Improvements to a cytometry system

Abstract

A flow cytometry system is provided. The system comprises: a flow cell or flow system configured to accommodate the flow of a fluid including one or more objects of interest; a light source configured to illuminate the object of interest; a plurality of tilted lenses each configured to capture a set of 2D images of the object as it flows through the flow cell or flow system and together configured to translocate the 2D images into a linear array of images perpendicular to the flow of the fluid; a sensor configured to integrate each set of 2D images; wherein the system is configured to read out and process the data from the linear array of images in order to determine one or more spatial parameters of the objects of interest. A method for 3D imaging an object is also provided.

Description

[0001]IMPROVEMENTS TO A CYTOMETRY SYSTEM FIELD OF THE INVENTION The present invention relates to a flow cytometry system and, in particular, to a flow cytometry system configured to read out and process data from a linear array of 2D images of an object of interest to determine one or more spatial parameters of the object of interest. BACKGROUND TO THE INVENTION Modern flow cytometers are able to analyse many thousands of particles per second, with a broad range of applications including molecular biology, pathology, immunology, virology, plant biology and marine biology. Imaging flow cytometry is a technique which combines the high-throughput nature of flow cytometry with the precise imaging capabilities of microscopy to image particles on an individual scale. The resulting images can be analysed and used to extract a vast amount of information, forming rich datasets which have the potential to impact and improve a wide range of applications. Known challenges in imaging flow cytometry include achieving imaging speeds that enable the read-out rate and particle flow rate to be synchronised. Typically, imaging flow cytometry systems require large 2D sensors comprising hundreds to thousands of pixels on each side, causing them to be slow and suffer from rolling shutter artefacts or global shutter loss of signal.2D images of a particle can be reshaped into a linear arrangement suitable for reading with faster detector technologies. Pixels are reassigned and transformed using a single fibre bundle placed at the imaging plane such that an incoming image can be optically reshaped into a thin strip and imaged with a sCMOS camera. Although this can improve imaging speed, transforming pixels leads to a loss in spatial data, and the digital reconstruction of the image after the transformation is both inefficient and distorted. In addition, another challenge in the field of imaging flow cytometry results from the large amount of data produced from imaging objects in flow. Owing to the high throughput nature of the technique, it is necessary to store large numbers of images quickly. The challenge arises in finding efficient methods of processing the images to obtain useful data. Therefore, there is a requirement for an improved flow cytometry system which can efficiently image flowing objects, such that the read-out rate is synchronised with the object’s flow rate. Such a technique should maintain spatial continuity of the images throughout the imaging process such that data can be processed with an improved accuracy and efficiency. It is against this background that the present invention has arisen. SUMMARY OF THE INVENTION According to the present invention, there is provided flow cytometry system comprising: a flow cell or flow system configured to accommodate the flow of a fluid including one or more objects of interest; a light source configured to illuminate the object of interest; a plurality of tilted lenses each configured to capture a set of 2D images of the object as it flows through the flow cell or flow system and together configured to translocate the 2D images into a linear array of images perpendicular to the flow of the fluid; a sensor configured to integrate each set of 2D images; wherein the system is configured to read out and process the data from the linear array of images in order to determine one or more spatial parameters of the objects of interest. The present invention enables 3D imaging of an object in flow by time delayed integration of a plurality of contiguous images of the object. Each optical element captures an image of the object, and the image is translocated, not transformed, into a linear array so that it can be efficiently read out. By translocating the images, the sequence of spatial data is maintained during the optical realignment process, which contrasts known methods of pixel remapping. Consequently, data can be processed with an improved efficiency and can be used to form 3D images of an object with minimised distortion. In some embodiments, the 2D images may be translocated into a linear array comprising one or more linear rows. Translocating the 2D images into a linear arrangement, enables them to be read out with faster, cheaper and smaller sensor technologies such that the read-out rate of the system can be synchronised with the flow rate of the object in solution. By realigning a set of 2D images of an object onto a linear space, the field can be sampled as quickly as the readout technology allows, with none of the drawbacks of large sensor arrays. In some embodiments, the present invention may result in a speed up in measurement by a factor of 3 to 100. In some embodiments, the light source may be a laser such as a continuous wave laser. In some embodiments, the light source may be an LED light source. In some embodiments, the light source may be a lamp. In some embodiments, the light source may be configured to illuminate the object of interest from below, and / or from above, and / or from the side of the flow system. In some embodiments, the light source may illuminate the object of interest such that the object scatters, reflects, refracts and / or emits light, and / or introduces a phase shift to the light. In some embodiments, a plurality of light sources may be provided which may emit light of differing wavelengths. These light sources may be spatially translated in the direction of flow in order to facilitate sequential imaging of the object flowing through the system. In such embodiments, a plurality of detectors may be configured in alignment to each of the light sources. In some embodiments, the system may comprise a light collection assembly. In some embodiments, the light collection assembly may comprise one or more lenses including a high numerical aperture objective lens. In some embodiments, the light collection assembly may comprise spectral and / or spatial filtering components. In some embodiments, the objective lens may capture the emitted, reflected, scattered, refracted and / or phase-shifted light from the illuminated object. In some embodiments, the objective lens may direct at least a portion of the light to a plurality of optical elements such as fibre bundles or tilted lenses. In some embodiments, the light collection assembly may be configured to direct a portion of the light from the illuminated object away from the plurality of optical elements. In some embodiments, the object of interest may be a biological and / or chemical entity such as a biomolecule. The object may be, but is not limited to, a nucleic acid such as DNA or RNA, a protein, a peptide or a polypeptide, a polysaccharide, an antibody or an antibody fragment thereof, an enzyme, a biomolecular complex, a cell or part of a cell or multiple cells, organoids, exosomes and / or a vesicle. As such, the imaging is at the single cell scale. Therefore, the system does not require a wide field of view. Instead of scanning across a wide field of view, the present invention integrates along the scan / flow direction to enhance the detected signal and / or record data sequentially from multiple light sources. A further facet of the system being configured to address an object of interest that is the size of a single cell, is that the whole object can be simultaneously captured in the field of view perpendicular to the flow direction because it is smaller in diameter than the field of view. In some embodiments, the flow cytometry system of the present invention may be used for light field imaging, also called plenoptic imaging or split-pupil imaging, in which a light path is subsampled into angular views. In some embodiments, the system can be configured to record fluorescence images, scatter images, brightfield images and / or quantitative phase contrast images. In some embodiments, the system may be used in Fourier light field imaging for flow cytometry and cell sorting. In some embodiments, the system may be used for multiplane imaging, wherein the light path is modified to redirect differing axially located signals into different lateral positions. In some embodiments, the system of the present invention may be used in other imaging techniques. In some embodiments, the sensor may be a time delayed integration (TDI) camera. In some embodiments, the sensor may be a rolling shutter camera sensor. In some embodiments, the sensor may be an area scan camera. In some embodiments, the sensor may be a line scanner. In some embodiments, the sensor may be a line-scan camera. In some embodiments, the sensor may be a linear or 2D array of single-point detectors, such as photomultiplier tubes or avalanche photo diodes. In some embodiments, processed data from the linear array may be used to determine one or more spatial parameters of the objects of interest. In some embodiments, the processing of the data may include the combination of the translocated images into a 3D image of the object of interest. In some embodiments, the system of the present invention is able to achieve 3D spatial cytometry through multiple synchronised 2D images, because the sequence of spatial data is maintained during the realignment process. This is in contrast to known methods of pixel remapping, which transform pixel data and thus do not maintain the sequence of spatial data during the remapping process. In some embodiments, the plurality of optical elements may be tilted lenses. In some embodiments, the plurality of tilted lenses may be configured to capture a set of 2D images of the object as it flows through the flow system. In some embodiments, the plurality of tilted lenses may be aligned such that the 2D images are translocated into a linear array. In the context of the present invention, a tilted lens may be implemented is a number of different ways. A tilted plane can be added to the curved lensing surface, whether spherical or non-spherical. Alternatively, a tilted plane can be added to the flat side of a lens. Furthermore, each lens may be comprised of two separate optical elements, namely a lens and then an optical element with a tilted plane such as a prism. In this scenario, a prism is provided for each lens so that the tilt of each lens can be set separately. Alternatively, an aperture can be cut from an off-axis curved lens surface. The aperture defines the position of the lens on the substrate, the optical axis of the lens surface defines where the image is formed. This is because the optical axis of the lens is not co-located with the centre of the aperture. This formation provides accurate computation of the optical configuration and can give improved results. In some embodiments, the system may comprise three or more tilted lenses. In some embodiments, the system may comprise any number of tilted lenses from 3 to 100. In some embodiments, the system may comprise seven tilted lenses. In some embodiments, the lenses may be arranged in a non-linear arrangement. In some embodiments, the plurality of tilted lenses may be arranged in a circular, hexagonal, square or random arrangement. In some embodiments, the lenses may be arranged in a close-packed array. In some embodiments, the lenses may not be arranged in a close-packed array and may be spaced apart from each other. In some embodiments, the plurality of tilted lenses may have a spherical or an aspherical lens surface. In some embodiments, each of the lenses may be tilted individually. In some embodiments, each lens may have a different tilted plane. In some embodiments, each of the lenses may be tilted in such a way that 2D images can be translocated via the tilted lenses without rotation or distortion of the original image. In some embodiments, the spacing of the 2D images in the linear array may be adjusted by adjusting the tilt angle of the plurality of tilted lenses. In some embodiments, the lenses may be tilted by between 0 and 15 degrees. In some embodiments, the lenses may be tilted such that the 2D images are shifted by a distance of up to approximately 5 cm. Alternatively, the lenses may each be provided with a prism to guide the image to the desired translocated position in the linear array. In such embodiments, the lenses may not be tilted, but the configuration of the prisms enables the translocation of the 2D images. In some embodiments, the plurality of tilted lenses may be configured in an arrangement in which there are a number of inner lenses and a number of outer lenses. In some embodiments, the inner and outer lenses may be arranged with spacing between the lenses. In some embodiments, one or more of the outer lenses may be tilted such that a portion of light is diverted away from the sensor. In some embodiments, the diverted light may be fed back into the system during the processing of the data for example in a gating process. This can improve the efficiency of the data processing by informing decisions regarding which data to store and analyse. In certain embodiments, in which the tilted lenses are not arranged in a close-packed array, the substrate between the lenses may be shaped to divert light in different directions, typically away from the primary detector, in order to reduce the image background. In some embodiments, the diverted light may be discarded i.e. ejected from the system without being recorded, detected or analysed. Alternatively, the diverted light may be sent onto separate detectors, such as single-point detectors which can, in turn, trigger the acquisition on the imaging detector. This can improve the efficiency of the data processing by informing decisions regarding which data to store and analyse. Alternatively or additionally, the light diverted to the separate detectors can be used for traditional or spectral cytometry to identify cell populations of interest. Furthermore, the light can be diverted onto a velocity sensor to synchronize the flow speed with the detector read out rate. In some embodiments, the plurality of optical elements may be an imaging fibre bundle array. Within the context of the present invention, the term “imaging fibre bundle array” should be understood to refer to a bundle of fibre bundles i.e. a plurality of fibre bundles. In some embodiments, each bundle of imaging fibres may comprise at least 100 individual fibres. In some embodiments, each bundle of imaging fibres may comprise at least 1000 individual fibres. In some embodiments, each bundle of imaging fibres may comprise at least 30000 individual fibres. In some embodiments, the imaging fibre bundle array may comprise 3 to 100 bundles of imaging fibres. In some embodiments, the imaging fibre bundle array may have a density of 102to 106fibres per mm2. In some embodiments, the imaging fibre bundle array may have a density of 17000 fibres / mm2. In some embodiments, the size of an individual imaging fibre may match the pixel size of the sensor, which may range from 1 to 100 µm. In some embodiments, the plurality of optical elements may include an imaging fibre bundle array and a microlens array. In some embodiments the microlens array may be configured to capture a set of 2D images of the object of interest as it flows through the flow system and direct each image into an imaging fibre bundle for optical realignment. In some embodiments, the arrangement and number of microlenses matches the arrangement and number of imaging fibre bundles in the imaging fibre bundle array. In some embodiments, the microlens array may comprise 3 to 100 microlenses. In some embodiments, the arrangement of the imaging fibre bundle array optically realigns and translocates a set of 2D images into a linear array. In some embodiments, the imaging fibre bundle array may have a first, non-linear input configuration and may have a second, linear output configuration. In some embodiments, the input configuration of the imaging fibre bundle array matches the sampling lattice of the microlenses. For example, in some embodiments, the microlens array and the input face of the imaging fibre bundle array may both have a hexagonal arrangement. In some embodiments, the shaping of the imaging fibre bundle array from the first to the second configuration may be achieved by bending or rotating the imaging fibre bundles. In some embodiments, the spatial relationship between each local individual imaging fibre within each bundle of fibres is maintained during the optical realignment. In some embodiments, the imaging fibre bundle array optically realigns the 2D images by rearranging the fibre bundles whilst maintaining the arrangement of the individual fibres in each bundle. This enables the 2D images to be translocated, and not transformed such that their local spatial continuity is maintained during the optical realignment process. In some embodiments, the imaging fibres may be single-mode fibres. In some embodiments, the imaging fibres may be coherent fibres. In some embodiments, the imaging fibres may be incoherent fibres. In some embodiments, the plurality of optical elements may be diffractive optical elements. In some embodiments, the plurality of optical elements may be mirrors, metasurfaces and / or holograms. In some embodiments, the system may comprise a plurality of sensors. In some embodiments, the system may comprise a plurality of time delayed integration (TDI) cameras. In some embodiments, the system may be configured such that each set of 2D images is integrated and read out by a different sensor. In some embodiments, each set of 2D images read out by a different sensor may be produced by a different light source. In some embodiments, operating a plurality of sensors may facilitate more efficient processing of the data from the linear array. In some embodiments, in which the plurality of optical elements comprises an imaging fibre bundle array, one or more of the imaging fibre bundles may translocate a 2D image to a separate sensor. In some embodiments, in which the plurality of optical elements comprises a plurality of tilted lenses, one or more of the tilted lenses may translocate a 2D image to a separate sensor. In some embodiments, the plurality of optical elements may have a hexagonal configuration. In some embodiments, the plurality of tilted lenses may have a hexagonal configuration. In some embodiments, the microlens array may have a hexagonal configuration and the imaging fibre bundle array may have a first, input hexagonal configuration. In some embodiments, the plurality of optical elements may be provided in a square lattice. In some embodiments, the plurality of optical elements may be randomly distributed. In some embodiments, the system may further comprise a spectral and / or spatial filter. In some embodiments, a spectral and / or spatial filter may be located between the light source and the plurality of optical elements. In some embodiments, a spectral and / or spatial filter may be located between the plurality of optical elements and the sensor. In some embodiments, the system may further comprise a grating. In some embodiments, the system may further comprise a series of dichroic mirrors. In some embodiments, a spectral filtering component may be used to split the light collected from the object of interest into a set of discrete colour bands. In some embodiments, a spectral filtering component may be located after the optical realignment system such that spectral data from the 2D images can be spread onto un-used camera chip space. In some embodiments, a spectral filtering component may be located after the optical realignment system such that different colours are translated orthogonal to the flow direction. This can maximise the use of sensor space and may enable multiple sets of 3D data to be formed. In some embodiments, spectral and / or spatial filtering components located between the light source and the plurality of optical elements can be used to direct a portion of the light from the illuminated object away from the plurality of optical elements. In some embodiments, this portion of light can be directed for example to a flow velocity detector or a spectral separator. In some embodiments, the spectral separator may direct light onto point detectors. In some embodiments, the point detectors may be used to acquire traditional or spectral flow cytometry data, for example for identifying and gating a cell population of interest. In some embodiments, the point detectors may be used to trigger when the sensor saves data to memory which may be advantageous in reducing data handling and in providing the output in a format familiar to users of typical cytometry systems. In some embodiments, a flow velocity detector may be configured to facilitate the readout of the sensor being synchronised with the flow. In some embodiments, the flow velocity detector may comprise a fast detector such as an avalanche photodiode (APD) that can readout a light signal. In some embodiments, the flow velocity detector may comprise an opaque to transparent periodic mask such as a binary grating that creates a strobe effect of the signal on the point detector, allowing velocity to be measured easily. In some embodiments, there may be provided a method for 3D imaging an object, the method comprising the steps of: flowing the object in a flow cell or flow system; illuminating the object; capturing 2D images of the object in each of a plurality of optical elements; performing time delayed integration on the 2D images from each of the optical elements; wherein the rate of the time delayed integration is aligned to the flow rate of the object through the flow system; processing the integrated images from each optical element in order to develop a 3D image of the object. In some embodiments, the method may further comprise the step of adjusting the parameters of the plurality of optical elements. In some embodiments, in which the plurality of optical elements comprises a plurality of tilted lenses, the method may further comprise the step of adjusting the tilt angle of the lenses. In some embodiments, the lenses may be tilted at an angle between 0 to 15 degrees. In some embodiments, the lenses may be tilted such that the translocated 2D images are shifted by a distance of up to approximately 10 cm. In some embodiments, in which the plurality of optical elements comprises an imaging fibre bundle array, the method may further comprise the step of reshaping the imaging fibre bundles and / or bending the imaging fibre bundles and / or rotating the imaging fibre bundles into a linear array. FIGURES Figure 1A shows a flow chart of the flow cytometry system of the present invention; Figure 1B shows a schematic diagram of the details of the primary optical path through the system of the present invention; Figures 2A to 2C show the effect of remapping the lightfield in accordance with the present invention; Figures 3A, 3B and 3C show a plurality of non-tilted lenses and the resultant image; Figures 4A, 4B and 4C show a plurality of tilted lenses according to the present invention and the resultant image; Figures 5A and 5B show that the space between tilted lenses can be itself tilted to divert background light; Figure 6 shows a substrate on which a plurality of microlens arrays can be provided; Figure 7 shows a flow diagram of the optical realignment system comprising an imaging fibre bundle array and microlens array; Figure 8A shows the input face of an imaging fibre bundle array; Figure 8B shows the output face of an imaging fibre bundle array; Figure 9 illustrates the reshaping of an imaging fibre bundle array; Figures 10A and 10B illustrate schematically the translocation of 2D images into a linear array according to the present invention; and Figure 11 illustrates the combination of the translocated 2D images into a 3D image of the object of interest. DETAILED DESCRIPTION The present invention is a flow cytometry system which enables by time delayed integration of a plurality of contiguous images of the object and subsequent processing of this data to either produce 3D imaging of an object in flow and / or to extract spatial data regarding the object of interest. Referring to Figure 1A, there is provided a flow chart illustrating the flow cytometry system 10. The system comprises a flow cell 14 which is configured to accommodate the flow of a fluid including one or more objects of interest. The object may be a biological and / or chemical entity such as a biomolecule. The object may be, but is not limited to, a nucleic acid such as DNA or RNA, a protein, a peptide or a polypeptide, a polysaccharide, an antibody or an antibody fragment thereof, an enzyme, a biomolecular complex, a cell or part of a cell or multiple cells, organoids, exosomes and / or a vesicle. The system 10 comprises a light source 12 configured to illuminate the object of interest. The light source 12 may be a laser, an LED light source, a lamp or any other suitable light source. The light source 12 can illuminate the object of interest in the flow cell 14 from below, and / or from above, and / or from the side of the flow cell 14. The light source 12 illuminates the object such that light is emitted, reflected, scattered, refracted and / or phase-shifted by the object and a set of 2D images of the object can be captured by the plurality of optical elements 18. The system 10 can be used for light field imaging, also called plenoptic imaging or split-pupil imaging, in which a light path is subsampled into angular views. The system 10 can be used for multiplane imaging, wherein the light path is modified to redirect differing axially located signals into different lateral positions. The system 10 includes a light collection assembly 16. The light collection assembly 16 can comprise one or more lenses including a high numerical aperture objective lens. The objective lens captures the emitted, reflected, scattered, refracted and / or phase-shifted light from the illuminated object and directs at least a portion of the light to the plurality of optical elements 18. The light collection assembly can also comprise spectral and / or spatial filtering components. As shown in Figure 1A, the spectral and / or spatial filtering components can be used to direct a portion of the light from the object away from the plurality of optical elements 18. This portion of light can be directed for example to a flow velocity detector 24. The flow velocity detector 24 can facilitate the readout of the sensor 22 being synchronised with the flow. The flow velocity detector 24 may comprise a fast detector such as an avalanche photodiode (APD) that can readout a light signal. The flow velocity detector 24 may comprise an opaque to transparent periodic mask such as a binary grating that creates a strobe effect of the signal on the point detector, allowing velocity to be measured easily. The spectral and / or spatial filtering components may direct a portion of the light from the object to a spectral separator 26. The spectral separator 26 may be a stack of dichroic mirrors. The spatial separator 26 may direct light onto point detectors 28. The point detectors 28 may be photomultiplier tubes (PMTs) or avalanche photodiodes (APDs). The plurality of optical elements 18 are each configured to capture a set of 2D images of the object as it flows through the flow system, and to translocate the 2D images of the object into a linear array. Thus, the plurality of optical elements 18 act as an optical realignment system. The 2D images are translocated perpendicular to the direction of flow, not transformed in the direction of flow. The translocation perpendicular to the direction of flow results in a linear array that can be efficiently integrated and read out by the sensor 22. As shown in Figure 1A, the system 10 may comprise a further spectral separator 20 located after the optical realignment of the 2D images by the plurality of optical elements 18 and before the sensor 22. The spectral separator 20 can split the linear array onto unused camera space. The spectral separator 20 may be a dichroic mirror stack. Figure 1B shows a further breakdown of the optical elements in the primary optical path of the system 10 of Figure 1A. Figure 1B shows schematically an object of interest 13 flowing through a flow cell 14 supported by a carrier fluid. The direction of flow through the flow cell 14 is marked by the arrow F. In Figure 1B, a plurality of light sources 12 are configured to illuminate the object of interest 13. The output of each light source 12 is combined via a plurality of dichroic mirrors or beam splitters 15 to provide a beam of excitation light 17 which is directed by a dichroic mirror 21 via an objective 19 to illuminate the object of interest 13 in the flow cell 14. Light that is emitted, reflected or scattered from the object of interest 13 returns through the objective 19 and follows the optical pathway A through the light collection assembly 16. The light passes through a dichroic mirror 21, a spectral filter 31, a tube lens 32, a spatial filter 33 and a relay lens 34. These components may be referred to collectively as spectral and / or spatial filtering components. The collected light is routed via a relay lens 35, a spectral separator 20, a further relay lens 29 and onto the sensor 22. The plurality of optical elements 18 can include: a) A plurality of tilted lenses. b) An imaging fibre bundle array and a microlens array c) Flat optics such as diffractive optical elements, mirrors, metasurfaces or holograms. The sensor 22 is configured to integrate each set of 2D images. The sensor may perform time delayed integration on the 2D images from each of the plurality of optical elements 18. The sensor 22 may be an sCMOS sensor. The sensor 22 may be a line scanner. The sensor 22 may be a time delayed integration (TDI) camera. The sensor 22 may be a rolling shutter camera. The sensor 22 has a linear arrangement and may comprise one or more linear rows. By maintaining the sequence of spatial data during the realignment process, the sensor 22 may be a faster, cheaper and smaller sensor than is compatible with known methods of flow cytometry. The sensor 22 is configured such that the rate of the time delayed integration is synchronised with the flow rate of the object through the flow cell 14. The system 10 may comprise a plurality of sensors 22 such as a plurality of TDI cameras. The system 10 can be configured such that each set of 2D images is read out by a different sensor 22. This can increase the efficiency of the read out and data processing. The flow cytometry system 10 is configured such that processed data from the linear array can be used to determine one or more spatial parameters of the objects of interest. Processing the data may include combining the translocated 2D images into a 3D image of the object of interest, as illustrated by Figure 11. The system 10 may further comprise a processing system 30 which may include a 3D reconstruction system, achieving 3D spatial cytometry through multiple synchronised 2D images. The system 10 is able to achieve 3D reconstruction of an image of the object of interest because the sequence of spatial data is maintained during the realignment process. In addition, the flow velocity detector 24 and the point detectors 28 can transmit data to the processing system 30 to be used in a gating process which helps to identify images of interest and refine the set of images used in the 3D reconstruction process. Thus, the portions of light which are directed away from the plurality of optical elements 18 can be used to enhance the flow cytometry system 10 and facilitate a more efficient processing system 30. Figures 2A to 2C show the effect of remapping the lightfield in accordance with the present invention. Figure 2A shows an object of interest 13 flowing through a flow cell 14 in flow direction F. The field of view of the light collection assembly 16 is illustrated by the rectilinear zone 50. A first illumination area 52 and a second illumination area 54 are shown. These two illumination areas could have different excitation wavelengths. The present invention may make use of one, two or more illumination areas in flow direction for sequential imaging. Figure 2B shows schematically a standard lightfield image 60 comprising a plurality of angular views of the field of view of the optical system. The multiple images overlap significantly and cannot be imaged with a line scanner or TDI camera. In contrast, Figure 2C shows a remapped lightfield image 80 in showing a readout 82 from the first illumination area 52 as captured by the line scanner or TDI camera 22. In addition, a readout 84 from the second illumination area 54 is illustrated as captured by a second line scanner or TDI camera 22. The two readouts 82, 84 can correspond to two separate cameras 22 or separate detection arrays on the same camera 22. The plurality of optical elements 18 may be a plurality of tilted lenses. Figure 3A shows a hexagonal array of non-tilted lenses 36. Figure 3C illustrates the focusing of light 40 as it passes through the hexagonal array of non-tilted lenses 36, and Figure 3B shows the resultant corresponding hexagonal array of images 37. In contrast, Figure 4A shows a hexagonal array of tilted lenses 38 and Figure 4C shows the focusing of light 40 as it passes through the hexagonal array of tilted lenses 38. Figure 4B shows the resultant linear array of images 39. Each of the lenses may be tilted in such a way that 2D images can be translocated via the tilted lenses 38 without rotation or distortion of the original image. Each of the lenses can be tilted individually and each lens may have a different tilted plane. The tilted lenses 38 are configured to capture a set of 2D images of the target object 13 as it flows through the flow cell 14. Although Figure 4A depicts seven tilted lenses 38, the system 10 may comprise any number of tilted lenses from 3 to 100. Although Figure 4A depicts the lenses 38 arranged with space in between each lens, the spacing of the lenses may be adjusted to adjust the spacing of the 2D images in the corresponding linear array 39. Although Figure 4A shows a hexagonal arrangement of the tilted lenses 38, the lenses may be arranged in a circular, square or any other outline shape. The tilted lenses 38 may have a spherical or an aspherical lens surface. As shown in Figure 5A, the plurality of tilted lenses 38 can be configured in an arrangement in which the space between lenses 41 is shaped to divert background light 42. The resulting image with diverted background light 42 is shown in Figure 5B. The space between lenses 41 may be tilted such that light is diverted away from the sensor 22, or onto point detectors for velocity detection 24 or conventional cytometry 28. This diverted light 42 may be used to improve the efficiency of the data processing through feeding into a gating process in the processing system 30. The system 10 can be implemented with a plurality of sets of tilted lenses 38, each set as illustrated in Figures 4A and 5A, arranged next to each other on a linear stage. The sensor 22 can be configured to switch between each set of tilted lenses 38 in order to achieve different outputs. The sets of tilted lenses 38 can be provided together on a single glass substrate 90 in any configuration that facilitates their translation including, but not limited to a linear configuration or a rotating filter wheel. Figure 6 shows a substrate 90 on which a plurality of microlens arrays 38 can be provided. The microlens arrays 38 have different parameters including different lenslet number, different positions and diameter of lenslets and different focal length and / or tilt. In an alternative example not shown in the accompanying drawings, the microlens arrays may all be identical to one another. The relayed back focal plane 92 of the objective 19 is shown positioned around one of the microlens array 38 on which the system is focussed. The arrow X shows the direction of translation of the substrate 90 relative to the back focal plane 92 of the objective 19 which enables the use of different microlens arrays 38 without other modifications to the optical system 10. The substrate 90 may be placed on a linear or rotational stage (not shown) to position the different microlens arrays 38 in the optical path. Alternatively, in an example not illustrated in the accompanying drawings, a plurality of substrates, each with a different microlens array, may be placed on a linear or rotational stage for the same purpose. This allows the system to switch between different imaging modes optimised for different applications. Alternatively, the plurality of optical elements 18 may be a microlens array 44 and an imaging fibre bundle array 46. As shown in Figure 7, the light collection assembly 16 directs light from the object of interest 13 to the microlens array 44. The microlens array 44 captures a set of 2D images of the object of interest 13 as it flows through the flow cell 14. The microlens array 44 directs a 2D image of the object into each imaging fibre bundle of an imaging fibre bundle array 46, where the 2D images are optically realigned and translocated into a linear array, which is subsequently integrated by the sensor 22. Referring to Figure 8, the imaging fibre bundle array 46 has a first, input configuration 48 shown in Figure 8A, and a second, output configuration 52 shown in Figure 8B. The imaging fibre bundle array 46 may comprise 3 to 100 bundles of imaging fibres. The number of imaging fibre bundles in the imaging fibre bundle array 46 corresponds to the number of microlenses in the microlens array 44. Referring to Figures 8 and 9, an example is shown in which the imaging fibre bundle array 46 comprises seven individual imaging fibre bundles 1, 2, 3, 4, 5, 6, 7. As shown in Figure 8A, the first, input configuration 48 may be arranged in a hexagonal array, and the microlens array 44 will be arranged in a corresponding hexagonal arrangement. As shown in Figure 8B, the second, output configuration 52 is a linear arrangement. As shown in Figure 9, the shaping of the imaging fibre bundle array 46 may be achieved by bending or rotating the imaging fibre bundles 1, 2, 3, 4, 5, 6, 7 from a first, input configuration 48 to a second, output configuration 52. Each bundle of imaging fibres 1, 2, 3, 4, 5, 6, 7 may comprise at least 100, at least 1000, at least 30000 individual fibres. The spatial relationship between the individual fibres in each of the imaging fibre bundles 1, 2, 3, 4, 5, 6, 7 is maintained as only the macroscopic bundles of imaging fibres are rearranged. In this way, the imaging fibre bundle array 46 can optically realign and translocate a set of 2D images, without losing the spatial continuity of the 2D images. The imaging fibre bundle array 46 may have a density of 102to 106fibres per mm2. The imaging fibre bundle array 46 may have a density of 17000 fibres / mm2. The size of an individual imaging fibre may match the pixel size of the sensor 22, which may range from 1 to 100 µm. The imaging fibres may be single-mode fibres. The imaging fibres may be coherent fibres or incoherent fibres. Referring to Figures 10A and 10B, the optical realignment of a set of 2D images 62 into a linear array 66 is illustrated schematically. It can be seen in Figures 10A and 10B that the 2D images 62 are translocated during the optical area realignment process 64. The 2D images 62 are translocated, not transformed, and the local coordinates of each 2D image e.g. (x1, y1), (x2, y2), (xN, yN) are maintained during the optical realignment process 64. As illustrated by arrow 68, the sensor 22 is configured to read out the data from the linear array at a rate synchronised with the flow rate of the object. Referring to Figure 10B, the realigned 2D images 66 may represent different z slices of a target object, and the system 10 may be used for multi-plane imaging as shown in example 70. The realigned 2D images 66 may represent different angular projections of a target object and the system 10 may be used for light field or tomographic imaging as shown in example 72. Figure 11 illustrates the 3D image reconstruction process. The realigned, translocated 2D images 66 are combined into a 3D image of the object of interest 74. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

CLAIMS 1. A flow cytometry system comprising: a flow cell or flow system configured to accommodate the flow of a fluid including one or more objects of interest; a light source configured to illuminate the object of interest; a plurality of tilted lenses each configured to capture a set of 2D images of the object as it flows through the flow cell or flow system and together configured to translocate the 2D images into a linear array of images perpendicular to the flow of the fluid; a sensor configured to integrate each set of 2D images; wherein the system is configured to read out and process the data from the linear array of images in order to determine one or more spatial parameters of the objects of interest.

2. The flow cytometry system according to claim 1, wherein the sensor is a time delayed integration camera.

3. The flow cytometry system according to claim 1, wherein the sensor is a line scanner 4. The flow cytometry system according to any of the preceding claims, wherein the processing of the data includes the combination of the translocated images into a 3D image of the object of interest.

5. The flow cytometry system according to any of the preceding claims, wherein the system comprises a plurality of sensors.

6. The flow cytometry system according to any of the preceding claims, wherein the plurality of optical elements each has a hexagonal configuration.

7. The flow cytometry system according any of the preceding claims, further comprising a spectral and / or spatial filter.

8. A method for 3D imaging an object and / or determining spatial parameters of an object, the method comprising the steps of: flowing the object in a flow cell or flow system; illuminating the object; capturing 2D images of the object in each of a plurality of tilted lenses; performing time delayed integration on the 2D images from each of the optical elements; wherein the rate of the time delayed integration is aligned to the flow rate of the object through the flow system; translocating the 2D images into a linear array; and processing the integrated images from each optical element sequentially in order to develop a 3D image of the object and / or determine spatial parameters of the object.

9. The method according to claim 8, further comprising the step of adjusting the parameters of the plurality of tilted lenses.

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