Interference Scattering Microscopy

Abstract

1. A method for measuring a property of an object, the method comprising: immersing a solid immersion lens in a solution containing the object, whereby the object interacts with a surface of the solid immersion lens; illuminating the surface of the solid immersion lens with an illumination source and detecting scattered light from the object using an interference scattering microscope; measuring a property of the object using the detected scattered light; removing the solid immersion lens from the solution; and thereafter washing the solid immersion lens, whereby the object is removed from the surface of the solid immersion lens.

Description

[Technical field]

[0001] The present invention relates to interferometric scattering microscopy (herein referred to as iSCAT), and in particular to methods for measuring properties of objects using iSCAT. [Background technology]

[0002] iSCAT has materialized as a powerful approach for both single particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single molecule 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 ultra-sensitive 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, non-conventional cameras, and complex sample illumination, limiting iSCAT's ability for robust and accurate detection, imaging, and characterization of objects as small as single molecules.

[0003] One application of iSCAT is Mass Photometry (MP), where coherent light scattering is used to quantify the mass of an object. Typically, MP measurements are performed at a measurement interface in an inverted microscope type geometry. This is achieved by placing a small amount of analyte on the measurement interface. Challenges arise when combining MP with a biochemical screening workflow, where a large number of samples are prepared by a robotic process in a multi-well plate and then read out in a plate reader type instrument. Automation of the MP measurement process can be achieved by replicating each well on a sample carrier by a robotic pipetting step, which can then be interfaced with the iSCAT microscope. However, this approach has limited throughput and introduces additional complexity to 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 in a multi-well plate. Summary of the Invention [Problem to be solved by the invention]

[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 can achieve cost-effectiveness and simplicity to enable 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] It is against this background that the present invention arose. [Means for solving the problem]

[0006] According to one aspect of the present invention there is provided a method for measuring a property of an object, the method comprising: immersing a solid immersion lens in a sample solution containing the object, whereby the object interacts with a surface of the solid immersion lens; illuminating a surface of the solid immersion lens with an illumination source and detecting light scattered from the object using an interference scattering microscope; measuring a property of the object using the detected scattered light; and Removing the solid immersion lens from the solution; and then cleaning the solid immersion lens, whereby the object is removed from the surface of the solid immersion lens. Includes.

[0007] The interaction between the object and the surface of the lens can be temporary, or the interaction can be a permanent interaction between the object and the surface of the lens. In some embodiments, the interaction can involve the object adsorbing to the surface of the solid immersion lens. In other embodiments, the interaction between the object and the surface of the solid immersion lens can be an electrostatic bonding interaction and / or the bond can be formed by a salt bridge. In some embodiments, the interaction can involve a hydrophobic interaction between the object and the surface of the solid immersion lens.

[0008] According to another aspect of the present invention there is provided a method for measuring a property of an object, the method comprising: immersing a solid immersion lens in a sample solution containing the object, whereby the object is adsorbed onto a surface of the solid immersion lens; illuminating a surface of the solid immersion lens with an illumination source and detecting light scattered from the object using an interference scattering microscope; measuring a property of the object using the detected scattered light; and Removing the solid immersion lens from the solution; and then cleaning the solid immersion lens, whereby the object is removed from the surface of the solid immersion lens. Includes.

[0009] The method of the present invention is an automated workflow that utilizes a solid immersion lens and an iSCAT microscope arranged in a configuration such that the solid immersion lens can be immersed in a solution. To facilitate immersion of the solid immersion lens, microscopes such as the iSCAT microscope are arranged in a vertical configuration, under which the sample solution and the wash solution can be moved. The iSCAT microscope of the present invention is configured such that the solid immersion lens is located above the sample and can be immersed in the solution from above. Compared to a conventional iSCAT microscope setup where the sample is located above the objective lens, the configuration of the present invention is inverted. Furthermore, the microscope does not require the sample to be placed on a surface for detection of the object, since the immersion lens effectively replaces the surface.

[0010] Previously, widespread use of iSCAT microscopes in biochemical workflows was hindered by the need to apply and / or grow samples directly on the optical surfaces of the measurement interface. The method of the invention allows a multi-well plate containing samples and / or washing solutions to be positioned under the iSCAT microscope so that a solid immersion lens can be directly immersed in the solutions. Thus, the method of the invention allows measurements to be performed directly in the wells of the multi-well plate, achieving compatibility with standard biochemical workflows. This allows, for example, high-throughput screening of biological samples.

[0011] In some embodiments, once the samples are added to the well plate, the plate may be maintained with temperature control and / or agitation, hi some embodiments, the agitation is stopped for the duration of the measurement.

[0012] In some embodiments, the multi-well plate may be moved upward until the solid immersion lens is immersed in the solution. The fixed focus of the solid immersion lens allows the measurement to begin immediately, allowing for measurements at true equilibrium. In some embodiments, after the duration of the measurement, the solid immersion lens is removed from the solution as the multi-well plate is lowered. In some embodiments, the duration of the measurement may be between 10 seconds and 5 minutes. In some embodiments, the duration of the measurement may be greater than 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, or 4 minutes. In some embodiments, the duration of the measurement may be less than 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, or 30 seconds.

[0013] The use of a solid immersion lens in the method of the invention 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 a conventional lens facilitates the collection of a larger number of scattered photons, thus increasing the sensitivity of the method. Furthermore, the high refractive index of a solid immersion lens means that the method of the invention is compatible with a wider range of solvents compared to conventional iSCAT microscopes. The high refractive index of a solid immersion lens allows a sufficient refractive index difference to be achieved even for high refractive index solvents. For example, the method of the invention can be used to measure organic polymers that tend to only be soluble in solvents with an RI of 1.4 or higher.

[0014] The method of the present invention also facilitates controlled cleaning of the solid immersion lens so that the solid immersion lens may be reused in subsequent measurements. The cleaning process is performed without user interaction, which allows the cleaning process to be optimized to ensure that the entirety of the object is removed from the surface of the solid immersion lens without damage to the lens or risk to the user. Enabling effective cleaning of the solid immersion lens obviates the requirement for disposable solid immersion lenses, ensuring that the method of the present invention is compatible with high throughput biochemical screening workflows.

[0015] In some embodiments, measuring a property of an object can include quantifying the mass of the object. In some embodiments, the methods of the present invention can be used to perform mass photometry, in which the mass of an object is quantified by coherent light scattering. In some embodiments, the mass can be quantified to within 5% mass error.

[0016] In some embodiments, measuring a property of the object may include measuring or quantifying a change in mass of the object. In some embodiments, cleaning the solid immersion lens can include immersing the solid immersion lens in at least one cleaning solution. Cleaning the solid immersion lens so that the object and any sample solution are removed from the surface can be accomplished in a variety of ways. The cleaning step is important to allow the same solid immersion lens to be used in subsequent measurements, avoiding the need for disposable lenses.

[0017] In some embodiments, the solid immersion lens may be repeatedly immersed in the same or different cleaning fluids to ensure that all of the objects have been removed from the lens surface. The immersions may or may not involve agitation of the cleaning fluid.

[0018] In some embodiments, the solid immersion lens may be cleaned by successive immersions in ethanol, isopropanol, or a similar alcohol, followed by immersion in water. In some embodiments, more reactive cleaning may be required depending on the objects that are adsorbed to or interact with the surface of the solid immersion lens. In some embodiments, the solid immersion lens may be immersed in an acid, such as hydrochloric acid and / or sulfuric acid, followed by immersion in water. In some embodiments, immersing the solid immersion lens in an oxidizing acid, such as sulfuric acid, may also functionalize the surface.

[0019] Because the method of the present invention is an automated workflow, the cleaning procedure can utilize highly reactive solutions and / or more complex cleaning procedures without risk to the user. This allows appropriate cleaning solutions to be selected that ensure complete removal of objects from the solid immersion lens during the cleaning step while preventing damage to the surface of the solid immersion lens. Thus, the cleaning step can remove even "permanent" interactions between the object and the lens.

[0020] In some embodiments, a washing solution can be held in the washing well. In some embodiments, the washing well can create a seal with the solid immersion lens. In some embodiments, the washing well can spray the washing solution onto the solid immersion lens.

[0021] 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.

[0022] In some embodiments, the method may further include the step of agitating the cleaning solution. In some embodiments, the method may include immersing the solid immersion lens in a cleaning solution and agitating the cleaning solution using a sonicator bath, thereby facilitating removal of objects from the surface of the solid immersion lens.

[0023] In some embodiments, the method may further include the step of 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. In some embodiments, the plasma may be used to functionalize the surface of the solid immersion lens.

[0024] In some embodiments, the solid immersion lens has a diameter of 1-5 mm. In some embodiments, the diameter of the solid immersion lens can be greater than 1, 1.5, 2, or 2.5 mm. In some embodiments, the diameter of the solid immersion lens can be less than 3, 2.5, 2, or 1.5 mm.

[0025] In some embodiments, the solid immersion lens may be attached to a lens assembly. In some embodiments, the solid immersion lens may be attached to a lens assembly such that the lens assembly is solvent resistant. This can avoid solution leaking into the lens assembly during immersion of the solid immersion lens in a solution. Design considerations for solvent resistance can include, but are not limited to, ensuring that the lens assembly overlaps the solid immersion lens, gluing or fixing the solid immersion lens into the assembly.

[0026] In some embodiments, the lens assembly has a diameter between 2 and 6 mm. In some embodiments, the lens assembly diameter can be greater than 2, 3, 4, or 5 mm. In some embodiments, the lens assembly diameter can be less than 6, 5, 4, or 3 mm.

[0027] In some embodiments, the lens assembly can include a focusing lens, hi some embodiments, a 1 mm diameter solid immersion lens can require, for example, a 4 mm diameter focusing lens.

[0028] In some embodiments, the solid immersion lens may have a length of 6-10 mm. In some embodiments, the length of the solid immersion lens may be greater than 6, 7, 8, or 9 mm. In some embodiments, the length of the solid immersion lens may be less than 10, 9, 8, or 7 mm. The design of the solid immersion lens is important for the suitability of the method of the present invention for use in biochemical screening workflows that hold samples in multi-well plates. For example, a 96-well plate has a well diameter of 6.94 mm and a maximum well depth of about 11 mm, depending on the bottom type. Thus, in some embodiments, it may be desirable for the solid immersion lens to have a small diameter cylindrical geometry. In some embodiments, the solid immersion lens may be less than 6 mm in length. However, a shorter lens may require a larger volume of sample to fill the well, which may not be practical.

[0029] In some embodiments, the solid immersion lens can be hemispherical or hyperhemispherical. In some embodiments, the solid immersion lens can be made of a diffractive optical element. In some embodiments, a hyperhemispherical solid immersion lens can be preferred. A hyperhemispherical 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 increases the collection of scattered photons and can result in improved sensitivity of the measurement.

[0030] In some embodiments, the interference microscope may further comprise at least one optical element configured to compensate for the aberrations of the solid immersion lens. In some embodiments, a solid immersion lens with a small diameter may exhibit significant deviations from the optimal form. A solid immersion lens with a perfectly spherical surface will have significant aberrations, chromatic aberrations and coma aberrations (off-axis). In some embodiments, an optical element that compensates for these aberrations may be integrated into the optical path. In some embodiments, the optical element may 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 modified relay lens system. An optical system that can compensate for the deviations of a small diameter solid immersion lens is advantageous because it allows high-resolution images to be captured by a solid immersion lens that can be easily immersed in the solution contained in the well plate.

[0031] In some embodiments, the solid immersion lens material can be diamond, or can be zirconia, or can be sapphire, or can be lithium niobate. In some embodiments, a high refractive index material such as diamond (η≈2.4) can be used. In some embodiments, a diamond super-hemispherical solid immersion lens material can be accompanied by an objective lens with a numerical aperture of 0.3. In some embodiments, it may be necessary to use a diamond solid immersion lens with a freeform optical element in the relay lens system. In some embodiments, this can be utilized to overcome irregularities in the shape of the solid immersion lens as a result of manufacturing issues.

[0032] In some embodiments, the solid immersion lens material can be glass. In some embodiments, a high index glass solid immersion lens can be used with an objective lens having a numerical aperture of 0.5. In some embodiments, a hemispherical solid immersion lens can be combined with a focusing objective lens having a high numerical aperture of 0.8+.

[0033] In some embodiments, the method may further include the step of functionalizing the surface of the solid immersion lens. In some embodiments, the surface of the solid immersion lens may be functionalized prior to immersing the solid immersion lens in the sample solution. Functionalization of the surface of the solid immersion lens may be achieved by incubating the solid immersion lens in an appropriate solution. In some embodiments, it may be desirable to functionalize the surface of the solid immersion lens to achieve analyte specificity.

[0034] The interaction between the lens and the object can be due to any type of interaction ranging from hydrophobic to covalent bond formation. The interaction between the lens and the object can be due to ionic / electrostatic interactions. These interactions are due to the physical properties of the object.

[0035] The surface of the lens may be functionalized. The surface may be passivated, activated, coated, treated, or derivatized. The surface may be a passivated surface. Passivation is the process of treating or coating a surface to increase or decrease chemical reactions and thus increase or decrease the number of interaction events.

[0036] The surface of the lens may be activated, coated, treated, and / or derivatized to modify the surface chemistry. Surface functionalization may be to allow for various interactions, such as chemical interactions (e.g., covalent bonding) or physical interactions (e.g., adsorption). If the surface is coated, derivatized, or modified, a thin surface modification may be desired. Too thick a modified surface layer may alter the light scattering properties of the surface. Modification of only the outermost few molecular layers (3-10 nm) may be desired.

[0037] Any suitable surface coating may be applied to the lens to modify the surface chemistry. The surface may be modified to make it more hydrophobic or more hydrophilic. In some embodiments, an ideal solid immersion lens material can exhibit strong but non-specific interactions with biomolecules. In some embodiments, the surface of the solid immersion lens can be conditioned by carboxylation and / or oxidation of the surface. In some embodiments, carboxylation and / or oxidation of diamond solid immersion lenses can improve the adsorption of analytes such as proteins. In some embodiments, the surface of the solid immersion lens can be carboxylated and / or oxidized by immersion in strong acids such as sulfuric acid, hydrochloric acid, and / or nitric acid. The solid immersion lens can also be immersed in a sodium hydroxide solution. In some embodiments, the surface of the solid immersion lens can be oxidized by treating the surface with plasma. In some embodiments where repeated measurements are performed on the same solid immersion lens, functionalization can be performed after a washing step and prior to immersion of the solid immersion lens in the sample solution. This ensures that the functionalization and critical surface properties of the solid immersion lens are maintained.

[0038] The sample may be any suitable sample, but preferably contains objects that are biomolecules. The sample is preferably a liquid, such as a solution or suspension. The solution or suspension may comprise any suitable solvent, in particular water. The sample may comprise an organic solvent. In particular the solvent, solution or suspension may have a higher refractive index than water. An advantage of the present invention is that the use of lens materials such as diamond allows the use of organic solvents and the like.

[0039] The sample may be a biological sample, such as a medical or veterinary sample. Such a biological sample may be any suitable type of body fluid, or may be a suspension of any suitable type of tissue. The sample may be an environmental sample. Such an environmental sample may be a water sample, or a sample taken from the environment and suspended in a solvent. The sample may be an industrial sample, such as a sample from a production process.

[0040] If the sample is suspected to be concentrated, it may be appropriately diluted, which may be with any suitable solvent, such as water. The object may be a biological or biomolecule or a chemical molecule.

[0041] In some embodiments, the object can be a protein, a peptide, a polypeptide, a lipoprotein, a glycoprotein, a lipid, a carbohydrate, an organic polymer, a protein complex, an antibody or an antibody fragment of an antibody, an enzyme, or the object can be a nucleic acid molecule such as DNA, RNA, a polysaccharide, or the object can be a virus or viral vector, e.g., an adenovirus and / or a lentivirus, a virus-like particle, or a small molecule, an exosome, a vesicle, an assembly complex, a nanoparticle, a chemical compound, an ion, or a quantum dot.

[0042] In some embodiments, the object can be a single molecule, a macromolecule, a supramolecule, or an association of molecules, macromolecules (such as polymers), and supramolecules. Examples of suitable macromolecules can include, but are not limited to, nucleic acid molecules, either 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.

[0043] In some embodiments, the object may be a multimolecular complex, including aggregates or other higher order aggregates of compounds including proteins, such as monomeric, dimeric, or trimeric species.

[0044] In some embodiments, the illumination light can be spatially and temporally coherent. In some embodiments, the surface of the solid immersion lens can be illuminated by a laser light source. In some embodiments, the 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 the laser beam can be efficiently coupled into and out of the microscope with minimal impact on the overall imaging performance.

[0045] In some embodiments, the coherent scattering microscope can further comprise a spatial filter. In some embodiments, the iSCAT microscope can 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, can be performed prior to detection of 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.

[0046] The spatial filter is a filter that measures the intensity within a given numerical aperture by 10 -2 The spatial filter is arranged to pass output light having a reduction in intensity to less than or equal to the incident intensity of 10. -4 For example, 10 -2 ~10 -4 The optical fiber may be arranged to pass output light having a falloff within the range of the incident intensity of 100 nm to 150 nm, so that a particular numerical aperture may be used to detect these weakly scattering objects.

[0047] The spatial filter selectively reduces the intensity of the illumination light over the 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, the spatial filter exploits 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 surface of the sample will preferentially scatter light to a high numerical aperture. Thus, the intensity reduction by the spatial filter at low numerical aperture will preferentially affect the illumination light and have a minimal effect on the scattered light, thereby maximizing imaging contrast.

[0048] This effect can be maximized by positioning the spatial filter so that its predetermined numerical aperture is the same or similar to the numerical aperture of the illumination light reflected from the sample site. The invention will now be further and in more detail described, purely by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0049] [Figure 1] A schematic diagram of the iSCAT microscope. [Diagram 2] 1 is a schematic diagram of an inverted iSCAT microscope with a solid immersion lens. [Figure 3A] FIG. 3 shows the microscope setup of FIG. 2 positioned above the sample solutions in a multi-well plate. [Figure 3B] FIG. 1 shows a solid immersion lens arranged to be immersed in a sample solution. [Figure 3C] FIG. 3 shows the microscope setup of FIG. 2 positioned above a wash solution in a multi-well plate. [Figure 3D] FIG. 1 shows a solid immersion lens positioned for immersion in a cleaning solution. [Figure 4] FIG. 1 shows a schematic lens system. [Diagram 5] FIG. 1 shows two lenses mounted within a barrel (dipping tip). [Figure 6] FIG. 1 shows a lens system immersed in a well containing a biological solution. [Figure 7] FIG. 1 shows a hemispherical SIL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The present invention presents a method for measuring properties of objects using an automated workflow and an inverted interference scattering microscope with a solid immersion lens. The method of the present invention can be used to measure properties of objects directly in a sample solution and is compatible with multi-well plates used in standard biochemical screening workflows.

[0051] 1 shows an iSCAT microscope configuration disclosed in WO 2018 / 011591. The disclosure of WO 2018 / 011591 is incorporated herein by reference, however, for the sake of completeness, the following description will present the components and functions of the iSCAT microscope of the present invention, which are common to those of WO 2018 / 011591 and shown in FIG. 1, and then will describe various improvements to said configuration provided by the present disclosure and provide example embodiments thereof.

[0052] 1 shows an iSCAT microscope 1 arranged as follows: The microscope 1 comprises the following components, whose structure is conventional in the field of microscopy, except for a spatial filter, which will be explained in more detail below:

[0053] The microscope 1 comprises a sample holder 2 for holding a sample 3 at a sample location. The sample 3 may be a liquid sample containing the object to be imaged, which will be described in more detail below. The sample holder 2 may take any form suitable for holding the sample 3. Typically, the sample holder 2 holds the sample 3 on a surface that forms an interface between the sample holder 2 and the sample 3. For example, the sample holder 2 may be a coverslip and / or may be made from glass. The sample 3 may be provided on the sample holder 2 in a straightforward manner, for example using a micropipette.

[0054] The microscope 1 further comprises an illumination source 4 and a detector 5 . The illumination source 4 is arranged to provide illumination light. The illumination light may be coherent light. For example, the illumination source 4 may be a laser. The wavelength of the illumination light may be selected depending on the nature of the sample 3 and / or the property to be inspected. In one example, the illumination light has a wavelength of 405 nm.

[0055] Optionally, the illumination light may be spatially modulated to eliminate speckle patterns arising from the coherent nature of the illumination and laser noise, e.g., as detailed in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935.

[0056] The detector 5 receives the output light upon reflection from the sample location. Typically, the microscope 1 can operate in a wide field mode, in which case the detector 5 can be an image sensor that captures an image of the sample 3. The microscope 1 can alternatively operate in a confocal mode, in which case the detector 5 can be an image sensor, or can be a point-like detector, such as a photodiode, in which case a scanning arrangement can be used to scan an area of ​​the sample 3 to build up an image. Examples of image sensors that can be used as the detector 5 include a complementary metal-oxide semiconductor (CMOS) image sensor or a charge-coupled device (CCD).

[0057] The microscope 1 further comprises an optical system 10 arranged between the sample holder 2, the illumination source 4 and the detector 5. The optical system 10 is arranged as follows: to direct illumination light to the sample location for illuminating the sample 3, to collect output light on reflection from the sample location and to direct the output light to the detector 5.

[0058] The optical system 10 includes an objective lens 11, which is a lens system disposed in front of the sample holder 2. The optical system 10 also includes a condenser lens 12 and a tube lens 13. The condenser lens 12 focuses the illumination light (shown by a continuous line in FIG. 1) from the light source 11 through the objective lens 11 onto the sample 3 at the sample location.

[0059] The objective lens 11 collects the output light, which includes both (a) the illumination light reflected from the sample location (shown as a continuous line in FIG. 1 ) and (b) the light scattered from the sample 3 at the sample location (shown as a dotted line in FIG. 1 ). The reflected light is mainly reflected from the interface between the sample holder 2 and the sample 3. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be of the order of 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects within the sample 3.

[0060] In a manner similar to conventional iSCAT, scattered light from objects at or near the surface of the sample constructively interferes with the reflected light and is therefore visible in the image captured by detector 5. This effect results in imaging contrast that is different from, and much smaller than, microscopes operating in transmission mode, where the illumination light reaching the detector is transmitted through the depth of the sample.

[0061] As shown in Figure 1, the reflected illumination and the scattered light have different directionality. In particular, the reflected illumination has a numerical aperture that results from the geometry of the light beam output by the light source 4 and the optical system 10. The scattered light is scattered over a wide range of angles, thereby filling a larger numerical aperture than the reflected illumination.

[0062] Tube lens 13 focuses the output light from objective lens 11 onto detector 5 . The optical system 10 also includes a beam splitter 14 arranged to split the optical paths for the illumination light from the light source 4 and the output light directed to the detector 5. Except for the provision of a spatial filter as described below, the beam splitter 14 can have a conventional structure that provides partial reflection and partial transmission of the light incident on the beam splitter 14. For example, the beam splitter 14 can be a plate, typically comprising a film, which can be metallic or dielectric, arranged at 45° to the optical path. Alternatively, the beam splitter 14 can be a cube beam splitter formed by a matched pair of prisms with a partially reflective film at the interface between the prisms. Alternatively, the beam splitter 14 can be a polarizing beam splitter used in combination with a quarter wave plate between the beam splitter 14 and the sample 3.

[0063] In the example shown in FIG. 1 , the light source 4 is offset from the optical path of the objective lens 11 so that the illumination light from the light source 4 is reflected by the beam splitter 14 into the objective lens 11; conversely, the detector 5 is aligned in the optical path of the objective lens 11 so that the output light from the sample location passes through the beam splitter 14 towards the detector 5.

[0064] In addition to the components described above, which may be of conventional construction, the microscope 1 includes a spatial filter 20. In the example shown in FIG. 1, the spatial filter 20 is formed on the beam splitter 14, thereby positioned behind the back aperture of the objective 11, and thereby directly behind the back focal plane 15 of the objective 11. The spatial filter 20 can therefore be implemented without entering the objective as in phase contrast microscopy. Placing the spatial filter directly behind the entrance aperture of the objective, rather than in a conjugate plane (e.g., as described below), has the obvious advantage of strongly suppressing back reflections arising from the large number of lenses in high numerical aperture microscope objectives. This in turn reduces imaging noise, lowers the incoherent background, and reduces the experimental complexity, the number of optics, and the optical path length, resulting in an increase in the stability of the optical setup and therefore the image quality.

[0065] However, this positioning is not essential and a spatial filter having equivalent functionality may be provided elsewhere, as described below. Spatial filter 20 is thereby positioned to filter the output light passing to detector 5. In the example shown in Figure 1, where detector 5 is aligned with the optical path of objective lens 11, spatial filter 20 is therefore transmissive.

[0066] The spatial filter 20 is partially transmissive and therefore passes the output light, including the reflected illumination light, but reduces its intensity. The spatial filter 20 is also aligned with the optical axis and has a predetermined aperture, thus providing an intensity reduction within a predetermined numerical aperture. Numerical aperture is defined herein in its usual manner as being a dimensionless quantity that characterizes a 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 equation NA=n·sin(θ), where θ is the half angle of collection and n is the refractive index of the material (e.g., the material of a component of the optical system 10) through which the output light passes.

[0067] FIG. 2 shows the apparatus shown in FIG. 1 in an inverted configuration and with the sample holder 2 replaced by a solid immersion lens 22. The microscope configuration is described as inverted because the detector 5 and the illumination source 4 are located above the solid immersion lens 22, whereas in FIG. 1 and in the case of a conventional iSCAT microscope, the detector 5 and the illumination source 4 are located below the sample holder 2. The inverted microscope configuration allows the solid immersion lens 22 to be easily immersed from above in the solution, in contrast to requiring the sample 3 to be placed on the sample holder 2 as shown in the microscope setup of FIG. 1. Compared to conventional lenses, the solid immersion lens 22 can achieve increased sensitivity and is suitable for use with a wider range of solvents. The solid immersion lens 22 can be glass, diamond, zirconia, sapphire, lithium niobate, or any other suitable material. The solid immersion lens 22 can be hemispherical, hyperhemispherical, or can be a diffractive optical element.

[0068] Figures 3A-3D show the automated method of the invention and the inverted microscope setup of Figure 2 used to measure properties of objects. The objects can be proteins, nucleic acid molecules, virus-like particles, single molecules, macromolecules, supramolecules, or associations of molecules, macromolecules (such as polymers), and supramolecules.

[0069] The inverted microscope geometry and the immersion of the solid immersion lens allow measurements to be made directly in the sample solution. The inverted microscope configuration allows the solution to be automatically moved under and aligned with the solid immersion lens 22, so that the solid immersion lens can be immersed in the solution in the required sequence. The method of the present invention is compatible with multi-well plates 46 and can therefore be integrated into standard biochemistry workflows. The multi-well plate 46 shown in Figures 3A-3D shows six wells merely as an example, and it should be understood that the method of the present invention is compatible with other well plate formats, including the commonly used 96-well plate format.

[0070] Furthermore, the method of the present invention also provides a controlled washing step so that the solid immersion lens 22 can be reused in subsequent measurements. Thus, the solid immersion lens 22 does not need to be disposable, which keeps the method of the present invention cost-effective and suitable for implementation in high throughput workflows.

[0071] As shown in Figure 3A, one example embodiment of the present invention shows both sample solution 44 and wash solutions 42 and 52 disposed within the same multi-well plate 46. Alternatively or additionally, some or all of the solutions may be held in separate containers that can be moved under and aligned with the solid immersion lens 22, as desired. For example, the multi-well plate 46 may contain only the sample solution 44, and one or more wash solutions 42 and 52 may be held in separate containers or well plates that can be moved under the solid immersion lens 22 between measurements. The well plate 46 may be maintained with temperature control and / or agitation.

[0072] Figure 3A shows a sample solution 44 in a multiwell plate 46 aligned under the solid immersion lens 22. The sample solution 44 contains a target object of interest. The multiwell plate 46 can be moved up towards the solid immersion lens 22, as indicated by the arrow 48 shown in Figure 3A.

[0073] As shown in FIG. 3B, the multi-well plate 46 can be moved up until the solid immersion lens 22 is immersed in the sample solution 44 containing the object to be measured. The solid immersion lens 22 is held immersed in the sample solution 44, which allows the object of interest to adsorb to the surface of the solid immersion lens 22. To facilitate the adsorption of the object of interest, the surface of the solid immersion lens 22 can be functionalized prior to immersion in the sample solution 44. Functionalization of the lens surface can be performed by immersing the solid immersion lens 22 in a sodium hydroxide solution, sulfuric acid, hydrochloric acid, and / or nitric acid. Functionalization of the lens surface can also be performed, for example, by plasma treatment. The surface of the solid immersion lens 22 can be carboxylated and / or oxidized.

[0074] The solid immersion lens 22 is held in the sample solution 44 for a predetermined amount of time. During this time, objects of interest are adsorbed to the surface of the solid immersion lens 22 and measurements are made using the iSCAT microscope 1. The surface of the solid immersion lens 22 is illuminated by an illumination source 4 and light scattered from the objects is detected by a detector 5 of the iSCAT microscope 1. The scattered light is used to measure properties of the objects adsorbed to the surface of the solid immersion lens 22. The duration that the solid immersion lens 22 is held in the sample solution 44 as shown in FIG. 3B can be between 10 seconds and 5 minutes.

[0075] After a predetermined time has elapsed and measurements of the objects have been made using the iSCAT microscope 1, the multiwell plate 46 may be lowered as indicated by arrow 50 in Figure 3B. The lowering of the multiwell plate 46 is such that the solid immersion lens 22 is removed from the sample solution 44. At this point, the objects of interest remain adsorbed to the surface of the solid immersion lens 22.

[0076] In order to prevent the solid immersion lens 22 having to be disposable, which would significantly increase the cost of the method of the present invention, the present invention includes a method for cleaning the solid immersion lens 22 after a measurement has been taken, whereby objects are removed from the surface of the solid immersion lens 22.

[0077] 3C, the multi-well plate 46 can be automatically moved so that the wash solution 42 is positioned beneath and aligned with the solid immersion lens 22. Alternatively, the wash solution 42 can be held in a separate container that can be automatically positioned beneath the solid immersion lens 22 between measurements.

[0078] The solid immersion lens 22 may be washed as shown in FIG. 3C by moving the multiwell plate 46 up as indicated by arrow 54. FIG. 3D shows the solid immersion lens 22 immersed in a washing solution 42. After a predetermined time, the multiwell plate 46 may be lowered as indicated by arrow 56 and the washed solid immersion lens 22 may then be immersed in a further sample solution 44 and used for further measurements. Alternatively, after removing the solid immersion lens 22 from the washing solution 42, the solid immersion lens 22 may then be immersed in one or more further washing solutions 52. This allows the washing routine to be adapted depending on the objects adsorbed on the surfaces of the sample solution 44 and the solid immersion lens 22, so that all of the objects and the sample solution 44 are removed from the solid immersion lens 22 and do not contaminate further measurements. The washing solutions 42 and / or 52 may include, but are not limited to, ethanol, isopropanol, water, hydrochloric acid, and / or sulfuric acid. After removal of the solid immersion lens 22 from the cleaning solution 42 and / or 52, an air flow over the lens surface may be used to dry the surface to remove any remaining cleaning solution.

[0079] Alternatively or additionally, the cleaning solutions 42 and / or 52 may be agitated, for example by a sonicator bath, to aid in the removal of objects from the solid immersion lens 22 surface.

[0080] Alternatively or additionally, the solid immersion lens 22 may be treated with plasma, for example by an ambient pressure plasma torch, between measurements to facilitate the cleaning process. Alternatively or additionally, the solid immersion lens 22 may be treated with plasma after the cleaning process to functionalize the surface of the solid immersion lens 22 to facilitate adsorption of target objects when the solid immersion lens 22 is immersed in the sample solution 44.

[0081] 4, a lens system 60 is shown having an aspheric lens 62 focusing into a super-hemispherical solid immersion lens (s-SIL) 64. In some embodiments, the hemispherical solid immersion lens and / or the Weierstrass lens can be spherical and then truncated to a thickness t=r×(1+1 / n), where r is the radius and n is the refractive index. For this thickness, the lens increases the NA of the input beam by n without adding spherical aberration. 2 With diamond (n=2.42), a 2.1 NA can be reached for a 0.36 NA aspheric lens. Alternative materials, high index glass (S-LAH79, n=2) or cubic zirconia (n=2.17), can exceed 1.8 NA for a 0.45 NA aspheric lens.

[0082] With reference to Figure 5, two lenses 62, 64 are mounted in a barrel 66, such as a stainless steel barrel, small enough that it can be immersed in one or more standard well plates (6.5-7 mm diameter). A 2 mm diameter hemispherical solid immersion 64 is shown in Figure 5, although other sizes are possible.

[0083] Measurements may be taken by immersing the lenses 62, 64 in a biological solution 68, such as the protein solution shown in Figure 6, contained within a well 70. The barrel (dipping tip) 66 may be resistant to water, saline, alcohol, and other solvents.

[0084] The well may be made of plastic, but also of other suitable materials known to those skilled in the art. At least the SIL 64 may then be cleaned by immersion and spraying with an aqueous solution and an organic solvent such as isopropanol. Additionally or alternatively, both lenses 62, 64 may be cleaned by immersion and spraying with an aqueous solution and an organic solvent such as isopropanol. Thus, the lens system needs to be well sealed and chemically resistant. There may be restrictions on the material used for the lens barrel to avoid contaminating the sample solution. Stainless steel 316 may be an option. In the case of diamond bonding, the hemispherical lens may be securely attached so that it may then be integrated with the rest of the optical system. The optical system may be used with a standard oil immersion objective with a partially transparent mask to attenuate part of the beam, as described herein.

[0085] 7, an alternative design to the solid immersion lens is provided. A hemispherical solid immersion lens 80 can be used, but with an NA of the input beam 82 of n 2 Instead of increasing by n, this may require a 0.8+ NA input lens that appears to be a multi-element system rather than a single aspheric lens. A small diameter, 0.5-1mm SIL may be required so the input objective does not need excessive working distance. EXAMPLES

[0086] [material and method] [optical] The operating wavelength range can be 515-535 nm. Chromatic aberration can limit the wavelength range for a 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, NA (1.6 / 1.8) for high index glass or cubic zirconia, diffraction limited imaging over the field, and a Strehl ratio > (0.9 / 0.95).

[0087] [Other optical properties] To reach the above NA in s-SIL, the NA of the aspheric lens can be about 0.35. A diameter of 4 mm can be provided to fit the tip. AR coating can be applied to the aspheric lens and / or relay lens, R<1%. Low stray light and avoid reflections sent back along the imaging path. Effective focal length of the SIL aspheric system to determine the magnification, 0.5-5 mm. n of s-SIL 2 Note the magnification. Low stress birefringence as polarization can be important.

[0088] 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. No fixed interface. Additionally or alternatively, the SIL can be made with Asphericon surface roughness of flat surface (2, <1) nm RMS.

[0089] [mechanical] The system can be constructed with a sealed immersion tip 6mm maximum diameter for a length of 10mm from the flat surface of the s-SIL. The s-SIL and aspheric lens should be securely mounted in place to achieve the optical performance listed above. Thermal expansion of the components should not interfere with focusing over temperatures ranging from 20° to 22° Celsius and 15° to 40° Celsius.

[0090] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. As used herein, "and / or" is construed 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 construed as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0091] 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.

[0092] While the present invention has been described by way of example with reference to certain 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 envisaged without departing from the scope of the invention as defined in the appended claims.

Claims

1. Immersing a solid immersion lens in a sample solution containing an object, wherein the object interacts with the surface of the solid immersion lens, irradiating the surface of the solid immersion lens with an irradiation source, and detecting scattered light from the object using an interference scattering microscope, measuring the characteristics of the object using the detected scattered light, removing the solid immersion lens from the solution, and then cleaning the solid immersion lens, wherein the object is removed from the surface of the solid immersion lens comprising a method for measuring the characteristics of an object.

2. Measuring the characteristics of the object includes quantifying the mass of the object, The method according to claim 1.

3. Measuring the characteristics of the object includes measuring or quantifying a change in the mass of the object, The method according to claim 1.

4. Cleaning the solid immersion lens includes immersing the solid immersion lens in at least one cleaning solution, The method according to claim 1.

5. Further comprising flowing air over the solid immersion lens, The method according to claim 1.

6. Further comprising stirring the cleaning solution, The method according to claim 4.

7. Further comprising applying plasma to the surface of the solid immersion lens, The method according to claim 1.

8. The solid immersion lens has a diameter of 1 to 5 mm, The method according to claim 1.

9. The solid immersion lens is attached to a lens assembly, The method according to claim 1.

10. The lens assembly has a diameter of 2 to 6 mm, The method according to claim 9.

11. The solid immersion lens has a length of 6 to 10 mm, The method according to claim 1.

12. The solid immersion lens is hemispherical or super-hemispherical, The method according to claim 1.

13. The interference microscope further comprises at least one optical element configured to compensate for the aberration of the solid immersion lens, The method according to claim 1.

14. The material of the solid immersion lens is diamond, or zirconia, or sapphire, or lithium niobate, The method according to claim 1.

15. The material of the solid immersion lens is glass. The method according to claim 1.

16. Further comprising functionalizing the surface of the solid immersion lens. The method according to claim 1.

17. The object is a protein, lipid, carbohydrate, organic polymer, nucleic acid molecule, virus, vesicle, complex aggregate, or virus-like particle. The method according to claim 1.

18. The irradiation light is spatially and temporally coherent. The method according to claim 1.

19. The interference scattering microscope further comprises a spatial filter. The method according to claim 1.

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