Improvements in or relating to flow devices

Abstract

A flow cell device is provided that includes a fluid passageway having at least one wall and at least one solid immersion lens optically connected to the fluid passageway. Apparatus and methods for measuring properties of an object are also provided.

Description

[Technical Field]

[0001] The present invention relates to improvements in or relating to flow cell devices, and in particular to devices, apparatus and methods for measuring properties of objects. [Background technology]

[0002] Interferometric scattering microscopy (referred to herein as iSCAT) has emerged as a powerful approach for both single-particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single-particle level. iSCAT is disclosed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935, and Ortega-Arroyo et al., "Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy," Physical Chemistry Chemical Physics 2012 14:15625-15636. Despite its considerable potential, the widespread application of iSCAT is limited by the requirement for custom microscopes, unconventional cameras, and complex sample illumination, limiting iSCAT's ability to robustly and accurately detect, image, and characterize objects as small as single molecules.

[0003] One application of iSCAT is mass photometry (MP), in which coherent light scattering is used to quantify the mass and / or concentration of an object. Typically, MP measurements are performed at a measurement interface within an inverted microscope geometry. This is achieved by placing a small amount of the object on the measurement interface. Challenges arise when combining MP with biochemical screening workflows in which multiple samples are prepared by a robotic process. Automation of the MP measurement process can be achieved by replicating each well on a sample carrier through a robotic pipetting step, which can then be interfaced with the iSCAT microscope. However, this approach has limited throughput and introduces additional complexity into the overall workflow by requiring additional robots. A major challenge when considering the cost-effectiveness of combining an automated MP process with biochemical screening is the large number of sample carriers required to replicate samples.

[0004] Thus, there is a requirement to provide an efficient, automated process for measuring samples with an iSCAT microscope, which is compatible with current biochemistry workflows and which can achieve cost-effectiveness and simplicity that allows the process to be widely implemented. There is a requirement for the process to achieve high sensitivity and resolution so that it is suitable for use in measurements at the single molecule level and when making measurements at interfaces such as those required for mass photometry measurements.

[0005] Furthermore, known methods for constructing solid immersion lens structures include providing a mold defining a lenticular cavity into which the solid immersion lens will be cast, casting a translucent liquid elastomeric material into the lens cavity, allowing the elastomeric material to harden to form a solid immersion lens portion, and removing the solid immersion lens portion from the mold. While this method provides inexpensive and rapid construction of solid immersion lenses, the process can be complex and labor-intensive. Furthermore, molding techniques are not suitable for solid immersion lenses made from materials with high refractive indices, such as profiled glass SIL lenses and / or diamond SIL lenses. Furthermore, SILs made from elastomeric materials can have a high refractive index, e.g., megawatts / cm at the surface. 2 Furthermore, SILs made from elastomeric materials would not be able to provide a flat surface or surface roughness on the nanometer scale because the elastomeric solid immersion lens would bend.

[0006] It is against this background that the present invention arose. Summary of the Invention [Means for solving the problem]

[0007] According to one aspect of the present invention, there is provided a flow cell device comprising a fluid passage having at least one wall and at least one solid immersion lens optically connected to the fluid passage. The flow device disclosed herein provides a simple device for measuring properties of objects in a fluid.

[0008] The flow cell device of the present invention provides an interface suitable for mass photometry measurements. The interface is provided by a solid immersion lens in contact with the fluid. Thus, the surface is in contact with the fluid and any objects within the fluid, allowing the objects to interact with or at the interface. Objects interacting with or near the interface can be measured by optical techniques such as mass photometry.

[0009] According to one aspect of the present invention, there is provided a flow cell device comprising a fluid passageway having at least one wall and at least one solid immersion lens optically connected to the fluid passageway, the solid immersion lens being made of a material having a refractive index substantially equal to or greater than that of glass.

[0010] Within the context of the present invention and unless otherwise specified, the terms "high" or "higher" refer to any material of a solid immersion lens that has a refractive index equal to or greater than that of a solid immersion lens made from glass. By way of example only, a diamond solid immersion lens has a higher refractive index than a glass solid immersion lens.

[0011] Within the context of the present invention and unless otherwise specified, the term "refractive index of glass" refers to the refractive index of medium, standard glass. Irregular glass and / or high refractive index glass surfaces or materials are not considered standard glass.

[0012] In some embodiments, the solid immersion lens can be a high refractive index glass material, with a refractive index greater than that of glass.

[0013] Solid immersion lenses made from materials with a high refractive index can be advantageous because they allow for a high numerical aperture of the lens. A high numerical aperture is beneficial because it increases the efficiency of collecting scattered photons, which can result in improved sensitivity of the measurement. Therefore, providing a solid immersion lens made from a material with a high refractive index can help improve or enhance the resolution of the image detected by the microscope.

[0014] In some embodiments, a flow cell device is provided comprising a fluid passageway having at least one wall and at least one solid immersion lens optically connected to the fluid passageway, the solid immersion lens being made of a material having a refractive index substantially equal to or greater than that of fused silica. For example, the solid immersion lens may be made of a material having a refractive index substantially equal to or greater than that of η=1.4.

[0015] According to an alternative aspect of the present invention, there is provided a flow cell device comprising a fluid passage having at least one wall and at least one solid immersion lens optically connected to the fluid passage, the solid immersion lens being a diamond solid immersion lens. The flow device disclosed herein provides a simple device for measuring properties of objects within a fluid.

[0016] Within the context of the present invention and unless otherwise specified, the term "optically connected" is used to describe when light is allowed to travel between the solid immersion lens and the fluid passage. In other words, the solid immersion lens is connected to the fluid passage in an optical manner. In some examples, the "optical connection" is achieved by the solid immersion lens being embedded within at least one wall of the fluid passage. Alternatively, the solid immersion lens may be attached to at least one wall of the fluid passage. There are various techniques that can be used to attach the solid immersion lens to the wall of the fluid passage, including, but not limited to, adhesive, pressing, cementing, or bonding techniques.

[0017] Within the context of the present invention, the term "object" as described herein should be understood to include any suitable object, such as, but not limited to, a biomolecule, a protein, a peptide, a polypeptide, a lipoprotein, a glycoprotein, a lipid, a carbohydrate, an organic polymer, a protein complex, an antibody or antibody fragment thereof, an enzyme, or the object may be a nucleic acid molecule, such as DNA, RNA, or a polysaccharide, or the object may be a virus, or a viral vector, such as an adenovirus and / or a lentivirus, or a virus-like particle, or a small molecule, an exosome, a vesicle, an assembly complex, a nanoparticle, such as a lipid nanoparticle, a liposome or exosome, a compound, an ion, or a quantum dot. In some embodiments, the object may be a single molecule, a macromolecule, or an association of molecules and macromolecules (such as a polymer). Examples of suitable polymers may include, but are not limited to, nucleic acid molecules, natural nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or artificial nucleic acids such as peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Molecular associations can include assemblies such as virus-like particles in which envelope or capsid proteins are associated.

[0018] In some embodiments, the object may be a multimolecular complex comprising aggregates or other higher order aggregates of components including proteins, such as monomeric, dimeric, and trimeric species.

[0019] Within the context of the present invention, the term "biomolecule" as described herein should be understood to include proteins, lipids, carbohydrates, lipoproteins, glycoproteins, organic polymers, nucleic acid molecules, viruses, vesicles, complex assemblies, or virus-like particles.

[0020] As understood within the context of the present invention, a fluid passageway can be any structural form known to those skilled in the art that is configured to allow the passage of a fluid flowing through or along the fluid passageway. The fluid passageway comprises at least one wall. The wall defines the interior area of ​​the fluid passageway and acts as a barrier to prevent leakage of fluid from the fluid passageway during use. In some embodiments, the fluid passageway may comprise one circular wall configured to allow the passage of a fluid flowing through the fluid passageway. In some embodiments, the fluid passageway comprises four side walls configured to provide an enclosed fluid passageway that contains the fluid within the fluid passageway. Other structural variations will be recognized by those skilled in the art.

[0021] The flow cell of the present invention includes a solid immersion lens (SIL) optically connected to the fluid passage. This reduces or eliminates any manufacturing variations between different parts. Furthermore, the flow cell device of the disclosed invention can help reduce manufacturing costs. Yet another advantage of the present invention is the reduction of noise / drift and usability issues that can arise from the use of oil and standard objective lenses.

[0022] Furthermore, the optical connection between the solid immersion lens and at least one wall of the fluid passage can be used to measure multiple properties of an object within the fluid, for example, light scattered from the object during illumination of the solid immersion lens can be detected to measure the mass of the object.

[0023] The solid immersion lens may be embedded in the wall of the fluid passage. A solid immersion lens embedded in the wall of the fluid passage forms a single unit which can help reduce manufacturing complexity and therefore cost.

[0024] Any material with a high refractive index can be suitable for a solid immersion lens. The solid immersion lens may be made of a material with a high refractive index. For example, the solid immersion lens may have a high refractive index glass surface. In particular, the solid immersion lens of the present invention may be made of one or more materials with a refractive index substantially equal to or greater than that of glass.

[0025] [Table 1]

[0026] In some embodiments, the solid immersion lens may be a diamond solid immersion lens. In some embodiments, the diamond solid immersion lens may be embedded in one wall of the fluid passage. Providing a diamond solid immersion lens may be desirable because it has a high refractive index, which allows for a high numerical aperture. Furthermore, diamond solid immersion lenses are relatively robust, which is particularly advantageous for multiple uses. The refractive index (RI) of a diamond solid immersion lens is substantially η=2.42.

[0027] In some embodiments, the solid immersion lens may be made from fused silica or any glass, including the high refractive index glasses modified zirconia, sapphire, lithium niobate, or potassium niobate. In some embodiments, the solid immersion lens may be made from a high refractive index glass material.

[0028] In some embodiments, the solid immersion lens may be made from a material with a refractive index substantially greater than η=1.5 or η=1.52.

[0029] In some embodiments, the solid immersion lens may be hemispherical or super-hemispherical. In some embodiments, the solid immersion lens may be a glass half-ball lens. In some embodiments, a super-hemispherical solid immersion lens may be preferred. A super-hemispherical solid immersion lens increases the numerical aperture of the system by η 2 (η is the refractive index of the solid immersion lens). A higher numerical aperture is beneficial because it can enhance the collection of scattered photons, resulting in improved sensitivity of the measurement.

[0030] In some embodiments, the solid immersion lens may have a diameter of 1 to 5 mm. In some embodiments, the solid immersion lens may have a diameter of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mm. In some embodiments, the solid immersion lens may have a diameter of less than 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, or 1.5 mm.

[0031] Additionally or alternatively, the solid immersion lens may have a length of 6-10 mm. In some embodiments, the solid immersion lens may have a length greater than 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 mm. In some embodiments, the solid immersion lens may have a length less than 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, or 6.5 mm.

[0032] In some embodiments, at least a portion of the surface of the solid immersion lens has a surface roughness of less than 1 nm RMS. Root mean square (RMS) is a standard measure of surface roughness known to those skilled in the art.

[0033] In some embodiments, the surface roughness may be between 0.1 and 50 nm. For example, the surface roughness of the SIL may be greater than 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, or 45 nm RMS. Alternatively, the surface roughness of the SIL may be less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 nm RMS.

[0034] In some embodiments, the surface roughness of the SIL may be 0.1-1 nm RMS, or the surface roughness may be greater than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 nm RMS. In some embodiments, at least a portion of the surface of the solid immersion lens has a surface roughness of less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nm RMS. Preferably, the surface roughness of the solid immersion lens is 0.5 nm RMS.

[0035] The solid immersion lens (SIL) may have a flat surface and a spherical surface for the sample interface. The solid immersion lens may have a minimal surface roughness of sub-nanometer RMS on the sample interface of the SIL. For iSCAT detection, this allows for accurate detection of small molecules, minimizes errors in the detected signal, and therefore optimizes mass resolution.

[0036] In some embodiments, the surface roughness may be less than 200 nm peak valley deviation relative to the roughness on a spherical surface.

[0037] In some cases, the solid immersion lens may be polished so that the lobe of the spherical side is centered in the optical path. Deviations from the spherical surface roughness of the solid immersion lens can be corrected using a freeform corrector plate. Freeform corrector plates and / or phase corrector plates can be used in optical systems to correct wavefront deformations such as spherical aberration. In this context, deviations from sphere on the curved side of the SIL through which the collected light passes can cause wavefront deformations, which can therefore be corrected using a custom freeform corrector plate.

[0038] In some cases where the solid immersion lens is made from diamond, the solid immersion lens may be polished so that the lobe on the sphere side is centered in the optical path. Polishing the {100} crystal facets of the diamond SIL allows for two things. First, it allows for the best possible ultra-fine surface roughness on the flat side or surface of the SIL. Second, it allows for the lobe to be centered on the sphere side or surface, which allows for more radially uniform surface for much of the scattered light, at high NA, to travel over. This can be advantageous because it can produce a radially symmetric point spread function, which in turn maximizes contrast. Any polishing technique may be used.

[0039] In some embodiments, the flat surface of the SIL, i.e., the sample interface, may be polished using, for example, micro-mechanical polishing techniques to remove grooves on the sample interface. The polishing procedure smooths the surface of the SIL and may advantageously reduce speckle effects.

[0040] The thickness and / or height of the solid immersion lens may have a tolerance of + / - sub 3 microns. Those skilled in the art will recognize that the thickness and / or height of the solid immersion may be adjusted or corrected for optimal performance in the optical systems disclosed herein.

[0041] In some embodiments, a coating may be provided on the surface of the solid immersion lens. In some embodiments, the coating may allow for specific or enhanced binding of objects to the solid immersion lens. In some embodiments, the surface of the solid immersion lens may be coated with antibodies configured to bind to target biomolecules. This may be useful for detection assays. In some embodiments, the coating may allow for temporary binding at the surface of the solid immersion lens. For example, the coating may introduce hydroxyl (OH) interactions with proteins that can be quantitatively disrupted by pH changes.

[0042] It will be appreciated that a coating may be applied to the interface between the solid immersion lens and the fluid. This coating may modify the interaction between the object and the solid immersion lens. The coating may decrease the interaction or may increase the interaction. The interface between the fluid and the solid immersion lens may be referred to as the "surface" of the solid immersion lens.

[0043] In some embodiments, the coating may passivate the surface of the solid immersion lens, causing the surface of the solid immersion lens to become chemically inert. In some embodiments, passivating the surface of the solid immersion lens prevents objects from reacting with and / or bonding to the surface of the immersion lens.

[0044] In some embodiments, the coating may functionalize the surface of the solid immersion lens. In some embodiments, the coating applied to the surface of the solid immersion lens allows for functionalization such that specific objects can interact with or bind to the solid immersion lens. For example, the coating may be (3-aminopropyl)triethoxysilane (APTES), which imparts a positive charge to the surface of the solid immersion lens so that negatively charged biomolecules such as DNA can bind to the solid immersion lens.

[0045] The flow cell may be made of any suitable material as disclosed above. The flow cell may be made of a material that is completely transparent to radiation of a selected wavelength so that optical measurements can be achieved at any desired location / region along the fluid path. Alternatively, the material used for the flow cell may be opaque (does not allow light to pass through). In such embodiments, the flow cell may include one or more windows to allow light to pass through. Such windows may be regions of transparent material.

[0046] In some embodiments, the fluid path may include one or more windows configured to enable optical measurements. The optical measurements through the windows can occur at different locations relative to the solid immersion lens. In some embodiments, the one or more windows may be used to detect radiation from the object, such as emitted light, reflected light, or scattered light. For example, fluorescence emitted from the object may be detected through one or more windows. In some embodiments, the one or more windows may facilitate obtaining additional information from the object. In some embodiments, multiple windows may be provided along the fluid path. This can be particularly useful for kinetic / time course experiments.

[0047] In some embodiments, at least one window is provided in one of the fluid passages. Light can pass through the window and enter the fluid passage of the flow cell. A reflective material, such as a mirror, can be provided in one wall of the fluid passage to allow light to be reflected back out of the flow cell device through the same window. In such embodiments, a detector can be positioned to detect reflected light.

[0048] In some embodiments, at least two windows are provided in one fluid passage to measure at least one property of an object over a defined path length. By way of example only, a first window can be provided in a first wall of the fluid passage, and a second window is provided in a second wall of the fluid passage. In absorption measurements, the path length is defined as the distance between the two walls of the fluid passage. Light enters through the first window and is then detected through the second window. In another example where a circular wall is provided, a window is provided in a first location on at least one wall of the fluid passage, and a second window is provided in a different location on the same wall of the fluid passage. The path length is defined by the distance between the first window and the second window. Light enters through the first window and is then detected through the second window.

[0049] An example of a property that may be measured through a window on the fluid path is absorption. From the absorption measurement, the concentration of the object can be determined. In this example, a detector such as a UV detector can detect light from the window for the absorption measurement. From the absorption measurement, the concentration of the object can be determined. If more than one measurement is taken, the concentration can be determined at various locations along the fluid path to check for any variations.

[0050] Other properties, such as ratios between objects within the fluid, may also be optically examined through any one or more windows, e.g., the specific absorption of protein to nucleic acid may be measured through a window. Additionally or alternatively, the fluorescent properties of the objects may also be determined by measurement through the window.

[0051] Additionally or alternatively, at least some region of the fluid passage may be transparent to radiation of a selected wavelength, which may be suitable for techniques such as absorption measurements. In use, light may enter the flow cell through the transparent region of the fluid passage. A reflective material may be provided within one wall of the fluid passage to reflect light out through the same transparent region of the fluid passage for detection (such as absorption measurements).

[0052] Further optical measurements can occur within the fluid passage upstream from the solid immersion lens. Additionally or alternatively, optical measurements can occur within the fluid passage downstream from the location of the solid immersion lens. This can be advantageous—for example, during use, the concentration of an object may be affected as the object flows through the flow cell device because the object may interact with the walls of the fluid passage and / or the passivated surface of the solid immersion lens. By performing absorption measurements both upstream and downstream from the solid immersion lens, the measurements can be used to check the concentration of the object within the fluid passage. This can help determine whether the concentration of the object has decreased due to interaction with the flow cell device (walls or lens).

[0053] In some embodiments, the multiple fluid passages comprise a flow cell device. In some embodiments, each of the multiple fluid passages may comprise at least one solid immersion lens. In some embodiments, providing multiple fluid passages may be advantageous because different conditions may be provided between each of the fluid passages for optical measurement of the object. In some embodiments, multiple solid immersion lenses may be provided within a single fluid passage.

[0054] In some embodiments, the solid immersion lens may be in contact with the fluid that comprises the object.

[0055] According to another aspect of the present invention, a flow cell according to the above aspect of the present invention; an illumination source for irradiating a surface of the solid immersion lens; a first detector for capturing light passing through the solid immersion lens; An apparatus is provided comprising:

[0056] The apparatus may be an interference scattering microscope. In some embodiments, the apparatus may include a first detector configured to detect light from an object through a solid immersion lens. In some embodiments, the apparatus may include a first detector configured to detect scattered light from an object on a surface of the solid immersion lens and / or scattered light from an object within the fluid sample.

[0057] In some embodiments, the device may further include a second detector configured to capture radiation transmitted through the window. In some embodiments, the device may further include one or more additional detectors. In some embodiments, the device may include multiple detectors. In some embodiments, by way of example only, the second detector may be used to detect fluorescence emitted from the object and through the window, or the second detector may be used to detect and measure absorption.

[0058] In some embodiments, a second detector may be provided at or near the window to capture radiation transmitted through the window. In some embodiments, the second detector may be a UV detector configured to measure the absorption of the object through the window, which may then be used to determine the concentration of the object in the flow cell.

[0059] In some embodiments, a second and / or additional detector may be provided at the upstream end of the flow cell. By way of example only, the second detector, which may be a UV detector, may be used to measure the absorption of objects in the fluid before the objects enter the flow cell. This is advantageous because measurements made by the UV detector may be used to optimize concentration measurements in the flow cell or to adjust or optimize certain parameters of other devices in the apparatus, such as mixing devices.

[0060] In some embodiments, the second detector may be configured to detect fluorescence of objects in the fluid passageway through the window.

[0061] The apparatus may further comprise a mixing device. In some embodiments, the mixing device can be any structural form capable of performing the function of mixing or diluting the objects. In its simplest form, the mixing device includes a sample inlet port configured to introduce a sample containing the objects into the mixing device, and a second inlet port configured to introduce a diluent or diluent fluid into the mixing device. Examples of diluents can be water or buffer, although those skilled in the art will recognize that other diluent fluids can be used. In some embodiments, the two inlet ports can be fluidly connected to a distribution channel or chamber where the two fluids enter and mix together.

[0062] Within the context of the present invention, a sample comprises an object. A sample can comprise an object in combination with another entity. A sample can be in a fluid flow.

[0063] In some embodiments, the apparatus may further comprise a liquid handling system. In some embodiments, the liquid handling system may select a designated volume of the object of interest for the flow cell device. In some embodiments, the liquid handling system may include one or more of the following: a pressure source, such as a syringe pump with a valve, a mixer, and / or an air displacement pipette. In some embodiments, the volume of the sample selected may be between 1 and 10 μL.

[0064] In some embodiments, the volume of the selected sample may be greater than 1, 2, 3, 4, 5, 6, 7, 8, or 9 μL. In some embodiments, the volume of the selected sample may be less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In some embodiments, the volume of the selected sample may be between 1 and 100 μL. In some embodiments, the volume of the selected sample may be greater than 1, 10, 20, 30, 40, 50, 60, 70, 80, or 90 μL. In some embodiments, the volume of the selected sample may be less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μL.

[0065] In some embodiments, in use, a liquid handling system may be provided upstream of the flow cell device. The liquid handling system may select a specified volume of sample, e.g., 1-10 μL. A pressure source, such as a syringe pump with a valve, may be activated to move the selected sample into the flow cell for measurement. In some embodiments, the liquid handling system may be temperature controlled.

[0066] The liquid handling system may be used to automate the flow of sample and wash solutions into the flow cell device. By automating the wash steps, the wash procedure can utilize aggressive solutions and / or more complex wash procedures without risk to the user. This allows an appropriate wash solution to be selected that ensures complete removal of objects from the solid immersion lens during the wash step while preventing damage to the surface of the solid immersion lens. The wash step can therefore remove even "permanent" interactions between the object and the lens.

[0067] According to another aspect of the present invention, there is provided a method for measuring a property of an object, the method comprising: Providing a fluid comprising an object in a flow cell device according to any of the aspects of the present invention; illuminating a surface of the solid immersion lens with an illumination source; detecting scattered, emitted, or reflected light from the object through a solid immersion lens; Using the detected light to measure properties of the object Includes:

[0068] In some embodiments, the illumination light may be spatially and temporally coherent. In some embodiments, the surface of the solid immersion lens may be illuminated by a laser light source. In some embodiments, a laser light source can achieve wide-field illumination within the microscope by focusing a collimated laser beam into the back focal plane of the imaging objective, suggesting that laser beams can be efficiently coupled into and out of the microscope with little impact on overall imaging performance.

[0069] Alternatively, the microscope may operate in a confocal mode, in which case the detector may be an image sensor or a point-like detector such as a photodiode, in which case a scanning arrangement may be used to scan an area of ​​the object to build up an image. Examples of image sensors that may be used as detectors include CMOS (complementary metal-oxide semiconductor) image sensors or CCDs (charge-coupled devices). Apparatuses are further disclosed in WO2018 / 011591, the contents of which are incorporated herein by reference.

[0070] A detector may be provided to detect scattered, emitted, or reflected light from the object through the solid immersion lens. In some embodiments, the detector may be part of a microscope setup. In particular, the detector may be part of an interference scattering microscope setup.

[0071] The interference microscope may further comprise at least one optical element configured to compensate for aberrations of the solid immersion lens. In some embodiments, solid immersion lenses with small diameters can exhibit significant deviations from optimal form. Solid immersion lenses with perfectly spherical surfaces will have significant aberrations, including chromatic aberrations and coma (off-axis). In some embodiments, optical elements that compensate for these aberrations can be integrated into the optical path. In some embodiments, the optical element can be a freeform element that matches the solid immersion lens or a flexible element such as a high-resolution spatial light modulator. In some embodiments, the optical element may include a ball lens and / or a corrected relay lens system. An optical system that can correct for deviations of small-diameter solid immersion lenses is advantageous because it allows high-resolution images to be captured.

[0072] In some embodiments, the optical element may include at least one aspheric lens. The aspheric lens is configured to manipulate or condition light onto a solid immersion lens in the flow cell device. The aspheric lens can be used to reduce or eliminate spherical aberration and also reduce other optical aberrations, thus improving image quality. Alternatively or additionally, light can be provided directly onto the solid immersion lens.

[0073] In some embodiments, the coherent scattering microscope may further comprise a spatial filter. In some embodiments, the iSCAT microscope may further comprise a spatial filter, and spatial filtering of the output light, including both light scattered from the sample location and illumination light reflected from the sample location, may be performed prior to detecting the output light. The spatial filtering passes the reflected illumination light, but with a greater reduction in intensity within a given numerical aperture than at larger numerical apertures. This increases imaging contrast for coherent illumination, especially for objects that are weak scatterers.

[0074] The spatial filter is a filter that measures the intensity within a given numerical aperture. -2 The spatial filter may be arranged to pass output light with a reduction in intensity to no more than 10 of the incident intensity within a given numerical aperture. -4 Incident intensity of more than, for example, 10 -2 From 10 -4 The aperture may be arranged to pass output light having a falloff in the range of incident intensity up to 10 ...

[0075] Spatial filters selectively reduce the intensity of illumination light over scattered light by exploiting the mismatch between the numerical aperture of the reflected illumination light and the numerical aperture of the light scattered from objects within the sample at the sample location. To do so, spatial filters exploit the different directionality of these two light sources. The reflected illumination light will typically have a relatively small numerical aperture, while sub-diffraction-sized objects near the sample's surface will preferentially scatter light to a high numerical aperture. Therefore, intensity reduction by a spatial filter at low numerical apertures primarily affects the illumination light and has a minor effect on the scattered light, thereby maximizing imaging contrast.

[0076] This effect may be maximized by positioning the spatial filter so that its predetermined numerical aperture is the same as or similar to the numerical aperture of the illumination light reflected from the sample location.

[0077] In some embodiments, measuring a property of the object may include quantifying the mass of the object. In some embodiments, measuring a property of the object may include measuring or quantifying a change in the mass of the object. In some embodiments, the methods of the present invention may be used to perform mass photometry, in which the mass of the object is quantified by coherent light scattering. In some embodiments, the mass may be quantified to a 5% mass error.

[0078] In some embodiments, the method may further include detecting scattered light from an object in the solution, the object having a diameter greater than 10 nm. In some embodiments, objects having a diameter greater than 10 nm can be detected while in solution within the fluid passage. In some embodiments, upon illuminating the solid immersion lens with an illumination source, measurement of the scattered light can be utilized to determine at least one property of the object in the solution. This can be advantageous because the object does not interact with the surface of the solid immersion lens, and therefore there is no requirement to remove and clean the object from the solid immersion lens after use. As such, the flow cell can be easily reusable for further optical measurements.

[0079] In some embodiments, the object may have a diameter greater than 20 nm. Objects having diameters greater than 10 nm, 15 nm, or 20 nm may be detected in the fluid flowing along the fluid passage. This means that there is no requirement for the object to interact with the surface of the solid immersion lens, and therefore no cleaning step would be necessary.

[0080] In some embodiments, an object may interact with the surface of the solid immersion lens. In some embodiments, the interaction between the object and the surface of the lens may involve the object adsorbing to the surface of the solid immersion lens. In some embodiments, the interaction between the object and the surface of the solid immersion lens may be an electrostatic bonding interaction and / or the bond may be formed by a salt bridge. In some embodiments, the interaction may involve a hydrophobic interaction between the object and the surface of the solid immersion lens. Furthermore, the interaction may be specific, i.e., the surface may be coated with a binding entity such as an antibody or an aptamer.

[0081] In some embodiments, the interaction of the object with the solid immersion lens occurs at an interface, i.e., the surface of the solid immersion lens that is in contact with the fluid. This interface receives light from a light source and allows for the detection of objects that interact with the solid immersion lens, thus providing the surface typically required in light scattering microscopy while functioning similarly as a lens.

[0082] The use of a solid immersion lens, which directly interacts with the object, allows both high sensitivity and a high refractive index (RI) at the measurement interface to be achieved. The higher numerical aperture of a solid immersion lens compared to conventional lenses facilitates the collection of a large number of scattering gratings, thus increasing the sensitivity of the method. Furthermore, the high refractive index of a solid immersion lens means that the method of the present invention is compatible with a wider range of solvents compared to conventional iSCAT microscopes. The high refractive index of a solid immersion lens allows sufficient refractive index difference to be achieved even for high refractive index solvents. For example, the method of the present invention can be used to measure organic polymers, which tend to be only soluble in solvents with an RI of 1.4 or higher.

[0083] Typically, the object interacts directly with the surface of the solid immersion lens. The object may be provided in a solution or in a fluid, for example as a suspension. In some cases, the charge on the surface of the solid immersion lens may be altered to allow interactions, such as hydroxyl (OH) or ionic interactions, to occur between the object and the solid immersion lens.

[0084] In some embodiments, the interaction between the object and the surface of the solid immersion lens can be indirect. Indirect interaction may occur when a silane coating is provided on the surface of the solid immersion lens. In this case, the object can only interact with the silane coating and not directly with the surface of the solid immersion lens.

[0085] In some embodiments, the method may further include detecting scattered light from the object on the surface of the solid immersion lens.

[0086] Therefore, scattered light may be detected from the interface between the solid immersion lens and the fluid.

[0087] In some embodiments, the method may further include coating the surface of the solid immersion lens. In some embodiments, the coating on the surface of the immersion lens may aid in the interaction between the object and the surface of the lens. For example, the object may be adsorbed onto the surface of the solid immersion lens. In this case, a subsequent cleaning step is required to remove the object from the surface of the solid immersion lens.

[0088] Alternatively, the coating of the solid immersion lens may prevent objects from interacting with the surface of the solid immersion lens. If the surface of the solid immersion lens is passivated, this prevents objects from interacting with the coating of the solid immersion lens. There is no requirement for a subsequent washing step. One or more objects may be measured in solution along the fluid path of the device.

[0089] In some embodiments, the method may further comprise the step of cleaning the solid immersion lens. In some embodiments, the method of the present invention also facilitates controlled cleaning of the solid immersion lens so that it can be reused in subsequent measurements.

[0090] In some embodiments, the method may further include cleaning the solid immersion lens such that the object is removed from the surface of the solid immersion lens. In some embodiments, the cleaning process is performed without user interaction, which allows the cleaning process to be optimized to ensure that the entire object is removed from the surface of the solid immersion lens without damage to the lens or risk to the user.

[0091] By allowing for effective cleaning of solid immersion lenses, the method can obviate the requirement for disposable solid immersion lenses, ensuring compatibility with high-throughput biochemical screening workflows.

[0092] In some embodiments, a coating may be provided to aid interaction between the object and the surface of the solid immersion lens. A cleaning step is then applied to remove the object and coating from the surface of the solid immersion lens. After the cleaning step, a different coating may be applied to passivate the surface of the solid immersion lens, so that the different coating prevents the object from interacting with the surface of the solid immersion lens.

[0093] In some embodiments, cleaning the solid immersion lens may include flowing at least one cleaning solution through the fluid passageway. In some embodiments, an appropriate cleaning solution can be selected that ensures complete removal of objects from the solid immersion lens while preventing damage to the surface of the solid immersion lens during the cleaning step. In some embodiments, cleaning the solid immersion lens may include flowing successive cleaning solutions through the fluid passageway. In some embodiments, the cleaning solutions may include, but are not limited to, ethanol, isopropanol or similar alcohols, and / or water.

[0094] In some embodiments, cleaning the solid immersion lens may include flowing at least one or more increasingly aggressive cleaning solutions through the fluid passageway, depending on the objects adsorbed to or interacting with the surface of the solid immersion lens. In some embodiments, cleaning the solid immersion lens may include flowing an acid, such as hydrochloric acid and / or sulfuric acid, followed by water through the fluid passageway. In some embodiments, flowing an oxidizing acid, such as sulfuric acid, over the solid immersion lens may also functionalize the surface. Additionally or alternatively, the step may include flowing a base, such as sodium hydroxide, to functionalize and / or clean the surface.

[0095] In some embodiments, the method may further include the step of agitating the cleaning solution. In some embodiments, the step of agitating the cleaning solution may include the use of a sonicator bath to facilitate removal of objects from the surface of the solid immersion lens. In some embodiments, the sonicator bath may be temperature controllable to facilitate agitation of the cleaning solution at a temperature best suited for removal of objects from the solid immersion lens.

[0096] In some embodiments, the method may further include flowing air over the solid immersion lens. In some embodiments, the solid immersion lens may be air-dried following the cleaning step. This prevents any cleaning solution on the solid immersion lens from contaminating the sample solution in a subsequent measurement.

[0097] In some embodiments, the method may further include applying a plasma to the surface of the solid immersion lens. In some embodiments, the plasma may be applied to the surface of the solid immersion lens by, for example, an ambient pressure plasma torch. In some embodiments, the plasma may be applied to the surface of the solid immersion lens as part of a cleaning process to remove objects adsorbed to the surface of the lens.

[0098] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0099] [Figure 1] Figure 1A shows a flow cell device according to the present invention; Figure 1B shows a solid immersion lens embedded in at least the wall of a fluid passage; Figure 1C shows a cross-sectional view of the flow cell device of Figure 1A with one solid immersion lens; and Figure 1D shows a cross-sectional view of the flow cell device of Figure 1A with multiple solid immersion lenses. [Figure 2] FIG. 10 shows a flow cell device further comprising a window. [Figure 3] FIG. 1 shows a flow cell device with multiple flow passages. [Figure 4] FIG. 1 shows a fluid mixing device. [Figure 5] FIG. 1 illustrates an apparatus for measuring properties of an object in a flow cell device. DETAILED DESCRIPTION OF THE INVENTION

[0100] In accordance with the present invention, there is provided a flow cell device, an apparatus and method for measuring properties of objects within a fluid within the flow cell device.

[0101] Referring to FIG. 1A, a flow cell device 10 is provided that includes a fluid passage 12 having an inlet 14 and an outlet 16. The fluid passage 12 of the flow cell device 10 has at least one wall 19 and at least one solid immersion lens 18 optically connected to the fluid passage 12. The at least one solid immersion lens 18 may be embedded in the wall 19 of the fluid passage 12, as shown in FIG. 1B. The flow cell device 10 may include one solid immersion lens 18, as shown in FIG. 1C. Alternatively, the flow cell device 10 may include two or more solid immersion lenses 18, as shown in FIG. 1D. By way of example only, FIG. 1B shows the wall 19 of the fluid passage 12 that includes three solid immersion lenses. The solid immersion lens 18 may be a diamond solid immersion lens, or may be made from fused silica or any glass, including high refractive index glass variants, zirconia, sapphire, or lithium niobate. The solid immersion lens 18 may be hemispherical, super hemispherical, or a glass half-ball lens. The solid immersion lens 18 may have a diameter of 1 to 5 mm and a length of 6 to 10 mm.

[0102] Flow cell device 10 can be used to measure properties of objects within a fluid in fluid passage 12. Fluid containing objects is introduced into flow cell device 10 via inlet 14 and flows along fluid passage 12 in the direction of arrow 22 to outlet 16. One or more solid immersion lenses 18 are illuminated, and objects within fluid passage 12 scatter light that can then be detected. The detected scattered light from the objects can be used to measure at least one property of the objects. The detected scattered light can be used, for example, to measure the mass of the objects.

[0103] The solid immersion lens 18 may be provided with a coating to enable specific or enhanced binding of objects to the solid immersion lens 18 within the fluid passage 12. The solid immersion lens 18 may be coated with an antibody configured to bind to a target biomolecule. The coating may enable temporary binding at the surface of the solid immersion lens 18. For example, the coating may introduce hydroxyl (OH) interactions with proteins that can be quantitatively disrupted by pH changes. The coating may passivate the surface of the solid immersion lens 18 so that it is chemically inert. Passivating the surface of the solid immersion lens 18 can prevent objects from reacting with and / or binding to the surface of the solid immersion lens 18. The coating may functionalize the surface of the solid immersion lens 18. The coating may allow specific objects to interact with or bind to the solid immersion lens 18. For example, the coating can be (3-aminopropyl)triethoxysilane (APTES), which imparts a positive charge to the surface of the solid immersion lens 18 so that negatively charged biomolecules such as DNA can bind to the solid immersion lens 18 .

[0104] Referring to FIG. 2, at least a portion of the fluid path 12 may be transparent to radiation of a selected wavelength. The fluid path 12 may include one or more windows 24 configured to enable optical measurements. By way of example only, the flow cell device 20 shown in FIG. 2 includes three windows 24. The windows 24 facilitate further measurements from objects within the fluid path 12. The windows 24 can be used to detect radiation from the objects. For example, fluorescence emitted from the objects may be detected through one or more windows 24. When at least two windows 24 are provided along a single fluid path 12, the properties of the objects can be measured over a defined path length, which can be useful for making absorption measurements and determining the concentration of the objects. This can help determine the presence of aggregation, such as protein aggregation. The one or more windows 24 also allow ratios between objects within the fluid, such as the ratio of specific absorption between proteins and nucleic acids, to be measured.

[0105] The object may be a biomolecule, a protein, a peptide, a polypeptide, a lipoprotein, a glycoprotein, a lipid, a carbohydrate, an organic polymer, a protein complex, an antibody or antibody fragment thereof, or an enzyme. Alternatively, the object may be a nucleic acid molecule such as DNA, RNA, or a polysaccharide. Alternatively, the object may be a virus, a viral vector such as an adenovirus and / or a lentivirus, a virus-like particle, or a small molecule, an exosome, a vesicle, a complex assembly, a nanoparticle, a chemical compound, an ion, or a quantum dot. The object may be a single molecule, a macromolecule, or an association of molecules and macromolecules (e.g., a polymer). Examples of suitable macromolecules include, but are not limited to, nucleic acid molecules, natural nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or artificial nucleic acids such as peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Molecular associations may include assemblies such as virus-like particles in which envelope or capsid proteins are associated.

[0106] The object may be a multimolecular complex comprising aggregates or other higher order aggregates of components including proteins such as monomeric, dimeric, and trimeric species.

[0107] 3, a flow cell device 30 includes multiple fluid paths 12a, 12b, and 12c. Each of the fluid paths 12a, 12b, and 12c includes an inlet 14a, 14b, and 14c and an outlet 16a, 16b, and 16c. Each of the fluid paths 12a, 12b, and 12c may include a solid immersion lens 18 optically connected to the fluid path 12a, 12b, and 12c. Each of the fluid paths 12a, 12b, and 12c may include different conditions or parameters for performing optical measurements.

[0108] Referring to FIG. 4, a mixing device 40 for mixing fluids prior to their input into the flow cell device 10, 20, or 30 via the inlet port 14 is shown. While FIG. 4 shows the mixing device in its simplest form, the mixing device 40 may take any structural form. The mixing device 40 includes a sample inlet port 42 configured to introduce a sample containing object into the mixing device 40, and a second inlet port 44 configured to introduce a diluent or diluent fluid into the mixing device 40. Examples of diluents can be water or buffer, although those skilled in the art will recognize that other diluent fluids can be used. The two inlet ports 42, 44 are fluidly connected to a distribution channel or chamber 46 where the two fluids enter and mix together.

[0109] 5, mixing device 40 and flow cell device 10 form part of an apparatus 50 for measuring a property of an object. Apparatus 50 may be used to quantify the mass of an object and / or measure or quantify a change in mass of an object. While flow cell device 10 is shown in FIG. 5, it should be understood that any of flow cell device embodiments 10, 20, or 30 described herein can form part of apparatus 50.

[0110] The apparatus 50 may be an interference scattering microscope 60 as shown in FIG. 5. The apparatus 50 includes an illumination source 62 positioned to provide illumination light to the surface of the solid immersion lens 18 of the flow cell device 10. The illumination light may be spatially and temporally coherent. The illumination source 62 may be a laser light source. The wavelength of the illumination light may be selected depending on the nature of the sample and / or the property being examined. In one example, the illumination light has a wavelength of 405 nm.

[0111] Optionally, the illumination light may be spatially modulated to eliminate speckle patterns arising from the coherent nature of the illumination and laser noise, as detailed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935.

[0112] The apparatus 50 includes a detector 64 configured to receive output light scattered, emitted, or reflected from objects within the fluid sample in the flow cell 10 through the solid immersion lens 18. Alternatively or additionally, the detector 64 may detect light scattered by objects within the flow cell device 10 on the surface of the solid immersion lens 18.

[0113] Typically, microscope 60 may operate in a wide-field mode, in which case detector 64 may be an image sensor that captures an image of the object. Microscope 60 may alternatively operate in a confocal mode, in which case detector 64 may be an image sensor or a point-like detector such as a photodiode, in which case a scanning arrangement may be used to scan an area of ​​the object to construct an image. Examples of image sensors that may be used as detector 64 include a CMOS (complementary metal-oxide semiconductor) image sensor or a CCD (charge-coupled device).

[0114] The microscope 60 further comprises an optical system 66 disposed between the flow cell device 10, the illumination source 62, and the detector 64. The optical system 62 is arranged as follows: to direct illumination light onto the solid immersion lens 18 to illuminate the object; to collect output light from the object; and to direct the output light to the detector 64.

[0115] The optical system 66 includes an objective lens 68, a condenser lens 72, and a tube lens 74. The condenser lens 72 focuses illumination light from the light source 62 (shown by a continuous line in FIG. 5 ) through the objective lens 68 onto the solid immersion lens 18 of the flow cell device 10. The objective lens 68 collects output light that includes both (a) illumination light reflected from the object location (shown by a continuous line in FIG. 5 ) and (b) light scattered from the object (shown by a dotted line in FIG. 5 ). The reflected light is primarily reflected from the interface between the solid immersion lens 18 and the object. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be on the order of 0.5% of the intensity of the incident illumination light.

[0116] In a manner similar to conventional iSCAT, scattered light from objects at or near the surface of the sample constructively interferes with reflected light and is therefore visible in the image captured by detector 64. This effect differs from microscopes operating in transmission, in which the illumination light reaching the detector is transmitted through the depth of the sample, resulting in much smaller imaging contrast.

[0117] As shown in Figure 5, the reflected illumination and scattered light have different directionality. In particular, the reflected illumination has a numerical aperture resulting from the geometry of the light beam output by light source 62 and optical system 66. Because the scattered light is scattered over a larger range of angles, it satisfies a larger numerical aperture than the reflected illumination. Tube lens 74 focuses the output light from objective lens 68 onto detector 64.

[0118] The optical system 74 also includes a beam splitter 76 positioned to split the optical paths for the illumination light from the light source 62 and the output light directed to the detector 64. Except for the provision of a spatial filter as described below, the beam splitter 74 may have any conventional structure that provides partial reflection and partial transmission of light incident on the beam splitter 74. For example, the beam splitter 74 may be a plate, typically comprising a film, which may be metallic or dielectric, positioned at 45° to the optical path.

[0119] Alternatively, beam splitter 74 may be a cube beam splitter formed by a matched pair of prisms with a partially reflective form at the interface between the prisms. Alternatively, beam splitter 74 may be a polarizing beam splitter used in combination with a quarter wave plate between beam splitter 74 and flow cell device 10.

[0120] In the example shown in Figure 5, the light source 62 is offset from the optical path of the objective lens 68, so that the illumination light from the light source 62 is reflected into the objective lens 68 by the beam splitter 74; conversely, the detector 64 is aligned with the optical path of the objective lens 68, so that the output light from the flow cell device 10 is transmitted through the beam splitter 74 to the detector 64.

[0121] In addition to the components described above, which may be of conventional construction, microscope 60 includes a spatial filter 78. In the example shown in FIG. 5, spatial filter 78 is formed on beam splitter 74, thereby positioning it behind the back aperture of objective lens 68 and thus immediately behind the back focal plane 82 of objective lens 68. As such, spatial filter 78 may be implemented without entering objective lens 68, as in phase-contrast microscopy. Positioning the spatial filter immediately behind the objective's entrance aperture, rather than at a conjugate plane (e.g., as described below), has the obvious advantage of strongly suppressing back reflections arising from the multiple lenses in high-numerical-aperture microscope objectives. This, in turn, reduces imaging noise, lowers incoherent background, reduces experimental complexity, the number of optical components, and optical path length, leading to improved stability of the optical setup and, therefore, image quality.

[0122] However, this location is not essential and a spatial filter having equivalent functionality may be provided elsewhere, as described below. Spatial filter 78 is positioned to filter the output light passing therethrough to detector 64. In the example shown in Figure 5, where detector 64 is aligned in the optical path of objective lens 68, spatial filter 78 is therefore transmissive.

[0123] The spatial filter 78 is partially transmissive and therefore passes the output light, including the reflected illumination light, but with a reduced intensity. The spatial filter 78 is similarly aligned with the optical axis and has a predetermined aperture, thereby providing an intensity reduction within a predetermined numerical aperture. Numerical aperture is defined herein in its conventional manner as a dimensionless quantity that characterizes the range of angles relative to the sample location from which the output light originates. In particular, the numerical aperture NA may be defined by the formula NA = n·sin(θ), where θ is the collection half angle and n is the refractive index of the material (e.g., the material of a component of the optical system 66) through which the output light passes.

[0124] A fluid comprising an object is placed within the flow cell device 10, the surface of the solid immersion lens 18 is illuminated by an illumination source 62, and light scattered, emitted, or reflected from the object through the solid immersion lens 18 is detected by a detector 64, which uses the detected light to measure a property of the object.

[0125] The object may interact with the surface of the solid immersion lens 18 by one or more of adsorption of the object onto the surface of the solid immersion lens 18, electrostatic bonding interactions, bonding via salt bridges, and hydrophobic interactions between the object and the surface of the solid immersion lens 18.

[0126] The interaction between the solid immersion lens 18 and the object may be direct or indirect. A direct interaction may occur when the charge on the surface of the solid immersion lens 18 is altered to allow a hydroxyl (OH) or ionic interaction to occur between the object and the solid immersion lens 18.

[0127] Indirect interaction between an object and the surface of the solid immersion lens 18 may occur when a silane coating is provided on the surface of the solid immersion lens and the object interacts with the silane coating rather than directly with the surface of the solid immersion lens 18.

[0128] Indirect interactions may also occur between the object and the surface of the solid immersion lens in some situations where a coating functionalizes the surface of the solid immersion lens.

[0129] The detector 64 may detect light scattered from objects on the surface of the solid immersion lens 18. The surface of the solid immersion lens 18 may be cleaned after use so that objects can be removed from the solid immersion lens 18 and the flow cell device 10 can be reused in a subsequent measurement. A cleaning fluid may be flowed through the fluid passages 12 of the flow cell device 10, requiring minimal user interaction to clean the flow cell device 10 for reuse. The cleaning fluid may be agitated, for example, by a sonicator bath, to facilitate removal of objects from the surface of the solid immersion lens 18. Air may then be flowed over the solid immersion lens 18 to dry it and prevent any cleaning solution on the solid immersion lens 18 from contaminating subsequent measurements. Alternatively or additionally, the surface of the solid immersion lens 18 may be exposed to plasma, for example, by an ambient pressure plasma torch.

[0130] If the object has a diameter greater than 10 nm, the apparatus 50 may be used to measure properties of an object in solution in the flow passage 12 of the flow cell device 10 without the object interacting with the surface of the solid immersion lens 18. The detector 64 may be configured so that it can be used to detect scattered light from the object while it is in solution in the fluid passage 12. The apparatus can be used to detect scattered light from the object in solution by the detector 64 and determine at least one property of the object in solution. Because the object does not interact with the surface of the solid immersion lens 18, there is no requirement to remove and clean the object from the solid immersion lens 18 after use.

[0131] In some embodiments, the objects may have diameters greater than 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 nm. The objects may be in the range of 10-1000 nm, 10-900 nm, 10-800 nm, 10-700 nm, 10-600 nm, 10-500 nm, 10-400 nm, 10-300 nm, 10-200 nm, 10-100 nm, or 10-50 nm.

[0132] 2 and 3, which include one or more windows 24, the apparatus 50 may further include an additional detector (not shown in the accompanying drawings). The additional detector may be configured to capture radiation transmitted through the one or more windows 24. The one or more additional detectors may be used to detect fluorescence emitted from the object and through the window 24, or the one or more additional detectors may be used to detect and measure absorption. One or more of the additional detectors may be a UV detector.

[0133] Alternatively or additionally, one or more additional detectors may be provided at the upstream end of the flow cell. If one or more of the upstream additional detectors is a UV detector, it may be used to measure the absorption of objects in the fluid before the objects enter the flow cell device 10. These measurements may be used to adjust or optimize certain parameters of other devices in the apparatus, such as the mixing device 40, to optimize concentration measurements in the flow cell 10. Additional detectors may also be provided downstream of the flow cell for additional measurements.

[0134] The apparatus 50 may include a liquid handling system 52 configured to select a specified volume of the sample of interest or wash solution for the flow cell device 10. As shown in FIG. 5, the liquid handling device 52 may be provided upstream of the flow cell device 10. The liquid handling system 52 may include one or more of the following: a pressure source, such as a syringe pump with a valve, a mixer, and / or an air displacement pipette. The pressure source, such as a syringe pump with a valve, may be activated to move the selected sample into the flow cell device 10 for measurement. The liquid handling system 52 may select between 1 and 10 μL of the sample. Alternatively or additionally, the liquid handling system 52 may select between 1 and 100 μL of the sample. [Example]

[0135] (material and method) (optical) The operating wavelength range can be between 515 and 535 nm. Chromatic aberration may limit the wavelength range for specified performance without refocusing to (0.2 / 5) nm. Good imaging performance can be defined as a field (15 μm / 30 μm) diameter centered on the optical axis, NA (1.8 / 2.1) for diamond, or NA (1.6 / 1.8) for high-index glass or cubic zirconia, diffraction-limited imaging across the field, and a Strehl ratio > (0.9 / 0.95).

[0136] (Other optical properties) To achieve the above NA in a solid immersion lens, the NA of the aspheric lens can be approximately 0.35. A 4mm diameter can be provided to fit the tip. An anti-reflection coating can be applied to the aspheric lens and / or relay lens, R<1%, to reduce stray light and avoid reflections sent back along the imaging path. The effective focal length of the solid immersion lens aspheric system is 0.5-5mm to determine the magnification ratio. The NA of the solid immersion lens 2Note the magnification: low stress birefringence as polarization can be important.

[0137] The laser damage threshold can be provided high enough to allow a 2W beam to occupy any 10% of the area of ​​the aspheric lens clear aperture. Additionally or alternatively, the solid immersion lens can be made with a flat surface (2<1) and an Asphericon surface roughness of 15nm RMS.

[0138] (mechanical) The system can be constructed with a solid immersion lens with a maximum diameter of 5 mm for a maximum length of 10 mm. The solid immersion lens and aspheric lens must be securely mounted in place to achieve the above optical performance. Thermal expansion of the components should not interfere with focusing over temperatures ranging from 20 to 22 degrees Celsius and 15 to 40 degrees Celsius.

[0139] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0140] As used herein, "and / or" is contemplated as a specific disclosure of each of the two specified features or components with or without the other feature or component. For example, "A and / or B" is contemplated as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0141] Unless the context dictates otherwise, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0142] While the present invention has been described by way of example with reference to several embodiments, it will be further recognized by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments may be devised without departing from the scope of the invention as defined in the appended claims.

Claims

1. A flow cell device comprising a fluid passage having at least one wall and at least one solid immersion lens optically connected to the fluid passage, the solid immersion lens being made of a material having a refractive index substantially equal to or greater than that of glass.

2. The flow cell device of claim 1 , wherein the solid immersion lens is embedded in the wall of the fluid passage.

3. 3. The flow cell device of claim 1 or 2, wherein the solid immersion lens is made from one of diamond, zirconia, sapphire, lithium niobate, or potassium niobate.

4. 4. The flow cell device of claim 1, wherein the solid immersion lens is made from a high refractive index glass.

5. 5. A flow cell device according to claim 1, wherein the solid immersion lens is made of a material having a refractive index greater than η=1.

5.

6. 6. A flow cell device according to claim 1, wherein the solid immersion lens is a diamond solid immersion lens.

7. 7. A flow cell device according to any one of claims 1 to 6, wherein the surface of the solid immersion lens has a surface roughness of less than 1 nm RMS.

8. 8. A flow cell device according to claim 1, wherein the surface of the solid immersion lens is provided with a coating.

9. 9. The flow cell device of claim 8, wherein the coating passivates the surface of the solid immersion lens.

10. The flow cell device of claim 8 , wherein the coating functionalizes the surface of the solid immersion lens.

11. 11. The flow cell device of claim 1, wherein the fluid passage comprises one or more windows configured to allow optical measurements.

12. 12. A flow cell device according to any one of claims 1 to 11, wherein the solid immersion lens is in contact with a fluid comprising the object.

13. A flow cell according to any one of claims 1 to 12; an illumination source for illuminating a surface of said solid immersion lens; a first detector for capturing light passing through said solid immersion lens; An apparatus comprising:

14. The apparatus of claim 13 , wherein the apparatus is a coherent scattering microscope.

15. 15. The apparatus of claim 13 or 14, further comprising a second detector configured to capture radiation transmitted through the window.

16. 16. The apparatus of any one of claims 13 to 15, further comprising a mixing device.

17. 17. Apparatus according to any one of claims 13 to 16, further comprising a liquid handling system.

18. 1. A method for measuring a property of an object, comprising: Providing a fluid comprising an object inside a flow cell device according to any one of claims 1 to 12; illuminating the surface of said solid immersion lens with an illumination source; detecting scattered, emitted, or reflected light from the object through the solid immersion lens; using the detected light to measure a property of the object; and A method comprising:

19. 20. The method of claim 18, further comprising detecting the scattered light from the object within the solution, the object having a diameter greater than 10 nm.

20. The method of claim 18 , wherein the object interacts with the surface of the solid immersion lens.

21. 21. The method of claim 20, further comprising detecting the scattered light from the object at the surface of the solid immersion lens.

22. 22. The method of claim 20 or 21, further comprising the step of coating the surface of the solid immersion lens.

23. 23. The method of any one of claims 20 to 22, further comprising the step of cleaning the solid immersion lens.

24. 24. The method of claim 23, wherein cleaning the solid immersion lens comprises flowing at least one cleaning solution through the fluid passageway.

25. 25. The method of claim 23 or 24, further comprising agitating the cleaning solution.

26. 26. A method according to any one of claims 20 to 25, further comprising the step of flowing air over the solid immersion lens.

27. 27. A method according to any one of claims 20 to 26, further comprising the step of applying a plasma to the surface of the solid immersion lens.

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