Analytical methods and related compositions and uses

Abstract

The present invention relates to methods for characterising particulate organic carbon in a sample, for example to quantify the amount of particulate organic carbon present in a sample comprising one or more cells or organisms, such as to quantify protein, lipid, or carbohydrate constituents within a sample. More particularly, the invention relates to methods for characterising particulate organic carbon and / or one or more constituents thereof in one or more cells, such as one protist, fungal, yeast or bacterial cells. The invention further relates to compositions and systems for characterising particulate organic carbon and / or one or more constituents thereof, and related uses thereof.

Description

[0001] ANALYTICAL METHODS AND RELATED COMPOSITIONS AND USES

[0002] TECHNICAL FIELD

[0003] The invention relates to methods for characterising particulate organic carbon in a sample, for example to quantify the amount of particulate organic carbon present in a biological sample or an environmental sample. More particularly, the invention relates to methods for characterising particulate organic carbon in one or more cells, such as one or more protist, yeast or bacterial cells. The invention further relates to compositions and systems for characterising particulate organic carbon (POC) constituents including lipid, protein, and carbohydrate, and related uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0006] Biological production systems play a critical role in pharmaceutical production. For example, microbial fermentation of yeast and bacteria is used in the biomanufacturing of pharmaceuticals and therapeutics, using various technologies and methods such as gene editing and recombinant DNA techniques. Biological production systems are also recognised as key enablers of future food production and the production of sustainable energy. For example, microalgae can be used to produce food, feed, fertiliser, nutraceuticals, and biofuels. In each of these applications, the biological system contains particulate organic carbon (POC), the total organic carbon in the biological system, which includes protein, lipid, and carbohydrate constituents.

[0007] In some systems a goal may be to maximise POC - for example, a desirable target in carbon sequestration technologies may be algae with maximised POC content, while in other systems production of a particular POC component may be desired, such as a microorganism in which a single POC constituent, such as lipid, is maximised.

[0008] Thus, methods to assess POC - for example to quantify the impact on production or efficiency of a given technological modification, or to assess overall productivity or particular outputs - for example to maximise the production of a specific POC constituent are critical to supporting the growth, development and commercialisation of biological production systems that use organisms such as a protist, yeast or bacteria.

[0009] POC measurement also has potential application beyond biological production systems, for example, in environmental monitoring, disease monitoring and public health initiatives, in aquaculture, in horticulture, and in agricultural systems, bioreactors and the like.

[0010] However, current methods of POC analysis are extremely slow, intensive, and costly - typically taking approximately 30 hours - involve multiple manual steps, and require specialised equipment and a trained analyst. Currently, protein, lipid and carbohydrate are determined by a variety of methods involving dyes, corrosive liquids, heating, and specialised equipment. When dyes are used, protein, lipid, and carbohydrate are determined manually through the addition of a dye specific to that constituent. In manual dyeing protocols the dyes are stored as separate liquids, which must be added accurately and in sequence, before manual measurement of the dye absorbance or intensity. These and other limitation have prevented the implementation of POC measurement beyond areas where it is considered essential and viable with nearby laboratory facilities. At present many production systems that would benefit from accurate and rapid POC determination are limited to environmental data to provide closed loop control, which leads to variability in quality and yield of the intended product. Because POC measurement times are currently many hours (typically 30 hours), these current systems must make assumptions on the organism's physiological state and estimate POC constituent quantities.

[0011] There is an ongoing need for improved methods of analysing POC, and its protein, lipid, and carbohydrate constituents in samples, including single-celled organisms and particles.

[0012] The present invention relates broadly to a method of analysing POC, protein, lipid, and carbohydrate, and to related systems and reagents, and / or seeks to at least provide a useful alternative to existing methods and / or the public with a useful choice.

[0013] SUMMARY OF THE INVENTION

[0014] The invention broadly relates to methods, compositions, devices, and systems to allow the rapid, accurate determination of POC and / or its organic constituents, and particularly to examples including automated examples that enable accurate and rapid provision of reagents, speeding up the process for example by storing and providing dyes and other reagents in a manner that enables the single-step addition of the sample.

[0015] Examples disclosed here combine this dyeing approach with automated image analysis, allowing the dyed cells and particles to be differentiated from the background and non-carbon particles (e.g., contaminants, dirt, sand, and non-target organisms), and allowing fast and accurate determination of cell count, cell density, and the amounts and relative ratios of the organic carbon constituents of POC (proteins, lipids and carbohydrates). This quasi-real-time measurement enables optimisation of production systems, to improve yield and quality of the biological product. The invention thus enables the automated, real time (typically 20 minutes) determination of POC and its components, in turn enabling real time closed loop control of organic production processes.

[0016] Accordingly, in a first aspect the invention relates to a method of characterising POC and / or the organic constituents of POC in a sample, the method comprising: contacting a sample with a matrix as disclosed herein, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the sample, and detecting said binding, wherein said binding is indicative of at least one characteristic of said particulate organic carbon in the sample.

[0017] In another aspect the invention relates to a matrix, said matrix comprising at least one hydrophilic water-soluble polymer and one or more of the group consisting of: at least one dye capable of binding to lipid; at least one dye capable of binding to protein; and at least one dye capable of binding to carbohydrate; and wherein said matrix is at least partially soluble in an aqueous solution.

[0018] In one example, the matrix comprises at least one hydrophilic water-soluble polymer and one or more of the group consisting of: at least one dye capable of binding to polar and / or non-polar lipids; at least one dye capable of binding to protein; and at least one dye capable of binding to carbohydrate; and wherein said matrix is at least partially soluble in an aqueous solution.

[0019] Another aspect of the invention relates to the prior mixing of dyes at specific quantities to allow quantitative analysis of POC and / or the organic constituents of POC in a sample.

[0020] Another aspect of the invention relates to the dyes being applied by different types and quantities in a support, such as a slide, a slide comprising at least one cavity such as a multiple cavity transparent slide, enabling differentiate analysis of POC constituents and quantities.

[0021] Another aspect of the present invention relates to a system for characterising particulate organic carbon in a sample, the system comprising: a light source; a receiver for receiving a support, wherein the support comprises a matrix as contemplated herein and is adapted for receiving a sample; and an image acquisition system, and an image analysis system.

[0022] In certain examples, the image analysis system is capable of quantifying POC and / or a constituent thereof in the sample, for example, within an individual cell present in the sample.

[0023] In certain examples, the image analysis system is capable of determining the number of cells in the sample.

[0024] In certain examples, the image analysis system is capable of determining cell volume.

[0025] In certain examples, the image analysis system is capable of determining spatial distribution and / or relative concentrations of protein, lipid, and / or carbohydrate within the sample. In certain examples, the image analysis system is capable of determining spatial distribution and / or relative concentrations of protein, lipid, and / or carbohydrate in a cell present in the sample.

[0026] In certain examples, the image analysis system is capable to determining the lipid storage type, for example, whether the lipid is dispersed or droplet.

[0027] In certain examples, the image analysis system extracts the signal from dyed cells and particles to differentiate the target cells from the background signal.

[0028] In certain examples, the image analysis system uses the morphology and / or spatial distribution of the POC components to identify a target species or particle and distinguishing it from non-target species or particles.

[0029] In certain examples, the image analysis system applies a species-specific calibration model to the extracted dye signal to quantify the amount of a POC constituent in an individual cell or sample.

[0030] In certain examples, the image analysis system distinguishes between polar and non-polar lipids based on the spatial distribution of the lipid dye and the response of the dye to external stimulation.

[0031] In one example, the invention relates to a system for characterising POC and / or a constituent thereof in a sample. In various examples, the system comprises: a light source; a receiver for receiving a support, wherein the support comprises a matrix as contemplated herein and is adapted for receiving a sample; and an image acquisition system comprising a camera or other detector capable of detecting photons, for example a detector capable of detecting the position of photons in a two-dimensional or three-dimensional arrangement; optionally an image analysis system.

[0032] Any of the examples described herein can relate to any of the aspects presented herein. In one example, the sample comprises one or more cells. In certain examples the one or more cells comprise one or more eukaryotic cells, such as one or more plant cells, one or more animal cells, one or more fungal cells, one or more yeast cells, one or more protistan cells, or any combination of two or more thereof. In other examples, the one or more cells comprise one or more prokaryotic cells, such as one or more bacterial cells, one or more archaeal cells, or any combination of two or more thereof.

[0033] In certain examples, the sample comprises one or more microorganisms, such as one or more cells from a single-celled prokaryotic or eukaryotic organism, such as a yeast, a protist, a fungus, a bacteria, an archaea, or any combination of two or more thereof.

[0034] In other examples, the one or more cells comprise larvae, zygotes, gametes including eggs, ova, oocytes, sperm, spat including spat from a shellfish or a mollusc, or the like.

[0035] In certain specifically contemplated examples, the sample comprises one or more cells from an aquatic organism, such as an aquatic organism used in aquaculture. For example, the one or more cells comprise one or more eggs or larvae from fish, such as one or more eggs or larvae from finfish, one or more eggs, larvae or spat from molluscs or shellfish, and / or one or more cells from algae, such as one or more microalgal cells.

[0036] In certain examples, the sample comprises one or more cells from a protist, for example, one or more cells of a protist selected from the group comprising Amoebozoa, Opisthokonta, Rhizaria, Archaeplastida, Chromalveolata, Excavata, Incertae sedis, and Eukaryota.

[0037] In certain examples, the protist is selected from the group comprising Mayorella spp, Platyamoeba spp., Vannella spp., Paramecium spp., Strombidium spp., Euplotes spp., Bodo spp. , Rhyncomonas spp., and Metromonas spp..

[0038] In certain examples, the sample comprises one or more cells from an organism selected from the group comprising: Arthrospira spp., Chlamydomonas spp., Chlorella spp., Dunaliella spp., Haematococcus spp., Nannochloropsis spp., Nitzschia spp., Pavlova spp., Picochlorum spp., Rhodomonas spp., Scenedesmus spp., and Tetraselmis spp..

[0039] In certain examples, the sample comprises one or more cells from an organism selected from the group comprising: Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella vulgaris, Dunaliella salina, Haematococcus pluvialis, Nannochloropsis oculata, Nitzschia closterium, Pavlova lutheri, Picochlorum oklahomensis, Rhodomonas salina, Scenedesmus obliquus, and Tetraselmis chuii.

[0040] In certain examples, the sample comprises one or more cells from an organism selected from the group comprising: Chlamydomonas reinhardtii, Chlorella vulgaris, Nitzschia closterium, Scenedesmus obliquus, and Tetraselmis chuii.

[0041] In certain examples, the sample comprises one or more cells from an organism selected from the group comprising: Arthrospira platensis, Chlorella sorokiniana, Dunaliella salina, Haematococcus pluvialis, and Nannochloropsis oculata.

[0042] In certain examples, the sample comprises one or more cells from an organism selected from the group comprising: Pavlova lutheri, Picochlorum oklahomensis, and Rhodomonas salina.

[0043] In certain examples, the sample comprises one or more cells selected from the groups including: Glaucophyta, Prasinophyta, Chlorophyta, Euglenophyta, Cryptophyta, Raphidophyta, Xanthophyta, Eustigmatophyta, Chrysophyta, Bacillariophyta, Haptophyta, Dinophyta, Cyanobacteria, amoebae, flagellates. In certain examples, the sample comprises one or more yeast cells, such as one or more cells from an organism selected from the group comprising Saccharomyces spp, such as S. cerevisiae including recombinant Saccharomyces cerevisiae and S. pastorianus; Pichia pastoris; Yarrowia lipolytica; and Hansenula polymorpha.

[0044] In certain examples, the sample comprises one or more bacterial or archaeal cells, such as one or more cells from an organism selected from the group comprising Streptomyces spp., Pseudomonas spp., Bacillus spp., Escherischia spp., such as E. coll, Arthrospira spp., Synechococcus spp., Methanopyrus kandleri, Picrophilus torridus

[0045] In certain examples, the sample comprises one or more cells from an organism selected from the group including: Salmon, Trout, Tilapia, Catfish, Carp, Milkfish, Turbot, Barramundi, Cobia, Yellowtail, Amberjack, Shrimp, Prawn, Crayfish, Oyster, Mussel, Clam, Abalone, Scallop, Seaweed, Sturgeon, Eel, Krill, Pollock, Tuna, Herring, Mackerel, Cod, and Haddock.

[0046] In still further examples, the sample comprises one or more carbon-containing particles, such as one or more plastic particles including but not limited to microplastics.

[0047] In certain examples, the one or more additional agents is selected from of the group consisting of: a) at least one cell-permeabilizing agent; b) at least one settling agent; c) at least one salt.

[0048] In certain examples, the one or more additional agents is selected from the group consisting of: Cetyltrimethylammonium bromide (CTAB), Sodium dodecyl sulfate, 3-((3- cholamidopropyl)dimethylammonio)-l-propanesulfonate (CHAPS), Saponin, Digitonin, Sulfatase, Lysozyme, EDTA, and Chitinase.

[0049] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0050] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g. "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer or value is also specifically contemplated.

[0051] Other aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred examples of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. The invention is exemplified in the following non limiting examples and with reference to the accompanying figures.

[0052] BRIEF DESCRIPTION OF THE FIGURES

[0053] Figure 1 presents micrographs of microalgal cells stained with the fluorescent dye RbITC (Figure 1A), and a graph showing intensity increasingly linearly with POC concentration (Figure IB), as described herein in Example 1.

[0054] Figure 2 presents a graph showing the results of a quantification of the total concentration of nonpolar lipids as described in Example 7 herein. Notably, fluorescent intensity of microalgal cells increases linearly as total non-polar lipid concentration increases.

[0055] Figure 3 presents a graph showing the results of a quantification of the total concentration of polar lipids as described in Example 7 herein. Notably, fluorescent intensity of microalgal cells increases linearly as total polar lipid concentration increases.

[0056] Figure 4 presents fluorescent micrographs of various microalgal and yeast cells dyed in a method contemplated herein as described in Example 7.

[0057] DETAILED DESCRIPTION

[0058] The present invention relates to methods for the characterisation of particulate organic carbon (POC), such as POC present in a sample such as a biological sample or an environmental sample. The invention further relates to systems and compositions for the characterisation of POC.

[0059] Existing methods of measuring POC are based on a manual technique developed in 1964 (Menzel et al., 1964). Briefly, a glass fibre filter is heated to 450 °C, then gravity or vacuum filtration of the culture is used to collect particulate matter on the ashed filters. The filter is dried and then exposed to concentrated acid fumes, before it is re-dried and packed into a tin capsule. Finally, the sample is combusted at 960 °C, converting the POC back to CO2, which is then measured. Each sample takes ~30 hours to process and analyse.

[0060] Described herein is a rapid (20 minutes) method for POC analysis that requires minimal sample handling. Briefly, protein, lipid, and carbohydrate constituents in a sample comprising cells and / or particles is quantified by dyeing individual cells and / or particles, with protein-selective, lipid-selective and / or carbohydrate-selective dyes, such as RbITC, BODIPY, and iodine respectively. As shown herein, the fluorescent intensity of the cells is proportional to the lipid concentration (see, for example, Figure 2), POC content is determined by measuring the fluorescent intensity or absorbance of dyed samples.

[0061] The methods, systems and compositions described herein enable the characterisation of POC by light and fluorescent microscopy, image capture and analysis, optionally together with data analysis and determination of one or more characteristics of POC, such as the quantification of the amount or concentration of POC and / or of one or more constituents of POC, the subcellular location or distribution of POC constituents, or the composition of POC.

[0062] In particular examples, the method employs a matrix that comprises essentially all chemical components required for analysis and which is applied to a support for receiving a liquid sample, such as a reservoir or well on a microwell plate. Sample addition, whether automated or via a user, is followed by image acquisition, typically with a camera and fluorescent and / or light microscopy. Image analysis is then performed, for example to provide one or more characterisations of POC.

[0063] The provision of a matrix that is at least partially soluble in an aqueous solution enables the one or more dyes to be effectively immobilised onto the support (e.g, a microwell plate), and the dye to be released quickly into solution when the liquid sample, such as an microalgae culture sample, is added. In certain examples, the matrix and the support are and remain optically transparent and / or do not interfere with image acquisition.

[0064] The methods, systems and compositions described herein are applicable to the characterisation of POC in a wide variety of samples. Specifically contemplated are biological samples, particularly those comprising one or more intact cells.

[0065] Various aspects of the invention are described in further detail in the following subsections. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice of the invention, examples of suitable methods and materials are described below. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0066] Selected definitions

[0067] The term "and / or" can mean "and" or "or".

[0068] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises", and the terms "including", "include" and "includes" are to be interpreted in the same manner.

[0069] The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

[0070] Methods

[0071] The methods, systems, and compositions contemplated herein enable the analysis of samples in a readily managed fluid environment to determine particulate organic carbon (POC) present in said sample.

[0072] In one aspect, the invention relates to a method of characterising particulate organic carbon in a sample, the method comprising: storing a pre-mixed and quantified matrix in a mechanism ready for sampling, contacting a sample with a matrix as disclosed herein, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the sample, and detecting said binding, detecting total POC quantity or the quantity of one or more constituents of POC, and optionally calculating appropriate ratios of detection to total quantity; wherein said binding is indicative of at least one characteristic of said particulate organic carbon or a constituent thereof in the sample.

[0073] In one aspect, the invention relates to a method of characterising particulate organic carbon in a sample, the method comprising: contacting a sample with a matrix as disclosed herein, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the sample, and detecting said binding, wherein said binding is indicative of at least one characteristic of said particulate organic carbon or a constituent thereof in the sample.

[0074] In one example, the invention relates to a method of characterising particulate organic carbon in a sample, the method comprising: contacting a sample with a matrix as disclosed herein, wherein said matrix is a solid matrix, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the solid matrix to bind particulate organic carbon present in the sample, and detecting said binding, wherein said binding is indicative of at least one characteristic of said particulate organic carbon or a constituent thereof in the sample.

[0075] As used herein, the phrase "characterising particulate organic carbon", "characterising POC", "to characterise particulate organic carbon", "to characterise POC", and grammatical equivalents thereof contemplate determining at least one characteristic of POC or a constituent thereof, including any one or more of whether POC is present in the sample, the amount of POC, protein, lipid, or carbohydrate present in a sample or any entity in a sample, the concentration of POC, protein, lipid, or carbohydrate present in a sample or any entity in a sample, the distribution of POC, protein, lipid, or carbohydrate present in a sample or any entity in a sample including the location of POC, protein, lipid, or carbohydrate present in a sample or any entity in a sample, and includes any changes to one or more of said characteristics, for example over time or across different samples.

[0076] In one example, the method of characterising particulate organic carbon is a method of detecting particulate organic carbon and / or one or more of the protein, lipid, or carbohydrate constituents thereof. For example, the characteristic of said particulate organic carbon is whether or not protein, lipid, or carbohydrate is present in said sample.

[0077] In one example, the method of characterising particulate organic carbon is a method of determining the location of POC, such as determining the subcellular location of POC, protein, lipid, and / or carbohydrate in one or more cells present in a sample.

[0078] The characteristics of POC, such as the amount or composition of POC, will vary depending on a number of factors, including whether the POC is present in the biological cell or particle. In relation to biological samples, POC, protein, lipid, and carbohydrate characteristics may vary depending on the particular organism present, the health of the organism, its growth stage or growth rate, environmental factors affecting the organism, such as the presence of other organisms such as competitors or pathogens, and the like.

[0079] The methods described herein have application in a range of industries, including biological production systems, such as the production of antibiotics from bacteria, insulin from yeast, and biofuels or food from microalgae. Certain characteristics of POC may be of particular interest to a given field or industry. For example, the relative composition of the protein, lipid, and carbohydrate composition of POC is of interest in certain applications of the methods contemplated herein, such as the biomanufacturing of pharmaceuticals. Examples of the invention enable optimisation of the biological production process, improving yields and manufacturing efficiency.

[0080] Advantageously, the methods described herein are amenable to determining various characteristics of POC of potential interest.

[0081] In various examples the methods described herein are directed to the characterisation of one or more of the POC constituents protein, lipid, and carbohydrates. Accordingly, in one example, the method of characterising particulate organic carbon is a method of characterising one or more constituents of POC. In one example, the one or more constituents of POC are selected from the group comprising: a lipid component, a carbohydrate component, a protein component, and any combination thereof.

[0082] Methods directed to determining one or more such components of POC, for example, the amount of protein present in a sample comprising one or more cells, or the amount of lipid present in one or more cells, are particularly contemplated.

[0083] In one example, when contacted with the matrix the sample is in an aqueous solution.

[0084] In certain examples, the matrix comprises substantially all reagents necessary to enable the characterisation of at least one characteristic of POC and / or a constituent thereof. For example, the matrix comprises substantially all reagents necessary to enable the characterisation of at least one characteristic of POC and / or a constituent thereof once it is contacted with the sample.

[0085] In certain examples, the matrix comprises substantially all reagents necessary to enable the characterisation of at least one constituent of POC. For example, the matrix comprises substantially all reagents necessary to enable the characterisation of at least one constituent of POC once it is contacted with the sample.

[0086] Accordingly, in one example the method of characterising particulate organic carbon in a sample consists essentially of or consists of: contacting a sample comprising an aqueous solution with a matrix as disclosed herein, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the sample, and detecting said binding by light or fluorescence microscopy, wherein said binding is indicative of at least one characteristic of said particulate organic carbon in the sample.

[0087] In one example, the method comprises contacting the sample with one or more additional reagents. For example, the method comprises, prior to, contemporaneous with, or after contacting a sample present in an aqueous solution with the matrix the addition to the aqueous solution of one or more additional reagents. In various examples, the one or more additional reagents is selected from the group comprising a dye, a buffer, a settling agent, a salt, a cell-permeabilising agent, or a combination therof.

[0088] Accordingly, in one example the method of characterising particulate organic carbon in a sample consists essentially of or consists of: contacting a sample comprising an aqueous solution with a matrix as disclosed herein, wherein said contacting comprises the addition of one or more additional reagents; maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon constituents present in the sample, and detecting said binding by light or fluorescence microscopy, wherein said binding is indicative of at least one characteristic of said particulate organic carbon in the sample.

[0089] In one example, said detecting comprises detection and / or recording by a camera or similar image acquisition device. In one example, the method comprises the iterative or continuous detection of fluorescence or absorbance over a period, for example to monitor changes in one or more characteristics of POC or a component thereof.

[0090] In one example, said binding is indicative or determinative of the identity of one or more cells present in said sample.

[0091] In various examples, one or more characteristics of particulate organic carbon present in a biological sample is indicative or determinative of the identity of one or more cells present in said sample. For example, one or more characteristics of a constituent of POC, such as one or more characteristics of protein, one or more characteristics of lipid, and / or one or more characteristics of carbohydrate, is indicative or determinative of the identity of one or more cells present in said sample.

[0092] In one example, said binding is indicative of cell count, such as the number of cells in the sample, including the number of cells of a given type in the sample. For example, said binding is indicative of cell count on a volume basis, or is indicative of cell count on a surface area basis.

[0093] In various examples, one or more characteristics of particulate organic carbon present in a biological sample is indicative or determinative of cell count. For example, one or more characteristics of a constituent of POC, such as one or more characteristics of protein, one or more characteristics of lipid, and / or one or more characteristics of carbohydrate, is indicative or determinative of cell count, such as the number of cells of a given type in the sample.

[0094] By selection of an appropriate dye, the methods, compositions and systems described herein can be used for non-POC measurements (e.g., nitrogen), for example to improve the predictive accuracy of the measurement of POC or one or more of its constituents, such as a decrease in nitrogen concentration or amount to forecast lipid accumulation and hence harvesting time.

[0095] In various examples, the one or more dyes is a fluorescent dye.

[0096] In various examples, the matrix is a solid matrix, such as solid matrix at least partially soluble in aqueous solution.

[0097] Polymers and polymer matrices

[0098] The methods, systems, and compositions contemplated herein enable the analysis of samples in a readily managed fluid environment, utilising (among other elements) a composition comprising a matrix, said matrix comprising one or more reagents of interest, such as one or more dyes useful in determining POC. In certain examples, the matrix is at least partially soluble in an aqueous solution. In certain examples, the matrix comprises at least one water soluble polymer.

[0099] In certain examples, the matrix comprises a hydrophilic water-soluble polymer.

[0100] In one example, the matrix comprises a polymer selected from the group comprising or consisting of a polyvinyl, such as a polyvinyl alcohol, a polyvinyl acetal; an agarose; and a cellulose. Specifically contemplated examples include polyvinyl alcohol (PVA) and hydroxypropyl cellulose.

[0101] Further examples of suitable water-soluble polymers contemplated herein include dextran, poly (acrylic acid sodium salt), poly (ethylene glycol), poly (methylacrylic acid sodium salt), poly(styrenesulfonic acid sodium salt), pullulan, or any combination thereof.

[0102] In certain examples, the at least one dye is a dye selected from the group comprising or consisting of: Nile Red, Oil Red 0, a Sudan dye, BODIPY, rhodamine B isothiocyanate, Coomassie Blue, fluorescein, acriflavine, iodine, 2-aminoacridone, primulin, Alcian blue and calcofluor.

[0103] In certain examples, the matrix composition comprises at least one dye useful in determining POC and one or more other reagents or components, such as a buffer, a cell-permeabilising agent, a settling agent, or a salt, including one or more examples of such agents as discussed herein. In some examples, the matrix is a solid matrix, such as a solid matrix comprising one or more water soluble polymers.

[0104] In some examples, the matrix is a solid matrix, such as a solid matrix comprising, consisting essentially of, or consisting of one or more water soluble polymers and one or more reagents of interest, such as one or more dyes as herein contemplated.

[0105] In some examples, the matrix is a film.

[0106] In some examples, the matrix is a solid delivery form, including for example a unit dosage form, such as a tablet, pill, capsule, or the like. In some examples the matrix is an extrusion.

[0107] In certain examples, the matrix comprises two or more layers, optionally with an interposing layer therebetween.

[0108] In various examples, one or more of the dyes is present in one of two or more layers, and one or more other agents is present in another of the two or more layers. In one example, the matrix comprises two or more layers, wherein one or more reagents, such as one or more reagents other than a dye, is present in the or one or more outer layers, and one or more dyes is present in an or the inner layer.

[0109] In one example, the matrix comprises at least two layers, wherein when present a cell- permeabilising agent is present in an or the outer layer, such that it is in contact with the sample before a reagent present in an or the inner layer. In one example, the matrix comprises at least two layers, wherein a cell-permeabilising agent is present in an or the outer layer, and the one or more dyes is present in an or the inner layer.

[0110] In some examples, the at least one dye and / or the one or more other reagents is applied to or printed on the matrix.

[0111] In some examples, the at least one dye and / or the one or more other reagents is encapsulated, embedded, or infused in the matrix, for example, the at least one dye and / or the one or more other reagents is encapsulated, embedded, or infused in the water-soluble polymer. In particularly contemplated examples, at least one dye and / or the one or more other reagents is encapsulated by the water-soluble polymer.

[0112] In some examples, the matrix composition further comprises at least one salt and / or at least one buffering agent.

[0113] In certain examples, when the sample comprises one or more cells from an organism selected from the group comprising: Arthrospira spp., such as Arthrospira platensis; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Chlorella spp., such as Chlorella sorokiniana and Chlorella vulgaris; Dunaliella spp., such as Dunaliella salina; Haematococcus spp., such as Haematococcus pluvialis; Nannochloropsis spp., such as Nannochloropsis oculate; Nitzschia spp., such as Nitzschia Closter! urn; Pavlova spp., such as Pavlova lutheri; Picochlorum spp., such as Picochlorum oklahomensis; Rhodomonas spp., such as Rhodomonas salina; Scenedesmus spp., such as Scenedesmus obliquus; and Tetraselmis spp., such as Tetraselmis chuii; the polymer matrix comprises one or more additional agents selected from the group consisting of: Cetyltrimethylammonium bromide (CTAB), Sodium dodecyl sulfate, Saponin, Lysozyme, EDTA, and Chitinase.

[0114] In certain examples, when the sample comprises one or more cells from an organism selected from Chlorella spp., such as Chlorella sorokiniana or Chlorella vulgaris, the polymer matrix comprises one or more additional agents selected from the group consisting of: Cetyltrimethylammonium bromide (CTAB), Sodium dodecyl sulfate, Saponin, EDTA, and Chitinase. In certain examples, when the sample comprises one or more cells from Chlorella spp., such as Chlorella sorokiniana or Chlorella vulgaris, the polymer matrix comprises, consists essentially of, or consists of: a) PVA, Nile Red, and saponin and / or chitinase; or b) PVA, Nile Red, and SDS, and / or saponin and / or EDTA, and / or chitinase; or c) PVA, RbITC, and CTAB; or d) PVA, RbITC, and SDS; or e) PVA and Acriflavine; and / or f) PVA and Iodine; and / or g) any combination of two or more of a) to f) above.

[0115] In one example, when the sample comprises one or more cells from Chlorella sorokiniana, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and SDS, and / or saponin and / or EDTA, and / or chitinase, for example, PVA, Nile Red, SDS, saponin, EDTA, and chitinase.

[0116] In one example, when the sample comprises one or more cells from Chlorella sorokiniana, the polymer matrix comprises, consists essentially of, or consists of PVA, RbITC, and SDS.

[0117] In one example, when the sample comprises one or more cells from Chlorella vulgaris, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and saponin and / or chitinase.

[0118] In one example, when the sample comprises one or more cells from Chlorella vulgaris, the polymer matrix comprises, consists essentially of, or consists of PVA, RbITC, and CTAB.

[0119] In certain examples, when the sample comprises one or more cells from an organism selected from: Arthrospira spp., such as Arthrospira platensis; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Haematococcus spp., such as Haematococcus pluvialis; Picochlorum spp., such as Picochlorum oklahomensis; and Scenedesmus spp., such as Scenedesmus obliquus; the polymer matrix comprises one or more additional agents selected from the group consisting of: Cetyltrimethylammonium bromide (CTAB), and Sodium dodecyl sulfate.

[0120] In certain examples, when the sample comprises one or more cells from an organism selected from: Arthrospira spp., such as Arthrospira platensis; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Haematococcus spp., such as Haematococcus pluvialis; Picochlorum spp., such as Picochlorum oklahomensis; and Scenedesmus spp., such as Scenedesmus obliquus; the polymer matrix comprises, consists essentially of, or consists of: a) PVA, Nile Red, and SDS; or b) PVA, Nile Red, and CTAB; or c) PVA, RbITC, and SDS; or d) PVA, RbITC, and CTAB; and / or e) any combination of two or more of a) to d) above.

[0121] In one example, when the sample comprises one or more cells from Arthrospira platensis, the polymer matrix comprises, consists essentially of, or consists of PVA, RbITC, and SDS.

[0122] In one example, when the sample comprises one or more cells from Chlamydomonas reinhardtii, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and SDS. In one example, when the sample comprises one or more cells from Chlamydomonas reinhardtii, the polymer matrix comprises, consists essentially of, or consists of PVA, RbITC, and SDS and / or CTAB, for example, PVA, RbITC, SDS, and CTAB.

[0123] In one example, when the sample comprises one or more cells from Haematococcus pluvialis, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and CTAB.

[0124] In one example, when the sample comprises one or more cells from Picochlorum oklahomensis, the polymer matrix comprises, consists essentially of, or consists of PVA, RbITC, and CTAB.

[0125] In one example, when the sample comprises one or more cells from Scenedesmus obliquus, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and SDS.

[0126] In certain examples, when the sample comprises one or more cells from an organism selected from the group comprising: Nitzschia spp., such as Nitzschia closterium, Pavlova spp., such as Pavlova lutheri, and Tetraselmis spp., such as Tetraselmis chuii; the polymer matrix comprises, consists essentially of, or consists of: a) PVA and Nile Red; or b) PVA and RbITC; or c) PVA and Iodine; and / or d) any combination of two or more of a) to c) above.

[0127] In one example, when the sample comprises one or more cells from Nitzschia closterium, the polymer matrix comprises, consists essentially of, or consists of PVA and Nile Red.

[0128] In one example, when the sample comprises one or more cells from Nitzschia closterium, the polymer matrix comprises, consists essentially of, or consists of PVA and RbITC.

[0129] In one example, when the sample comprises one or more cells from Pavlova lutheri, the polymer matrix comprises, consists essentially of, or consists of PVA and Nile Red.

[0130] In one example, when the sample comprises one or more cells from Pavlova lutheri, the polymer matrix comprises, consists essentially of, or consists of PVA and RbITC.

[0131] In one example, when the sample comprises one or more cells from Tetraselmis chuii, the polymer matrix comprises, consists essentially of, or consists of PVA and Nile Red.

[0132] In one example, when the sample comprises one or more cells from Tetraselmis chuii, the polymer matrix comprises, consists essentially of, or consists of PVA and RbITC.

[0133] In certain examples, when the sample comprises one or more cells from an organism selected from Rhodomonas spp., such as Rhodomonas salina, the polymer matrix comprises lysozyme.

[0134] In certain examples, when the sample comprises one or more cells from Rhodomonas spp., such as Rhodomonas salina, the polymer matrix comprises, consists essentially of, or consists of: a) PVA, Nile Red, and lysozyme; or b) PVA and RbITC; and / or c) any combination of two or more of a) or b) above.

[0135] In one example, when the sample comprises one or more cells from Rhodomonas salina, the polymer matrix comprises, consists essentially of, or consists of PVA, Nile Red, and lysozyme.

[0136] In one example, when the sample comprises one or more cells from Rhodomonas salina, the polymer matrix comprises, consists essentially of, or consists of PVA and RbITC

[0137] In one example, the invention relates to a matrix as herein discussed for use in the characterisation of POC and / or a constituent thereof, such as any one or more of protein, carbohydrate, and / or lipid. For example, the invention relates to a solid polymer matrix as herein discussed for use in the characterisation of POC and / or a constituent thereof, such as any one or more of protein, carbohydrate, and / or lipid.

[0138] In one example, the invention relates to a matrix as herein discussed when used for the characterisation of POC and / or a constituent thereof, such as any one or more of protein, carbohydrate, and / or lipid. For example, the invention relates to a solid polymer matrix as herein discussed when used in the characterisation of POC and / or a constituent thereof, such as any one or more of protein, carbohydrate, and / or lipid.

[0139] System

[0140] The invention relates in one aspect to a system for characterising POC in a sample. In various examples, the system comprises: a light source; a camera, or an image acquisition system comprising a detector capable of detecting photons; a receiver for receiving a support, wherein the support comprises a matrix as contemplated herein and is adapted for receiving a sample; and optionally an image analysis system.

[0141] When present the image analysis system is in communication with the image acquisition system, and generates one or more outputs representing one or more characteristics of POC and / or a constituent thereof.

[0142] In one example, one of said outputs is measurement data representing protein amount, content, or concentration.

[0143] In one example, one of said outputs is measurement data representing lipid amount, content, or concentration.

[0144] In one example, one of said outputs is measurement data representing carbohydrate amount, content, or concentration.

[0145] In one example, one of said outputs is measurement data representing protein distribution or location, such as distribution or location within an individual cell or population of cells present in the sample.

[0146] In one example, one of said outputs is measurement data representing lipid distribution or location, such as distribution or location within an individual cell or population of cells present in the sample.

[0147] In one example, one of said outputs is measurement data representing carbohydrate distribution or location, such as distribution or location within an individual cell or population of cells present in the sample.

[0148] In one example, one of said outputs is the identity of one or more cells present in the sample.

[0149] Any combination of two of more of the outputs discussed herein are contemplated.

[0150] In various examples, the system comprises a sonicator or an electrosonophore, for example to enhance or accelerate the uptake into a cell of one or more agents present in the polymer matrix or facilitate separation of cell aggregates.

[0151] In certain examples, the system is capable of holding and processing multiple samples. For example, in some examples, the system can hold and process at least 20 samples. In some examples, the system can hold and process at least 200 samples. In certain examples, the system is adapted to process one or more multiwell plates, such as one or more microtitre plates, wherein the one or more wells present are capable of receiving a liquid sample. Various light sources are well known in the art, and include incandescent, LED, and laser light sources.

[0152] It will be understood that the light source provides light having a wavelength, polarity, and / or intensity, or any combination thereof, required to excite the one or more dyes present. The light source in certain examples directly provides light of the required wavelength, polarity, and / or intensity, and in other examples does so via a filter, lens, diffraction element, mirror / mirror arrangement, or other optical element(s), as will be understood by those skilled in the art.

[0153] In one example, the light source provides visible light. In various examples, the light source provides light of wavelength from about 400 nm to about 750 nm.

[0154] In one example, the light source provides UV light. In various examples, the light source provides light of wavelength below about 400 nm, for example, from about 100 nm to about 400 nm.

[0155] In one example, the light source provides IR light. In various examples, the light source provides light of wavelength above about 750 nm, for example, from about 750 nm to about 1400 nm.

[0156] In various examples, the light source directly or via a filter provides light of wavelength from about 510nm to about 560nm.

[0157] In one example, the invention relates to a system as herein discussed for use in the characterisation of POC and / or one or more of its constituent components of protein, lipid and carbohydrates.

[0158] In one example, the invention relates to a system as herein discussed when used for the characterisation of POC and / or one or more of its constituent components of protein, lipid and carbohydrates.

[0159] In another aspect, the invention relates to a support comprising a matrix as herein described. In one example, the support provides a locus for receiving a sample, such as a liquid sample.

[0160] For example, the locus is a volume for receiving a liquid sample, and wherein the matrix is present on one or more surfaces of the support defining said volume.

[0161] In some examples, the matrix is present at or in said volume as a solid delivery form, including for example a unit dosage form, such as a tablet, pill, capsule, or the like. In some examples the matrix is an extrusion. In certain examples, the matrix is retained within said volume by a cover or lid.

[0162] In one example, the volume comprises a matrix comprising a dye capable of binding to protein. In one example, the volume comprises a matrix comprising a dye capable of binding to lipid. In one example, the volume comprises a matrix comprising a dye capable of binding to carbohydrate.

[0163] In various examples the support comprises two or more volumes for receiving a liquid sample, each of said volumes comprising a matrix as herein described, wherein at least one of said volumes comprises a matrix comprising a first dye, and one or more of said volumes comprises a matrix comprising a different dye. In one example, said first dye is capable of binding protein. In one example, said first dye is capable of binding carbohydrate. In one example, said first dye is capable of binding lipid. In one example, said different dye is capable of binding protein. In one example, said different dye is capable of binding carbohydrate. In one example, said different dye is capable of binding lipid.

[0164] In one example, the support is a multiwell plate.

[0165] In certain examples, each well in the multiwell plate comprises the same matrix as herein contemplated. In other examples, one or more wells in the multiwell plate comprise a matrix having a first composition, and one or more wells in the multiwell plate comprise a matrix having a different composition.

[0166] In one example, the multiwell plate comprises at least one well comprising a matrix comprising a dye capable of binding to protein.

[0167] In one example, the multiwell plate comprises at least one well comprising a matrix comprising a dye capable of binding to lipid.

[0168] In one example, the multiwell plate comprises at least one well comprising a matrix comprising a dye capable of binding to carbohydrate.

[0169] Specifically contemplated are multiwell plates in which at least one well comprises a matrix comprising a dye capable of binding to protein, and: at least one well comprises a matrix comprising a dye capable of binding to lipid; and / or at least one well comprises a matrix comprising a dye capable of binding to carbohydrate.

[0170] Also specifically contemplated are multiwell plates in which at least one well comprises a matrix comprising a dye capable of binding to lipid, and: at least one well comprises a matrix comprising a dye capable of binding to protein; and / or at least one well comprises a matrix comprising a dye capable of binding to carbohydrate.

[0171] Also specifically contemplated are multiwell plates in which at least one well comprises a matrix comprising a dye capable of binding to carbohydrate, and: at least one well comprises a matrix comprising a dye capable of binding to protein; and / or at least one well comprises a matrix comprising a dye capable of binding to lipid.

[0172] Other formats allowing multiple samples and / or multiple solid polymer matrices as herein described are also contemplated.

[0173] It will be apparent to those skilled in the art reading this disclosure that various supports are amenable to use in the methods and systems described herein. For example, microscope slides, whether plastic, glass or another at least partially transparent media are readily coated with a polymer matrix as contemplated herein.

[0174] The polymer matrix is in certain examples formulated with or applied together with one or more compounds or moieties to provide, for example, buffering or modulation of pH, agent release, such as surfactant or permeabilizing agent release, controlled or delayed release, for example controlled or delayed release of one or more dyes, control of buoyancy or surface tension, and the like.

[0175] In certain examples, the support comprises or is adapted to engage with a removable cover or lid, for example to prevent environmental exposure, contamination, or degradation for example during storage or shipping.

[0176] In certain examples, the support comprises one or more calibration aids, such as one or more markings or one or more objects for calibration, for example of a microscope or of image acquisition.

[0177] In one example, the invention relates to a support as herein discussed for use in the characterisation of POC.

[0178] In one example, the invention relates to a support as herein discussed when used for the characterisation of POC.

[0179] In one aspect, the invention relates to a method of preparing a support for use in the characterisation of POC and / or one or more constituents thereof, said method comprising applying to a support a matrix as herein contemplated. In various examples the support comprises at least one locus adapted to receive a sample, such as a liquid sample, and application of the matrix comprises application to at least a surface of said locus.

[0180] Data and image acquisition and analysis

[0181] In some examples, the system further comprises an image acquisition system, an image analysis system, or both an image acquisition system and image analysis system.

[0182] In certain examples the image acquisition system comprises a detector capable of detecting photons.

[0183] In certain examples the image acquisition system comprises a camera.

[0184] In specifically contemplated examples, the image acquisition system comprises a microscope, typically together with a camera or a photon detector, such as a digital imager, CCD, brightfield scanner, or the like. In one example, the image acquisition system comprises a Raman spectrometer, such as a Raman microspectrometer.

[0185] In certain examples, the image acquisition system acquires one or more images or data under or in response to a condition or set of conditions, (such as wavelength, electromagnetic field strength or orientation, temperature, displacement, stimulation, or the like). In one example, the image acquisition system acquires one or more images or data in response to a condition or set of conditions, (such as wavelength, electromagnetic field strength or orientation, temperature, displacement, stimulation, or the like), and one or more images or data acquired under or in response to a different condition or set of conditions.

[0186] In various examples, the condition is stimulation, for example by light of one or more wavelengths, polarities, or intensities, by electromagnetic radiation, by the presence, absence, strength, and / or modulation of one or more electromagnetic fields.

[0187] In certain examples, the image acquisition system acquires one or more images or data by, under, or in response to reflection, scattering, absorption, luminescence, pattern or pattern recognition, and / or displacement.

[0188] In one example, the system comprises internal calibration, or is capable of calibration using external calibration, for example via a marking or object present in or on the support, present in or on the polymer matrix, or added to the sample. Such calibration enables the image acquisition system and / or the image analysis system to work with different specifications of image acquisition equipment (resolution, pixelation, colour), such as a camera, different light sources, reflections, and different distances between support and camera, dye degradation.

[0189] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to one or more parameters indicative of a characteristic of POC and / or a constituent thereof. For example, the image analysis system converts one or more fluorescent or light images or data derived therefrom to information indicative of any one or more of: protein content, location, or distribution, lipid content, location or distribution, carbohydrate content, location, or distribution, cell or particle count, cell or particle volume, cell or particle identity, lipid storage type (e.g., dispersed or droplet), the proportion of viable or live cells, the relative ratios of the POC components with the sample, cell, or particle, and the identity of the cell or particle. When multiple measurements are made on a sample over time, the image analysis system can be used to quantify growth rate.

[0190] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to an output relating to the cell identity. For example, the output relates to information to determine the identity of one or more cells present in the sample, for example to distinguish cells of one population from those of another population, whether the population(s) is defined by a shared characteristic, such growth stage, metabolic status, or the like, or the population is defined taxonomically, phenotypically, or genetically.

[0191] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to an output relating to cell viability and / or integrity. For example, the output relates to information to determine the viability of one or more cells present in the sample, for example to quantify actively growing cells, to quantify quiescent or dead cells, and / or to distinguish actively growing cells from quiescent or dead cells.

[0192] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to an output relating to the amount, concentration, location, or distribution of POC and / or a constituent thereof in a cell lysate.

[0193] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to an output relating to the cellular amount, concentration, location, or distribution of POC and / or a constituent thereof.

[0194] In various examples, the image analysis system converts one or more fluorescent or light images or data derived therefrom to an output relating to the intracellular amount, concentration, location, or distribution of POC and / or a constituent thereof. In one example, the output relates to the intracellular amount, concentration, location, or distribution of protein. In one example, the output relates to the intracellular amount, concentration, location, or distribution of lipid. In one example, the output relates to the intracellular amount, concentration, location, or distribution of carbohydrate. In one example, the output relates to the intracellular amount, concentration, location, or distribution of any combination of two or more of protein, lipid, and carbohydrate.

[0195] In certain examples, the image analysis system processes one or more images or data acquired under or in response a condition or set of conditions, (such as wavelength, electromagnetic field strength or orientation, temperature, displacement, stimulation, or the like).

[0196] In various examples, the image analysis system processes two or more images or data acquired under differing conditions. For example, the image analysis system processes one or more images or data acquired under a first condition or set of conditions (such as wavelength, electromagnetic field strength or orientation, temperature, displacement, stimulation, or the like), and processes one or more images or data acquired under a different condition or set of conditions.

[0197] In various examples, the condition is stimulation, for example by light of one or more wavelengths, polarities, or intensities, by electromagnetic radiation, by the presence, absence, strength, and / or modulation of one or more electromagnetic fields.

[0198] In certain examples, the image analysis system processes one or more images or data acquired by, under, or in response to reflection, scattering, absorption, luminescence, pattern or pattern recognition, and / or displacement.

[0199] In various examples, the image analysis system comprises data, such as calibration data, for informing one or more outputs, such as one or more outputs relating to the amount and / or relative ratios of protein, lipid, and / or carbohydrate. In certain examples, the data informs an output relating to a variance between entities present in the sample, such as to inform one or more outputs that vary between the cells of one species and those of another, such as cells of a species known or suspected of being present in the sample. In one example, the data, such as calibration data, improves the accuracy of one or more outputs. For example, the data, such as calibration data, improves the accuracy of an output relating to the intracellular amount, concentration, location, or distribution of POC and / or a constituent thereof. In one example, the data, such as calibration data, improves the accuracy of an output relating to the intracellular amount, concentration, location, or distribution of protein. In one example, the data, such as calibration data, improves the accuracy of an output relating to the intracellular amount, concentration, location, or distribution of lipid. In one example, the data, such as calibration data, improves the accuracy of an output relating to the intracellular amount, concentration, location, or distribution of carbohydrate. In one example, the data, such as calibration data, improves the accuracy of an output relating to the intracellular amount, concentration, location, or distribution of any combination of two or more of protein, lipid, and carbohydrate.

[0200] In one example, the data, such as calibration data, improves the accuracy of an output relating to a characteristic that is differentially sensitive to intracellular amount, concentration, location or distribution.

[0201] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0202] EXAMPLES

[0203] Example 1: Characterising particulate organic carbon (POC) in biological samples

[0204] This example describes a rapid dye-based approach to measure POC in microalgal samples. Microalgae were chosen in part as they are of particular interest to the food and pharmaceutical industries, and in part as a model system for the development of compositions and methods suitable for the analysis of a wide range of biological samples and cell types.

[0205] One aim of certain examples of the methods described herein is to provide a rapid, cost- effective alternative to existing methods currently used to analyse POC in algae. Another aim of certain examples of the methods described herein is to characterise POC both in terms of total POC and in terms of the protein, lipid, and carbohydrate protein, lipid, and carbohydrate constituents of POC in biological samples, such as microalgal cells.

[0206] Samples comprising a commercial blend of microalgal cells (Figure 1A) or Pseudonitzschia sp. (Figure IB) were incubated with the protein-selective dye rhodamine B isothiocyanate (RbITC). Images acquired from a microscope were then analysed.

[0207] As shown in Figure 1A, sufficient RbITC was taken up by the cells to allow individual microalgae cells to be imaged. Furthermore, a linear relationship (Figure IB) between the intensity of the dye and the amount of POC in the sample was determined.

[0208] Example 2: Characterising particulate organic carbon (POC) in biological samples

[0209] This example describes further experiments investigating a method capable of providing data to allow characterization of protein, lipid, and carbohydrate separately, in addition to total POC characterization, and thus markedly improved over existing methods in which any quantitative measurements of protein, lipid, or carbohydrate are made on a bulk sample and typically require considerable sample processing. More specifically, the experiments described here relate to a rapid dye-based approach to measure POC in microalgal samples by fluorescent microscopy.

[0210] Method

[0211] Preliminary dye tests were done under a range of incubation conditions (e.g., heat and time) and on both microscope slides and in microwell plates. In each case, the dye was added to the microalgae sample, then allowed to react for 5 - 40 minutes. Visual examination of fluorescent (Nile Red, Bodipy, Fluorescein, RbITC) and visible light (Coomassie, Iodine) microscope images were used to assess the effectiveness of the dyeing of the desired protein, lipid, or carbohydrate constituent. This comparison was done with a range of microalgae, including those known to be challenging to dye and observe: Chlorella vulgaris (thick membranes with low dye permeability) and Spirulina (with a 3-D structure that makes quantitative fluorescence imaging difficult)

[0212] Results

[0213] The results showed that some dyes (e.g., RbITC, Nile Red, and iodine) had sufficient permeability, even without the addition of additives to increase dye uptake. These data suggest that in certain circumstances and with certain cell types dye uptake and imaging in the absence of other reagents may be adequate to at least partially characterise POC, protein, lipid, or carbohydrate.

[0214] Example 3: Further characterising particulate organic carbon (POC) in biological samples

[0215] This example describes further experiments investigating a method for rapidly characterising POC in microalgal samples.

[0216] Here, the effect of one or more additional reagents on assay efficacy was investigated.

[0217] Additional reagents included DMSO, glutaraldehyde, Tween, Triton, Sodium dodecyl sulfate, glycerol, saponin, cetrimonium bromide, and 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate. These additives were tested in combination with protein-specific dyes and lipid-specific dyes.

[0218] Varying combinations of dye and additive were added directly to microalgae cell cultures and visual examination of fluorescent (Nile Red, BODIPY, Fluorescein, RbITC) and visible light (Coomassie, Iodine) microscope images were used to assess dye uptake and assay efficacy.

[0219] Results

[0220] The results indicate that both Nile red, and RbITC, were very effective, providing reliable results across multiple species with a range of additional agents. Other dyes tested, such as fluorescein, provided useful dyeing and image enhancement in particular species and / or when one or more additional reagents, such as one or more cell-permeabilising agents, were present.

[0221] The results show that intracellular dyeing of protein and lipid can be enhanced in the presence of one or more additional agents. Furthermore, these data show that image acquisition and / or analysis can be improved in the presence of one or more additional agents.

[0222] Example 4: Solid polymer matrices for characterising POC in biological samples

[0223] This example describes further experiments investigating elements of methods for rapidly characterising POC in microalgal samples.

[0224] Here, the preparation of solid polymer matrices comprising dyes and other reagents, such as various solvents and surfactants, and their use in analytical methods for characterising POC is reported.

[0225] Polymer matrices comprising various combinations of dye and additional reagents encapsulated in PVA were prepared, and applied as a film layer to microwell plates. Three methods of preparing and applying the solid polymer matrices were employed:

[0226] • Direct attachment: Dyes were dissolved in a volatile solvent (e.g., absolute ethanol for RbITC, 90: 10 ethanol:DMSO for Nile Red) and solutions of known volume and concentration were applied and dried directly into the microwells.

[0227] • Sol-Gels: Dye was incorporated into either acid-catalysed (HCI) or base-catalysed (NaOH) solgel formulations.

[0228] • Dissolvable layers: Dye was incorporated into various concentrations (0.1% - 5 % w / v) of polyvinyl alcohol (PVA) and hydroxypropylmethylcellulose (HPMC) in water. In addition, a range of formulations were tested that also included additives (glycerol, Tween 80, Triton X- 100, and NaCI).

[0229] Visual examination of fluorescent (Nile Red, Bodipy, Fluorescein, RbITC) and visible light (Coomassie, Iodine) microscope images were used to assess dye uptake and assay efficacy.

[0230] Results

[0231] The assessment across a number of performance characteristics of the solid polymer matrices prepared by these methods and comprising a range of additional reagents was performed.

[0232] Direct attachment: this method of preparing solid polymer matrices was effective and provides matrices that are physically stable. Certain selected dyes (RbITC, Nile Red) exhibited low solubility in water when provided in polymer matrices prepared using this method. When necessary, the solubility of these dyes could be readily improved by addition of a solvent (e.g., ethanol or DMSO), for example during sample addition.

[0233] Sol-gels: The sol-gels prepared formed a porous and optically transparent layer on the bottom of the microwell plate. Dye release was generally less than from polymer matrices formed by the other methods reported here.

[0234] Dissolvable layers (HPMC and PVA): Solid polymer matrices prepared with PVA layers performed consistently well, drying into solid layers that adhered strongly to the microwell plate and dissolved readily in water (<2 minutes). Dissolvable preparations comprising HPMC were more viscous, exhibiting more limited and delayed dye release.

[0235] PVA and additives: The efficacy of solid polymer matrices comprising PVA and a range of additives to potentially improve assay performance, for example to enhance the permeability of dye into the microalgae cells, was then investigated.

[0236] • The surfactants Tween 80 and Triton X-100 were readily incorporated into the layer, drying and redissolving effectively.

[0237] • Glycerol and PVA formed a homogeneous layer having increased viscosity. This viscous layer dissolved more slowly and less completely than PVA alone when algal samples were added.

[0238] • NaCI significantly decreased dye settling time and adhered well to the microwells. However, the inclusion of NaCI impacted PVA dissolution.

[0239] These data indicate that solid polymer matrices prepared using a variety of methods are suitable for use in the methods and systems described herein. Matrices comprising PVA performed particularly well in these assessments, being easy to prepare, physically stable in the microwell, and fully and rapidly solubilised in the cell media (< 2 minutes). Additives, for example those directed to improving dye solubility or uptake to the microalgae cell such as Tween and Triton were readily incorporated into matices comprising PVA.

[0240] Example 5: Physical characteristics of solid polymer matrices for characterising POC

[0241] This example describes further experiments investigating solid polymer matrices for use in methods of characterising POC, such as methods for rapidly characterising POC in microalgal samples.

[0242] Here, the assessment of certain physical and chemical characteristics of various solid polymer matrices as contemplated herein is reported. Such assessment is important, as in order to ensure consistent performance, for example after distribution and storage, (a) the matrix comprising the dye ideally maintains physical integrity, for example when present on a support, even when agitated, and (b) the dye(s) and other reagents present should ideally remain chemically stable over time, including when exposed to various conditions, such as heat. In addition, the chemical stability of the dye / additive / PVA formulations in the liquid state (i.e., in the pre-coating state) ideally is known to ensure consistent coating of the support or casting or moulding of the matrix, for example into a solid delivery form such as a tablet.

[0243] Method

[0244] Polymer matrices comprising various combinations of dye and additional reagents encapsulated in PVA were prepared and applied as a film layer to microwell plates, as described above in Example 4.

[0245] Four types of stability tests were carried out:

[0246] • Physical stability: Direct attachment wells were subjected repeatedly to sonication and 0.5 m drops onto a hard surface. Any loose dye was vigorously shaken out and the remaining dye was redissolved and emission intensity measured. PVA and HPMC layers were repeatedly shaken, tilted, and scraped as a preliminary examination of physical durability.

[0247] • Stability in liquid form: Preliminary tests monitoring changes in emission intensity of dyes stored in solvent and in PVA solution at different temperatures were performed.

[0248] • Stability to heat: Dissolvable layers of various formulations were heated in the oven at 40 °C for up to 12 hours and visually inspected for changes. Preliminary tests of dye stability within PVA layers based on emission intensity were also performed.

[0249] • Shelf life: PVA + dye layers were stored in the freezer. At intervals over the course of 2 weeks, samples were removed, dissolved in water, and their emission intensities measured and compared.

[0250] Results

[0251] Physical stability: Direct attachment and dissolvable PVA and HPMC layers generated physically stable dye layers, although only the direct attachment method has been rigorously tested thus far. For this reason, PVA and HPMC are only marked as 'Good' for this parameter. Using glycerol as an additive prevented layers from drying, making this method of preparation of such matrices unsuitable.

[0252] Chemical stability in liquid form: Optimal chemical stability of dye solutions was achieved when the pre-cast solutions were stored in the freezer. Although some PVA / dye formulations have poor stability in solution, this was easily overcome by application of the liquid formulation to the microwell plates shortly after preparation, for example on the same day, rather than after storage.

[0253] Stability to heat: HPMC and PVA were both dried into microwells in the oven at 40 °C with no adverse effects. A preliminary test was done on the stability of dye in PVA layers when heated in the 40 °C oven for 12 hours, and no dye degradation was found.

[0254] Shelf-life: No detectable dye degradation was observed during short-term shelf-life tests (2 weeks).

[0255] Conclusion

[0256] The direct attachment and PVA layers were stable ('Good' to 'Very good'). In addition, the solid PVA and HPMC formulations were stable when exposed to heat (40 °C). Although tests showed that certain liquid dye formulations were not stable, this simply indicates that the solutions should be made fresh on the same day the microwell plates are coated.

[0257] Example 6: Image acquisition and analysis

[0258] This example describes further experiments investigating methods of characterising POC, such as methods for rapidly characterising POC in microalgal samples. In particular, this example describes the development of software and computational models for providing one or more characteristics of POC. Before models can be applied to convert fluorescent signals, such as fluorescent signals from Nile Red or RbITC dyes, to a protein, lipid, or carbohydrate concentration, (a) individual cells must be separated from the image background and (b) the signal from dye must be extracted from the fullcolour image. These tasks can be challenging when the background is a similar colour to the dye, or when the cell's natural fluorescence is similar to the dye signal. As reported in this example, successfully achieving steps (a) and (b) allowed the dye intensity and spatial distribution to be modelled to identify the target species and distinguish it from other non-target particles. Knowledge of the species, dye intensity, and dye spatial distribution can then be used to calculate the amount of POC constituents and determine the cell volume and cell count. To achieve accurate estimates of these parameters, the calibration models should incorporate knowledge of the target species physiology and morphology and the likely spatial distributions of POC constituents within the specific cell or particle type.

[0259] Method

[0260] Using Python, a GUI was developed that allows the user to (a) load a microscope image of dyed microalgae, (b) separate the image into colour channels, (c) remove the background from the image, (d) identify the microalgae species, and (e) extract and quantify the colour associated with the dye. For each dye / species combination, different colour spaces and image analysis techniques were compared to determine which approach provided the most reproducible results.

[0261] Results

[0262] While some approaches were promising, the extraction of cells using standard image analysis approaches is overly sensitive to image acquisition conditions. This leads to fair-to-poor reproducibility of the cell and dye extraction from the original image.

[0263] However, incorporating artificial intelligence approaches allowed both species identification and accurate separation of the dye signal from the background signal, enabling quantification of POC components, acquisition of cell counts, and species-specific determination of cell volume.

[0264] Conclusion

[0265] Standard image analysis techniques to separate the background and dye signals are overly sensitive to changes in image acquisition settings, which impacts reproducibility.

[0266] Artificial intelligence approaches have been adapted to extract the cell and dye signals from image, which enable the identification of the algal species and the delivery of parameters relevant to the growth cycle of the culture (e.g., number, size, and volume of microalgae cells).

[0267] Example 7: Assessment of polar and non-polar lipids, protein and carbohydrate in a range of microorganism species

[0268] This example presents the results of experiments investigating the use of solid polymer matrices as described herein to characterise POC in a wide variety of microalgal species and in a yeast species. Characterisation included the quantification of both polar and non-polar lipids, and a determination of protein dyeing, in these microalgae.

[0269] Solid polymer matrices were prepared as described above. The microalgae and yeast investigated in this example included Chlorella vulgaris, Tetraselmis chuii, Nitzschia closterium, Haematococcus pluvialis, Chlorella sorokiniana, Chlamydomonas reinhardtii, Pavlova lutheri, Scenedesmus obliquus, Nannochloropsis oculata, Picochlorum oklahomensis, and Saccharomyces sp.

[0270] Results

[0271] As can be seen in Figures 2 and 3, the total fluorescent intensity of the dyed cells increases as the concentration of non-polar (Figure 2) and polar lipids (Figure 3) in the sample increases. As can be seen in Figure 4, Nile Red, RbITC, Acriflavine, and Iodine were readily taken up by various microalgae and a yeast when incubated in the presence of solid polymer matrices comprising these dyes, enabling the ready characterisation of lipid, protein, and carbohydrate present in individual microalgal cells, yeast cells, and in the total sample.

[0272] Publications

[0273] MENZEL D. W. and R. F. VACCARO (1964) The measurement of dissolved organic and particulate carbon in seawater. Limnol. Oceanogr., 9, 138-142. WANGERSKY P. J. and D. C. GORDON, JR. 0965) Particulate carbonate, organic carbon and Mn 2+ in the open ocean. LimnoL Oceanogr., 10, 544-550.

[0274] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0275] Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0276] It should be noted that various changes and modifications to the presently preferred examples described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

[0277] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

[0278] Aspects of the invention have been described by way of example only, and it should be appreciated that variations, modifications and additions may be made without departing from the scope of the invention, for example when present the invention as defined in the indicative claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

[0279] Specifically contemplated examples

[0280] Paral. A method of characterising particulate organic carbon present in a sample, the method comprising: contacting a sample with a matrix, wherein the matrix comprises at least one hydrophilic water-soluble polymer and one or more of the group consisting of: a) at least one dye capable of binding to lipid; b) at least one dye capable of binding to protein; and c) at least one dye capable of binding to carbohydrate; and wherein said matrix is at least partially soluble in an aqueous solution; maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the biological sample, and detecting said binding, wherein said binding is indicative of one or more characteristics of said particulate organic carbon in the sample.

[0281] Para2. A method of determining the location, amount, the concentration, and / or the distribution of particulate organic carbon present in a biological sample, the method comprising: contacting a biological sample with a matrix as defined in numbered paragraph 1, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the matrix to bind particulate organic carbon present in the biological sample when present, and detecting said binding, wherein said binding is indicative of the location, the amount, the concentration, and / or the distribution of said particulate organic carbon in the biological sample.

[0282] Para3. The method according to numbered paragraph 2, wherein the biological sample comprises one or more protist cells.

[0283] Para4. A matrix, said matrix comprising at least one hydrophilic water-soluble polymer and one or more of the group consisting of: at least one dye capable of binding to lipid; at least one dye capable of binding to protein; and at least one dye capable of binding to carbohydrate; and wherein said matrix is at least partially soluble in an aqueous solution.

[0284] Para5. The matrix according to numbered paragraph 4, wherein the matrix comprises any combination of two or more of the group consisting of: at least one dye capable of binding to lipid; at least one dye capable of binding to protein; and at least one dye capable of binding to carbohydrate.

[0285] Para6. The matrix according to numbered paragraph 4 or 5, wherein the matrix comprises each of the group consisting of: at least one dye capable of binding to lipid; at least one dye capable of binding to protein; and at least one dye capable of binding to carbohydrate.

[0286] Para7. The matrix according to any one of numbered paragraphs 4 to 6 additionally comprising one or more of the group consisting of: a) at least one cell-permeabilizing agent; b) at least one settling agent; c) at least one salt.

[0287] Para8. The matrix according to any one of numbered paragraphs 4 to 7, wherein the at least one hydrophilic water-soluble polymer is selected from the group consisting of a polyvinyl, such as a polyvinyl alcohol, a polyvinyl acetal; an agarose; and a cellulose, such as hydroxypropyl methyl cellulose.

[0288] Para9. The matrix according to any one of numbered paragraphs 4 to 8, wherein the polymer is polyvinyl alcohol.

[0289] ParalO.The matrix according to any one of numbered paragraphs 4 to 9, wherein the matrix is a solid matrix, a semi solid matrix, a gel, a sol-gel, or a foam, or wherein the matrix comprises any combination of any two or more thereof. Parall.The matrix according to any one of numbered paragraphs 4 to 10, wherein one or more of the detectable labels is a dye.

[0290] Paral2.The matrix according to any one of the preceding numbered paragraphs, wherein the at least one dye is a fluorescent dye.

[0291] Paral3.The matrix according to any one of the preceding numbered paragraphs, wherein the dye capable of binding lipid is capable of: a) binding one or more polar lipids; or b) binding one or more neutral lipids; or c) binding both polar lipids and neutral lipids.

[0292] Paral4.The matrix according to any one of the preceding numbered paragraphs, wherein the dye capable of binding to lipid is selected from the group consisting of Nile Red, BODIPY, a Sudan dye, and Oil Red 0.

[0293] Paral5.The matrix according to any one of the preceding numbered paragraphs, wherein the dye capable of binding to protein is selected from the group consisting of rhodamine B isothiocyanate, Coomassie Blue, and fluorescein.

[0294] Paral6.The matrix according to any one of the preceding numbered paragraphs, wherein the dye capable of binding to carbohydrate is selected from the group consisting of acriflavine, iodine, 2-aminoacridone, primulin, and calcofluor.

[0295] Paral7.The matrix according to any one of the preceding numbered paragraphs, wherein: a) the at least one cell-permeabilizing agent is selected from the group consisting of a detergent, such as a saponin, digitonin, a non-ionic detergent such as Tween or Triton, or an ionic detergent such as sodium dodecyl sulphate, or a quaternary ammonium surfactant such as cetrimonium bromide (CTAB), or a zwitterionic surfactant such as 3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS); an organic solvent, such as DMSO, a formamide, a phosphoramide, an acetone, an alcohol or an ether; and an inorganic solvent; an enzyme such as lysozyme, chitinase or sulfatase and / or b) the at least one settling agent is selected from the group consisting of a glycerol; a glycol, such as a polyethylene glycol; and / or c) the at least one salt is selected from the group consisting of an organic acid salt, such as a citric acid salt, a malic acid salt, a potassium salt, a sodium salt such as NaCI, and / or any combination of two or more thereof.

[0296] Paral8.The method according to any one of the preceding claims, wherein when contacted with the sample, the matrix is in a unit dosage form, such as a tablet, pill, or capsule.

[0297] Paral9.A support comprising the matrix according to any one of the preceding numbered paragraphs.

[0298] Para20.The support according to numbered paragraph 19, wherein the support provides a locus for receiving a sample.

[0299] Para21.The support according to numbered paragraph 20, wherein the locus is a volume for receiving a liquid sample, and wherein the matrix is present on one or more surfaces of the support defining said volume. Para22.A system for characterising particulate organic carbon present in a sample, the system comprising: a) a light source; b) an image acquisition system comprising a detector capable of detecting photons; c) a receiver for receiving a support, wherein the support comprises a matrix as numbered paragraphed herein and is adapted for receiving the sample; and d) optionally an image analysis system.

[0300] Para23.The system according to numbered paragraph 22, wherein the characterisation of POC comprises determining any one or more of the location, amount, concentration, or distribution of particulate organic carbon present in the sample.

[0301] Para24.The method according to any preceding numbered paragraph, wherein the binding is indicative of: a) the total cell, organism, or particle count in the sample; and / or b) the number of viable or live cells or organisms in the sample; and / or c) the ratio of viable or live cells or organisms to dead cells or organisms in the sample; and / or d) the identity of one or more cells, organisms, or particles in the sample; and / or e) the presence or amount of one or more contaminants, such as one or more non-target cells, organisms, or particles in the sample; and / or f) any combination of any two or more of a) to e) above.

Claims

Claims1. A matrix, which contains a dye selective to a POC constituent, wherein the matrix comprises at least one polymer and one or more of the group consisting of: a) at least one dye capable of binding to lipid; b) at least one dye capable of binding to protein; and c) at least one dye capable of binding to carbohydrate; and wherein said matrix may be at least partially soluble in an aqueous solution.

2. The matrix of claim 1, wherein the matrix is a solid matrix, a semi solid matrix, a gel, a solgel, or a foam, or wherein the matrix comprises any combination of any two or more thereof.

3. A method of determining the location, amount, the concentration, and / or the distribution of particulate organic carbon constituents present in a sample, the method comprising: contacting a sample with a matrix as defined in claim 1, maintaining the sample under conditions conducive and for a time sufficient for one or more of the dyes present in the solid polymer matrix to bind particulate organic carbon present in the biological sample when present, and detecting said binding, wherein said binding is indicative of the location, the amount, the concentration, and / or the distribution of said particulate organic carbon in the biological sample."]4. The method according to claim 3, wherein the location and intensity or absorbance of the dye signal can be used to identify the species or particle and distinguish it from non-target cells or particles, where knowledge of the cell species and physiology and spatial location of POC components can be used to a) determine the amount and relative ratios of the POC components with the sample, cell, and / or particle; b) determine the total cell or particle count; c) determine the number or ratio of viable or live cells; d) recognise and quantify a contaminant (i.e. , non-target cell, particle, and / or organism).

5. The method according to claim 3 or claim 4, wherein the sample comprises one or more cells.

6. The method according to any one of claims 3 to 5, wherein the sample comprises one or more particles.

7. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises one or more dyes capable of binding to protein.

8. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises one or more dyes capable of binding to lipid.

9. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises one or more dyes capable of binding to carbohydrate.

10. The matrix or the method according to any one of the preceding claims, wherein the matrix releases the one or more dyes into the aqueous sample solution.

11. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises at least one cell-permeabilizing agent.

12. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises at least one settling agent.

13. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises at least one salt.

14. The matrix or the method according to any one of the preceding claims, wherein the matrix comprises at least one hydrophilic water-soluble polymer.

15. The matrix or the method according to claim 14, wherein the hydrophilic water-soluble polymer is selected from the group including polyvinyl, such as a polyvinyl alcohol, a polyvinyl acetal; an agarose; and a cellulose, such as hydroxypropyl methyl cellulose.

16. The matrix or the method according to any one of the preceding claims, wherein the polymer is polyvinyl alcohol.

17. The matrix or the method according to any one of the preceding claims, wherein the at least one dye is a fluorescent dye.

18. The matrix or the method according to any one of the preceding claims, wherein the at least one dye is an absorbent dye.

19. The matrix or the method according to any one of the preceding claims, wherein the dye capable of binding lipid is capable of binding one or more polar lipids.

20. The matrix or the method according to any one of the preceding claims, wherein the dye capable of binding lipid is capable of binding one or more neutral lipids.

21. The matrix or the method according to any one of the preceding claims, wherein the dye capable of binding lipid is capable of binding both polar lipids and neutral lipids.

22. The matrix according to any one of the preceding claims, wherein the lipid-binding dye is selected from the group consisting of Nile Red, BODIPY, a Sudan dye, and Oil Red 0.

23. The matrix or the method according to any one of the preceding claims, wherein the proteinbinding dye includes rhodamine B isothiocyanate, Coomassie Blue, and fluorescein.

24. The matrix or the method according to any one of the preceding claims, wherein the carbohydrate-binding dye includes acriflavine, iodine, 2-aminoacridone, Alcian blue, primulin, and calcofluor.

25. The matrix or the method according to any one of preceding claims, wherein: a) the at least one cell-permeabilizing agent is selected from detergents such as a saponin, digitonin, a non-ionic detergent such as Tween or Triton, or an ionic detergent such as sodium dodecyl sulphate, or a quaternary ammonium surfactant such as cetrimonium bromide (CTAB), or a zwitterionic surfactant such as 3-[ (3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS); an organic solvent,such as DMSO, a formamide, a phosphoramide, an acetone, an alcohol or an ether; and an inorganic solvent; an enzyme such as lysozyme, chitinase or sulfatase; and / or b) the at least one settling agent is selected from the group including glycerol; a glycol, such as a polyethylene glycol; and / or c) the at least one salt is selected from the group including organic acid salt, such as a citric acid salt, a malic acid salt, a potassium salt, a sodium salt such as NaCI, and / or any combination of two or more thereof.

26. A support comprising a matrix according to any one of the preceding claims.

27. The support according to claim 26, wherein the support provides a locus for receiving a sample.

28. The support according to claim 26 or 27, wherein the locus is a volume for receiving a liquid sample, and wherein the matrix is present on one or more surfaces of the support defining said volume.

29. The support according to any one of claims 26 to 28, wherein the locus is a volume for receiving a liquid sample, and wherein the matrix is applied to, present in or associated with said volume.

30. The support according to claim 29, wherein the matrix is in a unit dosage form, for example is a tablet, pill, or capsule.

31. A system for characterising particulate organic carbon present in a sample, the system comprising: a) an excitation or light source; b) a camera, or other detector capable of detecting photons; c) a receiver for receiving a support, wherein the support comprises a matrix as claimed in any preceding claimed s adapted for receiving the sample; and d) an image analysis system.

32. The system according to claim 31, wherein the characterisation of POC comprises determining any one or more of the amount, concentration, relative ratio, or distribution of a POC constituent present in the sample.

33. The system according to claim 31 or 32, wherein the characterisation of POC comprises determining intracellular or intraparticle location of the POC constituent present in the cell or particle.

34. The system according to any one of claims 31 to 33, wherein the system: a) determines the total cell or particle count; and / or b) determines the number of viable or live cells or organisms; and / or c) determines the ratio of viable or live cells or organisms to dead cells or organisms; and / or d) determines the identity of one or more cells, organisms, or particles; and / or e) recognises one or more contaminants, such as one or more non-target cells, organisms, or particles; and / orf) distinguishes the one or more cells, organisms, or particles of interest from one or more non-target components in the sample; and / or g) any combination of any two or more of a) to f) above.

Images