of Examples C1 or C3-C15, wherein the 2D light sheet is directed toward the channel in a second direction perpendicular to the flow direction so that a first scan of the 2D light sheet forms a first plane including the flow direction and the second direction; wherein an image plane of the optical detection system is arranged along a third direction perpendicular to the flow direction and the second direction so that a second scan of the 2D light forms a second plane parallel to the first plane and which varies along the third direction with the distance from the first plane.

[0151] Example C3 includes the system of Example C2, wherein the optical illumination system is configured to scan a first portion of the particle in a first plane at a first time point and scan a second portion of the particle in a second plane at a second time point while the particle flows along a flow direction, thereby generating a timing signal spatially mapped to the particle.

[0152] Example C4 includes the system of Example C2, wherein the apertures of the spatial filter are positioned along the second direction such that each aperture is configured to filter a 2D sheet of light corresponding to a voxel of the particle.

[0153] Example C5 includes the system of any of Examples C1-C4 or C6-C15, wherein the data processing unit is operable to generate a 3D image of the particles voxel by voxel.

[0154] Example C6 includes the system of any of Examples C1-C5 or C7-C15, wherein the image data of the particles comprises a spatial resolution of at least 1 μm in three axes and a field of view of 20 μm in three axes.

[0155] Example C7 includes the system of any of Examples C1-C6 or C8-C15, wherein the flow rate of the particles in the channel is at least 0.2 ms -1 , and wherein the system is operable to generate image data of particles at a throughput of 500 particles per second.

[0156] Example C8 includes a system of any of Examples C1-C7 or C9-C15, wherein the plurality of holes of the spatial filter are arranged in a pattern, wherein each hole of the pattern is evenly spaced along the flow direction and varies its position relative to another hole of the pattern along a second direction perpendicular to the flow direction.

[0157] Example C9 includes the system of any of Examples C1-C8 or C10-C15, wherein the light redirecting device includes an acousto-optic deflector (AOD).

[0158] Example C10 includes the system of any of Examples C1-C9 or C11-C15, wherein the light sheet generation unit is programmable by the data processing unit to generate the periodic angular variation at a rate of at least 200 kHz.

[0159] Example C11 includes the system of any of Examples C1-C10 or C12-C15, wherein the one or more photodetectors include a photomultiplier tube.

[0160] Example C12 includes the system of any of Examples C1-C11 or C13-C15, wherein the optical detection system includes an objective lens optically coupled to the spatial filter, and an emission filter optically coupled between the spatial filter and the photodetector.

[0161] Example C13 includes the system of Example C12, wherein the optical detection system further includes one or more light directing elements to direct light from the spatial filter to the plurality of photodetectors.

[0162] Example C14 includes the system of example C13, wherein the optical detection system includes two or more photomultiplier tubes to generate two or more corresponding signals based on two or more light bands or two or more types of light emitted or scattered by the cells.

[0163] Embodiment C15 includes the system of any of Examples C1-C14, wherein the particles comprise living cells.

[0164] In some embodiments according to the present technology (Example C16), a method for three-dimensional (3D) imaging of a moving particle includes: moving the particle along a first direction through a channel; scanning multiple portions of the particle portion by portion by directing separate two-dimensional (2D) sheet light to corresponding ranges of the channel as the particle moves through the channel; spatially filtering portions of the 2D sheet light on the scanned portions of the particle to allow detection of certain optical point signals passing through the spatial filter; detecting the spatially filtered light point signal corresponding to a single voxel in three dimensions mapped to a detection time point; and processing the light point signal to generate image data of the particle.

[0165] Example C17 includes the method of any of Examples C16 or C18-C24, including modifying light beams produced by the light source into separate 2D sheets of light.

[0166] Example C18 includes the method of Example C17, wherein scanning the multiple portions of the particle includes redirecting the light beam to different angles through a cylindrical lens, which modifies the redirected light beam into a 2D sheet of light.

[0167] Example C19 includes the method of any of Examples C16-C18 or C20-C24, wherein a first separate 2D sheet of light is directed toward a channel in a second direction perpendicular to the first direction so that a first scan of the 2D sheet of light forms a first plane including the first direction and the second direction; and wherein an image plane for detection is arranged along a third direction perpendicular to the first direction and the second direction so that a second scan of a second separate 2D sheet of light forms a second plane parallel to the first plane and changes along the third direction with a distance from the first plane.

[0168] Example C20 includes the method of any of Examples C16-C19 or C21-C24, comprising generating a 3D image of the particles based on pixel-by-pixel construction from the generated image data.

[0169] Example C21 includes the method of any of Examples C16-C20 or C22-C24, wherein the image data of the particles includes a spatial resolution of at least 1 μm in three axes and a field of view of 20 μm in three axes.

[0170] Example C22 includes the method of any of Examples C16-C21 or C22-C24, wherein the image data of the particles is generated at a throughput of 500 particles per second.

[0171] Example C23 includes the method of any of Examples C16-C22 or C24, wherein the particles include living cells.

[0172] Example C24 includes the method of any of Examples C16-C23 implemented on the system of any of Examples C1-C15.

[0173] In some embodiments according to the present technology (Example D1), a system for three-dimensional (3D) imaging of moving particles includes a particle moving device, the device including: a substrate to allow the particle to move along a travel path in a first direction; an optical illumination system for generating an asymmetric illumination area of ​​light within the range of the particle travel path, the optical illumination system scanning multiple parts of the particle at multiple time points while the particle moves, the asymmetric illumination area including an illumination dimension that is thinner than other illumination dimensions to form a shape similar to an illumination plane, the optical illumination system including: a light source for generating a light beam, the light source optically coupled to a light redirecting device to modify the light beam to a different angle by redirecting the light beam so that the asymmetric illumination area of ​​light points to the travel path of the particle; an optical detection system, coupled to the particle The particle moving device is optically connected and operable to obtain light signal data during movement of the particle along the travel path, wherein the light signal data is associated with different parts of the particle, and the different parts of the particle correspond to the volume of the particle, wherein the optical detection system includes: one or more photodetectors and a spatial filter located between the particle moving device and the one or more photodetectors, the spatial filter including a plurality of holes to selectively allow a portion of an asymmetric illumination area of ​​light on a scanned portion of the particle to pass through the spatial filter and be detected by the one or more photodetectors; and a data processing unit in communication with the optical detection system, the data processing unit including a processor configured to process the light signal data obtained by the optical detection system and generate data including information indicating 3D characteristics of the particle.

[0174] Example D2 includes a system of any one of Examples D1 or D3-D20, wherein an asymmetric illumination area of ​​light is directed toward a range of a travel path in a second direction perpendicular to the first direction, so that a first scan of the asymmetric illumination area of ​​light forms a first plane including the first direction and the second direction; and wherein an image plane of the optical detection system is arranged along a third direction perpendicular to the first direction and the second direction, so that a second scan of the asymmetric illumination area of ​​light forms a second plane parallel to the first plane and varying along the third direction with a distance from the first plane.

[0175] Example D3 includes the system of Example D2 or D4, wherein the optical illumination system is configured to scan a first portion of the particle in a first plane at a first point in time and scan a second portion of the particle in a second plane at a second point in time while the particle moves in a first direction, thereby generating a timing signal spatially mapped to the particle.

[0176] Example D4 includes the system of Example D2 or D3, wherein the apertures of the spatial filter are positioned along the second direction such that each aperture is configured to filter an asymmetric illumination region of light corresponding to a voxel of the particle.

[0177] Example D5 includes the system of any of Examples D1-D4 or D6-D20, wherein the data processing unit is operable to generate a 3D image of the particles voxel by voxel.

[0178] Example D6 includes the system of Example D5, wherein the 3D image data of the particles comprises a spatial resolution of at least 1 μm in three axes and a field of view of approximately 20 μm in three axes.

[0179] Example D7 includes the system of any of Examples D1-D6 or D8-D20, wherein the particle moving device includes a particle flow cell including a channel formed on a substrate to allow a fluid containing particles to flow through the channel as a travel path.

[0180] Example D8 includes the system of Example D7, wherein the flow velocity of the particles in the channel is at least 0.2 m·s -1 , and wherein the system is operable to generate particle data at a throughput of 500 particles per second.

[0181] Example D9 includes the system of any of Examples D1-D8 or D10-D20, wherein the particles are secured to a substrate and wherein the particle moving device includes a positioning system to move the substrate and the optical detection system relative to the optical illumination system so that the particles move along a travel path and are brought within the range of an asymmetric illumination area of ​​the scanning light.

[0182] Example D10 includes a system of any of Examples D1-D9 or D11-D20, wherein a plurality of holes of a spatial filter are arranged in a pattern, wherein each hole of the pattern is spaced apart along a first direction and varies its position relative to another hole of the pattern along a second direction perpendicular to the first direction.

[0183] Example D11 includes the system of any of Examples D1-D10 or D12-D20, wherein the light redirecting device includes an acousto-optic deflector (AOD).

[0184] Example D12 includes the system of Example D11, wherein the AOD is programmable by the data processing unit to produce periodic angular variations at a rate of at least 100 kHz.

[0185] Example D13 includes the system of any of Examples D1-D12 or D14-D20, wherein the light redirecting device comprises: a multiplexed array of scanning mirrors and / or a multiplexed array of scanning gratings.

[0186] Example D14 includes the system of any of Examples D1-D13 or D15-D20, wherein the light redirection device is optically coupled to one or more passive optical components selected from cylindrical lenses, lenses, prisms, and spatial filters to shape the redirected light along a path of travel.

[0187] Example D15 includes the system of any of Examples D1-D14 or D16-D20, wherein the one or more photodetectors include a photomultiplier tube.

[0188] Example D16 includes the system of any of Examples D1-D15 or D17-D20, wherein the optical detection system further comprises an objective lens optically coupled to the light redirecting device.

[0189] Example D17 includes the system of any of Examples D1-D16 or D18-D20, wherein the optical detection system includes an objective lens optically coupled to the spatial filter.

[0190] Example D18 includes the system of any of Examples D1-D17 or D19-D20, wherein the optical detection system further comprises: one or more light-guiding elements to direct light from the spatial filter to the plurality of photodetectors.

[0191] Example D19 includes the system of Example D18, wherein the optical detection system further comprises: two or more photodetectors to generate two or more corresponding signals based on two or more light bands or two or more types of light emitted or scattered by the cells.

[0192] Example D20 includes the system of any of Examples D1-D19, wherein the particles include living cells.

[0193] In some embodiments according to the present technology (Example D21), a method for three-dimensional (3D) imaging of a moving particle includes: moving the particle along a first direction; scanning multiple parts of the particle portion by portion by directing a separate asymmetric illumination area of ​​light to a corresponding range as the particle moves; spatially filtering portions of the asymmetric illumination area of ​​light on the scanned portions of the moving particle to allow certain light signals corresponding to the particle volume to be detected through the spatial filter; and detecting the spatially filtered light signal to obtain separate pixels in three dimensions mapped to the detection time point.

[0194] Example D22 includes the method of any of Examples D21 or D23-D32, including modifying light beams produced by the light source into separate asymmetric illumination areas of light.

[0195] Example D23 includes the method of any of Examples D21-D22 or D24-D32, wherein scanning the plurality of portions of the particle comprises redirecting the light beam to different angles and shaping the redirected light beam into an asymmetric illumination area of ​​the light.

[0196] Example D24 includes the method of any one of Examples D21-D23 or D25-D32, wherein a first separate asymmetric illumination area of ​​light is directed toward a path of travel of a particle in a second direction perpendicular to the first direction, so that a first scan of the first separate asymmetric illumination area of ​​light forms a first plane including the first direction and the second direction; and wherein an image plane for detection is arranged along a third direction perpendicular to the first direction and the second direction, so that a second scan of a second separate asymmetric illumination area of ​​light forms a second plane parallel to the first plane and that varies along the third direction with a distance from the first plane.

[0197] Example D25 includes the method of any of Examples D21-D24 or D26-D32, further comprising processing the light signal to generate data including information indicative of 3D characteristics of the particle.

[0198] Example D26 includes the method of any of Examples D21-D25 or D27-D32, comprising generating a 3D image of the particles based on voxel-by-voxel construction from the generated data.

[0199] Example D27 includes the method of any of Examples D21-D26 or D28-D32, wherein the data of the particles includes a spatial resolution of at least 1 μm in three axes and a field of view of 20 μm in three axes.

[0200] Example D28 includes the method of any of Examples D21-D27 or D29-D32, wherein the data of the particles is generated at a throughput of 500 particles per second.

[0201] Example D29 includes the method of any of Examples D21-D28 or D30-D32, wherein the particles are moved through a particle flow cell including a channel formed on a substrate so that a fluid containing the particles flows through the channel in a first direction.

[0202] Example D30 includes the method of any of Examples D21-D29 or D31-D32, wherein the particles are fixed to a substrate and the substrate is moved by a positioning system such that the particles move on the substrate in a first direction.

[0203] Example D31 includes the method of any of Examples D21-D30 or D32, wherein the particles include living cells.

[0204] Example D32 includes the method of any of Examples D21-D31 implemented on the system of any of Examples D1-D20.

[0205] The implementation of the subject matter and functional operations described in this patent document and the attached appendix can be implemented in various systems, digital electronic circuits or computer software, firmware or hardware, including the structures disclosed in this specification and their structural equivalents, or a combination of one or more thereof. The implementation of the subject matter described in this specification can be implemented as one or more computer program products, that is, one or more modules of computer program instructions encoded on a tangible and non-transitory computer-readable medium for execution by a data processing device or controlling the operation of a data processing device. The computer-readable medium can be a machine-readable storage device, a machine-readable storage substrate, a storage device, a material composition that affects a machine-readable propagation signal, or a combination of one or more thereof. The term "data processing unit" or "data processing device" covers all devices, devices and machines for processing data, including, for example, a programmable processor, a computer or multiple processors or a computer. In addition to hardware, the device may also include code that creates an execution environment for the computer program in question, for example, code constituting processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more thereof.

[0206] A computer program (also referred to as a program, software, software application, script, or code) may be written in any form of programming language, including compiled or interpreted languages, and may be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program does not necessarily correspond to a file in a file system. A program may be stored in a portion of a file that retains other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files storing one or more modules, subroutines, or portions of code). A computer program may be deployed to execute on a single computer or on multiple computers located at one site or distributed at multiple sites and interconnected by a communications network.

[0207] The processes and logic flows described in this specification may be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The processes and logic flows may also be performed by, and the apparatus may also be implemented as, special purpose logic circuitry, such as an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit).

[0208] For example, processors suitable for executing computer programs include general-purpose microprocessors and special-purpose microprocessors, as well as any one or more processors of any kind of digital computer. Typically, the processor will receive instructions and data from a read-only memory or a random access memory or both. The basic elements of a computer are a processor for executing instructions and one or more storage devices for storing instructions and data. Typically, a computer will also include or be operably coupled to one or more mass storage devices (e.g., magnetic magneto-optical disks or optical disks) to receive data from one or more mass storage devices or transfer data to one or more mass storage devices or both to receive data from one or more mass storage devices and transfer data to one or more mass storage devices for storing data. However, a computer need not have such devices. Computer-readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media, and storage devices, including semiconductor storage devices (e.g., EPROM, EEPROM, and flash memory devices). The processor and memory may be supplemented by or incorporated into a dedicated logic circuit.

[0209] As used herein, the term "about" when referring to a measurable value such as an amount or concentration is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0210] It is intended that the specification, together with the drawings, be considered exemplary only, wherein exemplary refers to examples. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, the use of "or" is intended to include "and / or" unless the context clearly indicates otherwise.

[0211] Although the patent document and the appendix contain many details, these details should not be interpreted as limiting any invention or the scope of what may be claimed, but should be regarded as descriptions of features that may be unique to a particular embodiment of a particular invention. In the context of separate embodiments, certain features described in the patent document and the appendix may also be implemented in combination in a single embodiment. Conversely, the various features described in the context of a single embodiment may also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although the above features may be described as working in certain combinations and even if these features are initially claimed, in some cases, one or more features in the claimed combination may be deleted from the combination, and the claimed combination may be directed to a sub-combination or a variation of the sub-combination.

[0212] Similarly, although operations are depicted in a particular order in the drawings, this should not be understood as requiring that such operations be performed in the particular order shown or in a sequential order or that all of the operations shown be performed to obtain the desired results. In addition, the separation of various system components in the embodiments described in this patent document and the appendix should not be understood as requiring such separation in all embodiments.

[0213] Only a few implementations and examples are described, and other implementations, optimizations, and variations may be made based on what is described and shown in this patent document and the attached appendices.

Claims

1. A system for three-dimensional (3D) imaging of moving particles, the system comprising: (i) a particle moving device comprising a substrate configured to allow particles to move along a travel path in a first direction; (ii) an optical illumination system configured to produce an asymmetric illumination area of ​​light in the range of the travel path of the particle, the optical illumination system scanning multiple portions of the particle at multiple points in time while the particle is moving, wherein said asymmetric illumination area of ​​light includes an illumination dimension that is thinner than the other illumination dimensions to form a shape similar to the illumination plane, The optical illumination system comprises: a light source configured to generate a light beam, the light beam being optically coupled to a light redirecting device, wherein the light redirecting device is configured to modify the light beam by redirecting the light beam to a different angle, and the light redirecting device is optically coupled to one or more passive optical components to shape the redirected light into the asymmetric illumination area of ​​light that illuminates the moving particle; (iii) an optical detection system configured to detect individual points in the particle at time points, thereby allowing space-to-time mapping on a one-to-one basis, wherein the optical detection system is optically connected to the particle moving device and is configured to obtain light signal data associated with different portions of the particle corresponding to the volume of the particle during movement of the particle along the travel path, The optical detection system comprises: one or more photodetectors and a spatial filter, wherein the spatial filter is located between the particle moving device and the one or more photodetectors, wherein the spatial filter comprises a plurality of apertures positioned along a second direction and arranged at an oblique angle to the first direction such that each aperture is configured to filter the asymmetric illumination region of light to correspond to a voxel of the particle, wherein the resolution in the first direction is determined by the distance between holes in the plurality of holes, and the resolution in the second direction is determined by the number of holes in the plurality of holes; and (iv) a data processing unit in communication with the optical detection system, the data processing unit comprising a processor configured to process the light signal data obtained by the optical detection system and generate data including information indicating the 3D characteristics of the particle to reconstruct a 3D image of the particle from the time domain signal, Wherein, the optical illumination system is configured to provide high-speed scanning along a scanning axis.

2. The system according to claim 1, wherein: The asymmetric illumination area of ​​light is directed to the range of the travel path in a second direction perpendicular to the first direction, so that a first scan of the asymmetric illumination area of ​​light forms a first plane including the first direction and the second direction; and wherein the image plane of the optical detection system is arranged along a third direction perpendicular to the first direction and the second direction, so that a second scan of the asymmetric illumination area of ​​light forms a second plane parallel to the first plane and varying along the third direction with the distance from the first plane.

3. The system according to claim 2, wherein: The optical illumination system is configured to scan a first portion of the particle in the first plane at a first time point and scan a second portion of the particle in the second plane at a second time point while the particle moves in the first direction, thereby generating a timing signal spatially mapped to the particle.

4. The system according to claim 1, wherein: The data processing unit is configured to generate a 3D image of the particle voxel by voxel.

5. The system according to claim 4, wherein: The 3D image data of the particles includes a spatial resolution of 1 μm or more in three axes and a field of view of 20 μm or more in the three axes.

6. The system according to claim 1, wherein: The particle moving device includes a particle flow cell including a channel formed on the substrate to allow a fluid containing the particles to flow through the channel as the traveling path.

7. The system according to claim 6, wherein: The flow rate of the particles in the channel is at least 0.2 ms -1 , and wherein the system is configured to generate the data of the particles at a throughput of 500 particles per second or greater.

8. The system according to claim 1, wherein: The particles are fixed to the substrate, and wherein the particle moving device includes a positioning system configured to move the substrate relative to the optical illumination system and the optical detection system so that the particles move along the travel path and are brought into the range where the asymmetric illumination area of ​​light is scanned.

9. The system according to claim 1, wherein: The plurality of apertures of the spatial filter are arranged in a pattern, wherein each aperture of the pattern is spaced apart along the first direction and a position of each aperture of the pattern varies relative to another aperture of the pattern along a second direction perpendicular to the first direction.

10. The system according to claim 1, wherein: The light redirecting device comprises an acousto-optic deflector (AOD).

11. The system according to claim 10, wherein: The AOD can be programmed by the data processing unit to produce periodic angular variations at a rate of at least 100 kHz.

12. The system of claim 1, wherein: The light redirecting means comprises a multiplexed array of scanning mirrors and / or a multiplexed array of scanning gratings.

13. A system according to any one of claims 10 to 12, wherein: The one or more passive optical components are selected from the group consisting of cylindrical lenses, lenses, prisms, and spatial filters to shape the redirected light in the travel path.

14. The system of claim 1, wherein: The one or more photodetectors include photomultiplier tubes.

15. The system of claim 1, wherein: The optical detection system also includes an objective lens optically coupled to the light redirecting device.

16. The system of claim 1, wherein: The optical detection system includes an objective lens optically coupled to the spatial filter.

17. The system of claim 1, wherein: The optical detection system further includes one or more light directing elements to direct the light from the spatial filter to a plurality of photodetectors.

18. The system of claim 17, wherein: The optical detection system also includes two or more photodetectors configured to generate two or more corresponding signals based on two or more light bands or two or more types of light emitted or scattered by the cells.

19. The system of claim 1, wherein: The particles include living cells.

20. A method for three-dimensional (3D) imaging of moving particles, the method comprising: moving the particle along a first direction; Scanning a plurality of portions of the particle portion by portion by directing asymmetric illumination areas of separate lights to corresponding ranges while the particle is moving; spatially filtering a portion of the asymmetric illumination area of ​​light on a scanned portion of the moving particle by a spatial filter, the spatial filter comprising a plurality of apertures positioned along a second direction and arranged at an oblique angle to a first direction, such that each aperture is configured to filter the asymmetric illumination area of ​​light to correspond to a voxel of the particle and allow certain light signals corresponding to a volume of the particle to be detected that pass through the spatial filter; detecting the spatially filtered light signal to obtain individual voxels in three dimensions mapped to the detection time point, The resolution in the first direction is determined by the distance between the holes in the plurality of holes, and the resolution in the second direction is determined by the number of holes in the plurality of holes.

21. The method according to claim 20, further comprising: The light beam generated by the light source is modified into an asymmetrical illumination area of ​​the separate light.

22. The method of claim 20, wherein scanning the plurality of portions of the particle comprises redirecting the light beam to different angles and shaping the redirected light beam into an asymmetric illumination area of ​​light.

23. The method according to claim 20, wherein: An asymmetric illumination area of ​​a first separate light is directed toward a travel path of the particle in a second direction perpendicular to the first direction, so that a first scan of the asymmetric illumination area of ​​the first separate light forms a first plane including the first direction and the second direction; and wherein an image plane for detection is arranged along a third direction perpendicular to the first direction and the second direction, so that a second scan of the asymmetric illumination area of ​​a second separate light forms a second plane parallel to the first plane and varying along the third direction with a distance from the first plane.

24. The method of claim 20, further comprising: The optical signal is processed to generate data including information indicative of the 3D characteristics of the particle.

25. The method according to claim 24, comprising: A 3D image of the particles is generated based on a voxel-by-voxel construction from the generated data.

26. The method of claim 24, wherein the data of the particles comprises: Spatial resolution of 1 μm or more in three axes and a field of view of 20 μm or more in the three axes.

27. The method of claim 24, wherein the data for the particles is generated at a throughput of 500 particles per second or greater.

28. The method of claim 20, wherein the particles are moved through a particle flow cell including a channel formed on a substrate so that a fluid containing the particles flows through the channel in the first direction.

29. The method of claim 20, wherein the particles are fixed to a substrate, and the substrate is moved by a positioning system such that the particles move along the first direction on the substrate.

30. The method of claim 20, wherein the particles comprise living cells.

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