Optical apparatus, system and method for determining a property of at least one particle

Abstract

An optical apparatus having an optical source arrangement to provide first and second source lights having different wavelengths, an optical sampling arrangement to receive the source lights, and direct respective portions of the source lights towards a fluid sample comprising at least one particle, provide, at a focusing region within the sample, first and second line illumination patterns based on the first and second portions, the illumination patterns being offset from one another, and combine, with a reference light, a first return light from the particle illuminated by the first illumination pattern and a second return light from the particle illuminated by the second illumination pattern, and an optical detection arrangement having at least one photodetector arrangement to detect resultant lights resulting from combination of the first and second return lights and the reference light for determining a property of the particle.

Description

[0001] OPTICAL APPARATUS, SYSTEM AND

[0002] METHOD FOR DETERMINING A PROPERTY OF AT LEAST ONE PARTICLE FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to an optical apparatus, a system having an optical apparatus, and a method for determining a property of at least one particle.

[0004] BACKGROUND

[0005] Size measurement of small particles including proteins, nucleic acids, viruses and nanoparticles, has significant applications in biotechnology. Techniques based on scattering of light can be used to measure particle size. Proteins are typically characterised by mass which is closely corelated to size and the measured scattering coefficient. Similarly for nucleic acids (RNA, DNA) the number of base pairs squared is proportional to scattering coefficient.

[0006] If a beam of light is propagated through a solution containing particles, the intensity of light scattered outside the beam increases with particle size or concentration. If the concentration is known in advance, this technique of static light scattering (SLS) allows size to be determined.

[0007] For particles with sizes near the wavelength of the light used, the scattered intensity varies more with angle, Mie scattering, compared to the dipole pattern Rayleigh scattering of smaller particles. Multi-Angle static Light Scattering (MALS) allows both average size and concentration of particles to be determined from the variation of scattering intensity detected by detectors at different angles from the input beam.

[0008] Dynamic light scattering (DLS) [Stetefeld J., et al, Biophysical Review 8:409-427 (2016)] observes the time variation of the scattered signal for a detector or in some cases multiple detectors at different angles. Scattering from a small number of large particles with slow diffusion, Brownian motion, will show large statistical fluctuations over a long timescale. Comparatively for a large number of small, fast diffusing, particles the scattering will fluctuate less from the average but over a faster timescale. Analysing the time correlations of the scattering signal allows calculation of the particles diffusion coefficient and hydrodynamic radius related to its physical size. Mixed populations of large and small particles can be analysed from their contributions to correlations on different timescales, but it is difficult to resolve populations differing by less than a factor of 3-5 in size.

[0009] Size Exclusion Chromatography (SEC-MALS) first fractionates the sample into populations with differing diffusion coefficient by flow through a gel column. Multi-Angle static Light Scattering can then be used to measure the size of each population. This is relatively slow and passage through the chromatography column may change the sample.

[0010] Interferometric scattering microscopy (iSCAT) is a technique for detection and tracking of particles binding to a glass coverslip or other surface. The interference between light scattered from an input beam and a fraction of the input beam reflected from the surface is detected. This has been further developed, with a spatially variable transmission filter to control the intensity of the reflected reference beam, as mass photometry (MP) [GB2552195A], Single protein molecules can be detected binding to a coverslip with the strength of the interference signal proportional to mass or size. As each individual particle is detected separately, MP has a much-improved ability to resolve populations of particles with small size differences compared to DLS. Size / mass of proteins, nucleic acids and viruses have all been measured with MP. The rate at which particles bind to the surface and are detected is related to the sample concentration but also the surface chemistry. As reliable particle binding to the measurement surface is required, most MP measurements use disposable single use surfaces. A reusable surface will have strict requirements on cleaning making MP more difficult to apply to a high throughput system. Solid immersion lenses with durable materials have been considered for reusable surface MP systems [WO2023012473A1] and may be integrated into a flow cell system [WO2024161138A1].

[0011] Needham L.-M., et. al. [bioRxiv Preprint doi: 10.1101 / 2023.03.24.534170] have demonstrated single molecule detection of small proteins in solution with fibre coupled Fabry-Perot microcavities. Information on the size and diffusion coefficient is obtained but the resolution is not clear. This technique uses very high reflectivity mirrors that may put strict requirements on the cleanliness of the cavity and sample.

[0012] ISICL (In situ coherent lidar) was developed for particle detection in semiconductor processing chambers by Hobbs [US5294806A, and Applied Optics Vol. 34, No. 9, 1579-1590 (1995)]. It builds on developments in lidar where a reference beam is interfered with the signal light for coherent detection. In one arm of a Michelson-type interferometer, the input beam is focused by a lens and scanned by a moving mirror. Some of the scattered light from any particle near the focus is collected by the lens and forms a signal beam to the detection system. The other arm of the interferometer uses a retroreflector to send a portion of the input as a reference beam to the detection system. The interference between the signal and reference is used to detect the presence of a particle at the focus. Scattering from moving particles shows a Doppler shift that modulates the interference and detecting the frequency of this modulation allows the particles axial velocity to be determined. Interference between the signal and reference occurs only for the collimated beam returned from near the focus of the lens limiting the detection to an enclosed volume. With a low numerical aperture (NA) lens that collects a small fraction of the available scattered light, this system can detect particles below 1 micrometre in size. The signal obtained will depend on particle size but also on how close it passes to the centre of the focus, limiting ISICL’s use for size measurement. The minimum detectable size is two orders of magnitude larger than a typical protein. Further, ISICL has limited size resolution.

[0013] It is an object of at least preferred embodiments of the present invention to provide one or more apparatus and / or one or more methods that address at least some of the problems of known techniques, and / or to at least provide the public with a useful alternative.

[0014] SUMMARY

[0015] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0016] In an aspect, the present disclosure broadly includes an optical apparatus comprising: an optical source arrangement configured to provide a first source light and a second source light, the first and second source lights having different wavelengths; an optical sampling arrangement optically coupled to the optical source arrangement to receive the first and second source lights, the optical sampling arrangement being configured to: direct a first portion of the first source light and a second portion of the second source light along a propagation axis towards a fluid sample comprising at least one particle; provide, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, and a second line illumination pattern based on the second portion, the first and second line illumination patterns being offset from one another and each having a long axis perpendicular to the propagation axis; and combine, with a reference light derived from the first and second source lights, a first return light from the at least one particle illuminated by the first line illumination pattern and a second return light from the at least one particle illuminated by the second line illumination pattern; and an optical detection arrangement optically coupled to the optical sampling arrangement, the optical detection arrangement comprising at least one photodetector arrangement configured to detect a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light for determining a property of the at least one particle.

[0017] In an aspect, the present disclosure broadly includes an optical apparatus having an optical source arrangement configured to provide a first source light and a second source light, the first and second source lights having different wavelengths; an optical sampling arrangement optically coupled to the optical source arrangement to receive the first and second source lights, the optical sampling arrangement being configured to: direct a first portion of the first source light and a second portion of the second source light towards a fluid sample comprising at least one particle; provide, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, and a second line illumination pattern based on the second portion, the first and second line illumination patterns being offset from one another; and combine, with a reference light derived from the first and second source lights, a first return light from the at least one particle illuminated by the first line illumination pattern and a second return light from the at least one particle illuminated by the second line illumination pattern; and an optical detection arrangement optically coupled to the optical sampling arrangement, the optical detection arrangement having at least one photodetector arrangement configured to detect a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light for determining a property of the at least one particle.

[0018] In various embodiments, the first and / or second return light comprise light scattered by the at least one particle from the first and / or second line illumination pattern, respectively. In various embodiments the at least one particle comprises one particle. In various embodiments, the long axis may refer to a longitudinal axis. In various embodiments, the line illumination pattern comprises the long axis and a short axis, wherein the long axis is at least approximately 2, 3, 5, 10 or 20 times longer than the short axis.

[0019] In various embodiments, the first and second illumination patterns are offset along the direction of the propagation axis.

[0020] In various embodiments, the first return light and the second return light are collected from light back scattered, forward scattered, or scattered to the side, optionally perpendicular, to the propagation axis.

[0021] In various embodiments, the first portion and the second portion are directed towards the fluid sample through a shared objective lens.

[0022] In various embodiments, the first and second line illumination patterns are generated by scanning of the first source light and second source light.

[0023] In various embodiments, the first source light and second source light comprise line illumination outputs configured to generate the line illumination patterns at the focussing region.

[0024] In various embodiments, the reference light comprises a first reference light and a second reference light, the first reference light associated with the first source light and the second reference light associated with the second source light. In various embodiments, the first resultant light results from the combination of the first return light and the first reference light and the second resultant light results from the combination of the second return light and the second reference light.

[0025] In various embodiments, the optical source arrangement may include a first multi spatial mode laser source configured to provide the first source light; and a second multi spatial mode laser source configured to provide the second source light.

[0026] In various embodiments, comprising a dual wavelength laser configured to provide the first multi-spatial mode laser source and the second multi-spatial mode laser source. In various embodiments, the first illumination pattern and the second line illumination patterns overlap. The at least one particle may be present in the first, second or both illumination patterns.

[0027] In various embodiments, a wavelength of the first source light and a wavelength of the second source light may be within a range of 200 - 2000 nm.

[0028] In various embodiments, each of the wavelength of the first source light and the wavelength of the second source light may be within a range of 400 - 500 nm.

[0029] In various embodiments, the different wavelengths may be offset from one another by about 5 - 500 nm.

[0030] In various embodiments, the first and second line illumination patterns may be offset from one another by about 10 - 5000 nm.

[0031] In various embodiments, the optical source arrangement may include an optical element configured to combine the first and second source lights.

[0032] In various embodiments, the optical element may include a dichroic mirror or a prism or a diffraction grating.

[0033] In various embodiments, the optical sampling arrangement may include an optical polarising arrangement configured to control respective amounts of the first portion of the first source light, the second portion of the second source light, and the reference light.

[0034] In various embodiments, the optical polarising arrangement may include: a polarising beam splitter configured to: split the first source light and the second source light into the first portion and the second portion; direct the first portion and the second portion towards the fluid sample; and combine the first return light and the second return light with the reference light; an optical polarising device in an optical propagation path between the optical source arrangement and the polarising beam splitter; a first polarisation control element (such as a first quarter waveplate element) in an optical propagation path between the polarising beam splitter and the fluid sample; and a second polarisation control element (such as a second quarter waveplate element) in an optical propagation path between the polarising beam splitter and an optical reflector arrangement configured to provide the reference light.

[0035] In various embodiments, the optical reflector arrangement may include a retroreflector.

[0036] In various embodiments, the optical reflector arrangement may include a dispersion compensator.

[0037] In various embodiments, the optical source arrangement may be configured to alternately provide the first source light and the second source light.

[0038] In various embodiments, the optical apparatus may be configured for phases of the reference light and the first and second return lights to be modulated relative to one another.

[0039] In various embodiments, the optical sampling arrangement may include an optical phase modulator configured to modulate the phase of the reference light relative to the phases of the first and second return lights. In various embodiments, the optical phase modulator is configured to modulate the phase of the first reference light and the second reference light differently.

[0040] In various embodiments, the optical detection arrangement may include another optical phase modulator configured to modulate the phases of the reference light and the first and second return lights relative to one another.

[0041] In various embodiments, the optical detection arrangement may include: a non-polarising beam splitter configured to receive the first resultant light and the second resultant light, and further configured to split each of the first resultant light and the second resultant light along a first optical propagation pathway and a second optical propagation pathway; a first polariser element in the first optical propagation pathway, the first polariser element being configured for an angle that is non-zero and non-90 degrees; a second polariser element in the second optical propagation pathway, the second polariser element being configured for an angle that is non-zero and non-90 degrees; and a polarisation element (such as a waveplate element) configured to introduce a phase difference of about 90° between the first optical propagation pathway and the second optical propagation pathway.

[0042] In various embodiments, the at least one photodetector arrangement may include: a first photodetector arrangement in the first optical propagation pathway; and a second photodetector arrangement in the second optical propagation pathway.

[0043] In various embodiments, the optical detection arrangement may include: a non-polarising beam splitter configured to receive the first resultant light and the second resultant light, and further configured to split each of the first resultant light and the second resultant light along a first optical propagation pathway and a second optical propagation pathway; a first polarising beam splitting device in the first optical propagation pathway, the first polarising beam splitting device being configured to: split the first resultant light into a plurality of first pairs of lights, wherein for each first pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective first section of the first line illumination pattern; and split the second resultant light into a plurality of second pairs of lights, wherein for each second pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective second section of the second illumination pattern; a second polarising beam splitting device in the second optical propagation pathway, the second polarising beam splitting device being configured to: split the first resultant light into a plurality of third pairs of lights, wherein for each third pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with the respective first section of the first illumination pattern; and split the second resultant light into a plurality of fourth pairs of lights, wherein for each fourth pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with the respective second section of the second illumination pattern; and polarisation control element (such as a waveplate element) configured to introduce a phase difference of about 90° between the first resultant lights in the first optical propagation pathway and the second optical propagation pathway, and between the second resultant lights in the first optical propagation pathway and the second optical propagation pathway. In various embodiments, the at least one photodetector arrangement may include: a first photodetector arrangement configured to detect the plurality of first pairs of lights and the plurality of second pairs of lights for determining the property of the at least one particle; and a second photodetector arrangement configured to detect the plurality of third pairs of lights and the plurality of fourth pairs of lights for determining the property of the at least one particle.

[0044] In various embodiments, the optical detection arrangement may further include an optical element configured to separate the first resultant light and the second resultant light from each other.

[0045] In various embodiments, the optical detection arrangement may include a polariser element arranged for the first resultant light and the second resultant light to pass through, the polariser element being configured for an angle that is non-zero and non-90 degrees.

[0046] In various embodiments, the at least one photodetector arrangement may include a plurality of detector elements, wherein a respective detector element of the plurality of detector elements is configured to receive a respective portion of at least one of the first resultant light or the second resultant light.

[0047] In various embodiments, the optical detection arrangement may include a polarising beam splitting device configured to: split the first resultant light into a plurality of first pairs of lights, wherein for each first pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective first section of the first line illumination pattern; and split the second resultant light into a plurality of second pairs of lights, wherein for each second pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective second section of the second illumination pattern, wherein the at least one photodetector arrangement is configured to detect the plurality of first pairs of lights and the plurality of second pairs of lights for determining the property of the at least one particle.

[0048] In various embodiments, the optical sampling arrangement may include an optical chromatic aberration arrangement configured to receive the first portion of the first source light and the second portion of the second source light, and further configured for the first and second line illumination patterns to be offset from one another at the focusing region within the fluid sample.

[0049] In various embodiments, the optical chromatic aberration arrangement may include an immersion lens.

[0050] In various embodiments, the optical apparatus may further include a flow cell device, the flow cell device having a flow channel configured to receive the fluid sample, and the immersion lens defines a side of the flow channel.

[0051] In various embodiments, the optical detection arrangement may include an array of microlens elements configured to receive the first resultant light and the second resultant light, wherein respective microlens elements are associated with respective sections of at least one of the first line illumination pattern or the second line illumination pattern.

[0052] In various embodiments, the optical detection arrangement may include an optical telephoto arrangement configured to receive the first and second resultant lights and direct the first and second resultant lights to a plane where the array of microlens elements is arranged.

[0053] In various embodiments, the optical source arrangement may be configured to alternately provide the first source light and the second source light.

[0054] In various embodiments, the optical apparatus may be configured for phases of the reference light and the first and second return lights to be modulated relative to one another.

[0055] In various embodiments, the optical sampling arrangement may include an optical phase modulator configured to modulate the phase of the reference light relative to the phases of the first and second return lights.

[0056] In various embodiments, the optical detection arrangement may include another optical phase modulator configured to modulate the phases of the reference light and the first and second return lights relative to one another. In various embodiments, the optical sampling arrangement may include a beam splitter (such as a non-polarising beam splitter), the beam splitter being configured to: split the first source light and the second source light into the first portion and the second portion; direct the first portion and the second portion towards the fluid sample; and combine the first return light and the second return light with the reference light.

[0057] In various embodiments, the optical sampling arrangement comprises a beam splitter, the beam splitter being configured to: direct the first portion of the first source light and the second portion of the second source light towards the fluid sample; direct a third portion of the first source light and a fourth portion of the second source light towards a reference arm configured to generate the reference light.

[0058] In various embodiments, the beam splitter is configured to combine the reference light (comprising first and second reference lights) returned from the reference arm with the first return light and the second return light.

[0059] In various embodiments, the at least one photodetector arrangement may include a plurality of detector elements, wherein a respective detector element of the plurality of detector elements is configured to receive a respective portion of at least one of the first resultant light or the second resultant light.

[0060] In an aspect, the present disclosure broadly includes a system having the optical apparatus described herein; and a processor configured to process electrical signals corresponding to the first resultant light and the second resultant light detected by the at least one photodetector arrangement for determining the property of the at least one particle.

[0061] In various embodiments, the processor may be configured to determine a size of the at least one particle based on processing of the electrical signals.

[0062] In various embodiments, for determining the size of the at least one particle, the processor may be configured to determine a scattering coefficient associated with the at least one particle based on the processing of the electrical signals. In various embodiments, for determining the scattering coefficient, the processor may be configured to determine a position of the at least one particle at the focusing region based on the processing of the electrical signals.

[0063] In various embodiments, for determining the size of the at least one particle, the processor may be configured to: for each first pair of the plurality of first pairs of lights, perform a first subtraction operation of electrical signals corresponding to the first pair of lights; for each second pair of the plurality of second pairs of lights, perform a second subtraction operation of electrical signals corresponding to the second pair of lights; for each third pair of the plurality of third pairs of lights, perform a third subtraction operation of electrical signals corresponding to the third pair of lights; for each fourth pair of the plurality of fourth pairs of lights, perform a fourth subtraction operation of electrical signals corresponding to the fourth pair of lights; and determine the size of the at least one particle based on results of the first to fourth subtraction operations for the plurality of first to fourth pairs of lights.

[0064] In various embodiments, for determining the size of the at least one particle, the processor may be configured to: for each first pair of the plurality of first pairs of lights, perform a first subtraction operation of electrical signals corresponding to the first pair of lights; for each second pair of the plurality of second pairs of lights, perform a second subtraction operation of electrical signals corresponding to the second pair of lights; and determine the size of the at least one particle based on results of the first and second subtraction operations for the plurality of first and second pairs of lights.

[0065] In an aspect, the present disclosure broadly includes a method for determining a property of at least one particle, the method including: directing a first portion of a first source light and a second portion of a second source light along a propagation axis towards a fluid sample comprising at least one particle, the first and second source lights having different wavelengths; providing, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, and a second line illumination pattern based on the second portion, the first and second line illumination patterns being offset from one another and each having a long axis substantially perpendicular to the propagation axis; combining, with a reference light derived from the first and second source lights, a first return light from the at least one particle illuminated by the first line illumination pattern and a second return light from the at least one particle illuminated by the second line illumination pattern; detecting a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light; and determining a property of the at least one particle based on electrical signals corresponding to the first resultant light and the second resultant light that are detected.

[0066] In various embodiments, a wavelength of the first source light and a wavelength of the second source light may be within a range of 200 - 2000 nm.

[0067] In various embodiments, each of the wavelength of the first source light and the wavelength of the second source light may be within a range of 400 - 500 nm.

[0068] In various embodiments, the different wavelengths may be offset from one another by about 5 - 500 nm.

[0069] In various embodiments, the first and second line illumination patterns may be offset from one another by about 10 - 5000 nm.

[0070] In various embodiments, the method may further include combining the first and second source lights prior to directing the first portion of the first source light and the second portion of the second source light towards the fluid sample.

[0071] In various embodiments, the method may further include controlling respective amounts of the first portion of the first source light, the second portion of the second source light, and the reference light.

[0072] In various embodiments, controlling the respective amounts may include: splitting, with a polarising beam splitter, the first source light and the second source light into the first portion and the second portion; directing, with the polarising beam splitter, the first portion and the second portion towards the fluid sample; combining, with the polarising beam splitter, the first return light and the second return light with the reference light; using an optical polarising device in an optical propagation path between an optical source arrangement configured to provide the first and second source lights and the polarising beam splitter; using a first quarter waveplate element in an optical propagation path between the polarising beam splitter and the fluid sample; and a second quarter waveplate element in an optical propagation path between the polarising beam splitter and an optical reflector arrangement configured to provide the reference light.

[0073] In various embodiments, the method may further include alternately providing the first source light and the second source light.

[0074] In various embodiments, the method may further include modulating phases of the reference light and the first and second return lights relative to one another.

[0075] In various embodiments, the method may further include splitting, with a non-polarising beam splitter, each of the first resultant light and the second resultant light along a first optical propagation pathway and a second optical propagation pathway; using a first polariser element in the first optical propagation pathway, the first polariser element being configured for an angle that is non-zero and non-90 degrees; using a second polariser element in the second optical propagation pathway, the second polariser element being configured for an angle that is non-zero and non-90 degrees; and introducing, with a waveplate element, a phase difference of about 90° between the first resultant lights in the first optical propagation pathway and the second optical propagation pathway, and between the second resultant lights in the first optical propagation pathway and the second optical propagation pathway.

[0076] In various embodiments, the method may further include splitting, with a non-polarising beam splitter, each of the first resultant light and the second resultant light along a first optical propagation pathway and a second optical propagation pathway; splitting, with a first polarising beam splitting device in the first optical propagation pathway, the first resultant light into a plurality of first pairs of lights, wherein for each first pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective first section of the first line illumination pattern; splitting, with the first polarising beam splitting device, the second resultant light into a plurality of second pairs of lights, wherein for each second pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective second section of the line illumination pattern; splitting, with a second polarising beam splitting device in the second optical propagation pathway, the first resultant light into a plurality of third pairs of lights, wherein for each third pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with the respective first section of the line illumination pattern; splitting, with the second polarising beam splitting device, the second resultant light into a plurality of fourth pairs of lights, wherein for each fourth pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective second section of the second line illumination pattern; introducing, with a waveplate element, a phase difference of about 90° between the first resultant lights in the first optical propagation pathway and the second optical propagation pathway, and between the second resultant lights in the first optical propagation pathway and the second optical propagation pathway, wherein detecting the first resultant light and the second resultant light comprises detecting the plurality of first pairs of lights, the plurality of second pairs of lights, the plurality of third pairs of lights and the plurality of fourth pairs of lights.

[0077] In various embodiments, detecting the plurality of first pairs of lights and the plurality of second pairs of lights may include detecting with a first photodetector arrangement, and detecting the plurality of third pairs of lights and the plurality of fourth pairs of lights may include detecting with a second photodetector arrangement.

[0078] In various embodiments, the method may further include separating the first resultant light and the second resultant light from each other prior to splitting each of the first resultant light and the second resultant light along the first optical propagation pathway and the second optical propagation pathway.

[0079] In various embodiments, the method may further include passing the first resultant light and the second resultant light through a polariser element, the polariser element being configured for an angle that is non-zero and non-90 degrees.

[0080] In various embodiments, the method may further include splitting, with a polarising beam splitting device, the first resultant light into a plurality of first pairs of lights, wherein for each first pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective first section of the first line illumination pattern; and splitting, with the polarising beam splitting device, the second resultant light into a plurality of second pairs of lights, wherein for each second pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and the lights are associated with a respective second section of the second line illumination pattern, wherein detecting the first resultant light and the second resultant light may include detecting the plurality of first pairs of lights and the plurality of second pairs of lights.

[0081] In various embodiments, providing the first and second line illumination patterns at the focusing region may include controlling a chromatic aberration of a lens system along the propagation axis toward the fluid sample (which may be referred to as the objection lens arrangementjto offset the first and second line illumination patterns from one another at the focusing region.

[0082] In various embodiments, the method may further include alternately providing the first source light and the second source light.

[0083] In various embodiments, the method may further include modulating phases of the reference light and the first and second return lights relative to one another.

[0084] In various embodiments, the method may further include splitting, with a non-polarising beam splitter, the first source light and the second source light into the first portion and the second portion; directing, with the non-polarising beam splitter, the first portion and the second portion towards the fluid sample; and combining, with the non-polarising beam splitter, the first return light and the second return light with the reference light.

[0085] In various embodiments, determining the property of the at least one particle may include determining any one or more of: a mass, a size, or a number of base pairs of the at least one particle based on the electrical signals.

[0086] In various embodiments, determining the size of the at least one particle may include determining a scattering coefficient associated with the at least one particle based on the electrical signals.

[0087] In various embodiments, determining the scattering coefficient may include determining a position of the at least one particle at the focusing region based on the electrical signals. In various embodiments, the method may further include for each first pair of the plurality of first pairs of lights, performing a first subtraction operation of electrical signals corresponding to the first pair of lights; for each second pair of the plurality of second pairs of lights, performing a second subtraction operation of electrical signals corresponding to the second pair of lights; for each third pair of the plurality of third pairs of lights, performing a third subtraction operation of electrical signals corresponding to the third pair of lights; and for each fourth pair of the plurality of fourth pairs of lights, performing a fourth subtraction operation of electrical signals corresponding to the fourth pair of lights, wherein determining the size of the at least one particle may include determining the size of the at least one particle based on results of the first to fourth subtraction operations for the plurality of first to fourth pairs of lights.

[0088] In various embodiments, the method may further include for each first pair of the plurality of first pairs of lights, performing a first subtraction operation of electrical signals corresponding to the first pair of lights; for each second pair of the plurality of second pairs of lights, performing a second subtraction operation of electrical signals corresponding to the second pair of lights; and wherein determining the size of the at least one particle may include determining the size of the at least one particle based on results of the first and second subtraction operations for the plurality of first pairs of lights and the plurality of second pairs of lights.

[0089] In various embodiments the system and / or method may be used to determine the size and / or mass of the one or more particles. If the particles are DNA or RNAthe system and / or method may be used to determine the number of base pairs.

[0090] In an aspect, the present disclosure broadly includes an optical apparatus comprising: an optical source arrangement configured to provide a first source light, the optical source arrangement configured to scan the first source light; an optical sampling arrangement optically coupled to the optical source arrangement to receive the first source lights, the optical sampling arrangement being configured to: direct a first portion of the first source light along a propagation axis towards a fluid sample comprising at least one particle; provide, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, the first line illumination pattern having a long axis perpendicular to the propagation axis, and the scanning of the first source light moving the first line illumination pattern perpendicular to the propagation axis and the long axis; and combine, with a reference light derived from the first source light, a first return light from the at least one particle illuminated by the first line illumination pattern and at least one second return light from the at least one particle illuminated by the first line illumination pattern offset from the first return light by the scanning of the first source light; and an optical detection arrangement optically coupled to the optical sampling arrangement, the optical detection arrangement comprising at least one photodetector arrangement configured to detect a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light for determining a property of the at least one particle. Any of the various embodiments described may be applied to this aspect.

[0091] In an aspect, the present disclosure broadly includes an optical apparatus comprising: an optical source arrangement configured to provide a first source light; an optical sampling arrangement optically coupled to the optical source arrangement to receive the first source lights, the optical sampling arrangement being configured to: direct a first portion of the first source light along a propagation axis towards a fluid sample comprising at least one particle; provide, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, the first line illumination pattern having a long axis perpendicular to the propagation axis; and combine, with a reference light derived from the first source light, a first return light from the at least one particle illuminated by the first line illumination pattern; and an optical detection arrangement optically coupled to the optical sampling arrangement, the optical detection arrangement comprising at least one photodetector arrangement configured to detect a first resultant light resulting from a combination of the first return light and the reference light for determining a property of the at least one particle. Any of the various embodiments described may be applied to this aspect.

[0092] In an aspect, the present disclosure broadly includes an method for determining a property of at least one particle in a fluid sample, the method comprising: providing, at a focusing region within the fluid sample, a first line illumination pattern, the first line illumination patterns having a long axis perpendicular to the propagation axis; determining a property of the at least one particle illuminated by the first line illumination pattern based on variations along the long axis of the first line illumination pattern, of a return light from a first region of the fluid sample illuminated by the first line illumination pattern. Any of the various embodiments described may be applied to this aspect.

[0093] In various embodiments, the return light comprises light scattered by the at least one particle.

[0094] In various embodiments, the first line illumination pattern is divided into a plurality of regions along the long axis, wherein each of the plurality of regions is analysed to determine the property of the at least one particle.

[0095] In various embodiments, the first line illumination pattern is generated by the output of a multi-spatial mode diode focussed on the focussing region.

[0096] In various embodiments, providing a second line illumination pattern, the second line illumination patterns having a long axis perpendicular to the propagation axis and being offset from the first illumination pattern. Optionally offset parallel to a propagation axis of a source light generating the illumination pattern.

[0097] BRIEF DESCRIPTION OF THE DRAWINGS

[0098] These and other features, aspects, and advantages of the present disclosure are described with reference to the drawings of certain embodiments, which are intended to schematically illustrate certain embodiments and not to limit the disclosure.

[0099] FIG. 1 shows a schematic diagram of an optical apparatus system according to various embodiments.

[0100] FIG. 2 shows a schematic diagram of the lens system of the optical apparatus of FIG. 1. FIG. 3 shows an enlarged schematic section view showing the location of the beam focus and flow channel for the sample solution for the optical apparatus of FIG. 1.

[0101] FIG. 4 shows a diagram illustrating how the foci of the two lasers are offset and an example particle trajectory.

[0102] FIG. 5 shows a diagram illustrating the line focus of the lasers with an example particle trajectory and how it is imaged to the microlens array.

[0103] FIG. 6 shows a schematic diagram of an optical apparatus system according to another embodiment. FIG. 7 shows a schematic diagram of an optical apparatus system according to a further embodiment.

[0104] FIG. 8 shows a schematic diagram of alternative optical arrangements for directing and detecting light on the sample.

[0105] FIG. 9 shows a diagram illustrating the line focus of the lasers showing the long axis of the line focus.

[0106] FIG. 10 shows a representation of the formation of the line focus using two multi-spatial mode diodes.

[0107] FIG. 11 shows a simulated output of particle position.

[0108] FIG. 12 shows a processed output of the detected signal.

[0109] FIG. 13 shows a Gaussian filtered output of FIG. 12.

[0110] DETAILED DESCRIPTION

[0111] Although certain examples are described below, those of skill in the art will appreciate that the disclosure extends beyond the specifically disclosed examples and / or uses and obvious modifications and equivalents thereof. Thus, it is intended that the scope of the disclosure herein disclosed should not be limited by any particular examples described below.

[0112] The present disclosure may relate to detection and size measurement of biomolecules with coherent lidar.

[0113] The present disclosure may provide a system in which improves by distinguishing between small particles passing through the centre of a beam focus and large particles passing through the edge of a beam focus. The disclosure provides systems and methods for detecting particles across, at least a 2D plane. The solution containing the particles to be detected / measured may flow perpendicularly to this 2D plane. The variation in signal strength for particles passing through different points on the plane may be determined by a calibration measurement and then compensated for. Position along the axis of the laser beam in this system may be determined by using signal amplitude and phase with an input beam comprising two wavelengths, for example as generated by two coaxial beams from lasers with different wavelengths. Position along the other dimension of the 2D plane, at right angles to the beam axis, may be determined from multiple detectors imaged to points along a line focus. As described below, it is not merely the use of two wavelengths which allows the detection and / or measurement, but also how the line foci of the wavelengths overlap. In particular, the present application describes how the scattering at the two wavelengths may be analysed in view of particular motion. Where the line foci are perpendicular to the flow of the particles and the input beam (rather than along the input beam) and / or the line foci are substantially similar in shape, this comparison between the scattering may be made. In some cases, the use of high NA lens systems allows for accurate detection due to the creation of smaller focus regions with higher power density and collection of a larger fraction of the scattered light. The larger fraction of scattered light may be received from a larger range of angles.

[0114] The present disclosure may provide an optical apparatus or device (e.g., a lidar system), for example, for detecting and / or measuring the size of one or more particles in a flow of liquid (or solution or liquid sample). As a non-limiting example, beams from two (high) power, multi spatial mode lasers with different wavelengths may be combined to form the input beam. The apparatus or system may form or define a Michelson interferometer using a polarising beam splitter and wave plates (or waveplate elements) in each arm of the interferometer to separate the transmitted and received beams. Most of the input power may go to the measurement arm of the interferometer and is brought by a lens system to a high numerical aperture (NA) focus in a flow cell containing the sample solution. Scattered light from particles within the sample solution passing through the focus (or focusing region) within the fluid sample may be collected by the high NA lens to form the signal beam. In the reference arm of the interferometer, a small fraction of the input beam may be reflected or retror ef ected to form the reference beam. The interference of the signal and reference beams may be used to detect the particles. Each multi spatial mode laser beam may form a line shaped rather than a gaussian focus. A telephoto lens may then image the returned signal beam and reference beam onto a linear microlens array. Particles passing through different points of the line focus give signals imaged to different microlenses and are detected separately. The high NA lens may be designed to have a controlled amount of longitudinal chromatic aberration so the foci of the beams from the two lasers are offset. A prism in the detection system splits the beams from the two lasers to different detectors. From the detected signal strengths for the two lasers and different microlens channels, the position, on a 2D plane, of where the particle is in the focus may be determined. The sample solution flows normal to that plane so particles pass through the focus. The strength of the detected signal provides information about the particle size. As the position the particle passes through the focus is known, differences in signal strength away from the centre of focus may be calibrated out.

[0115] The reference and signal beams are colinear but may have orthogonal polarisations relative to one another. As such, the signal beam and the reference beam may be considered as separate beams. A resultant light or interference signal may be formed when the signal and reference beams pass through a polariser at 45 degrees (or an angle that is not exactly horizontal or vertical) and may then be detected.

[0116] Lasers may be multi longitudinal mode emitting different wavelengths. Lasers may also be multi spatial mode (i.e., multi transverse mode) emitting a beam that may not be a single gaussian beam but something else that may be expressed as the sum of multiple gaussian beams. The multi spatial mode lasers (e.g., high power diode lasers) employed for the optical apparatus disclosed herein are multimode in both ways, and may provide the desired focus shape. The focus shape may be referred to as a line focus, where the multiple gaussian beams form foci along an axis, such that the overall focus shape is formed by a series of overlapping gaussian beams. The line focus may approach a rectangular shape with rounded corners. The long axis of the line focus is substantially perpendicular to the direction of the beam. For example, the long axis may be within 20 degrees of perpendicular to the direction of the beam, or within 10 or 5 degrees. In some cases, the line focus is an image of the elongated output window of the laser(s). It will be understood that the line focus may not be an exact image of the output window, or that the laser(s) may be configured in any suitable way to produce line foci at the sample. The line focus is a line perpendicular to the beam propagation direction (rather than the spot / point of a single mode laser), or an extended depth of field as generated by a low NA lens. In one example the line focus has a diffraction limited width in one dimension that is of the order of, for example, at least 3, 5, 10, or 20 times wider in the other dimension. Other relative dimensions are possible. In preferred beams the intensity is substantially constant along the line focus. In some cases, the disclosure broadly describes how the line focus can be images onto a linear array of detectors, or detector channels. This enables the detector to detect or measure scattering at different positions along the line focus.

[0117] FIG. 10 shows an example line focus. The example is generated by multi-spatial mode diode lasers. However, alternative lasers or methods may be used to obtain similar line foci. Further examples are described below. FIG. 10 expands the lasers source 114a and collimation lenses 116a of laser 110 as shown in FIG. 1. As shown in the expanded view, laser source 114a is a laser diode with a long axis of its output window orientated along the line marked LL. In contrast to a single mode laser which is typically considered to emit a near gaussian beam from a single point, the multi spatial mode laser diode 114a may be modelled as emitting gaussian beams from a large number (how many varies with the diode and output power) of points. For example, three representative points are shown as 1A1, 1BL, 1CL along line LL in FIG 10. Typically, there would be more points and / or the points would overlap. Line LL is imaged by the laser collimation optics and focusing lens to line LF1 at the focus in the sample. The points 1AL, 1BL, 1CL are imaged to diffraction limited focus spots 1A, IB, 1C on the line focus at the sample. The second laser source 114b is similar, so that the line that forms the long axis of laser source 114b output window is imaged to line focus LF2. The line focus of laser source 114b (shown by the areas 88, 89 along the axis LF1, LF2 of FIG. 10) is made up of a series of diffraction limited focus spots, for example, 2A, 2B, 2C, that are imaged from points (which may be approximately considered to be on laser source 1 Mb’s output window). The three diffraction limited spots shown in FIG. 10 are representative examples of the larger number of overlapping spots that form the line focus. The 1 / e2intensity boundaries 304, 306 are shown for the focused beams that form spots IB and 2B respectively, as an example. In some cases, these will be adjacent to or overlap with the 1 / e2intensity boundaries of the neighbouring spots, so as the line focus appears substantially continuous.

[0118] The disclosed optical apparatus and system may include one or more of the following: (i) two lasers of different wavelengths with their foci offset in position by chromatic aberration of a lens or lenses in the lens system 134 such as 140, 146, 148 or 150, (ii) multi spatial mode lasers where the focus may be a line (instead of a typical gaussian) that may be imaged onto a linear array of detection channels, (iii) flows the sample through the detection area at a known rate perpendicular to the beam axis. Where a particle passes through the focus may be determined and the variation of signal strength with position may be compensated, resulting in a more accurate size and / or scattering coefficient measurement.

[0119] The disclosed optical apparatus and system is adapted for a high NA water immersion lens to increase the signal from small objects and enable the detection of single molecules. The phase of the signal from the particle may vary with the position it passes through the focus within the sample solution. The disclosed optical apparatus may allow phase measurement of a relatively slow random particle motion parallel to the beam axis by I / Q detection or phase modulation.

[0120] In a version of the optical apparatus, the detected light may be split between two channels, one with a phase delay. Combining the results from the two channels allows the full available signal to be used and the phase, with additional position information, to be calculated. This process may be referred to as I / Q detection. I / Q detection is the process of detecting an in-phase (I) and a 90-degree phase shifted or quadrature (Q) component. In another version of the system the phase difference between the reference and signal beams is modulated and the resulting signal at the detector demodulated.

[0121] In a variant of the optical apparatus, rather than separating the two lasers beams in the detector system, the lasers may be pulsed on and off alternately so the signals may be separated electronically or in software.

[0122] In variants of the optical apparatus, the position of the focus within the fluid sample may be scanned in one or more axis. Enhanced position information may be obtained from the variation of the particles signal over the scan cycle.

[0123] A desirable instrument for biotechnology applications would preferably have good size resolution when directly detecting free particles in solution down to single protein molecule size. Simple integration with fluidic systems for high throughput measurements and manageable cleaning requirements are desirable. A predictable detection rate would allow accurate concentration measurements. These requirements are not met by known methods or known systems. The present disclosure provides techniques to achieve one or more of the described requirements with a lidar (light detection and ranging) system that detects particles and the position the particles cross two measurement beams.

[0124] A schematic diagram of an optical apparatus (e.g., a lidar system) 100 according to the techniques disclosed herein is shown in FIG. 1. The optical apparatus 100 may be used for particle detection and determination (or measurement) of a property (or characteristic), e.g., size, of at least one particle. As a non-limiting example, the optical apparatus 100 may include or may be based on an interferometer (e.g., a Michelson interferometer) with an input arm 102, a sample arm 104, a reference arm 106 and a detection arm 108. The term ‘Michelson interferometer’ as described herein may mean an interferometer with a single beamsplitter that separates the input beam into two beams that are returned and combined by the same beamsplitter.

[0125] In some embodiments, while technically challenging, a Mach-Zehnder interferometer may instead be employed in the optical apparatus 100 where two beamsplitters are used. One beamsplitter is used to split the input beam into two beams and a second beamsplitter to combine them.

[0126] The optical apparatus 100 includes two laser modules 110 and 112 provide or generate the input beams (or first and second (optical) source lights) 119a, 119b. Each module 110, 112 includes a multi spatial mode laser source (e.g., laser diode) 114a, 114b. As an example, the laser source 114a may provide or generate or output a first source light 119a, and the laser source 114b may provide or generate or output a second source light 119b.

[0127] Laser sources are preferred for single molecule detection and / or characterisation as laser sources provide sufficient power per unit area at the focus. For detection and / or characterisation of large(r) particles, LEDs may be employed as the light sources.

[0128] The output of each of the multi spatial mode laser (e.g., laser diode) 114a, 114b may be such that focusing it results in a diffraction limited spot in one dimension and an elongated line focus corresponding to the shape of the laser diodes output facet in the other dimension.

[0129] Both lasers 114a, 114b may have the same orientation to match subsequent components, drawn here such that the line focus is aligned to the plane of the diagram.

[0130] Each laser module 110, 112 may further include a collimation lens 116a, 116b. Each laser module 110, 112 may further include additional beam shaping optics 118a, 118b that may include, for example, anamorphic prisms or cylindrical lenses to adjust the beam shape of the source lights 119a, 119b to be more circular. The collimating lenses 116a, 116b, and, optionally, the additional lenses in the beam shaping optics 118a, 118b may be to focus the output of the laser diodes 114a, 114b at infinity. The two modules 110, 112, or the laser sources 114a, 114b have different wavelengths or different central wavelengths or different wavelength ranges. Part of the wavelength or wavelength range of the source light or output light 119a of the laser source 114a and part of the wavelength or wavelength range of the source light or output light 119b of the laser source 114b may overlap one another.

[0131] The output lights or beams (i.e., the source lights) 119a, 119b from the laser sources 114a, 114b may be (spatially) combined to a single beam 121 by an optical element, e.g., a dichroic mirror 120. The output lights 119a, 119b are to be made as nearly colinear as possible with a similar cross section by the dichroic mirror 120, and, so the output lights 119a, 119b, after interacting with the dichroic mirror 120, are effectively a single beam 121. Put in another way, after the dichroic mirror 120, it is a single beam from a spatial point of view, but two distinct beams from a spectral (wavelength) point of view. It should be appreciated that other suitable optical elements may be used to combine the first and second source lights 119a, 119b, for example, a prism or a diffraction grating in place of the dichroic mirror 120.

[0132] As it is preferable that there is accurate matching of the directions of the two beams 119a, 119b, an electronically adjustable mount for the dichroic mirror 120 or an element in the beam shaping optics 118a, 118b may be used for active beam direction control. Active beam direction control (which may be referred to as active alignment) of the two beams, 119a, 119b independently, may be used. Active beam direction control may be achieved using piezo actuators, or other electronically controlled elements moving dichroic 120 and / or elements of the beam shaping optics 118a, 118b, or additional mirrors folding beams 119a, 119b, for example. An alignment sensor in the detection arm 108 may provide a signal to an electronic or software feedback loop controlling the actuators. This sensor may use multiple photodiodes or a camera monitoring the position of a line image or parts of it from different microlens array elements.

[0133] In various embodiments, the laser modules 110, 112, including the laser sources 114a, 11b, as well as the dichroic mirror 120 may form or define an optical source arrangement.

[0134] Each of the multi spatial mode laser sources 114a, 114b may be or may include a blue laser with a wavelength in the range of about 400 - 500 nm, e.g., 400 - 480 nm or 440 - 500 nm. Preferably, the wavelengths may be within the range of about 440 - 475 nm as the lasers available in this wavelength range have a high power or the highest power.

[0135] Each of the multi spatial mode laser sources 114a, 114b may be or may include a blue diode laser, for example, made from gallium nitride or indium gallium nitride.

[0136] In various embodiments, the wavelengths of the laser sources 114a, 114b may differ or offset by about 5 - 500 nm from each other, for example, about 5 - 300 nm, about 5 - 200 nm, about 5 - 100 nm, about 5 - 50 nm, about 5 - 20 nm, about 50 - 500 nm, about 100 - 500 nm, about 300 - 500 nm, about 50 - 300 nm, about 50 - 100 nm, about 100 - 300 nm, or about 200 - 400 nm. Such an offset may allow the source lights 119a, 119b to be (more) easily combined and separated by a dichroic mirror or a prism.

[0137] As a non-limiting example, the (central) wavelength of the first source light 119a provided by the first multi spatial mode laser source 114a may be about 450 nm, while the (central) wavelength of the second source light 119b provided by the second multi spatial mode laser source 114b may be about 465 nm. As another example, the wavelength for the first source light 119a may be about 440 nm while the wavelength for the second source light 119b may be about 455 nm.

[0138] It should be appreciated that any one of the two laser sources 114a, 114b may provide a source light of a shorter wavelength while the other of the two laser sources 114a, 114b may provide a source light of a longer wavelength.

[0139] As non-limiting examples, the two laser sources 114a, 114b may be a Nichia NDB7Y75 laser (wavelength range of about 448 - 462 nm) and a Nichia NDB7Z75 laser (wavelength range of about 462 - 472 nm). Alternatively, one or both of the two laser sources 114a, 114b may be OSRAM PLPT9 450LB E lasers (available with different wavelengths in the range of about 440 - 455 nm).

[0140] It is noted that Rayleigh scattering is stronger at shorter wavelengths and this effect, together with the higher power of available laser diodes means more signal than other colours in the visible range. Further, it is noted that water is highly transparent in the blue wavelengths to avoid heating. While two high power diodes with different blue wavelengths may be used as described above, it should be appreciated that a wide range of laser wavelengths are suitable. Blue lasers are preferable as they are available with high power and low cost. Other combinations of ultraviolet (UV), visible or (near) infrared wavelengths may be used. As a non-limiting example, a combination of a blue laser with a green laser (e.g., 525 nm laser), or green laser (e.g., 525 nm laser) with a red laser (e.g., 633 nm laser). The wavelength difference between the lasers employed needs to be sufficient for the source lights being output to be separated with a prism or dichroic and by chromatic aberration of the focusing lens to be described below.

[0141] As described, UV, visible, and (near) infrared lasers may be employed in various embodiments. As non-limiting examples, each of the laser sources 114a, 114b may have a wavelength in the range of about 200 - 2000 nm, e.g., about 200 - 1500 nm, about 200 -1000 nm, about 200 - 500 nm, about 500 - 2000 nm, about 1000 - 2000 nm, about 1500 -2000 nm, about 500 - 1500 nm, about 500 - 1000 nm, about 400 - 1000 nm, or about 400 -800 nm.

[0142] As a non-limiting example, for red and near infra red laser sources, the wavelengths of the laser sources may differ or offset from each other by about 500 nm.

[0143] The power for each of the two laser sources 114a, 114b may be in the range of about 1 - 10 W for single molecule detection, for example, about 1 - 5 W, about 1 - 2 W, or about 5 - 10 W, e.g. about 1 W. Laser power in the range of about 0.1 - 1 W may be suitable for detection of larger particles. Laser power in the range of about 10 - 100 W may be suitable for an optical apparatus working or operating in the near infrared.

[0144] Non-limiting examples of laser sources may have a rated power of 5 W, such as Nichia NDB7Y75 and NDB7Z75 or OSRAM PLPT9450LB E.

[0145] The optical apparatus 100 further includes an optical sampling arrangement optically coupled to the optical source arrangement to receive the first and second source lights 119a, 119b or the beam 121. The optical sampling arrangement may include an optical polarising arrangement having a polariser and / or waveplate 122, a polarising beam splitter 124, and quarter waveplates (or waveplate elements) 130 and 132. The polariser and / or waveplate 122 may define an input polarisation controller.

[0146] The polariser (or optical polarising device) 122 polarises the beam 121 at a specific angle for the next section. Alternatively, a waveplate element (e.g., a half waveplate) may be used in place of the polariser 122. In further embodiments, a combination of polariser and waveplate element (e.g., a half waveplate) may be used in place of the polariser 122.

[0147] The polarising beam splitter 124 splits the beam 121 into an input beam 126 in the sample arm 104 and a beam 128 in the reference arm 106. The beam 126 is directed towards a fluid sample (e.g., a solution) 138 containing at least one particle. The beam 126 includes a portion of the source light 119a and a portion of the source light 119b.

[0148] The respective amounts (or powers) of the beams 126, 128 or the ratio of power of the beams 126, 128 that the polarising beam splitter 124 directs to the sample arm 104 and the reference arm 106 may be controlled. The ratio of power may be controlled with or by the input polarisation that is set or defined by the polariser 122, and, optionally, additionally by the non ideal behaviour of the polarising beam splitter 124. In various embodiments, most of the power goes to the sample arm 104 to get the maximum signal, while the reference beam in the reference arm 106 does not need high power.

[0149] By controlling the polarisation, the power or amount of light or beam that is reflected and transmitted by the polarising beam splitter 124 may be controlled. Using a quarter waveplate for circular polarisation may help to increase or maximise the power sent to the detection arm 108. All available signal or light, or as much signal as possible, is preferred to be provided in the sample arm 104. For the reference arm 106, high or full power may not be needed. A different polarisation sensitive element or rotating the quarter waveplate 132 may reduce the power in a controlled way.

[0150] As a non-limiting example, the ratio of power in the sample arm 104 to the reference arm 106 may be in the range of 10:1 to 1,000:1, for example, 10:1 to 500:1, 10:1 to 200:1, 10:1 to 100:1, 100:1 to 1,000:1, 200:1 to 1,000:1, 500:1 to 1,000:1, or about 100:1. It should be appreciated that a ratio of power of 1,000:1 to 100,000:1 may be helpful to control the power at the detector but may require a polarising beam splitter with a high performance.

[0151] Additionally or alternatively, the power of the beam 128 in the reference arm 106 may be controlled by attenuating the beam 128.

[0152] Quarter waveplates (or quarter waveplate elements) 130 and 132 convert the beams 126, 128 to circular polarisation that swaps from left / right circular if back reflected then on a second pass through the quarter waveplate 130, 132 become linearly polarised shifted by 90 degrees from its original polarisation.

[0153] Conversion to circular polarisation allows the return beam or return light from the sample 138 (or from an optical reflector arrangement to be described below) is directed by the polarising beam splitter 124 to the detection arm 108. Circular polarisation will swap from left / right circular if back reflected (mirror image). Then on returning through the quarter waveplate 130, 132 swap linear polarisation from the original (e.g., horizontal polarisation swaps to vertical, or vice versa).

[0154] The polarising beam splitter 124 with the quarter waveplates 130, 132 for circular polarisation may form or define a “beam separator” configuration that causes beams back reflected in either arm 104, 106 to be sent by the polarising beam splitter 124 to the detection arm 108 as the signal beam (or resultant light) from the sample arm 104 and the reference beam 106. Such a beam separator may minimise or avoid power loss at the beamsplitter 124. The orientation of the sample arm 104 and the reference arm 106 may be swapped differing in whether the high-power beam to the sample 138 is initially reflected or transmitted by the polarising beam splitter 124.

[0155] The 90-degree shift in the linear polarisation from the original polarisation described above allows the returned beam from the sample arm 104 (or the reference arm 106) to be directed by the polarising beam splitter 124 to the detection arm 108.

[0156] In various embodiments, the input beam to the sample arm 104 is vertically polarised as it is reflected from the polarising beam splitter 124, i.e., s-polarised as orientated as shown in FIG. 1. So, if the returned beam in the sample arm 104 is made horizontally polarised, it is p-polarised at the beam splitter 124 and transmitted to the detection arm 108.

[0157] Similarly, the input beam to the reference arm 106 is horizontally polarised, i.e., p-polarised, as transmitted through the polarising beam splitter 124. The returned beam in the reference arm 106 is made vertically polarised, i.e., s-polarised, and reflected to the detection arm 108. The reference arm 106 may be configured or designed to reduce the power sent to the detector.

[0158] In some embodiments, in applications where a large amount of power may not be required for illuminating or interacting with the sample 168 or the parti cle(s) contained therein, the optical polarising arrangement may be optional, and a non-polarising beam splitter may be used in place of the polarising beam splitter 124.

[0159] The optical sampling arrangement may include a lens system or lens arrangement 134, which, for example, may be or may define an objective lens arrangement. In the sample arm 104, the beam 126 is focused by the lens system 134 into a flow cell 136 containing the fluid sample (e.g., solution) 138. The focus or focusing region 142 is within the fluid sample 138. The lens system 134 may be an immersion lens (e.g., a water immersion lens) with the front element 140 of the lens system 134 forming one side of the flow cell 136, or one side of a flow channel 139 of the flow cell 136. An image of the laser output is formed in the sample solution 138 at the focus or focusing region 142 with a line form (or line illumination pattern) from each of the multimode lasers 114a, 114b. The lens system 134 may be or may define an optical chromatic aberration arrangement, or a focusing arrangement with chromatic aberration. The lens system 134 may be configured or arranged with a controlled axial (longitudinal) chromatic aberration sufficient so to focus the line images or line illumination patterns for the two laser wavelengths offset along the beam path axis (or optic axis or beam propagation direction). The chromatic aberration may result in an overlap of the two-line illumination patterns. The chromatic aberration may be selected to provide a desired or sufficient overlap. The chromatic aberration may depend on the wavelengths of the beams and / or the difference in wavelengths between the beams. The sample solution 138 may flow during the measurement with a direction orthogonal to the line focus and parallel to the axis 144. Light (back-)scattered or return light from particles at the focus 142 within the sample 138 is collected by the lens system 134 as the signal beam. To maximise the signal enabling detection of small particles, it is preferable or desirable that the lens system 134 has a high numerical aperture (NA) to reduce the size of the focused beam line images or line illumination patterns. A high NA means that the input light can be focused more tightly by the lens system 134, thereby increasing the power per unit area, and, hence, resulting in more scattered light or return light from the particle. Further, a high NA allows more or a higher fraction of the dipole emission pattern scattered light or return light being collected by the lens system 134. While a standard microscope objective lens may be used, such objective lens may not have the required level of chromatic aberration and the cement in commonly used multi element lens groups is vulnerable to damage with high beam power. The custom water immersion lens system 134 used in the optical apparatus 100 may be simplified compared to standard microscope objectives as a small field of view is required only covering the line image of the laser output. A specific amount of chromatic aberration to offset the focus of the two lasers line images or line illumination patterns is required but this may be a limited wavelength range compared to the chromatically corrected range of standard objectives, thus, simplifying the design of the lens system 134, and therefore, the optical apparatus 100.

[0160] In various embodiments, the two line illumination patterns may overlap each other. The line illumination patterns may be offset from each other although there may be an overlapping region between the line illumination patterns where a region of a first line illumination pattern overlaps with a region of a second line illumination pattern.

[0161] In various embodiments, the amount of chromatic aberration may be such that the two line illumination patterns provided or focused at the focusing region 142 may be offset (e.g., along the beam propagation axis) from each other by about 10 - 5000 nm, for example, about 10 - 3000 nm, about 10 - 1000 nm, about 10 - 500 nm, about 10 - 100 nm, about 100 -5000 nm, about 500 - 5000 nm, about 1000 - 5000 nm, about 2000 - 5000 nm, about 100 -1000 nm, or about 100 - 500 nm, e.g., about 100 nm, about 200 nm, about 300 nm, or about 500 nm.

[0162] The lens systems 134 shown in FIG. 1 is a non-limiting example of a possible implementation that can provide the necessary performance with a NA >1 using four optical elements. In the lens system 134, a super hemispherical ball front lens 140, and a positive crown glass element with an aspheric surface 146 provide most of the focusing [Robens C., et.al. Optics Letters Vol. 42, No. 6, 1043-1046 (2017)]. Chromatic aberration may be controlled by an air spaced doublet with a negative flint glass element 148 and a positive crown glass element 150 in the lens system 134. A crown glass is a low dispersion glass, while a flint glass is a high dispersion glass. It should be appreciated that other lens systems to provide diffraction limited focusing may be used, e.g., having different numbers and / or combinations of optical elements or lenses. Any lens system or lens arrangement with the required specific chromatic aberration and resistance to high laser power (e.g., no cemented interfaces) may be used. As shown in FIG. 1 the input beam 126 passes through the high NA lens system. Because both wavelengths (e.g. as generated by lasers 110, 112) are present in the input beam 126, both wavelengths pass through the high NA lens. This may increase the power per unit area at the line foci, providing greater scattered light for collection. A system which uses separate lenses, or requires a low NA lens to control the foci of the input beam, would not typically be able to achieve a similar power per unit area.

[0163] It should be appreciated that the quarter waveplate 130 may be placed within the lens system 134, for example, between the glass element 148 and the aspheric glass element 146. Reflections from lens surfaces before the quarter waveplate 130 will not have their polarisation rotated 90 degrees and are returned to the input arm 102. This may reduce stray light in the detection arm 108.

[0164] In some embodiments, the quarter waveplate 130 may be optional and may be removed.

[0165] In some embodiments, an optical element that causes a polarisation change, for example, of less than a 90-degree change from the original polarisation may send power to the detector but not necessarily all that is available.

[0166] The front element 140 is in contact with the sample solution 138 (and potentially any cleaning solutions) and should be durable to allow long term use. The focus 142 may be some tens or hundreds of micrometres deep in the sample solution 138. Fused silica is suitable for the front element 140, being chemically resistant, and its refractive index is relatively close to that of water, reducing the aberration in a high NA beam at the interface with a water based sample solution. Borosilicate and other glasses are also possible, cutting plane lines A-A’ mark where more detail will be shown in FIG. 3.

[0167] The lens system 134 may be or may define an optical chromatic aberration arrangement. The positive lenses with convex curvature surfaces and focusing capability in the lens system 134, such as the optical or lens elements 140, 146, 150 add positive chromatic aberration, while the optical or lens element 148 having concave surfaces and designed for diverging light beams adds opposite negative aberration. As a glass with high dispersion may be used for the negative lens element 148 and the positive lens elements 140, 146, 150 are made of a lower dispersion glass, the sum of the chromatic aberration may be near or close to zero, but sufficient to provide the aberration required. The aspheric lens element 146 and the ball lens element 140 have specific requirements (e.g., to achieve a high NA focus, durable material in contact with the sample solution 138) that limit how these lens elements 140, 146 may be adjusted. The lens elements 148, 150 may form or define a chromatic correction group to control the chromatic aberration. The shape and glass used for the lenses 148, 150 may be adjusted over a wide range to achieve the desired chromatic aberration for the optical apparatus 100.

[0168] FIG. 2 shows a schematic diagram of the lens system 134 of the optical apparatus 100. Anon-limiting example for the design of the lens system 134 may be based on the materials and properties as shown in Table 1 below. A standard software for lens design may be used to determine the performance of the lens system 134. Different designs may be determined following the same. TABLE 1 : Example of a design of a 4-element objective for lens system

[0169]

[0170] In various embodiments, instead of the lens element 150, two positive lens elements with convex curvature surfaces may be employed, with one lens element being arranged at the same position as the lens element 150, and another lens element being arranged between the negative lens 148 and the aspheric lens 146.

[0171] In some embodiments, a 5-element objective may be employed in place of the lens system 134, for example, the lens system 135 shown as insert in FIG. 1. The lens system or lens arrangement 135 may be or may define an optical chromatic aberration arrangement. Similar to the lens system 134, the lens system 135 includes a super hemispherical ball front lens 141 and a positive crown glass element with an aspheric surface 147 to provide most of the focusing. The chromatic correction group may be an air spaced triplet rather than the doublet used in the objective or lens system 134, with a negative flint glass element 151 and positive crown glass elements 149, 153. The triplet works in the same way as the doublet but using two positive elements 149, 153 may reduce the precision with which the various lens elements are to be positioned and reduces the curvature of the elements. The triplet may thus be easier to manufacture with acceptable performance and cost than the doublet. Using the triplet may make the lenses used especially the aspheric lens element 147 easier to manufacture and / or may reduce the sensitivity of the system to tolerances in the manufacture and placement of the chromatic group lenses. The lens elements 149, 153 may be identical, or differ in their shape and / or glass used to optimise performance. Other lens systems to provide diffraction limited high NA focusing with appropriate longitudinal chromatic aberration control may be used. A modification of an existing water immersion objective may be possible. There may be designs for 0.3 - 0.8 NA air objectives that may work with some modification and a water immersion ball lens added to increase the NA to >1 (i.e. numerical aperture greater than 1).

[0172] The optical sampling arrangement may include an optical reflector arrangement. The optical reflector arrangement may include a retroreflector in the reference arm 106 to back reflect the beam 128 to provide a reference light or reference beam. This beam will contain both wavelengths (e.g. as generated by lasers 110, 112) so may be referred to as first and second reference beams. For example, the wavelengths may be separated again in the detection system. As a non-limiting example, a cats-eye retroreflector including a lens system 152 with a mirror 154 or other reflective surface at its focus 156 may be used. This arrangement returns the reference light or beam with the laser output focused at infinity. The mirror or reflective surface 154 may have a low reflectivity property to control the power of the reference beam. As another non-limiting example, a corner cube retroreflector 161 may be used instead of the cats-eye retroreflector arrangement as shown in outlined dashed box 163. A dispersion compensator 158 may be arranged in the reference arm 106. The quarter waveplate 132 may be substituted for a different waveplate, polarisation control element, or polarisation dependent element to control the power of the reference beam.

[0173] For efficient combining of or interference between the returned lights (or signal beams) and the reference beam (or reference light), the difference in the optical path length between the sample arm 104 and the reference arm 106 should be small compared to the coherence lengths of the lasers 114a, 114b. High power multi spatial mode laser diodes are typically also multi longitudinal mode with coherence lengths of the order of 100 micrometres. The length of the reference arm 106 may be made adjustable by moving the retroreflector lens assembly 152 and the mirror 154 along the beam path. As different lenses are used in the sample and reference arms 104, 106, glass dispersion may cause the path length difference to vary over the range of the laser wavelengths used. The lenses in both arms 104, 106 may be matched using the same glass types and centre thicknesses to equalise the dispersion. Additionally or alternatively, a dispersion compensator 158, having one or more flat glass plates may be used to add dispersion to the reference arm 106. Rotating a plate of the dispersion compensator 158 about an axis orthogonal to the beam propagation axis to adjust its effective thickness may control the amount of dispersion introduced to the reference arm 106 to match the sample arm 104. Rotating the plate changes the angle of incidence of the beam on the plate, as illustrated by the dispersion compensator 158 being shown as tilted in FIG. 1. However, rotating the plate, and, therefore the dispersion compensator 158 may displace the reference beam sideways and may need to be taken into account in the alignment of the retroreflector in the reference arm 106.

[0174] The retroreflector lens assembly 152 may use some of same lens elements as the focusing objective to simplify production. Other lens elements of the retroreflector lens assembly 152 may be different to give it suitable optical properties. The retroreflector lens assembly 152 is an imaging lens that will likely have lower NA and a longer effective focal length than the objective lens 134. It will typically be designed to have diffraction limited imaging of the line focus for both laser beams. Typically, chromatic aberration should be controlled to a low enough level as to have insignificant effect on the lens performance. The beam of both lasers 110, 112 should be focused on the mirror surface 156 giving line images on surface 156 with no significant focus offset. Although the mirror surface 156 is shown on the far side (second surface) of mirror 154 in FIG. 1 alternatively the mirror surface 156 may be on the front side (first surface) of mirror 154. In some cases, either or both surfaces of a glass element without any coatings may work as low reflectivity mirrors suitable for mirror 154.

[0175] As described above, the polarising beam splitter 124 acts to direct the combined signal and reference beams to the detection arm 108. Return lights or back-scattered lights from a particle in the sample 138 resulting from the particle being illuminated by the source lights or interacting with the source lights in the sample arm 104 are combined with a reference light from the reference arm 106 at the polarising beam splitter 124. The resultant lights resulting from the combination (including, for example, interference) of each of the return lights and the reference light are directed by the polarising beam splitter 124 to the detection arm 108. As a non-limiting example, a first resultant light may result from a combination of the reference light and a first return light from a particle illuminated by a first line illumination pattern (e.g., associated with the laser source 114a) at the focusing region 142, and a second resultant light may result from a combination of the reference light and a second return light from a second region of the particle illuminated by a second line illumination pattern (e.g., associated with the laser source 114b) at the focusing region 142. The first region and the second region may overlap each other. This means that the first line illumination pattern and the second line illumination pattern may overlap each other. For a particle or part thereof that is located in the overlapping region of the first and second line illumination patterns, the particle is illuminated by both line illumination patterns that are associated with both the source lights 119a, 119b at the same time, thereby, resulting in first and second return lights at the same time.

[0176] The optical apparatus 100 further includes an optical detection arrangement optically coupled to the optical sampling arrangement to receive the resultant lights or resultant signals. The optical detection arrangement may include a telephoto lens (or optical telephoto arrangement) with a positive lens element 160 that may be a doublet and a negative lens element 162 to form a highly magnified image at plane 164. For the reference light or beam, this is the line image of the laser output. Scattered lights or returned lights from a particle at the focus 142 in the sample 138 forms a collimated signal beam that is focused to a spot image by the telephoto. If a particle is not at the focus 142, the signal beam may not be collimated and may not be focused at the image plane 164. The focus offset for the two laser wavelengths mean that a particle can’t be exactly focused for both at the same time.

[0177] A linear microlens array 166 may be arranged or placed at the image plane 164. Each microlens or microlens element 167 focuses light from a part of the line image or line illumination pattern including the scattered light or signal from that area or section of the image. Light from each microlens 167 may be detected separately in subsequent optics as channels of a ID image. For clarity in FIG. 1, only five microlenses 167 are shown, each microlens 167 focusing a spot to plane 168, though it should be appreciated that there may be any number of microlens elements 167 depending on requirements and / or configurations. Then, from the plane 168 on, only the optical path for the central microlens 167 is shown for clarity and ease of understanding, though it should be appreciated that each microlens 167 has its corresponding optical path. An array with 10 microlenses 167 may be used, for effectively a 10 pixel image, or to get a different number of channels, a different number from 2 to 100 microlenses 167 may be provided. Rectangular microlenses may be used to allow a channel spacing smaller than the diffraction limited spot size at the image plane 164 while the other dimension may be large enough to accommodate the full beam. The focused spots from each microlens 167 at the plane 168 may be collected by a lens 170. Lens 170 may be a doublet or other multi element lens to image plane 168 to the detector arrays 172, 174. Cylindrical lens elements may be used to correct distortion of the image by prism 176. The position and the focal length of the lens 170 may be selected to focus the light from each spot onto photodiodes or detector elements 173, 175 in (photodetector) arrays 172, 174. Light from each microlens 167 may be spread over 8 photodiodes 173, 175, 4 in each array 172, 174, by splitting the beams or (first and second) resultant lights 171 three times. First, a prism 176 (or other dichroic element) may be used to split or (spatially) separate the resultant lights 171 from the two lasers 114a, 114b with different wavelengths. Prism 176 may be a multi element compound prism. For example, Prism 176 may be formed from two or more prisms of different glass mounted together. A multi-element compound prism may provide the splitting of wavelengths required without too much distortion of the image formed by lens 170. Second, with a non-polarising beam splitter 178 to split the resultant lights 171 along a first optical propagation pathway and a second optical propagation pathway. Non-polarising beam splitter 178 may be a cube beam splitter rather than the plate shown. In some cases, the system may use controlled attenuation of reference light in the I (detector array 172) or Q (detector array 174) channel to correct for the beam splitters 178 non-ideal splitting (physical non-polarising beam splitters typically do not provide exactly 50-50 transmission reflection for both polarisations and / or over a range of wavelengths). Finally, with two Wollaston prisms 180, 182 or other polarising beam splitters (or polarising beam splitting devices) in the first and second optical propagation pathways.

[0178] The microlens array 166, the prism 176, the non-polarising beam splitter 178, the polarising beam splitting devices 180, 182, and the photodetector arrays (or photodetector arrangements) 172, 174 form part of the optical detection arrangement.

[0179] The signal (returned lights) and reference beams are polarised 90 degrees apart and so may not interfere directly.

[0180] The first and second resultant lights 171 are split or separated with polarising beam splitters 180, 182 at 45 degrees into a plurality of first, second, third and fourth pairs of lights corresponding to the source lights of different wavelengths. As non-limiting examples, the polarising beam splitting device 180 may split the first resultant light into a plurality of first pairs of lights, wherein for each first pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and may split the second resultant light into a plurality of second pairs of lights, wherein for each second pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another. As further non-limiting examples, the polarising beam splitting device 182 may split the first resultant light into a plurality of third pairs of lights, wherein for each third pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another, and may split the second resultant light into a plurality of fourth pairs of lights, wherein for each fourth pair, the lights are linearly polarised and at least substantially orthogonally polarised to one another. As a non-limiting example, for each pair of lights, the lights may be at 45 degrees and - 45 degrees or close to that.

[0181] The first to fourth pairs of lights are detected with the detector elements or photodiodes 173, 175, and the two photocurrents (or electrical signals) corresponding to or representative of each pair of the first to fourth pairs of lights are subtracted to give an interference signal. Put in another way, each light from each first pair, each second pair, each third pair and each fourth pair is detected by a separate photodiode element 173, 175, and the two currents corresponding to the lights of each pair are then subtracted. For example, each subtractor 186, 188, 190, 192 is shown in FIG. 1 connecting to two photodiode array elements 173, 175 to represent a subtraction operation of the photocurrents (or electrical signals) determined from or based on respective pairs of photodetector elements 173, 175. For example, the subtractor 192 connects to two elements 175, each element 175 with a light or beam that comes from a single beam splitting at the polarising beam splitter 182.

[0182] In various embodiments, the first to fourth pairs of lights may be incident on or detected with any pairs of detector elements 173, 175. For example, a respective first pair of lights may be detected by two adjacent (or neighbouring) detector elements 173, or two spaced apart detector elements 173. This similarly may apply to a respective second pair of lights, a respective third pair of lights, and a respective fourth pair of lights. Where a dichroic element is used to replace the prism 176 there may be four arrays of photodetector arrays 172, 174 (with half as many detector elements 173, 175 each). Each photodetector array 172, 174 may have its own polarising beamsplitter. There may be two sets of non-polarizing beam splitters after the dichroic, or one non-polarising beam splitter and two dichroics if the non-polarising beam splitter comes before the dichroic. In some cases, a plurality, or more than four, of photodetector arrays 172, 174 are used.

[0183] In some embodiments, for each pair of the first to fourth pairs of lights, it may be possible to detect (only) one of the lights with a single detector element 173, 175 for the purpose of determining a property of at least one particle. The output of the detector element 173, 175 is similar to the subtractor result but with a lower signal amplitude as approximately half of the signal is lost. As a result, photodiode arrays 173, 175 with half as many elements may be used.

[0184] The waveplate (or waveplate element) 184 in the path reflected from the non-polarising beamsplitter 178 is set for a 90 degree phase difference at the 45 degree polarising beam splitter 182. Detecting the subtracted photocurrents (or electrical signals) for pairs of photodiodes 173, 175 without (term I, array 172) and with the phase shift (term Q, array 174) allows the magnitude and phase of the interference signal to be calculated. This is I / Q demodulation. Each of the subtractors 186, 190 is associated with or represent an in-phase (I) term, and is in the section of the optical detection arrangement with beams transmitted through the non-polarising beam splitter 178. Each of the subtractors 188, 192 is associated with or represent a quadrature (Q) term, and is in the section of the optical detection arrangement with beams reflected from the non-polarising beam splitter 178 with a phase shift from the waveplate 184. The I and Q terms have a fixed 90 degree phase difference but can be added together to give a signal with any phase.

[0185] As non-limiting examples, the polarising beam splitter 180 and the photodetector array 172 may be in or along a first optical propagation pathway, while the waveplate element 184, the polarising beam splitter 182 and the photodetector array 174 may be in or along a second optical propagation pathway

[0186] The phase shift or phase difference between the resultant lights or beams 171 directed to the photodetector arrangements 172, 174 that is introduced by the waveplate (or waveplate element) 184 is preferably 90 degrees (90°), though it should be appreciated that any suitable variance from 90° (i.e., not exactly 90°) may be possible with suitable analysis software. With noiseless data, the magnitude of the interference signal may be determined based on [square root(I2+Q2)] while the phase may be determined based on arctan(Q / I). Lowpass filtering [square root(IA2 +QA2)] may provide the magnitude sufficiently well for a measurement of the scattering coefficient associated with the particle being determined. Phase may be needed if it is used for additional position information.

[0187] The separating or splitting of the resultant lights or beams 171 by the prism 176 allows separate detection for each wavelength. The resultant light with the shorter wavelength may deflect more from the prism 176 and may be associated with the I term 186 at the photodiode array 172 and the Q term 188 at the photodiode array 174. The resultant light with the longer wavelength may be associated with the I term 190 at the photodiode array 172 and the Q term 192 at the photodiode array 174.

[0188] For each microlens channel 167, the 4 pairs of photodiode currents associated with the microlens channel 167 are subtracted electronically to yield the I terms 186, 190 and the Q terms 188, 192, then digitised and recorded with a computer or processor 195. The processor 195 may be electrically coupled to the photodetector arrangements or arrays 172, 174. In the case of, for example, an array of 10 microlens channels 167, there are 10 1 terms (or signals) for the laser 114a, 10 Q terms (or signals) for the laser 114a, 10 I terms (or signals) for the laser 114b and 10 Q terms (or signals) for the laser 114b for a total of 40 terms to be digitised. Other numbers of microlens channels 167 similarly give 4 times the number of microlens channels (or pixels) 167 results to be digitised. It should be appreciated that each microlens channel 167 corresponds to one pixel of a ID image.

[0189] In various embodiments, a balanced detector may be employed for the subtraction operation.

[0190] If the power of the pair of lights and detector sensitivities are equal, subtracting two photocurrents removes the noise due to the lasers intensity fluctuations. If photocurrents II and 12 have identical noise, the operation II - 12 removes the noise. However, making the powers of the pair of lights and detector efficiencies equal is challenging and they may vary slowly with time.

[0191] A balanced detector is an analogue circuit that may output (Il - b*I2) or similar. Il and 12 are photocurrents as described above. The balance term b may be adjusted at low frequency (10 to 1000 Hz) by a feedback loop to make the average output of the circuit 0 [US5134276], High frequency signals different between the two photocurrents pass through the circuit. High frequency may refer to 1 kHz to 1MHz for the optical apparatus 100, and may be up to 100 MHz in the optical apparatus 101 with modulation to be described below.

[0192] The balance adjustments causes versions of 11 and 12 with equal DC / low frequency components to be subtracted. High frequency noise signals common to both photocurrents are removed in the same way as a subtractor with ideal beams and detectors.

[0193] Dividing one current by another has similar advantages to a balanced detector but analogue electronics division is noisy at high frequency. Division or subtraction in software requires the detection and digitisation to be fast and accurate for good performance in a way that may be limiting.

[0194] The returned and reference lights are colinear but have orthogonal polarisations, for example, horizontal and vertical polarisation respectively as orientated in the top view of FIG. 1. The result is a single beam with some polarisation state depending on the amplitude and phase of the returned and reference lights. Directly detecting this beam with a photodiode would not give an interference signal in the photocurrent. This is a feature of a Michelson interferometer using a polarising beam splitter. The photocurrent is proportional to the intensity, I, that is the square of the absolute value of vector sum of the electric fields, i.e., I = |sum E|2. If the returned and reference lights are polarised 90 degrees apart the same is true of the electric fields and the intensity is the sum of the intensities of the returned and reference lights. There is no interference signal that depends on the relative phase of the returned and reference lights.

[0195] For two beams with the same polarisation, an interference signal in the intensity determined by the relative phase may be generated. For example, if the phase of equal power beams is 180 degrees apart, the electric fields cancel, the vector sum of E and, thus, the intensity is zero, and there is a dark fringe. If the polarisations were 90 degrees apart, the electric fields could not cancel to zero.

[0196] After splitting there are two lights or beams, one with components of both signal and reference polarised at 45 degrees. The other light or beam has both signal and reference components polarised at - 45 degrees. These beams both show phase dependent interference signals. Polarisation beam splitters are asymmetric such that the phase difference signal to reference differs by 180 degrees (pi radians) between the two beams. So, if one shows constructive interference, a dark fringe, the other shows destructive interference, a bright fringe. Subtracting the intensities of the two beams as measured by the photodiodes gives the full available interference signal.

[0197] While the optical detection arrangement is described based on the microlens array 166, it should be appreciated that the microlens array 166 is a non-limiting example, and other devices or elements may instead be used. For example, an array of small mirror elements or a single mirror with a faceted surface may be used to provide a similar function of dividing the beam focused onto the plane 164 into sub beams corresponding to different positions on the plane 164.

[0198] In some embodiments, the microlens array 166 may be removed and the optical elements of the optical detection arrangement, for example, the prism 176, the beam splitter 178, polarising beam splitting devices or elements 180,182 and the waveplate element 184 may be arranged before the plane 164. Multiple copies of the image plane 164 may then be directly imaged onto the detector arrays 172, 174. However, such an arrangement makes positioning of the photodiode arrays 172, 174 critical and may waste light falling between the photodetector elements 173, 175 on the arrays 172, 174.

[0199] In some embodiments, a respective portion or region of each of the first and second resultant lights 171 may be detected with a single detector element 173, 175, without being split into pairs of lights described above and without the need for the subtraction operation described above, where the output of the detector element 173, 175 is similar to the subtractor result but with a lower signal amplitude as approximately half of the signal is lost. As such, (only) one detector output is obtained for each portion of the first resultant light and (only) one detector output is obtained for each portion of the second resultant light. For such embodiments, the polarising beamsplitters 180, 182 may be replaced by polarisers (or polariser elements) at 45 degrees, or any angle that is not 0 or 90 degrees (e.g., 0.1 degree to 89.9 degrees, 0.5 degree to 89.5 degrees, or 1 degree to 89 degrees), and photodetector or photodiode arrays 172, 174 with half as many detector elements 173, 175 may be used. The lower signal amplitude and lack of the subtraction operations suppression of fluctuations in the laser power make such an arrangement less preferable as the single detectors output is more sensitive to laser intensity noise compared to subtracting two photocurrents (or electrical signals) from two detector elements 173, 175 for each of the first to fourth pairs of lights. Nevertheless, such an arrangement may be employed, for example, if simplicity of the optical detection arrangement is desired.

[0200] In FIG. 3, a non-limiting example of how the flow cell assembly 136 and front lens 140 may be configured is shown. This is an enlarged schematic cross section where the cutting plane was marked A-A’ in FIG. 1. A flow channel 139 is formed between the surface of the front lens 140 and the flow cell device 136 (or a surface or portion thereof). As an example, the hemispherical ball front lens 140 may define a side of the flow channel 139. The lens 140 and the flow cell 136 may be permanently bonded or have an elastomeric seal between them. The input beam 126 is focused into the flow channel 139 containing the sample particle solution 138. The input beam 126 includes a portion of the source light 119a and a portion of the source light 119b. Light is scattered by particles in the input beam 126 and some of the scattered light from particles at the focus 142 reverses the input beam path to form the signal beam or return light. The remaining unscattered light 202 exits through a transparent section of the flow cell 136 and is not used further. Fluidic connections may be made to the input end 204 and the output end 206 of the channel 139 so sample 138 may be flowed through the channel 139 at a controlled rate. The flow channel 139 may be thick enough, along the axis of the input beam 126, for the beam 126 to be focused at a point of about 10s or 100s of micrometres from the walls of the flow cell 136. The flow channel 139 may also be thick enough normal to the direction of flow of the sample 138 and the beam axis that the width of the beam 126 away from the focus 142 may be accommodated. Enlarged areas indicated by dashed oval 31 and the cutting plane B-B’ respectively, are shown in FIG. 4 and FIG. 5.

[0201] To more accurately measure or determine the size of a detected particle, the position the detected particle crosses the focus 142 is determined. Otherwise, the signal of a small particle passing through the centre of the focus 142 may potentially not be distinguished from a large particle passing through the edge of the focus 142. The large particles’ larger scattering coefficient may be balanced by the lower sensitivity away from the focus 142 to give a similar signal. The following section uses FIGS. 4 and 5 as non-limiting examples to explain the position determination and measurement techniques. FIGS. 4 and 5 are cross-sectional views of the axis 144 of flow in or out of the page in FIG. 1. FIG. 4 is a diagram showing the detail at the focus or focusing region 142 around the area marked 31 in FIG. 3. The optic axis 302 and the 1 / e2intensity boundaries 304, 306, for the source lights or beams 119a, 119b from the two lasers 114a, 114b are shown in FIG. 4. The optic axis 302 refers to the axis of symmetry of the lens system 134. The optic axis 302 may be designed to be colinear with the input beam 126. The optic axis may be aligned with the propagation axis of the input beam 126 (see FIGS. 1 and 3), where the input beam has been adjusted to be aligned to the optic axis 302. The controlled amount of longitudinal chromatic aberration causes the beam waist 308, 310 (foci), illustrated for clarity and ease of understanding, for the two lasers 114a, 114b to be offset. For a focusing lens with under corrected chromatic aberration, the intensity boundary 304 is associated with the source light with the shorter wavelength and the intensity boundary 306 is associated with the source light with the longer wavelength, while for overcorrected chromatic aberration, it would be the other way round. An example trajectory 312 is shown of a particle 316 moving with the solution flow 314 in the flow channel 139 (see FIG. 3) and random diffusion (Brownian motion). The particle 316 crosses the focus or line illumination patterns (see, for example, FIG. 5) of the source lights or beams and scatters light 318. The scattered light or return light 318 (only) gives an interference signal if the particle 316 is near the focus limiting the detection to an enclosed volume as light scattered from particles away from the focus may not or will not form a collimated return beam. The volumes where a particle 316 may produce 1 / e2of the maximum signal are marked for clarity and ease of understanding as 320, 322 for the two lasers 114a, 114b. The volumes 320, 322 are offset, as for the foci 308, 310 and the associated line illumination patterns, with an overlapping region 324. For a particle 316 detected in the overlapping region 324, the position of the particle 316 along the optic axis 302 may be determined by the relative magnitude of the resultant lights or interference signals for the two lasers 114a, 114b.

[0202] Additional axial position information may be obtained from the phase of the interference signal which is determined by the optical path difference between the sample and reference arms 104, 106 of the interferometer modulo 2K. The path length may not be known and may likely drift over time. But position information from the magnitude of the resultant lights or interference signals for many particles may be used to calibrate the path length difference and update the path length difference as it drifts. Combining position information from the phase and magnitude may give a more accurate result, e.g., by using a Kalman filter or similar. The detected phase may be determined by the optical path difference between the sample and reference arms 104, 106. As a non-limiting example, if one particle gives a signal with phase 0, another particle placed about 85 nm along the axis has phase 180 degrees or TI radians (e.g., 85= 340nm / 4 450 nm light is 340 nm wavelength in water). With an 85 nm position, the path difference is 170 nm as the light propagates forward and back. This is half a wavelength or the difference between constructive and destructive interference. Other positions give phases in proportion to position along the axis. The use of phase may be more sensitive than the positions found from the relative signal magnitude from the two lasers 114a, 114b described above. It should be noted that 0, + / -2n , + / -4K ....radians or 0 ,+ / -360 ,+ / -720.... degrees phase are all the same, and resolving these ambiguities is phase unwrapping. Also, the position for 0 phase depends on the pathlengths of the sample and reference arms 104, 106. Temperature changes (and potentially other effects) may cause the length and / or refractive index of components or elements in both arms 104, 106 to drift. So, the path length difference for any position in the measurement area may drift with time. As such, continuous calibration of the phase data may be required. The position data from signal magnitudes may be used for calibration and unwrapping.

[0203] Position data from the relative signal magnitudes may have limited accuracy (e.g., more noise in the position result in nm) as the magnitudes may change slowly over distances of hundreds of nm. Phase data may be more sensitive but drifts with time and has the 2TI phase ambiguity. As an example, Kalman filtering (e.g., implemented in software) may be used for combining multiple information sources with different noise and drift for an optimum result. A more accurate position data allow a more accurate compensation of the variation of signal strength with position and result in a more accurate measurement of the scattering coefficient.

[0204] FIG. 5 shows a diagram illustrating how the position of a particle 316 is determined along an axis 350 normal to the direction of the solution flow 314 and the optic axis 302. FIG. 5 shows the view indicated by the cutting plane B-B’ in FIG. 3 and FIG. 5 has the same scale as FIG.

[0205] 4. As discussed, when describing FIG. 1 above, the multi spatial mode lasers 114a, 114b used form a line focus 351 defined by the offset line illumination patterns associated with the lasers 114a, 114b. The 1 / e2intensity of the line focus 351 is marked for clarity and ease of understanding as 352, near diffraction limited gaussian in one dimension and an extended line along axis 350. A particle 316 is shown with its trajectory 312 passing through the focus with the solution flow 314 and Brownian motion. Scattered light or return light from particles 316 at the focus 351 may be imaged onto a microlens array (e.g., array 166 in FIG. 1). A grid 354 is shown in FIG. 5 to illustrate which areas of the line focus 351 are imaged onto 8 microlens elements. Each microlens element directs light to a resultant / interference signal magnitude and phase detection system (or optical detection arrangement) for both wavelengths as described above. For the particle 316, a strong signal may be seen in the microlens channel 356, with weaker signals in the microlens channels 358, 360 and no signal in the microlens channel 362. The point spread function may be calculated or observed from many particle detections and used to fit an observed set of channel signal magnitudes to position along the axis 350. An arbitrary number (8) of microlens elements 356, 358, 360, 362 are shown here as a non-limiting example. Rectangular microlens elements 356, 358, 360, 362 may allow the beam to be divided up along the axis 350 with sub diffraction limited resolution while collecting all the beam in the other dimension as shown by the non-square grid 354.

[0206] FIG. 9 shows a third view of the focus region, perpendicular to FIG. 4 and FIG. 5. FIG. 9 is at a similar scale to the previous figures to show the orientation of the line focus 351. FIG. 9 has the same orientation as FIG. 1, with optic axis 302 of the focusing objective 134 aligned parallel to input beam 126 of FIG. 1. The 1 / e2intensity boundaries 304, 306 are shown for the source lights or beams 119a, 119b from the two lasers 114a, 114b. The intensity boundaries 304, 306 represent the outer edges of the laser beams. In practice the lasers (e.g. if using laser diodes) may comprise a plurality of laser points along the line). These may be imaged to a plurality of focus spots along the sample (within the 1 / e2intensity region 322 - horizontally as shown in FIG. 9). It should be understood that the line focus 351 may comprise the combination of these diffraction limited focus spots.

[0207] The solution containing the particles flows in the direction of axis 314 perpendicular to (into) the plane of FIG. 9. A particle, labelled 316, will scatter light from the input beam 126. Therefore, the particle may be detected within the line focus volumes 320, 322 generated by the two input lasers 114a, 114b. These focal volumes 320, 322 are shown as the limits where a particle will produce 1 / e2of the maximum signal. Considering these with the same 1 / e2signal limits 320, 322 as shown in FIG. 4 it is noted that the focal volumes 320, 322 are elongated perpendicular to the optic axis 302 with a line focus parallel to the plane of FIG. 9. As shown the direction of flow, the orientation of the line focus and the optic axis 302 are perpendicular to one another. This allows the system to monitor across a portion of the sample as it flows through the input beam. Monitoring the portion of the sample (e.g. across the line focus volume), rather than a single beam spot, allows the relative scattering from particles moving across the portion to be detected. The relative scattering may be used to improve detection or measurement of particles because it allows at least 2-dimensional information to be measured. This may distinguish between large particles and multiple small particles, for example. The relative scattering at positions along the line focus may aid measurement of the position at which the particle crossed the focus.

[0208] As also shown in FIG. 4, the two focal volumes 320, 322 shown in FIG. 9 (which may also be referred to as the two line focus regions) are offset along the optic axis 302 due to the axial (longitudinal) chromatic aberration of the objective lens 134. The two line foci may be considered as images of the output windows (facets) of the two input lasers 114a, 114b. The scattered light from the particle 316 is collected by the objective and focused onto the microlens array (166 in FIG. 1). In FIG. 5 grid 354 was shown indicating where light from particles 316 at the focus 352 may be imaged to different microlens array channels (or detectors). A similar grid may be imaged to the detector (e.g. a microlens array) from, for example, planes 354. The plane of focus for imaging to the microlens array is perpendicular to the drawing plane of FIG. 9 and thus appears as two lines (one for each of the two lasers wavelengths separated along the optic axis 302 by the axial (longitudinal) chromatic aberration). As described above, the use of two lasers 114a, 114b or two wavelengths in the input beam 124, provides multiple signals from the particles 316 providing improved measurement accuracy. The distance of the offset between the planes 354 (i.e. the vertical distance of FIG. 9) may be selected to optimise position sensing of particles depending on the lasers are lenses used. A high NA lens system 134 means the depth of focus is small and planes 354 must be close together to be able to detect a single particle with both line foci 320, 322 simultaneously. With a lower NA and / or longer wavelength lasers the depth of focus is larger and planes 354 may be further apart. In some cases the planes are offset at least by a threshold distance (for example relative to the depth of field, or greater than a fraction (e.g.

[0209] 1 / 10 or 1 / 4 or 1 / 2) of the depth of field) to ensure there sufficient difference between the signals of the two line foci 320, 321 enabling position sensing.

[0210] With the above method particles moving in the flow are detected as the particles cross the focus and the position of the particles in the focus determined. A calibration of how the resultant or interference signal changes with position may be made using particles of known size(s). The scattering coefficient of each particle detected may then be calculated indicating its size.

[0211] The magnitudes of the detected signal may be compared to those for a sample of a particle of known scattering coefficient or known size or known mass (e.g., protein, DNA and nano particle standard samples). The relation between the scattering coefficient and the size and / or mass may be calculated by assuming a sphere of known refractive index. Typical refractive index and density giving mass / size relation for proteins and other samples are generally known.

[0212] One or more of two similar calibrations may be used.

[0213] One calibration may be used to determine the position of a particle given magnitudes of the different microlens channel signals. Relative signal magnitudes are to be considered. If a particle has twice the signal magnitudes of another (smaller) particle in all microlens channels, the position is the same.

[0214] Another calibration may be used to determine the scattering coefficient from the signal magnitudes for a detected particle. The magnitudes of the signals from all microlens channels may be added together. Another option is fitting the estimated point spread function for each particles data and recording the data. The result may then be compared to the signal from a calibration protein or other particle solution of known scattering coefficient / size / mass.

[0215] Compensation of non ideal sensitivity difference between different microlens channel positions may be needed.

[0216] Additional or alternative to moving the particles 316 through the line focus 351 by flowing the solution, the focus position may be scanned. An electro-optic, acousto-optic, moving mirror or other scanner may be placed in the beam or optical propagation path between the lasers 114a, 114b and the sample 138. The beam would be moved back and forth aligned and parallel to the flow axis 314. The variation of the signal with the scanning motion may allow the particle position on the axis 314 to be determined. Adding this to the above described position measurements may give the particles’ 3D position and may improve the accuracy of the size measurement or determination.

[0217] A single spatial mode laser that focuses to a diffraction limited spot may be rapidly scanned in a line. This may achieve a similar line focus to the multi spatial mode lasers described above. In some examples this provides an alternative type of laser which may be used for the system. Instead of a laser 110, 112 which produces a line focus (e.g. a diode laser with a plurality of emitting points - multi spatial mode diodes) a laser with a single (or a plurality) of emitting points may be scanned across a region equivalent to the line focus. This scanning could be automated or controlled. For example, there are high power (10W+) diode pumped solid state (DPSS) lasers are available that may be suitable for a high-performance system. High power DPSS lasers are expensive and suitable beam scanners such as acousto-optic deflectors (AOD’s) are also expensive, but if particular accuracy is required the high power lasers may provide increased accuracy. In some cases, the focus of the beam may be scanned as well as (or as an alternative to) along the line. This may allow accurate position detection along the optic axis to be determined with a single laser from the variation of signal with focus modulation. In these examples the effects, or parts thereof, of the two laser 110, 112, two wavelength system is being generated through scanning of a laser. It is expected that a laser which produces a line focus will be more efficient in some cases. Although the disclosure primarily focuses on position measurement over a 2-D plane (defined by the direction of the optic axis 302 and the direction of the line focus 350 see FIG. 5) beam scanning may also be used to provide a third dimension position measurement, along optic axis 302, for example. A system with beam scanning over three dimensions may be able to track the position of a particle for some time to improve the accuracy with which its properties (e.g. size) can be determined.

[0218] A schematic diagram of another embodiment of an optical apparatus (e.g., a lidar system) 101 is shown in FIG. 6. The optical apparatus 101 may be used for particle detection and determination (or measurement) of a property (or characteristic), e.g., size, of at least one particle. In the optical apparatus 101, modulation is used to reduce the number of detectors used. It modifies the optical apparatus 100 of FIG. 1 to simplify the detection arm 108. The optical apparatus 101 may operate as described for the optical apparatus 100 FIG. 1 and identical parts or elements of the optical apparatus 101 share the same reference numbers as for the optical apparatus 100 and description thereof is omitted here for conciseness. The lens system 134 or the lens system 135 may be employed in the optical apparatus 101.

[0219] Two laser modules 110, 112, with laser sources 114a, 114b provide the source lights or input beams 119a, 119b combined to form a single beam 121 by the dichroic mirror 120 as described previously. For this optical apparatus 101, the two lasers 114a, 114b are modulated on and off alternately with a period much less than the time for a particle to pass through the focus. This enables the signals from the two wavelengths to be distinguished without needing to split or separate the resultant lights or beams over separate detector arrays, i.e., timedivision multiplexing,

[0220] To avoid the use of separate detectors for the I and Q terms, the relative phase of the signal beams or return lights and the reference light or beam is modulated at a different frequency to the laser on / off modulation. This may be a scan or triangular wave covering a range of 2TI which is demodulated to I and Q terms, or a sinusoidal phase modulation, which may be simpler to implement at high frequency, may be used where with appropriate amplitude, the I and Q terms may be determined from the amplitude of the sidebands at 1 and 2 times the modulation frequency respectively. The phase modulation may be achieved in one or more of the following ways:

[0221] 1) moving (represented by double-headed arrow 402) the reference arm mirror 154 to change the optical path length. For example, a movement of 112.5 nm adds 225 nm to the round-trip path length or a 7t phase shift with a 450 nm wavelength laser. As a non-limiting example, a resonant piezo actuator may be used to move the mirror at high frequency. The surface of a quartz oscillator may function as the mirror 154 while moving at MHz frequencies.

[0222] 2) An optical phase modulator such as a Pockels cell, Kerr cell or photo-elastic modulator 404 may be arranged in the reference arm 106. Placing the modulator 404 in the sample arm 104 is equivalent but requires the modulator 404 to withstand high laser power.

[0223] 3) The phase modulators described above are commonly polarisation dependent (except the Kerr cell) so may be placed as a modulator 406 in the detection arm 108 to modulate the relative phase of the signal and reference beams. Liquid crystal based phase modulators may be used but may have limited frequency response. It may be possible to use one in various embodiments of the optical apparatus configured or designed for large slowly moving particles.

[0224] In the optical apparatus 101, a polarising beamsplitter 408 set at 45 degrees splits or separates the resultant light or beam 171 from each microlens channel 167 of the microlens array 166 to a pair of lights to be detected at two detectors or detector elements 411 of a single photodetector array (e.g., photodiode array) 410. As a non-limiting example, when the laser source 114a is switched or modulated on and the laser source 114b is off, a combination (including, for example, interference) of a return light from a particle in the sample 138 illuminated by a line illumination pattern associated with a first source light 119a at the focusing region 142, and a reference light, results in a first resultant light that is then split by the polarising beamsplitter 408 into a plurality of first pairs of lights. When the laser source 114b is switched or modulated on and the laser source 114a is off, a combination (including, for example, interference) of a return light from the same particle in the sample 138 illuminated by a line illumination pattern associated with a second source light 119b at the focusing region 142, and a reference light, results in a second resultant light that is then split by the polarising beamsplitter 408 into a plurality of second pairs of lights.

[0225] The photocurrents (or electrical signals) determined from each pair of detectors 411 are subtracted, as shown by the subtractor 412, as described for the optical apparatus 100. For each microlens channel 167, the subtracted photocurrents may be demodulated electronically or in software, for example, in the processor 195, to provide the I and Q terms for each laser source 114a, 114b. The processor 195 may be electrically coupled to the photodetector arrangement or array 410. So, for the optical apparatus 101, only 2 photodetectors or photodiodes 411 are needed per microlens channel 167 to get the same results as obtained with 8 photodiodes per channel spread over two arrays in the optical apparatus 100. Some elements or components from the optical apparatus 100 of FIG. 1 are not needed for the optical apparatus 101 such as the prism 176, non-polarising beamsplitter 178, waveplate 184, polarising beam splitter 182 and photodiode array 174 of the optical apparatus 100.

[0226] Similar to the optical apparatus 100, the first and second pairs of lights may be incident on or detected with any pairs of detector elements 411. For example, a respective first pair of lights may be detected by two adjacent (or neighbouring) detector elements 411, or two spaced apart detector elements 411. This similarly may apply to a respective second pair of lights being detected by two adjacent or spaced apart detector elements 411.

[0227] As described above, for the optical apparatus 101, only 2 photodiodes 411 are needed per microlens channel 167 using both phase modulation and laser on / off modulation to obtain the same results as obtained with 8 photodiodes 173, 175 per microlens channel 167 spread over two photodetector arrays 172, 174 in the optical apparatus 100.

[0228] In some embodiments, either the laser on / off modulation or phase modulation may be employed, with 4 detector elements needed for each microlens channel.

[0229] For example, an optical apparatus may be configured or operated with laser on / off modulation without the phase modulation. 4 photodetector or photodiode elements per microlens channel spread over two photodetector arrays are needed. I and Q detection may be as described in the context of the optical apparatus 100. Each detector element and I / Q determination works for both wavelengths as the laser sources modulate on / off alternately.

[0230] It is also possible for an optical apparatus to be configured or operated with phase modulation without laser on / off modulation. 4 photodetector or photodiode elements per microlens channel in a single photodetector array are needed. Phase may be measured from the modulation signal as described in the context of the optical apparatus 101. A prism or other dichroic element may be employed to split or separate the two resultant lights corresponding to the two lasers to different detector elements as described in the context of the optical apparatus 100.

[0231] Similar to the optical apparatus 100, for the optical apparatus 101, a respective portion or region of each of the first and second resultant lights 171 may be detected with a single detector element 411, without being split into corresponding first and second pairs of lights and without the need for the subtraction operation. As such, (only) one detector output is obtained for each portion of the first resultant light and (only) one detector output is obtained for each portion of the second resultant light. The polarising beamsplitter 408 may be replaced by a polariser (or polariser element) at 45 degrees, or any angle that is not 0 or 90 degrees (e.g., 0.1 degree to 89.9 degrees, 0.5 degree to 89.5 degrees, or 1 degree to 89 degrees), and the photodetector or photodiode array 410 with half as many detector elements 411 may be used.

[0232] As described above in the context of the optical apparatus 100, 101, the input beam 126 is focused into the sample solution 138 and light scattered from particles at the focus 142 is collected by the lens system 134 as the signal beam or return light. The lens system 134 may be defined or called the objective lens as the lens system 134 focuses onto the sample (object) 138. To maximise the signal enabling detection of small particles, it is preferable or desirable that the lens system 134 has a high numerical aperture (NA) to reduce the size of the focused beam line image (or line illumination pattern), thereby increasing the power per unit area and increasing the fraction of the dipole emission pattern scattered light that is collected by the lens system 134. In Various embodiments, an NA of about 0.3 - 1.3 may be provided. An NA in the range of about 1 - 1.3 may provide sufficiently good performance. An NA of about 0.7 - 1 NA may be sufficient for single molecule detection. A lower NA of about 0.3 - 0.7 may be used for detection of larger particles. A custom water immersion objective lens may be employed as the lens system 134.

[0233] The longitudinal chromatic aberration of the objective or lens system 134 may preferably be controlled so as to achieve a specific amount of longitudinal aberration so the foci (or the (focused) line illumination patterns) of the source lights or beams 119a, 119b from the two lasers 114a, 114b are offset as needed for the position sensing as described above in the context of FIG. 4. Typical glasses have significant dispersion that for a monochromatic design of an objective may result in more chromatic aberration than needed. Achieving the required level of chromatic aberration may thus require a compensation that removes most of the aberration leaving (only) what is required. Control of the longitudinal chromatic aberration is preferable as the laser sources (e.g., high-power laser diodes) 114a, 114b are multi longitudinal mode with bandwidths of about 1 - 5 nm. As the focus position changes with wavelength, the total phase shift for the sample arm 104, between a particle near the focus and the beam splitter 124, may also change. If the phase shift difference is large enough over the laser bandwidth, the interferometric contrast when the return light from the particle is combined or interfered with the reference light or beam and detected may be reduced. The foci offset (or offset between the line illumination patterns) may be small 50 - 500 nm (and may be to 2000+ nm for lower NA optical apparatus or systems) and the bandwidth of the lasers may be smaller than, for example, the 10 - 50 nm wavelength difference between the two lasers 114a, 114b. So, correcting the longitudinal chromatic aberration to the required offset may be adequate to give sufficient interferometric contrast across each laser’s bandwidth.

[0234] While use of a standard commercial microscope objective may be possible, the cement in commonly used multi element lens groups may be vulnerable to damage with the high power lasers, e.g., about 1 - 10 W, to be used. Also, a standard objective is not likely to have the required level of chromatic aberration. A custom objective, e.g., lens system 134, may be simplified compared to standard commercial microscope objectives as a small field of view is required (only) to cover the line image of the laser output. Standard microscope objectives are corrected for aberrations that are intrinsically zero in the centre of the field of view (on axis) but increase away from the centre to achieve a large usable field of view (>100 pm where about 1 - 2 pm may be needed to cover the line image). These aberrations, for example, distortion, coma, astigmatism (assuming the lens is cylindrically symmetrical) and transverse chromatic (lateral, variation in image position rather than focus with wavelength) have less relevance or importance for the custom objective used in various embodiments. Designing the custom objective for a small field of view (almost on axis) needed generally involves controlling spherical aberration including, for example, higher order terms and longitudinal chromatic aberration. The limited requirements for aberration correction mean a simpler design than standard commercial objectives, with fewer lens elements, is possible.

[0235] In optical apparatus 100, the objective lens 134 includes (only) four elements 140, 146, 148, 150 as a possible implementation that may provide an NA > 1. A near aplanatic (e.g., free of spherical aberration) super hemispherical ball front lens 140, and a positive (convex curvature surfaces, focusing) glass element with an aspheric surface 146 provide most of the focusing (optical power). The two lens elements 140, 146 may together provide high NA focusing with narrowband light. The front element 140 may be configured or arranged to be in contact with the sample solution 138 and should be durable to allow long term use. The focus 142 may be some tens or hundreds of micrometres deep in the sample solution 138. The lens element 146 may be made from crown glass and the aspheric surface of the lens element 146 may correct spherical aberration, for example, from the lens element 146 itself and also aberration from the rest of the objective or lens system 134. The lens elements 140, 146 may introduce (significant) longitudinal chromatic aberration, more than may be needed. Chromatic aberration may be controlled by a chromatic correction group having an air spaced doublet with a negative (concave surfaces, diverging) flint glass element 148 and a positive crown glass element 150. The positive lenses 140, 146, 150 in the objective 134 add positive chromatic aberration while the negative element 148 adds opposite negative aberration.

[0236] In some embodiments, a 5-element objective may be employed in place of the lens system 134, for example, the lens system 135 as shown in FIG. 1 and described above.

[0237] In various embodiments, to obtain a sufficiently good measurement while efficiently using a high power low cost laser source (e.g., diode laser), multi spatial mode laser sources with line focus are used for position sensing to improve size resolution, and longitudinal chromatic aberration is employed with two different laser sources, with different wavelengths) for position sensing to improve size resolution.

[0238] A schematic diagram of a further embodiment of an optical apparatus (e.g., a lidar system) 103 is shown in FIG. 7. The optical apparatus 103 may be used for particle detection and determination (or measurement) of a property (or characteristic), e.g., size, of at least one particle. In the optical apparatus 103, a non-polarising beam splitter 702 may be used in the optical sampling arrangement or the interferometer thereof, replacing the polarising beamsplitter 124 employed in the optical apparatus 100 (FIG. 1) and the optical apparatus 101 (FIG. 6). The optical apparatus 103 is more typical for a Michelson interferometer with all beams in the input arm 102, the sample arm 104, the reference arm 106 and the detection arm 108 having substantially same polarisation. The lens system 134 or the lens system 135 may be employed in the optical apparatus 103. As the optical apparatus 103 is similar in layout to the optical apparatus 101 of FIG. 6, for conciseness, only the differences compared to the optical apparatus 103 are described.

[0239] The input polarisation controller 122 of FIG. 6 is not needed in the optical apparatus 103, with the linear polarisation of the outputs of the laser sources (e.g., diode lasers) 114a, 114b acceptable and the exact polarisation angle may not be required (e.g., any polarisation state including elliptical may work). The non-polarising beam splitter 702 sends the beam 121 to the sample arm 104 and the reference arm 106. An approximately 50%:50% (reflection: transmission) beam splitter may be desirable and most efficient. However, a range of about 20%: 80% to about 80%:20% (e.g., about 20%:80% to about 60%:40%, about 20%:80% to about 50%:50%, about 40%:60% to about 80%:20%, about 50%:50% to about 80%:20%, or about 40%:60% to about 50%:50%) may be suitable and a reflectivity in the range of about 1% to 99% (e.g., about 1% to 80%, about 1% to 50%, about 1% to 30%, about 10% to 99%, about 30% to 99%, about 50% to 99%, about 20% to 80%, or about 40% to 60%) may be suitable.

[0240] The beams sent to both arms 104, 106 have the same polarisation and, as quarter wave plates (e.g., 130, 132 in FIG. 6) are not included, remain in their initial polarisation state. The nonpolarising beam splitter 702 may send some of the return and reference lights (or signal and reference beams) to the detection arm 108 and some back towards the laser sources 114a, 114b. The return and reference lights sent to the detection arm 108 have the same polarisation as the initial light or beam 121.

[0241] Generally, an optical apparatus or system with a non-polarising beam splitter may do no better than send 50% of the input power to the sample, then 50% of the signal from the sample to the detector (or 90% then 10%, or 30% then 70%, or other combinations adding to 100%). So, such an apparatus may lose at least 75% (50% loss twice) of the available power and may be less preferable than the optical apparatus 100, 101 of FIGS. 1 and 6, but may still be implemented as described in the context of the optical apparatus 103. A low reflectivity of <10% or <1%, e.g., 0.1% may be required for the mirror or reflective surface 154 in the reference arm 106 as the optical apparatus 103 is without the use of polarisation to control the reference beam power that is provided in the optical apparatus 100, 101.

[0242] As for the optical apparatus 101, phase modulation and / or alternate on / off modulation of the two laser sources 114a, 114b may be used, e.g., to simplify the optical detection arrangement or the detection optics. As for the optical apparatus 101, the reference arm mirror 154 may be moved (represented by double-headed arrow 402) and / or an optical phase modulator 404 may be used. A phase modulator (e.g., 406; FIG. 6) in the detection arm 108 may not be desirable as the return and reference lights have the same polarisation. As the return and reference lights have the same polarisation, full interference may be seen between the return and reference lights without the need for a polarising beam splitter (e.g., 408; FIG. 6) in the optical detection arrangement. The photodetector array (or photo diode array) 704 used for detection may have fewer detector or photodiode elements 706 than in the optical apparatus 101 as (only) one detector element 706 is needed per microlens element or channel 167 of the microlens array 166. For each detector element 706, a signal, that with its DC component removed is like that from subtracting pairs of photodiode currents (as represented by the subtractor 412 in FIG. 6), is recorded and may be analysed by the processor 195.

[0243] As (only) a single photodiode element 706 may be needed per microlens 167, the photodiode array 704 may, in some embodiments, optionally be placed at the image plane 164 and the microlens array 166 and the lens 170 omitted. This is simpler but it may be preferable to use the microlens array 166 if the active areas of the photodiodes elements 706 are small compared to their spacing in the array 704. The lens 170 may be useful without including the microlens array 166 to increase the magnification from that provided by the optical telephoto arrangement having the telephoto lens 160, 162 by reimaging plane 164 onto the photodetector array 704.

[0244] In various embodiments of the optical apparatus 100, 101, 103, one or more folding mirrors (not shown in the figures) may be added to make the apparatus 100, 101, 103 more compact. A folding mirror is a mirror placed in a long straight beam path to turn it into two shorter paths. It ideally has no effect on the operation of the apparatus 100, 101, 103 but helps the apparatus 100, 101, 103 to fit in a reasonably sized box. Preferably, the detection arm 108 and / or the input arm 103 has at least one folding mirror.

[0245] The optical apparatus 100, 101, 103 has been described using a Michelson interferometer layout (e.g., with the use of a single beam splitter 124 to separate the reference 128 and signal 126 beams and to recombine the returned light and the reference light). However alternative optical apparatus may be used for detecting and / or measuring the size of one or more particles as described herein. For example, a Mach-Zehnder interferometer layout may be used. Representative diagrams of alternative layouts are shown in FIG. 8. The lens systems have been simplified for clarity, with only some parts shown to make the configuration clear. Layout 801 is a simplified diagram of the Michelson interferometer as shown in FIG. 1, for comparison. Layout 801 has input arm 102, sample arm 104, reference arm 106 and detection arm 108. Beam splitter 124 splits the input beam 121 from the lasers (not shown) to beams in the sample 104 and reference 106 arms. In the sample arm 104 an objective lens system 134 brings light to a focus in the flow channel 138 with the sample solution containing particles. The objective lens system 134 also collects the scattered light and collimates it to a beam directed back towards the beam splitter 124. Some of the light returned from the mirror 154 in the reference arm 106 and from the sample arm 104 is combined by the beam splitter 124 and sent to the detection arm 108. The detection arm 108 is connected to a detection system, not shown. The use of the same beam splitter 124 to separate the sample and reference beams then recombine the returned light is a characteristic of a Michelson interferometer.

[0246] Layout 803 is an example of a possible Mach-Zehnder interferometer type layout. In a Mach-Zender type of layout two separate beam splitters are used, a first to separating the reference and signal beams and a second to recombine the returned light and the reference light. The input arm 802 may be the same as previously described. The input arm 802 may be arranged such that a line focus of the lasers is orientated perpendicular to the drawing plane rather than parallel to it. Abeam splitter 124 sends light from the input beam 121 to the sample arm 804 and reference arm 806. A characteristic of a Mach-Zehnder interferometer is that a second beam splitter 810 is used to combine light from the sample 804 and reference 806 arms and send them to the detection arm 808. The second beam splitter 810 collects light scattered sideways (e.g. perpendicularly) from the particles in the solution. In some cases, the light may be collected from scattering at an angle of between 0 and 180 degrees from the sample beam. In some cases, the angle is substantially 90 degrees.

[0247] A detection system of layout 803 may be in either (or both) of the output beams of beam splitter 810. For example, the detection system may be in the part of the detection arm 808 where sample light is reflected from beam splitter 810 and / or in the part of the detection arm 808 where sample light is transmitted through beam splitter 810. In some cases, a detection system in both parts of detection arm 808 will give optimum performance, however this may increase complexity. As shown in FIG. 8 objective lens 816 focuses light into the flow channel 822 and a second objective lens 818 collects scattered light from the sample and collimates it to a beam sent to beam splitter 810. In some cases, these objectives 816, 818 will to be of a lower NA than an equivalent Michelson layout due to space requirements. In reference arm 806 a mirror 812 sends the reference light to beam splitter 810. The line focus from lens 816 must be perpendicular to the plane of the FIG. 8 such that the lens 816 can form an image of it. Consequently, in the flow channel the flow must be across the line focus, that is in the plane of the drawing. In one example, this is achieved by having the flow from bottom left of FIG. 8 to top right. Beam splitters 124 and 810 may be non-polarising or polarizing. If polarising beam splitters are used wave plates may be placed in the sample 804 and detection 806 arms to control the polarisation and thus power reflected / transmitted by beam splitter 810.

[0248] Layout 805 is another example of a possible Mach-Zehnder interferometer type layout. Layout 805 is similar to Layout 803 except that the objective lens 820 that collects the scattered light is arranged opposite the input objective 816. The input beam 121 therefore passes through the sample solution. A sample solution containing particles flows left to right (or vice versa) in flow channel 824. In layout 805 the objective lens 820 collects forward scattered light and the full power of the part of the input beam 121 sent to the sample arm 804 by beam splitter 124. In some cases, such as when using axial (longitudinal) chromatic aberration to separate the foci of two lasers objectives 816 and 820, the objectives will need to have opposite aberration effect on the wavelengths (one under corrected and the other over corrected). Layout 805 may have applications to large particles where a low input power, suitable to be directly detected, is sufficient. Particles with size comparable to the wavelength of the lasers will show Mie scattering behaviour with more intense forward scatted light than other directions improving the available signal.

[0249] The example layouts shown illustrate example methods of generating reference and sample signals. As discussed, a Michelson interferometer arrangement may be used with a single beam splitter and using back scattered light from the sample, or alternative systems may be used to capture reflected or scattered light, such as the Mach-Zehnder interferometer type layouts using perpendicularly (i.e. perpendicular to the propagation direction of the light entering the sample scattered light or light passing through the sample. In some cases, the light may be scattered at an angle between back scattering and forward scattering.

[0250] Examples have been provided for samples in water, or water-based liquids. The sample solution may not be water-based. Other possible solutions include organic solvents (for example hydrocarbons, alcohols, acetone) and / or any one or more other liquids that may support particles. The sample solution used may affect the design of the objective lens 142. For example, the objective lens 142 may need to be adjusted as a liquid with a different refractive index will change the aberrations. Low absorption of the light used by the solution is desirable and the wavelength of the lasers 110, 112 may need to be selected based, at least in part, on absorption parameters. In a further example, particles in a flow of gas (for example air or a combination of one or more gases) could also be measured with a modified objective 142.

[0251] In a further example an alternative system could use a single laser 110 with a line focus. Consider a system as shown in FIGs 1, 6, or 7 where the second laser module 112 and dichroic 120 are removed. The detection arm 108 may also be simplified. For example, the FIG. 1 system detection arm 108 can be simplified by removing the prism 176 and half the photodiode elements 173, 175 in detection arrays 172, 174 as there is now only one laser 110 and a single wavelength. For the FIG. 6 and 7 systems the laser does not need to be pulsed on and off as there is no time division multiplexing of two lasers. In this single laser 110 example position information along the optic axis (302 in FIG. 4) is not obtained from a measurement of a difference in the signals between the two lasers. Instead of the use of signal difference and phase difference only phase is available. However, there will still be position information along the optic axis 302 from the phase of the detected signal. Additional processing, or assumptions may be required to adjust for drift and 2TI phase ambiguity. Because the line focus 352 is still imaged to the detector channels (e.g. microlens array detector channels 167) the system will obtain position information along the line focus 352 as was described in FIG. 5. The reduced position information may not be able to accurately correct for the variation in signal strength for a particle 361 of a given size passing through different parts of the focus region 352. However, some compensation for signal strength variation will be possible. In some cases, if many particles 361 are observed, a statistical analysis may allow the distribution of particle sizes in the sample solution to be determined.

[0252] In a further alternative a signal laser 110 may be used with two laser sources 114a, 114b (i.e. a dual wavelength laser), or another technique to provide a single input beam 121 with two wavelengths. In some cases, three or more wavelengths may be used.

[0253] The analysis steps and / or signal processing for various embodiments will now be described by way of the following non-limiting examples. Suitable adaptations or modifications may be made to the analysis steps and / or signal processing for various embodiments of the optical apparatus 100, 101, 103. Alternative processing methods may be applied. The applied processing method may depend on the particle being measured and / or the size or speed of the particle being measured. Alternative analysis methods may be developed for particular particles, such as DNA and / or proteins.

[0254] The (electrical) signals to be analysed come from pairs of detector or photodiode elements (e.g., 173, 175, 411) where each element of the pair detects one light of a pair of lights separated or split from a resultant light or beam (e.g., 171) by a polarising beam splitter (e.g., beam splitters 180, 182, 408). Each pair of detector or photodiode currents is subtracted, for example, using a balanced detector (balanced subtractor) to minimise or avoid laser noise giving a false signal if the pair of photocurrents are unequal. The result of subtracting each pair of photocurrents is an I (in-phase) or Q (quadrature) signal.

[0255] Using the optical apparatus 100 as an example, the pair of detector elements with their currents subtracted as labelled with the subtractor 186 is the I signal, e.g., Il, for the shorter wavelength laser. The pair of detector elements with their currents subtracted as labelled with the subtractor 188 is the Q signal, e.g., QI, for the shorter wavelength laser. The pair of detector elements with their currents subtracted as labelled with the subtractor 190 is the I signal, e.g., 12, for the longer wavelength laser. The pair of detector elements with their currents subtracted as labelled with the subtractor 192 is the Q signal, e.g., Q2, for the longer wavelength laser. There are corresponding signals for each microlens array channel so the signals are: Iln, Qin, I2n, Q2n, with n = 1,2, 3, 4... labelling the microlens array channel.

[0256] The signals Iln, Qin, I2n, Q2n, from the subtractors (e.g., balanced detectors) are analogue signals, e.g., analogue voltages. The following analysis steps use these signals to detect particles passing through the focus and for each one derive a value, S, the scattering signal. S is related to the scattering coefficient and mass or size for each detected particle as described below.

[0257] Step 1 - The signals Iln, Qin, I2n, Q2n, are low pass filtered with an analogue filter (e.g., may be an RC, LC, or active filter). The filter bandwidth is higher than the change in signal due to the motion of the particle (of the order of 100kHz). For embodiments of the optical apparatus using modulation to reduce the number of detectors, described with reference to FIG. 6, the filter bandwidth includes the modulation frequencies (may be up to 10s of MHz).

[0258] Step 2 - The filtered signals may then be digitised with analogue to digital converters (ADCs) with a sampling rate sufficient to avoid aliasing. Minimum frequency possible is the Nyquist rate or 2x highest frequency components with sufficient or significant amplitude after the filtering in step 1. The resulting digital signals are processed by a processor or computer system in the following steps. The processor may include FPGA or GPU to components to aid signal processing. Low pass digital filtering may be applied after digitising.

[0259] Step 3 - For each microlens channel, the signal magnitudes Mln= ((Un)2+ (Qin)2)0'5, and M2n= ((I2n)2+ (Q2n)2)05are calculated. They may be lowpass digitally filtered. For embodiments of the optical apparatus using modulation to reduce the number of detectors Mlnand M2nare demodulated from the output of the subtractors (e.g., one subtractor 412 is shown in FIG. 6). The magnitudes of the I and Q terms may be adjusted to correct for differences in sensitivity of the I and Q detection arms. For example, a potential cause of different sensitivity would be the non-polarising beam splitter 178 not splitting the signal and / or reference light equally between the I and Q detection arms. Other adjustments may be made for known differences in the optical systems for I and Q.

[0260] Step 4 - A spatial and temporal filter is applied to Mlnand M2nto give fMlnand fM2n. The spatial component filters the signal by microlens array channel number n = 1,2, 3, 4... with a response that may be a gaussian or matched filter with a width determined by the calculated point spread function for the part of the optical imaging from the focus in the sample solution onto the microlens array. The temporal component may be a gaussian or matched filter with a width determined by the expected time for a particle moving with solution flow to cross the focus. The purpose of these signals is to select the signal from a particle and remove noise. The parameters of the filters may be adjusted based on analysis of previous measurements to maximise the signal to noise ratio.

[0261] Step 5 - Points (time and space along the microlens array) where fMlnand fM2nexceed threshold values are recorded as particle detections. If adjacent points exceed the threshold, the largest may be selected as the detection point. The thresholds may be set as multiples of the standard deviation (sigma) of the signal level in a sample with no particles. For example, a 5 sigma threshold which gives a probability of a false detection on noise of the order of 1:1,000,000. As there are many time points (of the order of 10 -100 kHz for each microlens array channel), a low probability of false detection per point may be needed to keep the number of false detections for the whole optical apparatus or system per second low (<1 false count per second is a reasonable target). The thresholds may be adjusted to give a desired compromise between detecting small particles and the false detection rate.

[0262] Step 6 - The position of where the particle was imaged on the microlens array (e.g., array 166) may be found by fitting a gaussian curve or the expected point spread function to the Mlnand M2nvalues at the time point where the particle is detected. The peak of the fitted curve is recorded as the maximum Mlnand M2nvalues, e.g., mMlnand mM2n. The position of the fitted curve is recorded as chPos (micro lens array channel position). See, for example, FIG. 5 and the corresponding description.

[0263] Step 7 - The ratio of mMlnand mM2nis calculated as Rml2. This ratio is a measure of the position where the particle passed through the beams along the propagation axis. Rml2 is used as is as a measurement of position in arbitrary nonlinear units. See, for example, FIG. 4. and the corresponding description.

[0264] Converting Rm 12 to position in nanometres may be done, but may not be necessary as a result in arbitrary units may be adequate for the processing steps below to find S. A conversion may be made by comparing the measured Rm 12 value to the expected Rm 12 values for different positions calculated from the axial point spread function of the interference contrast. The electric field of the reference and signal beams may be calculated numerically if the input beam and lens properties are known. The signal beam and its field will vary with particle position (or position of the mirror to be placed at the focus for the calibration to be described below). The interference signal intensity change that will be detected by the photodiodes may be calculated from the field as a function of focus, i.e., the axial point spread function.

[0265] The point spread function for each of the two lasers 114a, 114b may be calculated from the parameters of the input and reference beams, or it may be measured by removing the flow cell and placing a mirror at the beam focus. Moving the mirror along the axis may cause an interference signal changing in amplitude with axial position to be observed. The axial dependence of the signal with mirror position is the point spread function. The point spread functions indicate the expected interference signal strengths or mMlnand mM2nvalues for laser 114a and 114b respectively. Once the axial point spread functions are known, the functions may be used to calculate the expected Rm 12 as the ratio of the magnitudes of the point spread functions for the two lasers. This provides the expected Rml2 for any position as needed to convert a measured Rm 12 to position in nanometres (or micrometres or any other unit).

[0266] Step 8 - Rml2, mMlnand mM2nare recorded for each particle detection event. Thresholds may be applied to remove detections where mMlnand / or mM2nare too low to reliably indicate a detected particle. A threshold may also be applied for Rml2 to remove large and / or small values that may indicate a particle was too far from the focus (e.g., Rm 12 = 1 at a point centred between the two lasers foci. It increases moving in one direction and decreases in the other direction).

[0267] Step 9 - Finding S, the scattering signal from Rm 12, mMlnand mM2nmay be done with the aid of a previous calibration measurement. This calibration may be made by a measurement of a calibration solution containing many particles with uniform scattering coefficient. Different values of mMlnand mM2nmay be acquired corresponding to particles that passed through the focus in different axial positions. The axial positions the particles passed through the focus have been recorded as Rml2. It is defined that S = mMln+ mM2nwhen Rml2 =1. Calibration functions cl (Rm 12) and c2(Rml2) may be used where cl and c2 describe functions that may be polynomial or modified gaussian, and the bracketed Rml2 indicates these functions have Rm 12 as a variable. The calibration functions may be such that S = [mMln * cl(Rml2)] + [mM2n* c2(Rml2)] for any Rml2 (position) within the range where consistent results may be obtained. The threshold values of Rml2 mentioned in step 8 may be such as to restrict the positions (e.g., range of Rml2 values) where particle detections are recorded to the range where this calibration is valid. This may mean (only) using particle detections near (axial direction) the centre of focus. The functions cl(Rml2) and c2(Rml2) may be found as fits to the uniform particle data to make S as equal as possible for all observed events. Using cl(Rml2) and c2(Rml2) as found from the uniform particle calibration measurement for any sample to be analysed, S is calculated as S = [mMln* cl(Rml2)] + [mM2n* c2(Rml2)].

[0268] A correction to S based on the position the particle was imaged to the microlens array channel, chPos, as found in step 6, may be applied. The parameters of this correction may be found from how S changes with chPos for the uniform particle calibration measurement. It is likely that the observed S may be high at the centre of the line focus and drop off at the edges. The power may be lower at the edge and the objective focus less well.

[0269] Step 10a - The scattering coefficient of the particle may be found as S2times a calibration constant. S is proportional to the amplitude of the combined or interferometric signals as measured by the photodiode currents. S may therefore be proportional to the amplitude of the electric field of the signal beam or return light scattered from the particle. The intensity of the signal beam is proportional to the square of its electric field or proportional to S2. The calibration constant may be found from the measured S for particles of known scattering coefficient, e.g., the uniform particles from the calibration for step 9.

[0270] S is the device or instrument output. The various steps 10a, 10b, and 10c describe how the scattering signal S may be interpreted as scattering coefficient, protein mass or size.

[0271] Step 10b - For proteins, mass is proportional to S times a mass calibration constant. The mass calibration constant may be found from calibration measurements of proteins of known mass. (It has been demonstrated for mass photometry and other scattering measurements that the scattering coefficient of a protein is proportional to the square of its mass to a good approximation). As S is proportional to the square root of the scattering coefficient, S is proportional to the protein mass. A similar calibration with a different constant may relate S to DNA / RNAmass or number of base pairs.

[0272] Step 10c - For particles of a uniform composition and spherical shape, the Rayleigh scattering coefficient is proportional to the radius6(radius to the 6thpower or volume squared). So, the scattering coefficient provides a measure of radius, volume or generally size. To a reasonable approximation, the Rayleigh scattering coefficient of non-spherical particles is proportional to the volume squared if the non-spherical particles are much smaller than the wavelength of light. After a calibration measurement, of known size particles, the size may be found as a calibration constant times S for volume or a different constant times S1 / 3for radius.

[0273] The size calibration constant described in Step 10a and the mass calibration constant described in Step 10b may be fairly stable. However, in some situations, the size and mass calibration constants may need to be recalibrated with a known particle sample on a regular basis (e.g., daily / hourly basis) or if the temperature changes by a certain amount.

[0274] The above analysis assumes that only a single particle is detected at any one time. This may be controlled. For example, the concentration of particles may be controlled, or the solution diluted sufficiently that the presence of two or more particles, in locations where they may be detected simultaneously is unlikely. In some cases, the analysis may compare the signal detected to that expected from a single particle and exclude multi particle detections. The analysis may be extended so that properties of two or more particles simultaneously detected at significantly different points on the line focus may be correctly measured. Improved analysis may thus extend the ability of the system to measure solutions containing high concentrations of particles.

[0275] The above is a non-limiting example of a process to find mass / size / scattering coefficient from the detected particle data. The use of phase data to improve the axial position, Rml2, result may improve calibration accuracy. For example, phase data may be the main source of position information for calculating the size / mass of a particle. The difference of signal from the two offset laser foci may be used to correct for phase drift and 2TI phase ambiguity. The difference in wavelength of the two lasers will mean that the spacing of points with 2TI phase difference is different potentially providing a way to correct for the 2TI phase ambiguity in addition to the difference in signal amplitude.

[0276] As well as size measurement, other useful information about particles may be obtained from analysis of the apparatus or system data.

[0277] The concentration of particles in the sample solution may be determined from the flow rate and number of detections per second. The flow rate and area covered by where particles can be detected provides the volume of solution measured per second. Dividing the number of detections per second by this volume gives the concentration of particles per unit volume.

[0278] The diffusion coefficient indicating how fast the particle moves with Brownian motion may be determined. The fluctuations in the detected position and / or phase may indicate a particle’s motion at fast time scales. The detected positions may change small amounts as the particle moves rapidly and randomly with Brownian motion. The mean squared position displacement over a given timescale may define the diffusion coefficient.

[0279] The diffusion coefficient may aid distinguishing particles with the same overall size but different shape. Particles with the same overall size or mass but different shape may have similar scattering coefficients but different diffusion coefficients. So, a mixture of two types of particles may appear indistinguishable when the size / scattering coefficient is measured. But a measurement of diffusion can show two distinct populations.

[0280] A correlation calculation may also be possible measuring the autocorrelation of position measurements over time. If the correlations in position extend to long timescales, this means the particle is diffusing slowly. If the correlations in position are limited to short timescales, this means that the particle is diffusing faster with a large diffusion coefficient.

[0281] Large particles may show Mie scattering with a more complex dependence of scattering intensity with angle than the dipole emission Rayleigh scattering of small particles altering the detected signal. The point spread function of the particle signals may be altered both axially (focus direction) and along the microlens array. This may be detected by comparing the observed signals to the expected point spread function for small particles (measured or calculated). The degree of change in the point spread function may contain information on the size or shape of the particle.

[0282] The optical apparatus and techniques disclosed herein may be suitable for detection and size determination of particles of various sizes. For example, protein complexes in the range of 5 -20 nm diameter spheres or equivalent volume if nonspherical (such range being typical to large proteins) may be detected. Detection may be extended to small 2 - 5 nm diameter proteins and 1 nm or less high refractive index nanoparticles by optimising the performance of the apparatus or system. As further non-limiting examples, virus particles, and other larger particles in the range of 10 - 200 nm diameter may be detected. Large 100 - lOOOnm particles that exhibit Mie scattering may also be detected.

[0283] While the method described above is illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events are not to be interpreted in a limiting sense. For example, some steps may occur in different orders and / or concurrently with other steps or events apart from those illustrated and / or described herein. In addition, not all illustrated steps may be required to implement one or more aspects or embodiments described herein. Also, one or more of the steps depicted herein may be carried out in one or more separate acts and / or phases.

[0284] The method may be configured to detect protein molecules, RNA and / or DNA, for example. Consider the example of a single protein molecule. The single protein molecule will scatter only a small amount of light which will result in small changes in the signal from the detection system, as described above. Example sources of noise in the signal include shot noise and noise from the random Brownian motion of the molecule. The Brownian motion also means that the flow rate cannot be too slow. If the flow rate approaches or is less than the movement caused by the Brownian motion during the measurement time the particle cannot be assumed to pass through the focus in a known direction. This is because, for small measurement times, the Brownian motion movement may be fast relative to the flow rate) If a particle moves with the flow to the edge of the focus then moves away from the focus against the flow direction it may never reach the centre of focus where the signal would be strongest. Therefore, a smaller maximum signal would be recorded and the particle size would be under estimated. In some cases, the signal to noise ratio for detecting a single molecule may be low.

[0285] The described apparatus and method may be configured to detect single protein molecules. Simplified simulations are provided to show an example of how a small particle may produce a signal distinguishable from noise. Some aspects of the apparatus and method are omitted from these simulations, for example different microchannel detection channels and the variation of sensitivity with position along the optic axis 302. The simulations assume the particle moves with flow and diffusion (random Brownian motion) through the focus 142 and with diffusion only along the optic axis 302. Motion along the third axis (along the line focus 351) is not modelled and the particle is assumed to be in a position that is imaged to some of the microlens array 166 detection channels. The variation in the I / Q signals with phase changes as the particle moves on the optic axis 302 is calculated.

[0286] A lens system 134 with a 45° half angle, slightly less than NA = 1, is modelled. This will collect 19% of the light from a dipole pattern Rayleigh scatterer at the focus 142. A total input power of 1 W at 450 nm with ideal detection efficiency is modelled. This is equivalent to 2 W power and 50% efficiency due to non-ideal optics plus the limited quantum efficiency of the photodiode detectors 173, 175. This power is assumed to be distributed over a 2 um long line focus 142 with its short axis a Gaussian intensity distribution 1 / e2width 300 nm. The reference beam power is set to 5 uW per photodiode.

[0287] The particle has a scattering cross section of 1.5 x 10'24m2, that for a 6 nm diameter sphere of refractive index 1.43 in water, which should be similar to a medium sized protein. The motion with diffusion is calculated from a diffusion coefficient of 6.5 x 10'11nfs’1also typical for a medium sized protein. These are rough values for bovine serum albumin (BSA) a commonly used protein.

[0288] The particle is started 1 um from the centre of focus 142 in the direction of flow of the sample 138 and randomly within one wavelength (340 nm in water) of centre on the optic axis 302. It is then moved with a 5 ns time step moving with diffusion and a flow rate of 0.001 ms'1. For each step the intensity and phase of the scattered light at the detector 173, 175 are calculated. With this and the reference beam power the I / Q signals are calculated. Gaussian random noise is added calculated from the shot noise of the reference beam. A 200 kHz low pass filter is applied, the data decimated to 500 kHz sampling, then M = (I2+Q2)05is calculated. The simulation runs for 2 ms in which time the flow should move the particle past the focus 142.

[0289] FIG. 11 shows the position of a typical particle along the flow and optic axis. FIG. 12 shows the magnitude of the interference signal M for the same particle. There is significant noise from the shot noise of the reference beam but a peak in the signal can be seen at 1000 us where the particle crosses the focus 142. A gaussian filter, standard deviation 35 us, is then applied to signal M as a peak detector. In FIG. 13 the gaussian filtered signal shows a clear peak where the particle crosses the focus. A series of one hundred simulations were run and the gaussian filtered signal was analysed to determine if the particle was detected. The noise level of a blank simulation with no particle was used to set a detection threshold. For each of the one hundred simulations the particle is considered to be detected if the gaussian filtered signal exceeds the threshold. Setting a threshold of five times the standard deviation above the mean will give a high degree of confidence (for example, >99.9999 %) and a low rate of false positives. With this threshold the particle was found to be detected in 95 out of 100 runs. The simulation parameters are examples and alternative methods of detection, methods of analysis and / or systems may improve performance.

[0290] The term ‘comprising’ as used in this specification and claims means ‘consisting at least in part of’ or ‘including, but not limited to’ such that it is to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense. When interpreting each statement in this specification and claims that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

[0291] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0292] The term ‘and / or’ means ‘and’ or ‘or’, or both.

[0293] As used herein, the phrase of the form of “at least one of A or B” may include A or B or both A and B. Correspondingly, the phrase of the form of “at least one of A or B or C”, or including further listed items, may include any and all combinations of one or more of the associated listed items.

[0294] The use of ‘(s)’ following a noun means the plural and / or singular forms of the noun. Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” as used herein represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result. For example, the terms “approximately”, “about”, “generally,” and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, and within less than 0.01% of the stated amount.

[0295] The processor described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic component, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, circuit, and / or state machine. A processor may also be implemented as a combination of computing components, e.g., a combination of a DSP and a microprocessor, a number of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0296] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0297] The scope of the present disclosure is not intended to be limited by the specific disclosures of embodiments in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

Claims

CLAIMS1. An optical apparatus comprising:an optical source arrangement configured to provide a first source light and a second source light, the first and second source lights having different wavelengths;an optical sampling arrangement optically coupled to the optical source arrangement to receive the first and second source lights, the optical sampling arrangement being configured to:direct a first portion of the first source light and a second portion of the second source light along a propagation axis towards a fluid sample comprising at least one particle;provide, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, and a second line illumination pattern based on the second portion, the first and second line illumination patterns being offset from one another and each having a long axis perpendicular to the propagation axis; and combine, with a reference light derived from the first and second source lights, a first return light from the at least one particle illuminated by the first line illumination pattern and a second return light from the at least one particle illuminated by the second line illumination pattern; andan optical detection arrangement optically coupled to the optical sampling arrangement, the optical detection arrangement comprising at least one photodetector arrangement configured to detect a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light for determining a property of the at least one particle.

2. The optical apparatus as claimed in claim 1, wherein the optical source arrangement comprises:a first multi spatial mode laser source configured to provide the first source light; and a second multi spatial mode laser source configured to provide the second source light.

3. The optical apparatus as claimed in claim 1 or 2, wherein a wavelength of the first source light and a wavelength of the second source light are within a range of 200 - 2000 nm or within a range of 400 - 500 nm.

4. The optical apparatus as claimed in any one of claims 1 to 3, wherein the different wavelengths are offset from one another by about 5 - 500 nm, and / or wherein the first and second line illumination patterns are offset from one another by about 10 - 5000 nm.

5. The optical apparatus as claimed in any one of claims 1 to 4, wherein the optical source arrangement comprises an optical element configured to combine the first and second source lights, optionally wherein the optical element comprises a dichroic mirror or a prism or a diffraction grating.

6. The optical apparatus as claimed in any one of claims 1 to 5, wherein the optical sampling arrangement comprises an optical polarising arrangement configured to control respective amounts of the first portion of the first source light, the second portion of the second source light, and the reference light.

7. The optical apparatus as claimed in claim 6, wherein the optical polarising arrangement comprises:a polarising beam splitter configured to:split the first source light and the second source light into the first portion and the second portion;direct the first portion and the second portion towards the fluid sample; and combine the first return light and the second return light with the reference light;an optical polarising device in an optical propagation path between the optical source arrangement and the polarising beam splitter;a first polarisation control element in an optical propagation path between the polarising beam splitter and the fluid sample; anda second polarisation control element in an optical propagation path between the polarising beam splitter and an optical reflector arrangement configured to provide the reference light.

8. The optical apparatus as claimed in claim 7, wherein the optical reflector arrangement comprises a retroreflector and / or the optical reflector arrangement comprises a dispersion compensator.

9. The optical apparatus as claimed in any one of claims 1 to 8, wherein the optical source arrangement is configured to alternately provide the first source light and the second source light, and / or wherein the optical apparatus is configured for phases of the reference light and the first and second return lights to be modulated relative to one another.

10. The optical apparatus as claimed in claim 9, wherein the optical sampling arrangement comprises an optical phase modulator configured to modulate the phase of the reference light relative to the phases of the first and second return lights.

11. The optical apparatus as claimed in any one of claims 1 to 10, wherein the optical detection arrangement comprises:a non-polarising beam splitter configured to receive the first resultant light and the second resultant light, and further configured to split each of the first resultant light and the second resultant light along a first optical propagation pathway and a second optical propagation pathway;a first polariser element in the first optical propagation pathway, the first polariser element being configured for an angle that is non-zero and non-90 degrees;a second polariser element in the second optical propagation pathway, the second polariser element being configured for an angle that is non-zero and non-90 degrees; and a polarisation control element configured to introduce a phase difference of about 90° between the first optical propagation pathway and the second optical propagation pathway.

12. The optical apparatus as claimed in any one of claims 1 to 11, wherein the optical detection arrangement comprises an array of microlens elements configured to receive the first resultant light and the second resultant light, wherein respective microlens elements are associated with respective sections of at least one of the first line illumination pattern and / or the second line illumination pattern.

13. The optical apparatus as claimed in any one of claims 1 to 12, wherein the optical sampling arrangement comprises a beam splitter, the beam splitter being configured to:direct the first portion of the first source light and the second portion of the second source light towards the fluid sample;direct a third portion of the first source light and a fourth portion of the second source light towards a reference arm configured to generate the reference light.

14. The optical apparatus as claimed in claim 13 wherein the beam splitter is configured to combine the reference light returned from the reference arm with the first return light and the second return light.

15. A system comprising:the optical apparatus as claimed in any one of claims 1 to 14; anda processor configured to process electrical signals corresponding to the first resultant light and the second resultant light detected by the at least one photodetector arrangement for determining the property of the at least one particle, optionally wherein the processor is configured to determine a size of the at least one particle based on processing of the electrical signals.

16. The system as claimed in claim 15, wherein, for determining the size of the at least one particle, the processor is configured to:determine a scattering coefficient associated with the at least one particle based on the processing of the electrical signal, optionally wherein, for determining the scattering coefficient, the processor is configured to determine a position of the at least one particle at the focusing region based on the processing of the electrical signals.

17. A method for determining a property of at least one particle, the method comprising:directing a first portion of a first source light and a second portion of a second source light along a propagation axis towards a fluid sample comprising at least one particle, the first and second source lights having different wavelengths;providing, at a focusing region within the fluid sample, a first line illumination pattern based on the first portion, and a second line illumination pattern based on the second portion, the first and second line illumination patterns being offset from one another and each having a long axis perpendicular to the propagation axis;combining, with a reference light derived from the first and second source lights, a first return light from the at least one particle illuminated by the first line illumination pattern and a second return light from the at least one particle illuminated by the second line illumination pattern;detecting a first resultant light resulting from a combination of the first return light and the reference light and a second resultant light resulting from a combination of the second return light and the reference light; anddetermining a property of the at least one particle based on electrical signals corresponding to the first resultant light and the second resultant light that are detected.

18. The method as claimed in claim 17, further comprising combining the first and second source lights prior to directing the first portion of the first source light and the second portion of the second source light towards the fluid sample.

19. The method as claimed in any one of claims 17 or 18, further comprising controlling respective amounts of the first portion of the first source light, the second portion of the second source light, and the reference light, and / or further comprising alternately providing the first source light and the second source light and / or further comprising modulating phases of the reference light and the first and second return lights relative to one another.

20. The method as claimed in any one of claims 17 to 19, wherein providing the first and second line illumination patterns at the focusing region comprises controlling a chromatic aberration of a lens system along the propagation axis toward the fluid sample to offset the first and second line illumination patterns from one another at the focusing region, and / or wherein determining the property of the at least one particle comprises determining any one or more of a mass, a size, or the number of base pairs of the at least one particle based on the electrical signals, optionally wherein determining the size of the at least one particle comprises determining a scattering coefficient associated with the at least one particle based on the electrical signals.

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