WO2016034859A1

WO2016034859A1 - A method of preparing an analytical sample comprising a particulate analyte for use in microscopy

Info

Publication number: WO2016034859A1
Application number: PCT/GB2015/052482
Authority: WO
Inventors: John LUCOCQ
Original assignee: University of St Andrews
Current assignee: University of St Andrews
Priority date: 2014-09-01
Filing date: 2015-08-27
Publication date: 2016-03-10
Anticipated expiration: 2017-03-01
Also published as: GB201415409D0

Abstract

A method of preparing an analytical sample comprising a particulate analyte, for use in microscopy. Disclosed is a method of preparing an analytical sample comprising a particulate analyte, for use in microscopy. A liquid composition is prepared which comprises an embedding medium and the particulate analyte. The liquid composition is consolidated so as to form an analytical sample in which the particulate analyte is immobilized and dispersed within the embedding medium. The resulting analytical sample includes a dispersion of the particulate analyte within an embedding medium, which is representative of the particulate analyte when present in the liquid composition. Optionally, the analytical sample may be stained and/or calibrated using a particulate calibrant. The method is not susceptible to perturbations which might otherwise result in the properties of the analyte in the analytical sample, such as the particle size, particle size distribution, concentration or relative concentration of particles within the particulate analyte differing from those of the analyte in the liquid composition.

Description

A METHOD OF PREPARING AN ANALYTICAL SAMPLE COMPRISING A PARTICULATE ANALYTE FOR USE IN MICROSCOPY

Field of the Invention Embodiments disclosed herein relate to the characterisation of particulate analytes, and in particular to methods and apparatus for the analysis of particle morphology and quantitation of parameters including particle size, size distribution and concentration.

Background to the Invention

In recent years analysis the qualitative and quantitative analysis of small micro- and nanoscale particles and structures has become increasingly important in a range of disciplines. For example, in biological and medical laboratory and experimental settings, synthetic nanoparticles are increasingly used as drug carriers^1,2, diagnostic, analytic and imaging tools³, therapeutic reagents^4"7, or as platforms for membrane proteins used in structural and functional studies^8,9. In nanomedicine, a wide range of NPs occur naturally and include pathogenic viruses and extracellular vesicles, of which a subset called exosomes are emerging markers of human disease and carriers of therapeutic agents¹⁰. Similarly, in fields as diverse as cosmetics, materials, foodstuffs, paints and pigments and the like, the properties of particles, including their shape, how they aggregate, sizes and size distributions can profoundly effect the performance of products in which they are used.

All these applications demand accurate, unbiased and precise analysis and/or quantification. In some applications, overall sensitivity is of increasing importance, so as to be able to detect, for example, on the smallest possible particles, or particles at lower concentrations.

Established techniques for quantitative analysis of small particles, in particular nanoparticles, are based mainly on indirect measurements of the physical properties of compositions containing the particles¹¹. But these methods are hampered by limited resolution, large sample size, as well as low sensitivity and lack of structural/molecular detail. A widespread approach is nanoparticle-tracking analysis¹² (NTA), which infers particle characteristics from the pattern and speed of particle movement. NTA's resolution is limited to roughly 50 nm and minimal sample volume is restricted to 100s of microliters. The sensitivity approximates to 10⁸ particles ml^"1 and readouts appear to be operator and lab dependent^12"14. Significantly, particle structure and levels of aggregation are not directly accessible using NTA. Other current techniques include flow cytometry, which has a maximum resolution of only 100 nm although applicability can be enhanced by identifying molecular components of NPs by fluorescence labeling^13,15. Another is dynamic light scattering (DLS or photon correlation microscopy), which infers particle size from Brownian motion based intensity fluctuations^16,17. Although DLS cannot count absolute particle concentration, size- distributions are accessible indirectly using mathematical models. Finally resistive pulse sensing measures changes in conductance in membranes with defined pore sizes. In this case, while the resolution reaches tens of nanometers, the utility is affected by operational problems such as pore-clogging or the need to pre-select membrane pore size. A more direct method for high resolution particle characterisation is negative stain transmission electron microscopy¹⁸ (TEM). In this approach particles are adsorbed onto an EM grid and heavy metal stain applied¹¹. This method is capable of visualising details below 1 nm, as well as the size and extent of aggregation, and can reveal features such as the presence of lipid membranes. Specific molecular components may be mapped using immunogold labelling. Notably, however, this approach is reliant on adsorption of particles onto the EM grid, which may be influenced by diffusion onto the surface and inherent binding strength. Consequently, the sample retained on the substrate may not accurately reflect the properties of particles in a liquid composition. These techniques cannot, for example, be used for quantitation because the efficiency of adsorption is unknown. Although attempts have been made to overcome this problem using calibration beads applied to EM grids by centrifugation, spraying or drying along with the particles^19"24, the results have low precision, procedures are lengthy and rely on assumptions about the homogeneity of the beads and their number. Consequently, TEM has typically been viewed as both a lengthy and technically demanding method of analysing small particles, producing results of variable quality. The approach is considered to be semi-quantitative at best^13,15.

Accordingly, there remains a need to address or ameliorate one or more of the foregoing disadvantages and limitations. Summary of the Invention

According to an aspect of the invention there is provided a method of preparing an analytical sample comprising a particulate analyte, for use in microscopy, the method comprising;

preparing a liquid composition comprising an embedding medium and the particulate analyte; and

consolidating the liquid composition so as to form an analytical sample in which the particulate analyte is immobilized and dispersed within the embedding medium.

The method provides for "whole sample" microscopy. That is to say the analytical sample includes a dispersion of the particulate analyte within an embedding medium, which is representative of the particulate analyte when present in the liquid composition. Conventional microscopy samples include steps such as centrifugation and/or surface adsorption, prior to use of an embedding medium to encapsulate the surface bound particles. The method of the present invention is not susceptible to perturbations associated with these steps, which might otherwise result in the properties of the analyte in the analytical sample, such as the particle size, particle size distribution, concentration or relative concentration of particles within the particulate analyte differing from those of the analyte in the liquid composition.

The liquid composition may comprise a dispersion of the particulate analyte. The liquid composition may be colloidal. The liquid composition may comprise a suspension of the particulate analyte.

The particles of the particulate analyte may be solid or liquid particles. The liquid composition may comprise an emulsion, e.g. of the particulate analyte.

The method may comprise depositing the liquid composition (typically a portion thereof) on a substrate, such as a microscopy slide, a TEM grid, or the like. The invention extends in another aspect to a substrate, such as such as a microscopy slide or plate, a TEM grid or the like, having an analytical sample thereon, the analytical sample comprising a particulate analyte which is immobilized and dispersed within an embedding medium. The analytical sample may be formed by a method in accordance with other aspects of the invention described herein. The invention also extends to a microscope (such as an optical microscope or an electron microscope) comprising a said analytical sample supported on a substrate.

The liquid composition may be deposited by spin coating, droplet loading, spraying or the like. The liquid composition may be deposited on a substrate and a further substrate subsequently applied, to retain the liquid composition there between.

It has also been found that depositing the liquid composition on a substrate results in low levels of absorption or adsorption to the substrate prior to consolidation of the sample, which might otherwise perturb the results of quantitative or semi-quantitative microscopy of the analytical sample.

The particulate analyte may comprise microparticles and/or nanoparticles. By microparticles, we include particles or structures having at least one length dimension, and more typically two or all three length dimensions, of the order of up to 1 -100 μιη. By nanoparticles, we include particles or structures having at least one length dimension, and more typically two or all three length dimensions, of the order of up to 1 -1000 nm.

The particulate analyte may be monodisperse or polydisperse.

The particulate analyte may comprise more than one type of particle; for example a range of particles sizes, morphologies, types (e.g. type of biological particle) or compositions (e.g. chemical composition). For example, the particulate analyte may be obtained from or comprise a biological fluid such as serum, comprising a mixture of particles. The particulate analyte may comprise a mixture of particles from an aerosol.

The particulate analyte may comprise additional components, such as micro- or nano- scale structures (e.g. membranes and the like) and/or structures formed from aggregated particles. The particulate analyte may comprise particles or structure of greater than nano- or micro-scale, which may interact with smaller particles in the particulate analyte. The liquid composition may comprise a solvent, e.g. water.

The liquid composition may comprise one or more co-solvents, e.g. an alcohol or polyol co-solvent of an aqueous liquid composition. The embedding medium (and/or other components of the liquid composition) may be dissolved in a solvent.

Consolidation of the liquid composition may include any process by which the liquid composition is treated so as to form a phase in which the particulate analyte is sufficiently immobilized for the purposes of microscopic analysis.

Consolidation of the liquid composition may comprise drying (for example to remove a solvent, or a portion of a solvent), freezing, gelification, and/or curing, so as to form a solid or gel in which the particulate analyte is sufficiently immobilized for the purposes of microscopic analysis.

Consolidating the liquid composition may comprise heating. Heating may dry the liquid composition (e.g. removing some or all of a solvent). The method may comprise placing the liquid composition at a reduced pressure, so as to dry it. The method may comprise freeze-drying the liquid composition.

The embedding medium may cause the liquid composition to consolidate, when a portion (or substantially all) of the solvent is removed by drying. The embedding medium may cure (e.g. oligomerise, polymerise and/or cross link), when heated and/or irradiated (e.g. by electromagnetic radiation), so as to solidify or gelify the liquid composition.

The method may comprise lowering the temperature of the liquid composition, so as to freeze the liquid composition. A solvent (e.g. water, DMSO) may serve as an embedding medium, when the liquid composition is frozen. In some embodiments, the method may comprise contacting the liquid composition with a cold substrate, for example by spraying, such that the liquid composition freezes. Consolidating the liquid composition may give rise to an analytical sample which is disordered. For example, the embedding medium and any residual solvent may be amorphous and/or the particulate analyte may be randomly dispersed throughout the analytical sample. The analytical sample may comprise some degree of order. For example, the analytical sample may be at least partially crystalline and/or in the distribution of the particulate analyte comprises some degree of order (e.g. in terms of particle orientation).

The embedding medium may be selected according to the microscopy technique to be conducted, and/or for compatibility with other components of the liquid composition.

The embedding medium may be selected so as to be transparent or substantially transparent to the radiation used in that technique. For example, a saccharide or polysaccharide embedding medium may be substantially transparent to an electron beam used for TEM or STEM analyses.

The embedding medium may be selected for compatibility with a particular solvent and/or with the particulate analyte. For example, a hydrophilic embedding medium (e.g. a polysaccharide such as methyl cellulose) may be selected for use with an aqueous liquid composition. Similarly, a lipophilic or amphiphilic embedding medium (e.g. an organic polymer) may be selected for use with an organic solvent. For example, a polyethylene glycol embedding medium may be used with a polar organic solvent such as an alcohol or chloroform, or a water-alcohol co-solvent mixture. It may be desirable to use a cryoprotectant embedding medium, e.g. DMSO or a polyol such as ethylene glycol or polyethylene glycol, in use with biological samples, so avoid damage during freezing.

The embedding medium may be selected to avoid aggregation of the particulate analyte. For example, a polysaccharide embedding medium such as methylcellulose has been found to be particularly useful for forming stable dispersions of biological particles and for preventing aggregation before the particles are immobilised, even at significant ionic strengths; as found for example in buffered solutions, or which might result from addition of a stain.

The concentration of embedding medium within a solvent may also be selected for compatibility with the particulate analyte and/or other components of the liquid composition. For example, liquid composition having a comparatively high ionic strength (such as a biological sample in aqueous media) may promote aggregation of the particulate analyte, other particulates present in the liquid composition (e.g. a calibrant, as described below) and may promote precipitation of salts, aggregated particulates and the like.

The concentration of embedding medium may be in the range of 0.05% to around 2%, or around 0.05% to around 1 .5%, or around 0.1% to around 1 %. The concentration of embedding medium may for example be around 0.15%, 0.5% or 1 %.

By "aggregation" we include processes whereby individual particles associate to form larger structures, including association caused by weak physical (e.g. electrostatic) binding between particles. Aggregation may be reversible or irreversible and may include phenomena such as flocculation, coagulation or agglomeration. Aggregation may cause precipitation of associated particles from a liquid composition/component.

The embedding medium may be cellulose, or a derivative such as methyl cellulose.

The liquid composition may be prepared mixing together two or more components. For example, the method may comprise dispersing a solid component (e.g. a particulate analyte, or a solute such as an embedding medium) in a liquid component. The liquid component may be a solution or a solvent, for example.

Alternatively, or in addition, the liquid composition may be prepared by mixing together two or more liquid components. For example, the method may comprise mixing a solution of an embedding medium (or a liquid embedding medium) with a suspension comprising the particulate analyte. The method may comprise lowering the ionic strength of the liquid composition, or a liquid component used to prepare the liquid composition.

The method may for example comprise dialysis, electrodialysis, ion-exchange or reverse osmosis of the liquid composition or component, and the like, so as to lower the ionic strength.

The method may comprise changing the liquid conditions (e.g. by lowering or elevating temperature, changing pH or the like) so as to induce precipitation of a solute. The method may comprise removing the precipitate and subsequently returning to the previous liquid conditions.

Lowering the ionic strength includes any method by which the concentration or relative concentrations of the non-ionic components of the liquid composition, and in particular the particulate analyte, are substantially or entirely unaffected. Lowering the ionic strength may, for example, exclude diluting the liquid composition, or a decrease in ionic strength caused by a component of the liquid composition (e.g. a stain) precipitating out of solution. The ionic strength of the liquid composition/component may be reduced by contacting a first side of a semi-permeable membrane (e.g. a cellulose membrane) with said liquid composition/component and contacting the second side of the semi-permeable membrane with a low-ionic strength liquid. The low-ionic strength liquid may be based on the same solvent, or the same type of solvent as the liquid component/composition. For example, for an aqueous liquid component/composition, the low-ionic strength liquid may be water (e.g. deionised water). The liquid composition/component may be drawn into a capillary comprising or formed from the semi-permeable membrane. The method may comprise sealing one or both ends of the capillary. The method may comprise immersing at least a part, or all, of the capillary in the low ionic strength liquid. The capillary may be immersed for between around 10 minutes to around 1 hour (e.g. around 30 minutes). The method may comprise extruding the liquid composition/component from the capillary (e.g. by progressively compressing the capillary along its length). The method may comprise extruding the liquid composition onto a substrate. The method may comprise extruding the liquid composition into apparatus for deposition onto a substrate (such as a micropipette tip).

The method may comprise staining the analytical sample. The method may comprise staining the liquid composition, or a liquid component used to prepare the liquid composition. The liquid composition may comprise a stain.

The method may comprise preparing a liquid composition comprising; the embedding medium, the suspension of the particulate analyte and a stain. A stain may improve the contrast or sensitivity of microscopic imaging technique with which an analytical sample is analysed. The method may comprise the use of any suitable stain. The stain may be selected according to the microscopy technique in question and/or the particulate analyte. For example, the stain may comprise a material for absorbing a particular wavelength(s) of light. The stain may comprise a chromophore, or a heavy metal atom or cluster, for absorbing electrons.

The stain may comprise a solution of a heavy metal salt, including but not limited to a solution of a salt of uranium, tungsten, gadolinium (e.g. gadolinium(lll) chloride or acetate), samarium (e.g. samarium(lll) acetate), molybdenum (e.g. ammonium molybdate or molybdenum blue stain) or osmium (e.g. osmium tetroxide). The stain may comprise a polyoxometallate solution (e.g. of a transition metal such as tungsten, molybdenum, vanadium etc.) The stain may be a positive stain. The stain may associate with the particulate analyte (e.g. absorb into, adsorb onto and/or chemically or physically bind to particles of the particulate analyte) to a greater degree than the stain associates with other components in the analytical sample and/or the liquid composition.

The method may comprise staining the particulate analyte.

Positive staining may require use of lower stain concentrations than negative staining. For example, negative staining may require between around 1 -5% of a stain in the liquid composition, whereas positive staining require between around 0.1 -0.5% of the same stain. Accordingly, positive staining may lower the overall ionic strength of the liquid composition. Positive staining may also provide for improved imaging of analytical samples prepared in accordance with the present invention, since the consolidated embedding medium remains largely transparent.

Of course, it is to be understood that the absolute stain concentration required may vary depending on the effectiveness of the stain in terms of its visibility/contrast in use of the microscopy technique in questions and, in the case of positive staining, associating with (e.g. binding to) the particulate analyte. For example, a stain such as phosphotungstic acid has a comparatively high molecular weight and each molecular unit comprises twelve heavy metal centres, whereas a stain such as uranyl acetate has a much smaller molecular weight and only one heavy metal centre per molecular unit. Consequently, a suitable uranyl acetate stain may be present at a concentration of around 0.1 -0.5% w/v (e.g. around 0.3%) whereas a suitable phosphotungstic acid stain may be present at a concentration of around 0.03-0.2% w/v (e.g. around 0.1 %).

The method may comprise contacting the particulate analyte with the stain. The method may for example comprise contacting the stain, or a solution of the stain, with a liquid suspension of the particulate analyte. The particulate analyte may be contacted with a solution of the stain.

The method may comprise labelling the particulate analyte with the stain. The stain may be a negative stain. That is to say, the stain may associate with the particulate analyte to a lesser degree than with other components in the analytical sample, such as the solvent or the embedding medium. The stain may be a selective stain. In embodiments in which the particulate analyte comprises more than one type of particle, the stain may bind preferentially to one or more said particle types. For example, the stain may comprise a targeting moiety, antibody or the like, adapted to bind specifically to a biological target or receptor in the particulate analyte.

The inventors have found that certain stains in particular are prone to cause aggregation and/or precipitation at high ionic strengths. The method may comprise preparing a liquid composition, reducing its ionic strength and then staining the liquid composition, prior to consolidation.

Reducing the ionic strength of the liquid composition in this way is of particular utility in connection with biological samples, which, as obtained (e.g. from a subject) may have comparatively high ionic strength. Indeed a high ionic strength may be required for preservation and/or storage of a biological sample, requiring the addition of buffer solution and the like.

In another aspect, the invention extends to a method of preparing a biological sample for analysis, the method comprising;

preparing a liquid composition comprising the biological sample; and

lowering the ionic strength of the liquid composition.

Addition of further components of a liquid composition, as required for its analysis, may increase the ionic strength of the solution. Alternatively, or also, components of a liquid composition (be they particulates, or solutes, an analyte) may not be compatible with high ionic strength compositions, and for example may be prone to precipitation, aggregation and/or decomposition (e.g. in the case of micellar compositions such as liposomes). Reduction of ionic strength may mitigate against effects such as these.

The method may comprise contacting the liquid composition with a stain (i.e. staining the liquid composition), such as a heavy metal stain. A stain may be prone to precipitation and/or aggregation, in compositions having comparatively high ionic strengths, which may be prevented by lowering the ionic strength.

The liquid composition may be contacted with the stain after lowering the ionic strength.

In some embodiments, the stain may be present before the ionic strength is lowered. Lowering the ionic strength may for example cause a stain to re-solubilise. The method may comprise contacting the liquid composition with a particulate composition (such a stain or a calibrant composition). A liquid composition comprising a suspended or dispersed particulate may become unstable above a certain ionic strength. Such effects may be prevented by lowering the ionic strength of the liquid composition.

The liquid composition may be aqueous. The liquid composition may comprise one or more co-solvents.

The biological sample may take the form of a liquid composition. The liquid composition may be prepared by purifying, diluting and/or buffering a biological sample.

The biological sample may comprise a particulate analyte.

The method may comprise acquiring the biological sample, for example from a subject and/or by incubating a biological material. Biological sample may for example comprise a blood sample or a sample of another body fluid, a cell or tissue culture or the like.

The liquid composition may comprise an embedding medium. The method may comprise consolidating the stained liquid composition so as to form an analytical sample in which the particulate analyte is immobilized and dispersed within the embedding medium.

The method may provide a sample for use in microscopy. The method may comprise lowering the ionic strength for example by dialysis, electrodialysis, ion-exchange, osmosis or reverse osmosis of the liquid composition or component, and the like. The ionic strength of the liquid composition/component may be reduced by contacting a first side of a semi-permeable membrane (e.g. a cellulose membrane) with said liquid composition/component and contacting the second side of the semi-permeable membrane with a low-ionic strength liquid. The liquid composition/component may be drawn into a capillary comprising or formed from the semi-permeable membrane. The method may comprise sealing one or both ends of the capillary.

The method may comprise immersing at least a part, or all, of the capillary in the low ionic strength liquid. The capillary may be immersed for between around 10 minutes to around 1 hour (e.g. around 30 minutes).

The method may comprise calibrating the analytical sample. The method may comprise contacting the liquid composition with a particulate calibrant in a known amount or concentration, so as to calibrate the liquid composition and thereby calibrate the analytical sample.

The method may comprise preparing a liquid composition comprising the embedding medium, the particulate analyte and a particulate calibrant at a predetermined concentration.

A particulate calibrant may for example comprise a colloid, for example of inorganic colloidal particles (e.g. metal or metal oxide particles, such as colloidal gold) or organic colloidal particles (e.g. a hydrocolloid formed from polymeric particles, or a micellar solution). The particulate calibrant may comprise particles/beads of an organic polymer (e.g. polystyrene) or an inorganic polymer (e.g. silica). Calibration of the analytical sample in this way enables the quantitation of particulate analyte concentration(s) based on the ratio of analyte:calibrant particles present in the analytical sample. The particulate calibrant may be monodisperse.

The method may comprise contacting the particulate analyte (or a liquid component comprising the particulate analyte) with a calibration composition comprising a known concentration of the particulate calibrant.

The calibration composition may comprise a stain. Thus, staining and calibrating may be conducted in the same step.

The calibration composition may comprise the embedding medium and/or a solvent (noting that other liquid components from which the liquid composition is formed may also comprise the embedding medium).

The method may comprise preparing a calibration composition comprising;

a known concentration of the particulate calibrant;

an embedding medium; and

optionally, a solvent and/or a stain.

The liquid composition, or liquid components used to prepare the liquid composition, may comprise one or more further components, such as one or more surfactants (to stabilise a suspension or dispersion), or ionic salts (to buffer the pH of a solution), solvents or co-solvents.

The various components of the liquid composition may be brought together in any order, as required.

In another aspect, the invention extends to a liquid calibration composition for use in quantitative or semi-quantitative microscopy, comprising;

a known concentration of a particulate calibrant;

an embedding medium;

and, optionally, a solvent and/or a stain. The invention also extends in a still further aspect to a method of preparing a liquid calibration composition for use in quantitative or semi-quantitative microscopy, comprising;

a known concentration of the particulate calibrant;

an embedding medium; and

optionally, a solvent and/or a stain.

The calibration composition may be prepared in bulk quantities, which may facilitate consistent calibration across multiple analytical samples.

The calibration composition may comprise a dispersion of the particulate calibrant. The calibrant composition may be colloidal. The calibrant composition may comprise a suspension of the particulate calibrant.

The calibration composition may be adapted to have a long shelf life. The inventors have found, for example, that stable aqueous suspensions of certain metal clusters, such as gold, may be formed in the presence of embedding media such as a polysaccharide (e.g. methylcellulose). Moreover, such suspensions may be stable in the presence of other species, such as stains, buffers or other species which might increase the ionic strength of the calibration composition.

The method may comprise calibrating the calibration composition. The calibration composition may be calibrated gravimetrically (e.g. based on the mass of particulate calibrant, or changes in mass of a composition to which a particulate calibrant is added), spectroscopically (e.g. based on optical properties such as absorption, transmission or scattering of light by a calibrant composition, or a composition to which a particulate calibrant is added), or microscopically (e.g. by counting the number of calibrant particles within a predetermined volume of a sample, using a microscopy technique).

The method may comprise systematic uniform random sampling (which may also be known as systematic sampling) of the calibration composition. The method may comprise depositing the calibration composition on a substrate and/or consolidating the calibration composition, generally as described above in relation to the liquid composition. The method may comprise depositing a predetermined volume of the calibration composition on a substrate.

In one embodiment, the method comprises systematically sampling the consolidated calibration composition by microscopy (e.g. TEM). The consolidated calibration composition may be systematically sampled by counting calibrant particles in randomly selected sampling areas. The calibration composition may then be calibrated based on the ratio of the total area sampled to the total area of the consolidated calibration composition on the substrate and the predetermined volume of calibration composition deposited on the substrate.

The method may comprise counting calibrant particles along a line (or multiple lines) across the consolidated calibration composition on the substrate.

A sampled area may fall within an acceptance distance of a said line.

Counting may comprise applying an acceptance criterion or criteria. For example, counting may comprise including calibrant particles falling entirely and/or partially within a sample area (e.g. within an acceptance distance of a line). Counting may comprise excluding calibrant particles falling entirely and/or partially outside of a sample area (e.g. the acceptance distance). In yet another aspect of the invention, there is provided a method of analysing a particulate analyte, comprising preparing an analytical sample accordance with the method of other aspects of the invention, and analysing the analytical sample using a microscopy technique. The microscopy technique may be an electron microscopy technique such as TEM or STEM or scanning electron microscopy. The microscopy technique may by an optical microscopy technique such as UV, superesolution or X-Ray microscopy.

The microscopy technique may be a transmission, reflection or scattering microscopy technique. The microscopy technique may comprise an optical absorption or emission technique, such as fluorescence microscopy. The method may comprise calibrating the analytical sample, for example by providing a particulate calibrant, as described above.

The method may comprise depositing the liquid composition (typically a portion thereof) on a substrate, such as a microscopy slide, a TEM grid, or the like.

The method may comprise microscopically analysing nano- and/or micro-particles.

The method may comprise quantitatively or semi-quantitatively analysing one or more properties of the particulate analyte.

The method may comprise systematically sampling the analytical sample.

The method may comprise measuring the relative concentrations of different particle types of the particulate analyte. The method may comprise measuring the absolute concentration of particles in the particulate analyte.

Measurement of absolute concentrations of particles in the particulate analyte may comprise determining the ratio of analyte:calibrant particles present in the analytical sample.

The analytical sample may be systematically sampled by counting particles in randomly selected sampling areas, for example to determine the ratio of analyte:calibrant particles present. The absolute concentrations of particles in the particulate analyte may then be calculated based on a knowledge of the absolute concentration of the calibrant particles present.

The method may comprise counting particles along a line (or multiple lines) across the analytical sample on the substrate. A sample area may fall within an acceptance distance of a said line. Counting may comprise including particles falling entirely and/or partially within the acceptance distance. Counting may comprise excluding particles falling entirely and/or partially outside of the acceptance distance.

The method may comprise a method of diagnosis, treatment or prophylaxis of a subject. The presence of certain types of particle or changes in the concentrations or relative concentrations of certain types of particles in an analytical sample comprising a biological analyte may be indicative of a medical or veterinary condition. Accordingly, the method may comprise for example acquiring a biological sample from a subject, preparing an analytical sample therefrom, in accordance with aspects of the invention, and analysing the analytical sample, typically quantitatively or semi-quantitatively. Further preferred and optional features of each aspect of the invention correspond to further preferred and optional features of each other aspect of the invention.

Description of the Drawings These and other aspects of the present invention will now be further described by way of example only, with reference to the following figures in which:

Figure 1 schematically shows the preparation of analytical sample of a particulate analyte in a methylcellulose embedding medium, for TEM analysis.

Figure 2 shows example TEM images of analytical samples prepared in accordance with the method of Figure 1 . Figure 2 (b)-(e) show a range of nanoparticles imaged at low magnification and Figure (f)-(i) show images at high magnification. Scale bars of images (b-e) are 100 nm and the scale bars of images (f-i) are 50 nm. The type of nanoparticles are indicated in the Figure.

Figure 3 shows surface concentration of liposomes on a TEM grid. Liposomes were incubated in aqueous conditions with and without 1 % methylcellulose. Each solution was deposited onto a TEM grid and each grid was washed with deionized water. Following washing each grid was stained using a 1 % methylcellulose and 0.3% uranyl acetate solution and imaged. Particle counting was conducted of the TEM images to determine the surface density of liposomes.

Figure 4 shows a schematic cross section of (a) asymmetric particles on a substrate, encapsulated in a thin layer of a negatively stained embedding medium; (b) positively stained asymmetric particles on a substrate in a thicker layer of an embedding medium.

Figure 5 shows orientation analyses of analytical samples of a nanodisc analyte. Liquid compositions of the nanodiscs prepared having methylcellulose concentrations of 0.1 % to 0.5%, and a uranyl acetate stain. 0.5 μΙ droplets of each composition were loaded onto EM grids and allowed to dry. TEM images were acquired of each analytical sample and orientation classified into three categories; (a) flat (b) tilted and (c) upright. Relative concentrations of each classification are plotted against methylcellulose concentration in the figure.

Figure 6 schematically depicts the calibration of a calibrant composition. Total numbers of gold particles per millilitre were estimated by systematic sampling at the hexagon grid corners as shown. Figure 7 shows the results of the calibration of a gold nanoparticle calibration composition, 15 nm diameter gold particles were prepared using the citrate reduction method of Frens³⁰ and stored in 1 % MC. Droplets were applied to pioloform EM support grid films. Total numbers of gold particles per millilitre were estimated by systematic sampling at the hexagon grid corners as shown schematically in Figure 6. The total number of gold particles is estimated by multiplying the sampled gold particles with the ratio of total droplet area to sampled area (both estimated using point sampling stereology³⁴). Each data point is from a separate loading experiment (n = 8, error bar standard error of the mean). Figure 8 shows the quantification of nanoparticle concentrations using gold particle calibration, (a) During scanning the green arrow tip traces an acceptance line and the red arrow tip a forbidden line. All particles (liposomes in this case) entirely included in the scanning band (bracket) and those crossed by the acceptance line (green arrow tip), were sampled/counted. All particles crossed by the forbidden line (red arrow tip) were excluded (examples marked by and X respectively), (b) Single equatorial scans match closely estimates for gold/NP ratio obtained from multiple scans (multiple scans 283 to 467 particles counted in total; single scan, approximately 100 particles counted). Counts from the same grid compared (n = 3 in each case, mean value and bars standard error of mean). Scale bar, 500 nm.

Figure 9 shows results of quantitative TEM determination of liposome concentration and size distribution, (a) Scaling between liposome dilution and estimates of liposome particle number. In each case, approximately 100 each of gold calibration and liposomes were counted (time for each scan 10-15 minutes). Values are means of 4 independent experiments and error bars are coefficients of variance, (b) Liposome size distribution. Liposomes were sampled using a scanning band and sized by a line intersection estimator. Data are from three experiments (approximately 100 vesicles counted per experiment; error bars are standard error of the mean). Figure 10 shows the steps of a microdialysis procedure to reduce ionic strength, using semipermeable cellulose kidney dialysis tubing, (a) The sample was drawn into the straw by capillary action, (b) Ends of the straw were sealed using an assembly made from interlocking truncated micropipette tips, (c) The assembly was transferred into the dialysate. (d) The dialyzed sample was extruded onto parafilm and could then be added into the staining mix before loading and drying of a 0.5 μΙ droplet on an EM grid.

Figure 1 1 (a) shows results of quantification of Influenza A virus concentration. The virus preparation was mixed with gold/MC calibration solution, dialyzed by the microdialysis method (see text) and stained using a MC/UA calibration composition. Estimates of virus particle number were compared with plaque forming units, determined on the same samples (n = 3; error bars, standard error of the mean).

Figure 1 1 (b) shows an example TEM image of isolated, non-aggregated gold nanoparticles (arrows) and virus particles. Scale bar, 100 nm.

Figure 12 depicts an example embodiment of a method in accordance with the invention. A gold/methylcellulose (Au/MC) calibration composition is mixed with an NP containing component to form a calibrated liquid composition. Optionally, the composition is dialyzed in microdialysis straws to lower ionic strength. The composition (or dialysate) is mixed with a heavy metal stain (e.g. uranyl acetate) and a portion deposited onto a substrate (e.g. a plastic coated TEM support grid) for counting, sizing or further characterization by microscopy.

Detailed Description of Example Embodiments

In an embodiment, suspensions of NPs were stably incorporated into a thin film of hydrophilic embedding medium containing heavy metal stain along with a recognizable calibration particle for counting. The embedding medium was methylcellulose (MC). MC has previously been used for embedding samples such as ultrathin cryosections^{25 27} and has also been employed to visualize NPs which have been preadsorbed to a substrate such as an electron microscope (EM) support²⁸. Since preadsorption cannot be used for quantitation (because of unknown adhesion efficiencies, as described above) the inventors have developed a new way to prepare a sample comprising a NP analyte.

NPs were mixed with MC prior to staining with uranyl acetate (UA).

A liquid composition comprising 1 .5 μΙ of 1 % methylcellulose (MC), 1 μΙ of an 0.3% uranyl acetate (UA) stain and 7.5 μΙ of nanoparticle suspension was prepared. 0.5μΙ of the liquid composition was loaded onto standard pioloform coated EM grid support and allowed to dry before examination in the TEM. The preparative steps are shown schematically in Figure 1 . Figure 2 shows example TEM images of analytical samples prepared in accordance with this method. In this context, uranyl acetate provided excellent display of structural details including membranes of liposomes, capsids of viruses and the rim regions of nanodiscs, as shown in Figure 2(b)-(i). The uranyl acetate concentration before drying was around ten times more dilute than that used for section contrasting²⁷. The images reflect "positive staining" of the particulate analyte.

It is postulated that the staining mechanism may involve local adsorption of the heavy metal stain onto the NPs. Positive and negative staining may yield complimentary structural information. For example, in some circumstances, TEM acquired using the positive staining method may show structural information in relation to the particulate analyte (e.g. of biological structures) not normally visible using more conventional "negative" staining (in which biological structures are highlighted by surrounding lakes of stain). In the examples shown the Figure 2, membranes were clearly visible around the liposomes.

It has also been found that MC as an embedding medium gives rise to comparatively small drying artefacts. A tilting analysis of particles embedded in 0.15% MC, in which particle height from the substrate is calculated trigonometrically, revealed compression after drying of vesicles of as little as 30% of the initial particle height.

The present invention, in which the particulate analyte is provided in a liquid composition together with the embedding medium is also compatible with cryogenic electron microscopy, in which the liquid composition is consolidated by freezing. This technique has the capacity to further reduce or entirely eliminate drying artefacts and, in addition, provide "snapshots" of processes occurring in the liquid phase, such as assem bly/d isassem bly²⁹.

As mentioned above, a further advantage of the present invention is the provision of images representative of all of the particles in the NP population. This is essential, for example, for accurate and precise quantitation. The mix of NP/MC and uranyl acetate provided consistent display of particles and an optimum final concentration of MC 0.15% ensured display particles from edge to centre of the droplet, without obscuring or loss of particle profiles.

This was in contrast to conventional negative staining in which large areas of the preparation were concealed by excessive contrast/stain.

A reduced tendency for the nanoparticles to aggregate or bind to a substrate has also been observed. The low level of adsorption to the substrate was demonstrated by quantifying the residual binding of absorbed particles in the absence or presence of the embedding medium, MC. A liquid composition comprising the embedding medium and particulate analyte was deposited on a substrate (a standard TEM grid) and then the substrate was washed with deionized water. A TEM grid was also prepared by depositing an otherwise identical composition lacking the MC embedding medium and then washing.

After washing, the each of the TEM grids were re-stained in MC/uranyl acetate and imaged, to look for bound particles. It was observed that MC prevented binding to the support film by 96.1 % (Figure 3).

The ability of MC to maintain NPs in suspension during drying raised the possibility of controlling the orientation of asymmetric NPs before imaging. One problem with negative staining is the analytical sample must be extremely thin, or the stain within the embedding medium may obscure the analyte. Such analytical samples may select for or restrict the orientation of asymmetric particles, which tend to lie along their longest axis (illustrated in Figure 4a). This effect reduces available spatial information.

The present method results in MC films with a final thickness of approximately 200 nm, with the particulate analyte distributed throughout the layer. This contrasts with convention techniques which result in far thinner MC films (by approximately 1 to 2 orders of magnitude), in which the nanoparticles preferentially bind to the substrate.

This additional sample thickness possible using the methods described herein is sufficient to accommodate all orientations of asymmetric particles (illustrated in Figure 4b). In addition, positive staining may improve the visibility of analyte particles in thicker samples.

This effect was investigated by acquiring images of analytical samples prepared using a range of different MC concentrations, to probe the effect of drying over on the orientation of asymmetric NPs (12 nm diameter nanodiscs).

All other factors being equal, liquid compositions comprising a lower concentration of the embedding medium resulted in the formation of thinner analytical samples, after drying. It was observed that the thinnest analytical samples, which were prepared using liquid compositions comprising 0.1 % MC, favoured enface orientations. Thicker samples, where were prepared using up to 0.5% MC, favoured tilted or upright orientations (Figure 5). The apparent freedom of these particles to orient during drying is additional evidence for restricted binding to the support film in the presence of MC.

Calibration of the liquid composition, and quantitative analysis of particle concentrations will now be described. In order to determine absolute NP concentrations, 15 nm gold particles at a known concentration (prepared with the citrate reduction method³⁰) were incorporated into the liquid composition (the MC/NP/stain mix). In alternative embodiment, other calibrants may be used. Ratio counts between gold and the NPs could then be used to determine NP numbers.

A gold calibration composition was prepared in 1 % MC. Inclusion of an embedding medium in the calibration composition, which was found to restrict cation-induced aggregation of the particulate calibrant (the 15 nm gold particles) in standard assays.³¹

Furthermore, improved compatibility of the gold calibration composition with salts found in laboratory buffers and body fluids was observed.

The gold/MC calibration composition was prepared in large volumes and can be stored indefinitely at 4°C.

To calibrate the calibration composition (i.e. to determine the concentration of gold particles in the calibration composition), the gold particles were stained with uranyl acetate prior to loading 0.5 μΙ of the mix onto a EM grid. The dried circular droplets were then sampled exhaustively using systematic uniform random sampling³² (SURS) and the nanoparticle number in the experimental 'population' was determined using a fractionation principle in which the number of sampled gold particles is multiplied by the ratio of total droplet area to sampled area (as shown schematically in Figure 6). The concentration of gold particles was found to be 4.4 x 10¹⁰ particles ml^"1 (CE, 6.47%; n=8), as shown in Figure 7. The 15nm gold particle calibration composition was used to determine liposome concentrations by ratio estimation (by the method illustrated in Figure 8). Liposome preparations are in widespread use for drug/reagent delivery and as carriers for study of protein and lipid function³³. Liposomes were mixed first with MC/gold calibration solution and then uranyl acetate, before loading 0.5 μΙ onto support grids. In this system neither liposomes nor gold particles showed evidence of aggregation and they did not interact substantially with each other. Using a live digital camera display we used unbiased counting rules³⁴ applied to a scanning band, the width of which was reduced to balance the counts at approximately 100 for each type of particle (Figure 8a).

The gold/NP ratio was determined using multiple scans positioned in a systematic uniform random pattern and the ration used to calculate liposome concentration (Figure 8b).

Next, a single scan across the grid equator was conducted (Figure 8b). The equatorial scan took 10 minutes and results were found to be within 1 .7% of those obtained from multiple scans.

Results are shown in Figure 9, together with results of size distribution analysis. The same liposome preparation was investigated across a range of dilutions that covered approximately 4 orders of magnitude. The average coefficient of variation was 6.3% but at the lowest concentrations ranged from 1 .9 to 4.1 % (Figure 9a). Scaling was strikingly close to linear (R² = 1 ; p < 0.001 ).

To assess the sensitivity we took another scanning approach, which included areas of the droplet with the thickest film and highest concentration of nanoparticles. On this basis and counting 50 particles each of gold and nanoparticles and limiting the time taken to 60 minutes, we found a minimum estimable concentration of nanoparticles to be approximately 5 x 10⁶ ml^"1.

NP size distributions were also evaluated on these samples. Liposomes were again selected using unbiased counting rules applied to a scanning band (Figure 9b). Selected liposomes were then translocated through a systematic spaced series of dots placed across the digital camera display. The number of intercepts of each NP with the dot edges was used to estimate the NP caliper diameter (number of intercepts x spacing of the dot edges = estimated caliper diameter). Random placement of the object or the line array ensured unbiasedness of the estimator. For each preparation results from 100 liposomes were grouped into ten 30 nm size bands (Figure 9b). Evaluation of each liposome preparation took 10 minutes and over three experiments the average coefficient of error for size estimates over all size bands under 210nm was 8%. The distributions from the three experiments were not distinguishable as determined by contingency/Chi square (p > 0.25, df 16) or by correlation analysis (possible pairs of frequency distributions each had R² > 0.81 , p < 0.005, n = 10).

As discussed above, in some circumstances it may be beneficial or required to reduce ionic strength of the liquid composition, or of a component from which it is prepared. Ionic strength reduction by way of microdialysis will now be described.

Many NP preparations are suspended in buffers, culture supernatants or body fluids. These contain salt and phosphate ions which can crystallize or cause precipitation of certain components of a liquid composition, such as a heavy metal stain.

Figure 10 shows a method of reducing the ionic strength of a composition, in which ionic species are removed by microdialysing with fluids such as deionized water. The method employs cellulose dialysis straws having an internal diameter of approximately 200 microns and a molecular weight cut-off of 10,000 Da.

A liquid composition including a NP analyte, a gold nanoparticiculate calibrant and a methylcellulose embedding medium was dialyzed against deionized water before mixing with heavy metal stain. Three to five microliters of the liquid composition was loaded into the straws by capillary action before sealing of the straw ends using truncated micropipette tips (although alternative means for sealing the straws may also be employed). After 30 minutes' dialysis, the dialysate was extruded onto parafilm by drawing the straw between the parafilm and a sharp instrument such as an edge of forceps blades or a scalpel. Heavy metal stain added before loading onto an EM grid and quantitation by ratio counting on a single equatorial scan.

After dialysis neither gold particle nor NP analyte (virus) aggregation was observed. The microdialysis procedure therefore allows microliter samples containing NPs to be processed for quantitation. These samples could include for example body fluids, culture supernatants or samples containing buffers.

To test the feasibility of the dialysis procedure, NP concentrations in three preparations of influenza virus particles were measured. This was done using single equatorial scans and ratio counts of approximately 100 gold and 100 virus particles, with each count requiring between 5 and 10 minutes. Results are shown in Figure 1 1 .

Infectivity was also assayed in parallel using plaque forming unit (PFU) assays, allowing the particle to infectivity ratio to be determined. The particle/infectivity ratio was estimated to be 69.9:1 (coefficient of variation for particle number was 15.4% and for PFU was 47.5%; showing particle number estimates varied less than PFU for a particular virus particle preparation). The new method improves greatly on the widely used haemagglutination (HA) assay³⁵ which is not only indirect, but is also well known to be imprecise, and relies on assumptions about thresholds of particle aggregation³⁶. Our estimates of virus particle number were much more precise and rapid compared to the HA assay³⁸ and suggest the ratio of particles to infective units might be substantially higher than previous estimates³⁷.

Figure 12 schematically depicts an example experimental protocol in which a particulate analyte liquid component is calibrated (by contacting with a particulate calibrant), dialysed, so as to lower ionic strength, then mixed with a stain/embedding medium, deposited on a substrate and consolidated, for example by drying, to form an analytical sample, suitable for quantitation using microscopy.

In conclusion disclosed herein is a rapid, accurate and precise method for counting and sizing nanoparticles, improving greatly on widely used biophysical methods. The embedding medium provides for a liquid composition comprising NPs in homogeneous suspension or dispersion, which may be consolidated to form a uniform dispersion of the NPs in the resulting analytical sample.

Whilst, in the embodiments described above, images were analysed manually, automated feature-recognition image analysis may be employed, to remove reliance on operator-based counting/sizing, for example using algorithms as described by Wang, R., Pokhariya, H., McKenna, S.J. and Lucocq, J. Recognition of immunogold markers in electron micrographs. J. Struct. Biol. 2011 , 176, 151 -158 (reference 38). The methods may also include specific labelling of molecular components of a particulate analyte, which is a powerful method for categorizing nanoparticles¹³ and/or membrane bound vesicles³⁹. Affinity labelling techniques using multiple sizes or shapes of electron dense markers⁴⁰ provide for correlation of size or detailed structure with molecular composition.

Compared to particle tracking analysis, the method improves sensitivity by two orders of magnitude and reduces sample size from 100s to just a few microliters.

By incorporating microdialysis, microliter samples from culture supernatants or body fluids such as tears, joints, body cavities or central nervous system, can be analysed. Preparation, counts or size distributions take a few minutes and require little training, and use standard electron microscopy to provide nanometre resolution of structural features. The method facilitates direct analysis of tiny samples of synthetic and naturally occurring NPs widely used in laboratory studies and theranostics.

Whilst certain specific embodiments have been described, the various variations may be possible without departing from the spirit and scope of the invention. For example, the method and compositions may be applied to analysis of microparticles. Alternative particulate calibrants and/or stains may be employed. The method may be used to characterise non-biological analytes, or particulate analytes dispersed/suspended in non-aqueous media. Moreover, the invention is suitable for use with a variety of microscopy techniques. Experimental procedures

2% w/v methylcellulose (25 centipoise, Sigma Aldrich, Dorset, UK) was prepared as described in Griffiths et al²⁷. Gold particles were prepared by citrate reduction according to Frens³⁰ and stored at 4°C. To make the calibration solution, freshly made colloid was mixed 1 :1 with 2% methylcellulose. To remove citrate and sodium ions, the mix was subsequently centrifuged for 30 min at 2,000 x g at 4°C and the sedimented particles resuspended in fresh 1 % methylcellulose. Nanodiscs were prepared as described in Ward⁴¹ , and liposomes according to Marius⁴². Standard plastic support films were made using 1 % pioloform (Agar Scientific, Stansted, Essex, UK) and copper hexagonal 100-mesh grids applied to the film and picked up using a sheet of parafilm followed by air drying.

Stain mixes and application to EM grid

Basic NP staining mixture (without incorporating gold calibration or pre-dialysis) contained 1 .5 μΙ of 1 % methylcellulose mixed with 7.5 μΙ of nanoparticle solution and 1 μΙ of 0.3% uranyl acetate (values are given in μΙ to illustrate minimal volumes but larger volumes were also used in proportion). Alternatively, freshly prepared 0.1 % phosphotungstic acid (w/v) was used instead of uranyl acetate. When calibration was required, 1 .5 volumes of gold particle-calibration solution (containing 1 % methylcellulose) were added to 1 .5 volumes of nanoparticle solution, 1 volume of 0.3% uranyl acetate and 6 volumes of deionized water. When dialysis was required, equal volumes of calibration solution and nanoparticle solution were mixed and capillary dialysis carried out as detailed below. To load droplets of staining-mix, the grids were held in self-closing fine tipped forceps and were loaded, under binocular control, with 0.5 μΙ of staining-mix using a standard 2 μΙ micropipette. Another effective way is to transfer 0.5 μΙ droplets to grids that have not been separated from the parafilm used to pick up the grids/pioloform film. It was important that the stain mix was kept on ice prior to droplet loading to reduce the viscosity of the methylcellulose and thereby facilitate accurate pipetting of microliter volumes. The grids were then air-dried at ambient temperature and examined in a JEOL 1200 EX transmission electron microscope operated at 80kv and imaging carried out using a GATAN Orius 200 digital camera (GATAN, Abingdon, Oxon, UK) Analogous stain/nanoparticle mixes were prepared using ruthenium, samarium, molybdenum and gadolinium stains (all at final concentrations between 0.005 and 0.05 % w/v). Capillary tube dialysis

The renal hemodialysis cassette contains a large number of parallel capillary straws and these were best extracted by pulling a few of them from the bundle, using a pair of forceps. If the tubes were kept straight without kinks, aspiration of the nanoparticle calibration mix into the capillary tube was rapid and consistent. Immediately after drawing it out from the cassette, the straw was cut with a clean scalpel at each end. Then each end was passed through the narrower aperture of a truncated micropipette tip and the mix aspirated by capillary action. This was done quickly to prevent drying of the mix inside the tube. To seal the tube another pipette tip was then inserted into the wider aperture of the first tip in order to trap the capillary. The whole assembly was subsequently immersed in a dialyzing solution. After dialysis the assembly and capillary tube was rapidly dried using filter paper and one-end of the capillary cut with a fresh curved scalpel blade. The tube was then laid down on a parafilm sheet and a pair of curved forceps was drawn along the length of the tube to expel a drop of the dialysate onto the parafilm. The drop was retrieved using a micropipette and stored in an Eppendorf centrifuge tube at 4°C, until use.

Counting and Sizing procedures These procedures were carried out directly on a digital camera display. Counting was done by manual translocation of the specimen in a single direction and particles selected using unbiased counting rules applied to the edges of a counting band defined on the digital camera display. The edges of the band were defined by two spots positioned inside opposite sides of the image frame, creating a guard area outside the counting band. The guard area allowed morphological characterization or size estimation even of the largest nanoparticles. During scanning one edge of one of the spots defined an acceptance line and the edge of the other spot defined a forbidden line. Any particle that fell completely within the lines, or was crossed by the acceptance line, was sampled/accepted for counting. Any particle that intersected the forbidden line was not sampled or accepted for counting⁴³. To evaluate the influence of methylcellulose on the binding of nanoparticles to the support film, liposomes were mixed 1 :1 with either 2% methylcellulose or deionized water and the mix allowed to adsorb onto an EM support grid for 2 minutes. The grids were washed three times on droplets of deionized water (2 minutes per step) followed by contrasting with 3% (w/v) uranyl acetate/ 2% methylcellulose in a ratio of 1 :9; prior to air drying in a wire loop (according to Griffiths et al²⁷). The number of liposomes per unit area was assessed in micrographs taken systematic random at 2,000 x. Counting of liposomes was carried in rectangular quadrats measuring 2.1 μηι x 2.1 μιη to which unbiased counting rules were applied³ and counts related to the area.

For estimating particle caliper diameter, a systematic series of dots with regular spacing was placed orthogonal to the travel direction of the microscope stage. The spacing was set to about half the size of the smallest nanoparticle and the extent of the array was greater than the width of the counting band so that any peripheral particles sampled by the counting band were sized. Consistently, one extreme edge of each dot was assigned for counting intersections and the number of edges that crossed each particle during translocation of the specimen was counted. An unbiased estimate of the particle caliper distance is I x d where I is the number of intersections and d the real spacing between the dots. The counting can be done during scanning and the particle sizes categorized into the size groups based on the number of intersections and plotted using Microsoft Excel.

To compare a random point process to the distribution of calibration nanoparticles a series of 20 SURS micrographs were collected for each nanoparticle (magnification 3000 x for liposomes and 4000 x for influenza A). The images were opened in Photoshop CS6 and gold particle counts made using the forbidden line unbiased counting rule as described above (quadrat areas 9.49 μιη² ίθΓ liposomes and 6.15 μιη² for influenza A). The gold calibration particles were categorized as being localized over empty parts of the support film or associated with the nanoparticles (particles less than one particle width distant from the nanoparticle were classified as associated). For the distribution of random points the same micrographs were overlaid with a randomly placed square systematic grid lattice (spacing 2.05 μιη for liposomes and 1 .55 μιη influenza A) resulting in 1 to 4 random points per grid placement. The random points were categorized according to the same criteria used for the calibration particles. The process of random grid placement was repeated until an equivalent number of random points compared to gold particles for the corresponding micrograph was reached.

To assess the linearity of nanoparticle numbers across different concentrations, a dilution series of liposomes was prepared in deionized water using 10 μΙ of neat liposome solution for every dilution (1/50, 1/100, 1/200 and 1/1000). For each sample 4 staining mixes were prepared and 0.5 μΙ per mix loaded onto individual EM grids. The grids were then quantified and the liposome number for each sample estimated (see above) and plotted in Microsoft Excel.

Cells, Virus and Plaque Assays

MDCK and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, supplemented with 10% fetal calf serum; Invitrogen, Paisley, UK) at 37 °C in a 5% C0₂ atmosphere. Influenza A/WSN/33 wild-type (rWSN wt) was generated using plasmid-based reverse genetics as previously described⁴⁴. Briefly, 293T cells were transfected with eight virus genome-encoding plasmids (pHH-21 -based; encoding genes under the control of the human RNA polymerase I promoter) and four protein expression plasmids encoding the genes for the viral polymerase subunits (PB1 , PB2, PA) as well as nucleoprotein (NP) under the control of a CMV polymerase II promoter. At 16 h post-transfection the cells were co-cultured with MDCK cells in serum-free DMEM containing 2.5 μg/mL N-acetyl trypsin (Sigma Aldrich, Dorset, UK). Virus- containing supernatant was harvested three days post-transfection, viruses propagated twice through MDCK cells followed by plaque assay titration on MDCK cells. MDCK cells in six-well plates were infected with serial 10-fold dilutions of each virus in serum- free DMEM for 1 h at 37°C. Cells were overlaid with DMEM-1 % agarose supplemented with 2 μg/ml N-acetyl trypsin and incubated at 37°C for 48 h. Cells were fixed in 5% formaldehyde for 1 h at room temperature. Plaques were visualized by immunostaining as previously described⁴⁵. Adeno-associated virus was prepared according to McClure et al⁴⁶.

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Claims

CLAIMS:

1. A method of preparing an analytical sample comprising a particulate analyte, for use in microscopy, the method comprising;

2. A method according to claim 1 , comprising depositing the liquid composition on a substrate.

3. A method according to claim 1 or 2, wherein the liquid composition comprises microparticles and/or nanoparticles.

4. A method according to any preceding claim, wherein the liquid composition comprises a solvent;

the embedding medium; and/or

optionally one or more other components of the liquid composition, is/are dissolved in the solvent.

5. A method according to claim 4 wherein the solvent is water.

6. A method according to any preceding claim, wherein the embedding medium is a polysaccharide such as methylcellulose.

7. A method according to any preceding claim, wherein consolidation of the liquid composition comprises one or more of:

drying, freezing, gelifying, curing, irradiating, heating, placing the liquid composition at reduced pressure;

so as to form a solid or gel in which the particulate analyte is sufficiently immobilized for the purposes of microscopic analysis.

8. A method according to any preceding claim, comprising lowering the ionic strength of the liquid composition, or of a liquid component used to prepare the liquid composition.

A method according to claim 8, comprising dialysis of the liquid composition component.

A method according to claim 9, comprising drawing the liquid composition or component into a capillary comprising or formed from a semi-permeable membrane; sealing one or both ends of the capillary; and immersing at least a part of the capillary a the low ionic strength liquid.

1 1. A method according to any preceding claim, comprising staining the liquid composition, or a liquid component used to prepare the liquid composition.

12. A method according to claim 1 1 , comprising preparing a liquid composition comprising; the embedding medium, the suspension of the particulate analyte and a stain.

A method according to claim 1 1 or 12, wherein the stain comprises a solution of a heavy metal salt, including but not limited to; a solution of a uranium salt or a tungsten salt.

A method according to any one of claims 1 1 -13, comprising positively staining the particulate analyte.

15. A method of preparing a biological sample for analysis, the method comprising; preparing a liquid composition comprising the biological sample;

lowering the ionic strength of the liquid composition; and

contacting the liquid composition with a stain.

16. A method according to claim 15, wherein the biological sample comprises a particulate analyte, and/or wherein the liquid composition comprises an embedding medium.

17. A method according to any preceding claim, comprising contacting the liquid composition with a particulate calibrant in a known amount or concentration, so as to calibrate the liquid composition and thereby calibrate the analytical sample.

18. A method according to any preceding claim comprising preparing a liquid composition comprising the embedding medium, the particulate analyte and a particulate calibrant at a predetermined concentration.

A method according to claim 17 or 18, wherein the particulate calibrant comprises colloidal metal or metal oxide particles.

A method according to any one of claims 17 to 19, comprising contacting the particulate analyte, or a liquid component comprising the particulate analyte, with a calibration composition comprising a known concentration of the particulate calibrant and optionally a stain.

A method according to claim 20, wherein the calibration composition comprises an embedding medium and/or a solvent and/or a stain.

A method of preparing a liquid calibration composition for use in quantitative or semi-quantitative microscopy, comprising;

a known concentration of a particulate calibrant;

an embedding medium; and

optionally, a solvent and/or a stain.

A method according to any one of claims 20 to 22, comprising calibrating the calibration composition.

A method according to claim 23, comprising depositing a predetermined volume of the calibration composition on a substrate, optionally consolidating the calibration composition; and

systematically sampling the consolidated calibration composition by microscopy.

A liquid calibration composition for use in quantitative or semi-quantitative microscopy, comprising;

a known concentration of a particulate calibrant;

an embedding medium;

and, optionally, a solvent and/or a stain.

A method of analysing a particulate analyte, comprising preparing an analytical sample in accordance with the method of any one of claims 1 -21 and analysing the analytical sample using a microscopy technique.

A method according to claim 26, wherein the microscopy technique is an electron microscopy technique such as TEM or STEM.

A method according to claim 26 or 27, comprising systematically sampling the analytical sample.

A method according to claim 28, comprising measuring the relative concentrations of different particle types of the particulate analyte.

A method according to claim 28 or 29, wherein preparing the analytical sample comprises contacting the liquid composition with a particulate calibrant in a known amount or concentration, wherein the method of analysing the particulate analyte comprises measuring the absolute concentration of particles in the particulate analyte.

A method according to any one of claims 26-30, comprising counting particles along a line, or multiple lines, across the analytical sample on a substrate.

A method according to claim 31 , wherein counting comprises including particles falling entirely and/or partially within the acceptance distance and excluding particles falling entirely and/or partially outside of the acceptance distance.

33. A substrate, such as such as a microscopy slide or plate, or a TEM grid, having an analytical sample thereon, the analytical sample comprising a particulate analyte which is immobilized and dispersed within an embedding medium.