Improvements in or relating to single particle light scattering

Abstract

A method of reducing the effect of optical distortion within single particle light scattering apparatus, the method comprising the steps of: (i) imaging a calibrant particle using light scattering apparatus to determine a position of the calibrant particle and a calibrant characteristic of the calibrant particle; (ii) moving the calibrant particle laterally with respect to an optical axis of the light scattering apparatus and repeating step (i) to determine a change in the position and calibrant characteristic; (iii) calculating a correction factor based on the change in the position and the calibrant characteristic; and (iv) applying the correction factor to a test characteristic of a test particle imaged by the light scattering apparatus.

Description

[0001] IMPROVEMENTS IN OR RELATING TO SINGLE PARTICLE LIGHT SCATTERING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to improvements in or relating to single particle light scattering and, more specifically, to the calculation of a correction factor and the application thereof to at least one characteristic of a test particle imaged using single particle light scattering, wherein the correction factor accounts for optical distortion across the field of view of the light scattering imaging apparatus.

[0004] BACKGROUND TO THE INVENTION

[0005] Single particle light scattering is an imaging technique that uses a light source to emit light onto a sample comprising one or more particles. An imaging device is then used to record the angular distribution of light that is reflected by the sample and / or transmitted therethrough. The angular distribution of the reflected light can be measured and used to determine certain characteristics of the sample. For example, one characteristic that may be determined is the size of a particle within the sample, wherein the size may be determined based on the contrast of the imaged particle.

[0006] However, optical distortions across the field of view of single particle light scattering apparatus lead identical particles in different physical positions to manifest differently. In some apparatus, a measured characteristic of a particle reduces roughly in proportion to a gentle quadratic curve centered on the point of best focus. In some single particle light scattering apparatus, the distortion is sufficiently severe to dominate the characteristic measurement uncertainty.

[0007] It is against this background that the present invention has arisen.

[0008] SUMMARY OF THE INVENTION

[0009] According to the present invention there is provided a method of reducing the effect of optical distortion within single particle light scattering apparatus, the method comprising the steps of: (i) imaging a calibrant particle using light scattering apparatus to determine a position of the calibrant particle and a calibrant characteristic of the calibrant particle; (ii) moving the calibrant particle laterally with respect to an optical axis of the light scattering apparatus and repeating step (i) to determine a change in the position and calibrant characteristic; (iii) calculating a correction factor based on the change in the position and the calibrant characteristic; and (iv) applying the correction factor to a test characteristic of a test particle imaged by the light scattering apparatus. Calculating and applying a correction factor enables the single particle light scattering apparatus to account for any optical distortion across its field of view. This optical distortion may be caused by the field curvature of a lens within the apparatus. The lens may be the objective lens. As such, the present method enables the single particle light scattering apparatus to image one or more test particles and determine at least one characteristic thereof with increased accuracy.

[0010] This method is required because real world calibrant samples do not provide identical calibrant particles to characterize the optical distortion of the light scattering apparatus. As such, in the present method, the correction factor is calculated based on at least one matched pair of calibrant particle observations. This is achieved by imaging the calibrant particle in two different positions and then comparing the same particle in those positions.

[0011] The calibrant particle may be a single particle. The calibrant particle may be isolated. Alternatively, the calibrant particle may comprise two or more linked, bound, associated, connected, or overlapping particles. Similarly, the test particle may be a single particle. The test particle may be isolated. Alternatively, the test particle may comprise two or more linked, bound, associated, connected, or overlapping particles.

[0012] The calibrant particle may comprise lateral (X, Y) and axial (Z) coordinates configured to define its position with respect to the light scattering apparatus. The coordinates may be Cartesian coordinates. The lateral coordinates may define the position of the calibrant particle on a substantially flat plane (i.e., a lateral plane). The axial coordinate may define the position of the calibrant particle along an axis substantially perpendicular to the substantially flat (lateral) plane. The axial distance between the calibrant particle and the objective lens may be measured along an axis parallel to the optical axis. Moving the calibrant particle laterally with respect to the optical axis of the imaging apparatus comprises varying at least one lateral coordinate (X, Y) of the particle. In some embodiments, moving the calibrant particle laterally with respect to the optical axis of the imaging apparatus comprises varying each of the lateral coordinates (X, Y) of the particle.

[0013] The calibrant characteristic may be: contrast; brightness; diameter; height; shape; mass; or size. In some embodiments, the calibrant characteristic is maximum size; maximum contrast; or maximum brightness.

[0014] The test characteristic of the test particle may be the same as the calibrant characteristic of the calibrant particle. However, in some embodiments, the test characteristic of the test particle may be different from the calibrant characteristic of the calibrant particle. For example, the calibrant characteristic may be diameter, whereas the test characteristic may be size. Other combinations of characteristics are also possible. The correction factor may comprise an adjustment factor configured to account for this difference is characteristic.

[0015] In some embodiments, step (i) comprises imaging the calibrant particle using the light scattering apparatus at a plurality of focal lengths to determine the position of the calibrant particle and the calibrant characteristic of the calibrant particle.

[0016] Imaging the calibrant particle at a plurality of focal lengths enables characteristics such as maximum brightness and / or contrast to be determined. As such, imaging the calibrant particle at a plurality of focal lengths may be used to determine a maximum calibrant characteristic of the calibrant particle. In turn, this may be used to determine the most optimal lateral plane within which to image and reimage the particle. However, any suitable method may be used to determine the optimal lateral plane within which to image and re-image the particle. That said, imaging the calibrant particle at a plurality of focal lengths to determine its position and the calibrant characteristic of the calibrant particle reduces the likelihood of error occurring during this determination.

[0017] The focal length may be varied by moving at least one of the particles to be imaged and a lens of the imaging apparatus along the focal axis of the light scattering apparatus. Varying the focal length varies the axial (Z) coordinate of the particle.

[0018] In some embodiments, the test particle may be the calibrant particle. In other words, the correction factor may be applied to a test characteristic of the calibrant particle. The test characteristic of the calibrant particle may be the calibrant characteristic of the calibrant particle.

[0019] Alternatively, or in addition, the correction factor may be applied to a test characteristic of another particle imaged by the light scattering apparatus. As such, in some embodiments, the test particle is distinct from the calibrant particle.

[0020] For example, the correction factor may be calculated based on a first particle within a test sample. The correction factor may then be applied to the first particle and / or a second particle within the test sample. The second particle within the test sample may be imaged simultaneously, or subsequently, to the first particle. More specifically, a surface may comprise a sample having a plurality of particles. The correction factor may be calculated based on a first particle and applied to the remaining particles within the sample. The first particle may be the calibrant particle. The surface may form part of a sample holder. The sample holder may be an element of a light scattering apparatus. The sample holder may be a high surface-to-volume chamber. Alternatively, or in addition, the correction factor may be calculated based on a calibrant particle within a calibrant sample. The correction factor may then be applied to at least one test particle within at least one test sample. The at least one test particle within the test sample(s) may be imaged simultaneously or subsequently to the calibrant particle within the calibrant sample. For example, a surface may comprise at least one calibrant sample having a calibrant particle and one or more test sample each having at least one test particle. The correction factor may be calculated based on the calibrant particle and applied to the one or more test particle.

[0021] In some embodiments, the method comprises imaging a surface, such as a cover slip or slide, comprising a plurality of wells, wherein each well comprise a sample. At least one well may be a calibrant well comprising a calibrant sample comprising a calibrant particle. The remaining wells may be test wells comprising test samples comprising test particles. The test sample may be substantially the same as the calibrant sample. As such, the test particles may be substantially the same as the calibrant particle. The correction factor may be calculated based on a calibrant particle within a calibrant well. Once calculated, the correction factor may be applied to the test characteristics of each test particle within each test well. In some embodiments, the method comprises imaging a surface comprising one calibrant well and thirteen sample wells.

[0022] In some embodiments, step (i) comprises determining a plurality of calibrant characteristics of the calibrant particle; step (ii) comprises determining at least one change in the plurality of calibrant characteristics; step (iii) comprises calculating the correction factor based on at least one change in the plurality of calibrant characteristics; and step (iv) comprises applying the correction factor to one or more test characteristic of a test particle imaged by the light scattering apparatus.

[0023] The one or more test characteristic of the test particle may be the same as the one or more calibrant characteristic of the calibrant particle that is determined to have changed in step (ii).

[0024] In some embodiments, the calibrant particle is a nanoparticle. For example, the size of the calibrant particle may be up to 50 nanometres (nm). However, in some embodiments, the size of the calibrant particle is greater than 50, 60, 80, 100, 120, 150, or 200 nm. Similarly, the test particle may also be a nanoparticle. Its size may also be as previously described.

[0025] In some embodiments, the single particle light scattering apparatus is an interferometric scattering microscope. However, any suitable light scattering microscope may be used including, but not limited to, a single particle light scattering microscope comprising a spatial filter or a mass photometer. For example, a suitable microscope or photometer may comprise: a sample holder for holding a surface for receiving the calibrant particle; an illumination source arranged to provide illuminating light; a detector; and an optical system being arranged to direct illuminating light onto the calibrant particle and being arranged to collect output light in reflection, the output light comprising both light scattered from the calibrant particle and illuminating light reflected from the calibrant particle, and direct the output light to the detector.

[0026] A suitable light scattering microscope may further comprise a spatial filter positioned to filter the output light. The spatial filter may be arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures. Such a spatial filter advantageously maximises image contrast, as described in PCT / GB2017 / 052070, and also in Cole et al (ACS Photonics, 2017, 4(2), pp 211-216).

[0027] In some embodiments, imaging the calibrant particle comprises imaging at least a 10 x 10 micrometre area comprising the particle. More specifically, in some embodiments, imaging the calibrant particle comprises imaging at least a 20 x 20; 30 x 30; 40 x 40; 50 x 50; or 100 x 100 micrometre area comprising the calibrant particle. As such, in some embodiments, an area greater than 100 x 100 micrometres may be imaged in each of steps (i), (ii) and (iv). The imaged area may comprise a plurality of particles. As such, a plurality of calibrant particles may be imaged. Similarly, the same area may be imaged when imaging the test particle. As such, a plurality of test particles may also be imaged.

[0028] In some embodiments, the method further comprising the step of: repeating steps (i)-(iii) at least every hour to calculate a new correction factor for step (iv). However, any time interval may be used. For example, the time interval may be at least every 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours. The new correction factor may account for any environmental variations, such as temperature or humidity, for example.

[0029] In some embodiments, steps (i) - (iii) comprise: (i) imaging a plurality of calibrant particles using the light scattering imaging apparatus at a plurality of focal lengths to determine a calibrant characteristic of each particle; (ii) moving each calibrant particle laterally with respect to the optical axis of the imaging apparatus and repeating step (i) to determine a change in the calibrant characteristic of each calibrant particle; and (iii) calculating the correction factor based on the change in the calibrant characteristic of each calibrant particle.

[0030] As such, the correction factor may be calculated based on the change in position and the calibrant characteristic of each calibrant particle. Calculating the correction factor based on a plurality of calibrant particles further increases the accuracy of the test characteristic of a test particle. In some embodiments, at least 35% of the calibrant particles imaged in step (i) are re-imaged in step (ii). Moving the calibrant particles laterally such that at least 35%, and more preferably 50%, of the particles imaged in step (i) are re-imaged in step (ii) may comprise moving the particles such that those particles visible in the top third, or half, of an image generated in step (i) are moved to the bottom (or middle) third, or bottom half, of an image generated in step (ii). This may comprise varying the Y coordinate of the calibrant particles whilst substantially maintaining the X coordinates. Alternatively, or in addition, the particles could be moved left to right, for example. This may comprise varying the X coordinate of the calibrant particles whilst substantially maintaining the Y coordinates.

[0031] In some embodiments, at least 35% of the area imaged in step (i) is re-imaged in step (ii). More preferably, at least 50% of the area imaged in step (i) is re-imaged in step (ii).

[0032] In some embodiments, the plurality of calibrant particles are heterogeneous. For example, the heterogeneous calibrant particles may be the test particles.

[0033] In some embodiments, the method comprises the steps of: imaging a calibrant particle using a single particle light scattering apparatus at a plurality of focal lengths to generate a first series of images; moving the calibrant particle laterally with respect to an optical axis of the light scattering apparatus and re-imaging the calibrant particle at a plurality of focal lengths to generate a second series of images; calculating a translation vector configured to define a movement of the calibrant particle from a first position in a first image in the first series of images to a second position in a second image in the second series of images; determining a change in a characteristic of the calibrant particle between the first image and the second image; calculating a correction factor based on the translation vector and the change in the characteristic; and applying the correction factor to a test characteristic of a test particle imaged by the light scattering apparatus.

[0034] The method may comprise imaging a plurality of calibrant particles using the single particle light scattering apparatus at a plurality of focal lengths to generate a first series of images; moving the calibrant particles laterally with respect to the optical axis of the light scattering apparatus and reimaging the calibrant particles at a plurality of focal lengths to generate a second series of images; identifying a plurality of calibrant particles that are present in both the first series of images and the second series of images; calculating, for each calibrant particle present in the first and second series of images, a translation vector configured to define a movement of the particle from a first position in a first image in the first series of images to a second position in a second image in the second series of images; determining, for each calibrant particle present in the first and second series of images, a change in a calibrant characteristic of the particle between the first image and the second image; and calculating the correction factor based on the translation vector and change in calibrant characteristic of each particle present in the first and second series of images. At least 35%, preferably 50%, of the calibrant particles present the first series of images may be present in the second series of images.

[0035] The method may involve a particle present in a sample. The particle may be a calibrant particle and / or a test particle.

[0036] The sample is preferably liquid, such as a solution or suspension. The solution or suspension may contain any suitable solvent, particularly water. The sample may contain an organic solvent.

[0037] The sample may be a biological sample, such as a medical or veterinary sample. Such biological sample may be a bodily fluid of any appropriate type or may be a suspension of a tissue of any appropriate type. The sample may be an environmental sample. Such environmental sample may be a water sample, or a sample taken from an environment and suspended in a solvent. The sample may be an industrial sample, such as a sample from a production process.

[0038] If the sample is suspected of being concentrated, it may be appropriately diluted. Dilution may use any appropriate solvent, such as water.

[0039] The particle may be a biological molecule or a biomolecule, or a chemical molecule.

[0040] In some embodiments, the particle may 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 thereof, an enzyme or it may be a nucleic acid molecule such as DNA, RNA, a polysaccharide, or it may be a virus, or a viral vector such as an adenovirus, adeno-associated virus (AAV) and / or a lentivirus, a virus-like particle, or a small molecule, an exosome, a vesicle, an assembly complex, a nanoparticle, a compound, an ion or a quantum dot.

[0041] In some embodiments, the particle may be a single molecule, a macromolecule, a supermolecule, or an association of molecules, macromolecules (such as polymers) and supermolecules. Examples of suitable macromolecules may 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). Associations of molecules can include assemblies such as virus like particles where envelope or capsid proteins are associated.

[0042] In some embodiments, the particle may be multi-molecular complexes comprising agglomerations of components, including proteins, such as monomer, dimer and trimer species, or other higher order agglomerations. In some embodiments, steps (i) and (ii) comprise: (i) imaging a surface in a first location using the light scattering apparatus to produce two different images, wherein, in the first image, the surface comprises the calibrant particle, and, in the second image, the surface does not comprise the calibrant particle, and using the two images to determine the position of the calibrant particle and the calibrant characteristic of the calibrant particle; and (ii) moving the surface laterally with respect to the optical axis of the light scattering apparatus and repeating step (i) to determine a change in the position and calibrant characteristic.

[0043] The surface may be the previously disclosed surface. The order in which the images within steps (i) and (ii) are produced is not important and may be varied. For example, one could first image the surface in the first location in the absence of the calibrant particle, then add the calibrant particle to the surface and generate a second image comprising the calibrant particle, before moving the surface and re-imaging the surface in the new location, and then, finally, inducing the particle to leave the surface in order to image the surface in the absence of the calibrant particle in the second / new location. Alternatively, one could first image the surface in the absence of the calibrant particle in two locations, and then subsequently image the same (or substantially similar) locations after the calibrant particle has been added to the surface. One could also image the surface in the presence of the calibrant particle in two locations, induce the particle to leave the surface, and then subsequently image the same (or substantially similar) locations after the calibrant particle has been removed from the surface. All scenarios require imaging the surface in a first location to produce two different images; moving the surface laterally; and re-imaging the surface in a new location to produce another two images. As such, the claimed method covers all scenarios. The same applies if a plurality of focal lengths are used and / or a plurality of calibrant particles are imaged etc.

[0044] In some embodiments, in step (i), determining the position and / or calibrant characteristic may comprise modifying the image comprising the calibrant particle based on at least one feature within the image not comprising the calibrant particle. For example, at least one feature present in the image not comprising the calibrant particle may be removed from the image comprising the calibrant particle. In some embodiments, all features present in the image not comprising the calibrant particle may be removed from the image comprising the calibrant particle. This enables the image containing the calibrant particle to be corrected for 'background' features. The image containing the calibrant particle, once corrected, may be used to determine the position and / or calibrant characteristic. The invention will now be further and more particularly described, by way of example only, with reference to the accompanying drawings.

[0045] FIGURES

[0046] Figure 1 shows the uncorrected characteristic measurements for a number of different particles in different positions;

[0047] Figure 2 shows the characteristic measurements for a calibrant particle in different positions; and

[0048] Figure 3 shows the corrected characteristic measurements for a number of different particles in different positions.

[0049] DETAILED DESCRIPTION

[0050] Figure 1 shows the uncorrected characteristic measurements for a number of different particles in different positions. These characteristics were determined using single particle light scattering apparatus. The variation in characteristic measurements is due to a combination of the different characteristics of the particles and the optical distortion of the single particle light scattering apparatus. As such, there is a need to reduce the effect of optical distortion within the single particle light scattering apparatus.

[0051] Figure 2 shows the characteristic measurements for a calibrant particle in different positions. These characteristic measurements were determined using the single particle light scattering apparatus used to image the particles in figure 1. The 'X' marks represent different measurements of a calibrant characteristic of the calibrant particle in different locations. However, given that the same characteristic of the same particle is being measured in each position, the difference between the measured characteristic in each position can be (predominantly) attributed to the optical distortion of the single particle light scattering apparatus. The dashed line represents the inferred distortion, which is used to calculate a correction factor. As such, the correction factor is based on the change in the position of the calibrant particle and the change in the measured characteristic of the calibrant particle.

[0052] For example, in some embodiments, the correction factor may be calculated using a multiplication of a 'gold standard' characteristic value by some parabolic function, or in the first version by an addition in logarithm space. To illustrate this process with a one dimensional example, the method may be derived as follows, subject to the relevant small value approximation for logarithms, where a, b and c represent quadratic coefficients, x and x + v represent the position of a calibrant particle when imaged in steps (i) and (ii) of the method, respectively; and C2represent characteristic values for the calibrant particle when imaged in steps (i) and (ii) of the method, respectively; and CGrepresents the true characteristic value if the optical distortion were removed: o log (Ci) = log(CG) + ax2+ bx + c o log (C2) = log(CG) + a(x + v)2+ b(x + v) + c o log (C2) — log (C- = a(2xv + v2) + b(v~) + c

[0053] Note that this construction removes the unknown true characteristic values (which vary for different particles) and allows for fitting the quadratic coefficients. Moreover, note that the use of logarithms and subtraction is not core to the invention. Any calculation which uses the paired measurements to control for the unknown true characteristic value may be used.

[0054] Figure 3 shows the corrected characteristic measurements for a number of different particles in different positions. These particles are the same particles that were imaged to produce figure 1. Moreover, these characteristic measurements were determined using the single particle light scattering apparatus used to image the particles in figure 1. However, in figure 3, the correction factor calculated based on figure 2 has been applied to the measured characteristics of each particle. As such, figure 3 shows a more accurate impression of the characteristics of the different particles being imaged.

[0055] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0056] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments that are described. It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

CLAIMS1. A method of reducing the effect of optical distortion within single particle light scattering apparatus, the method comprising the steps of:(i) imaging a calibrant particle using light scattering apparatus to determine a position of the calibrant particle and a calibrant characteristic of the calibrant particle;(ii) moving the calibrant particle laterally with respect to an optical axis of the light scattering apparatus and repeating step (i) to determine a change in the position and calibrant characteristic;(iii) calculating a correction factor based on the change in the position and the calibrant characteristic; and(iv) applying the correction factor to a test characteristic of a test particle imaged by the light scattering apparatus.

2. The method according to claim 1, wherein the calibrant characteristic is:I. contrast;II. brightness;III. diameter;IV. heightV. shape;VI. mass; orVII. size;3. The method according to any preceding claim, wherein step (i) comprises:(i) imaging the calibrant particle using the light scattering apparatus at a plurality of focal lengths to determine the position of the calibrant particle and the calibrant characteristic of the calibrant particle.

4. The method according to any preceding claim, wherein the test particle is the calibrant particle.

5. The method according to any preceding claim, wherein step (i) comprises determining a plurality of calibrant characteristics of the calibrant particle; step (ii) comprises determining at least one change in the plurality of calibrant characteristics; step (iii) comprises calculating the correction factor based on at least one change in the plurality of calibrant characteristics; and step (iv) comprises applying the correction factor to one or more test characteristic of a test particle imaged by the light scattering apparatus.

6. The method according to any preceding claim, wherein the calibrant particle is a nanoparticle.

7. The method according to any preceding claim, wherein the single particle light scattering apparatus is an interferometric scattering microscope.

8. The method according to any preceding claim, wherein imaging the calibrant particle comprises imaging at least a 10 x 10 micrometre area comprising the particle.

9. The method according to any preceding claim, further comprising the step of: repeating steps (i)-(iii) at least every hour to calculate a new correction factor for step (iv).

10. The method according to any preceding claim, wherein steps (i) - (iii) comprise:(i) imaging a plurality of calibrant particles using the light scattering imaging apparatus at a plurality of focal lengths to determine a calibrant characteristic of each particle;(ii) moving each calibrant particle laterally with respect to the optical axis of the imaging apparatus and repeating step (i) to determine a change in the calibrant characteristic of each calibrant particle; and(iii) calculating the correction factor based on the change in the calibrant characteristic of each calibrant particle.

11. The method according to claim 10, wherein at least 35% of the calibrant particles imaged in step (i) are re-imaged in step (ii).

12. The method according to claim 10 or 11, wherein the plurality of calibrant particles are heterogeneous.

13. The method according to any preceding claim, wherein steps (i) and (ii) comprise:(i) imaging a surface in a first location using the light scattering apparatus to produce two different images, wherein, in the first image, the surface comprises the calibrant particle, and, in the second image, the surface does not comprise the calibrant particle, and using the two images to determine the position of the calibrant particle and the calibrant characteristic of the calibrant particle; and(ii) moving the surface laterally with respect to the optical axis of the light scattering apparatus and repeating step (i) to determine a change in the position and calibrant characteristic.

14. The method according to claim 13, wherein, in step (i), determining the position and / or calibrant characteristic comprises modifying the image comprising the calibrant particle based on at least one feature within the image not comprising the calibrant particle.

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