Optical reference targets

Abstract

An optical reference target (2) is provided. The target comprises a reference structure (6) provided by a polymer fibre. Polymer fibres can be created with predictable, controllable and uniform diameters, widths and / or thicknesses at a micro-scale or nano-scale at relatively low cost. Polymer fibres can also be manufactured to have other predictable, controllable and uniform properties such as refractive index (RI). The optical reference target can thus provide a reliable calibration and / or characterisation tool at very small scales and for a wide variety of optical imaging systems.

Description

[0001] Optical Reference Targets

[0002] BACKGROUND OF THE INVENTION

[0003] This invention relates to optical reference targets, such as but not limited to microscopy resolution targets for assessing the spatial resolution of a microscope and refractive index targets for assessing the ability of a microscope to detect refractive indices.

[0004] There are many types of optical imaging systems available, including transmissionbased, scattering-based and refractive index-based imaging systems. Imaging systems can be label-free (i.e. in which light reflected from or transmitted through a sample is detected directly, without the addition of dyes or other labels to the sample) and / or fluorescence-based (i.e. in which a sample is imaged by detecting light from fluorescent particles, e.g. a fluorescent dye added to the sample).

[0005] Calibrating and / or characterising the performance of optical imaging system typically involves the use of a reference target, to provide a known benchmark of one or more properties against which the imaging system output may be compared. For instance, a resolution target has features with known dimensions and may be used to establish the smallest unambiguous feature that can be resolved by the imaging system and / or to identify aberrations in the images. An optical resolution target (e.g. for calibrating a microscope) may have dark features coated onto a contrasting (e.g. light or transparent) substrate, or vice-versa. The size and shape of the features in images can be used to calibrate and characterise the performance of the imaging system.

[0006] Super-resolution microscopes are available with sub-250 nm resolutions. However, it can be complex and costly to manufacture nanometre-scale reference targets needed to calibrate and characterise such microscopes. Moreover, conventional resolution targets that use contrasting coatings are of limited use for calibrating super-resolution label-free microscopes which map refractive indices within a sample, because the surface coatings may not have a measurable Rl. Conventional reference targets are also poorly suited for calibrating imaging systems designed to image fluorescence.

[0007] Some alternative reference targets are available, such as polystyrene beads. These are relatively simply to make, have a known and measurable refractive index (Rl) and can be loaded with fluorescent dyes. However, their symmetric spherical shape is not representative of real-world biological structures, which can limit their usefulness in characterising imaging systems for biological imaging.

[0008] An improved approach to optical reference targets may be desired.

[0009] SUMMARY OF THE INVENTION

[0010] From a first aspect, the invention provides an optical reference target comprising a reference structure provided by a polymer fibre.

[0011] Thus, it will be seen that, in accordance with embodiments of the invention, a polymer fibre is used to provide a reference structure for calibrating or characterising optical imaging performance (e.g. microscope performance). Polymer fibres can be created with predictable, controllable and uniform diameters, widths and / or thicknesses at a micro-scale or nano-scale at relatively low cost. Polymer fibres can also be manufactured to have other predictable, controllable and uniform properties such as refractive index (Rl). The overall density of polymer fibres in the optical reference target may also be controlled. The optical reference target can thus provide a reliable calibration and / or characterisation tool at very small scales and for a wide variety of optical imaging systems.

[0012] The polymer fibre providing the reference structure means that one or more properties of the polymer fibre itself (e.g. one or more dimensions of the fibre, or a refractive index of the fibre) provides a benchmark that can be used to calibrate and / or characterise an optical imaging system. For instance, the polymer fibre does not simply form a structural backing on which conventional reference patterns are printed, but rather directly provides one or more benchmark properties. The reference structure may consist of the polymer fibre. The applicant has recognised that fibres (i.e. elongate filaments) can resemble biological structures more authentically than conventional reference targets, meaning that optical reference targets according to embodiments of the present invention may aid the accurate calibration and characterisation of systems intended for biological imaging. An optical reference target that provides characterizable and controllable biological-like structures may be highly advantageous because authentic biological samples are heterogeneous and cannot provide a fully characterised reference. Biological samples can also be difficult to obtain. However, embodiments are not limited to such uses, and may be used to calibrate other types of imaging systems also.

[0013] In a set of embodiments the optical reference target comprises a microscopy reference target, i.e. suitable for calibrating and / or characterising the performance of a microscope.

[0014] A single polymer fibre providing a single reference structure may be sufficient for some optical reference targets. However, in a set of embodiments, the optical reference target comprises a plurality of reference structures. The optical reference target may comprise a plurality of polymer fibres. The plurality of reference structures may be provided by a corresponding plurality of polymer fibres. Additionally or alternatively, a single long polymer fibre may provide multiple reference structures (e.g. multiple locations along the fibre where a reference width can be measured), e.g. by looping and / or folding the fibre to cross the target several times. In some instances such a single long polymer fibre may effectively comprise several fibre portions that may be considered as distinct polymer fibres.

[0015] The optical reference target may comprise a resolution target. The polymer fibre may have one or more known dimensions (e.g. pre-determined and / or precharacterised dimensions). The polymer fibre may have a constant dimension (e.g. diameter, width or thickness) along its length.

[0016] The optical reference target may comprise a refractive index target. The polymer fibre may have a known refractive index (e.g. a pre-determined and / or precharacterised refractive index). The polymer fibre may have a constant refractive index along its length. The polymer fibre may have a refractive index of between 1.1 and 2.3, between 1.2 and 2.1 or between 1.3 and 1.9.

[0017] The optical reference target may comprise reference structures with different properties, e.g. provided by polymer fibres with different diameters and / or refractive indices.

[0018] In some embodiments where the optical reference target comprises a plurality of polymer fibres, some or all of the polymer fibres may share one or more common characteristics. Some or all of the polymer fibres may share common structural or material properties. Some or all of the one or more common characteristics may be predetermined or pre-characterised. For instance, some or all of the polymer fibres may all have the same diameter, width or thickness. Some or all of the polymer fibres may all be formed from the same material. Some or all of the polymer fibres may all have the same refractive index.

[0019] Some or all of the polymer fibres may be interconnected. The target may comprise a network of polymer fibres. Each polymer fibre of the network may be in contact with at least one other polymer fibre. The polymer fibres may form a web that extends in three dimensions.

[0020] In some sets of embodiments, some or all of the polymer fibres may be aligned. For instance, some or all of the fibres may extend in a common direction (i.e. parallel to or within a few degrees of a common axis). Aligning the polymer fibres may cause the optical reference target to be isotropic. Additionally or alternatively, some or all of the fibres may extend in different directions, e.g. such that the optical reference target is anisotropic. The fibres may extend in random directions. In some embodiments, one or more additives within the polymer fibre(s) have an orientation and these may also be aligned or non-aligned, i.e. to provide controllable levels of isotropy at the sub-fibre level.

[0021] Controlling the isotropy of the optical reference target may be useful for tuning a polarisation sensitivity of the target, e.g. suitable for calibrating and / or characterising polarisation light microscopes. For instance, the optical reference target may be polarisation sensitive (i.e. to respond differently to) or polarisation insensitive (i.e. to respond similarly to differently-polarised incident light). In some embodiments, the optical reference target may be sensitive to different linear polarisations (e.g. by aligning the fibres and / or additives with a common direction). In some embodiments, the optical reference target may be sensitive to different circular or elliptical polarisations.

[0022] In some sets of embodiments, some or all of the polymer fibres may be distributed randomly, i.e. with spacings and / or orientations that generally follow a random distribution. Some or all of the fibres may follow non-linear paths. A random or partially-random distribution of fibres may provide a reference target that is more representative of structures seen in nature, e.g. biological structures. In contrast, conventional reference targets typically include patterns and / or symmetries that are unlike structures seen in nature (e.g. rows of lines or gratings), which can induce artefacts or errors when they are used to characterise or calibrate an imaging system (e.g. a microscope) intended for imaging non-patterned and / or non- symmetric subjects.

[0023] The polymer fibre(s) may have various structural morphologies. One or more polymer fibre(s) in the optical reference target may comprise a looped fibre structure or a straight fibre structure. One or more polymer fibre(s) in the optical reference target may comprise one or more integral bead structures, i.e. a section of the fibre with a larger diameter than the primary fibre structure. The optical reference target may, additionally or alternatively, comprise one or more reference structures provided by separate polymer bead structures (i.e. separate to the polymer fibre). A bead structure may be generally spherical or ovoid. Alternatively, a bead structure may be generally spindle-shaped (fusiform).

[0024] One or more polymer fibre(s) in the optical reference target may comprise a ribbon (i.e. with a width that is greater and optionally much greater (e.g. five or more times, or ten or more times greater) than a thickness).

[0025] The polymer fibre(s) may be a microfiber. A microfibre may have a maximum or mean characteristic dimension (e.g. diameter, width or thickness) on the micrometre or near- micro metre scale. In a set of embodiments one or more of the polymer fibres has a maximum or mean characteristic dimension of 5 mm or less, 2 mm or less, 1 mm or less, 500 pm or less, 250 pm or less, 100 pm or less, or 50 pm or less. The polymer fibre(s) may be a nanofiber. A nanofiber may have a maximum or mean characteristic dimension (e.g. diameter, width or thickness) on the nanometre or near-nanometre scale. In a set of embodiments one or more of the polymer fibres has a maximum or mean characteristic dimension (e.g. diameter) of 20 pm or less, 10 pm or less, 5 pm or less, 1 pm or less, e.g. down to 100 nm or even less.

[0026] The use of polymer means that the reference structure can have a wide range of properties (e.g. refractive indices). The polymer fibre(s) may be made from a single type of polymer, or alternatively may comprise a mixture of different types of polymer. The polymer of the polymer fibre(s) may feature little or no crosslinking. The polymer fibre(s) may comprise little or no structural constituents other than the polymer material itself (i.e. the polymer fibre(s) may consist of polymer and optionally one or more non-structural additives). The polymer from which the polymer fibre(s) is formed may be polymerized by bulk casting without needing any secondary chemical reactions.

[0027] The polymer fibre(s) may have a glass transition temperature of between 0 °C and 50 °C, e.g. between 25 °C and 35 °C or approximately 30 °C. The polymer fibre(s) may comprise polymer with a biological, biocompatible, or a purely inorganic composition. In a set of embodiments, the polymer fibre(s) comprises one or more of: Polymethyl methacrylate (PMMA), Polycaprolactone (PCL), Polyacrylonitrile (PAN), Polyvinylidene fluoride (PVDF), Polylactic acid (PLA), poly(N- isopropylacrylamide) (PNIPA), or a lipid-based polymer.

[0028] The polymer fibre(s) may have one or more uniform properties (e.g. a consistent refractive index (Rl) throughout the polymer fibre). The polymer fibre(s) may be formed of a homogeneous polymer. This can help to ensure that properties of the fibre(s) (e.g. Rl) are consistent and predictable. The use of a homogenous polymer can allow properties (e.g. Rl) of the fibres to be easily and precisely characterised — e.g. by mixing the polymer to have a desired properties and / or by measuring properties of a bulk polymer before it is used to form the fibre(s). The polymer fibre(s) may be arranged to be substantially suspended in a fluid medium (e.g. in air) along its length — e.g. for all of its length except at points where it contacts another polymer fibre and / or a support structure of the target. In other words, the polymer fibre(s) may be substantially self-supporting.

[0029] The optical reference target may comprise one or more additives. The one or more additives may be provided within the polymer fibre(s) (e.g. dispersed homogeneously in the polymer). One or more additives may have a controlled concentration and / or an even distribution. The optical reference target (e.g. the polymer fibre(s)) may comprise additives such as fluorophores (e.g. fluorescent dyes), stains / markers, magnetic materials, ferroelectric particles, carbon nanotubes, metal (e.g. gold) particles, metal (e.g. gold) nanoparticles, pigments, salts, minerals, metal oxides, nanoparticles and / or biological or biochemical materials such as DNA, lipids, proteins, bacteria or viruses.

[0030] In a set of embodiments, the optical reference target comprises one or more additives for Raman spectroscopy, e.g. molecules with known structural (e.g. vibrational) modes. In some such embodiments, the polymer fibre is substantially transparent to wavelengths of light corresponding to Raman spectral lines of the additive(s).

[0031] In a set of embodiments, the optical reference target (e.g. the polymer fibre) comprises a fluorescent additive (e.g. fluorophores) and / or a reflective additive (e.g. metal particles). This may allow the optical reference target to be used with fluorescence and / or reflection-based imaging systems (e.g. microscopes).

[0032] Including fluorophores and / or reflective particles in the optical reference target can enable the same target to be used with transmission-based imaging (e.g. in which the Rl of the target is detected), and also for fluorescence and / or reflectance-based imaging (e.g. in which light emitted from fluorophores, or reflected from the sample, is detected). In other words, the same optical reference target may be suitable for use with label-free and fluorescence imaging (e.g. for simultaneous correlative imaging). The same target being compatible with different imaging modalities can provide greater versatility and may also avoid misalignment issues that can occur when switching between different reference targets for different imaging modes. Such embodiments may also be useful for calibrating imaging systems (e.g. microscopes) capable of correlative imaging (also known as multimodal imaging), in which the same sample is imaged using different imaging modes and the results combined to provide additional information about the sample. For instance, an optical reference target with reference structures that have known properties relating to both imaging modes (e.g. a known fluorophore concentration and a known Rl) may be used to characterise and / or optimise a microscope using correlative imaging.

[0033] Correlative imaging may be used to identify dynamic biological activities happening at very small scales of space and time. However, it is important that the two modalities are in good alignment. As such, it will be recognised that reference targets according to embodiments of the invention in which the same reference structure (i.e. the polymer fibre) supports multiple imaging modes can be very useful for calibrating correlative imaging systems.

[0034] The optical reference target may comprise a support structure for the polymer fibre(s) — e.g. arranged as a frame around a perimeter of a network of the fibres. In a set of embodiments, the optical reference target comprises a substrate on which the polymer fibre(s) are disposed. The substrate may be transparent to one or more wavelengths of light. For instance, the optical reference target may comprise a glass slide on which the polymer fibre(s) are disposed. However, this is not essential and as mentioned above in some embodiments the polymer fibre(s) are self-supporting, without any need for a substrate.

[0035] From a second aspect, the invention provides a method of manufacturing an optical reference target comprising: forming a polymer fibre using a physical manufacturing process; and arranging the polymer fibre to provide a reference structure of an optical reference target. In some embodiments, the method may comprise forming a plurality of polymer fibres using a physical manufacturing process. The method may comprise arranging one or more polymer fibres to provide a plurality of reference structures.

[0036] Forming the fibre(s) using a physical (i.e. not chemical) manufacturing process may allow improved control over the structure of the fibre(s), e.g. such that a characteristic dimension (e.g. diameter) of the fibre(s) can be tuned more accurately to a desired value. Using physical rather than chemical methods to form the fibre(s) may also reduce manufacturing complexity and aid repeatability.

[0037] In a set of embodiments, forming the fibre(s) comprises one or more processes from the following: electrospinning, melt blowing, interfacial polymerization, drawing, spinneret-based tuneable engineered parameters (STEP), template synthesis, phase separation, self-assembly, and freeze drying (FD).

[0038] Forming the fibre(s) may comprise an electrospinning process. In such embodiments a voltage is applied between a liquid polymer and a target. The resulting electric field charges a droplet of liquid polymer (e.g. melted polymer or polymer in solution) and draws it into an elongate shape, which is then attracted towards the target and solidifies to form the polymer fibre. The fibre may collect on the target. The target may comprise a substrate of the finished optical reference target. Alternatively, the electrospinning apparatus may be arranged to collect the polymer fibre(s) away from the target. For instance, the target may comprise two parallel plates, with polymer fibres collecting in a span between the plates.

[0039] When an electrospinning process is used, one or more properties of the polymer fibre(s) (e.g. diameter, density) may be controlled by controlling the applied voltage, and / or the distance between the liquid polymer and the target.

[0040] Forming the fibre(s) may comprise a melt-blowing process. In such embodiments a liquid polymer melt is extruded through a die alongside a moving gas (e.g. air) to form fibre(s), which are carried by the gas and collected on the surface of a suitable collector in the form of a web. In such embodiments, the diameter of the fibre(s) may be controlled by controlling the throughput rate, melt viscosity, melt temperature, gas temperature and / or gas velocity. Forming the fibre(s) may comprise a drawing process. In such embodiments a droplet of a polymer solution is deposited on a substrate and allowed to evaporate. As the droplet evaporates, it becomes more concentrated at the edge due to capillary flow. A micropipette is then dipped into the droplet near the contact line with the substrate and withdrawn to draw out a polymer fibre.

[0041] The polymer fibre(s) may be formed to have one or more desired properties. For instance, the method may comprise selecting one or more desired properties for the optical reference target (e.g. a desired reference structure dimension (e.g. diameter, width or thickness) and / or refractive index), and then forming the polymer fibre(s) to have said one or more desired properties. For instance, polymer material(s) used to form the polymer fibre(s) may be selected and / or tailored to achieve one or more desired properties. Additionally or alternatively, the physical process used to form the polymer fibres may be selected and / or tailored to achieve one or more desired properties. For instance, a viscosity of liquid polymer and / or a voltage used in an electrospinning process may be selected to obtain a desired diameter of polymer fibre in the optical reference target.

[0042] As explained above, the polymer fibre(s) may be selected and / or tailored to have one or more desired properties. Many properties of polymers are the same at the macro, micro and even nano level, and so in a set of embodiments one or more properties of the polymer fibre(s) may correspond to properties of bulk polymer from which the polymer fibre(s) are formed.

[0043] The applicant has recognised that this may facilitate reliable manufacture of polymer fibres with well-characterised properties. In a set of embodiments, the method of manufacturing the optical reference target comprises: providing a bulk quantity of polymer material; determining one or more properties of the bulk quantity of polymer material; and forming the polymer fibre from the bulk quantity of polymer material.

[0044] Determining properties of the bulk quantity of polymer material may be much more convenient and / or accurate than determining properties of the finished fibre (e.g. because the finished fibres may be very small). Because many polymer properties are essentially the same at the bulk and fibre level this approach may enable convenient and / or accurate characterisation of the finished polymer fibre. For instance, the method may comprise determining a refractive index of the bulk quantity of polymer material, thereby producing an accurate estimate for the refractive index of the resulting polymer fibre without needing to measure the polymer fibre’s Rl directly.

[0045] In some embodiments, determining one or more properties of the bulk quantity of polymer material comprises measuring said one or more properties. For instance, the method may comprise measuring a viscosity, refractive index and / or additive concentration of the bulk quantity of polymer material.

[0046] Additionally or alternatively, determining one or more properties of the bulk quantity of polymer material may comprise modifying the bulk quantity of polymer material to change one or more properties, e.g. towards a desired value or range. Modifying the bulk quantity of polymer material may comprise adding one or more additives, e.g. to increase a refractive index of the polymer material. Making adjustments to polymer properties at the bulk level may facilitate increased accuracy of those adjustments, because a small proportional change in a polymer property may correspond to a relatively large absolute change at the bulk level.

[0047] The bulk quantity of polymer material may be provided in a polymer solution, e.g. a liquid solution.

[0048] The bulk quantity of polymer material may comprise at least 1 g, at least 2 g, at least 5 g, at least 10 g or at least 20 g of polymer material. In embodiments where the bulk quantity of polymer material is provided in a polymer solution, the polymer solution may have a volume of 5 ml or more, 10 ml or more, 15 ml or more, 20 ml or more or 25 ml or more.

[0049] The method may comprise adding one or more additives to the bulk quantity of polymer material. Adding an additive may comprise a dispersion, dissolution, emulsification, stirring, cross-linking and / or chemical bonding process. Preparing the bulk quantity of polymer material may comprise magnetic stirring or ultrasonic mixing.

[0050] From a third aspect, the invention provides a method of calibrating an imaging system (e.g. a microscope system) using an optical reference target comprising a reference structure provided by a polymer fibre.

[0051] It will be appreciated that, because the polymer fibre itself provides the reference structure, i.e. rather than a test pattern printed or coated onto a flat surface, the optical reference target can provide calibration and / or characterisation information for imaging systems that may be poorly suited to conventional reference targets. The imaging system may comprise a microscope system. The microscope system may comprise a transmission microscope. The microscope system may comprise a refractive-index-based microscope, i.e. one which directly or indirectly maps the refractive index of a sample. The microscope system may comprise a bright field microscope, a phase contrast microscope, a quantitative phase imaging microscope, a tomography microscope, or a holography microscope. The optical reference target can also be used to calibrate other types of imaging systems such as those based on detecting reflected light or fluorescence, e.g. by including suitable reflecting and / or fluorescing materials in the polymer fibre(s).

[0052] Calibrating the imaging system may comprise capturing an image of the optical reference target and comparing one or more features of the image with corresponding known properties of the reference structure. For instance, an apparent width of the polymer fibre in an image of the optical reference target may be compared to a known width of the polymer fibre, e.g. to establish a magnification level. Additionally or alternatively, an apparent refractive index of the polymer fibre may be compared to a known refractive index of the polymer fibre.

[0053] Calibrating the imaging system may comprise characterising the performance of the imaging system, e.g. determining a spatial resolution of the imaging system (i.e. the smallest resolvable feature size in images produced by the imaging system), a refractive index sensitivity and / or accuracy of the imaging system and / or identifying any aberrations in images captured by the imaging system. In a set of embodiments, the optical reference target may be used to determine the spatial resolution of the imaging system, by determining a smallest resolvable reference structure. For instance, calibrating the imaging system may comprise imaging multiple reference structures provided by different polymer fibres with different widths (e.g. in the optical reference target or across a plurality of optical reference targets), and identifying the smallest resolvable reference structure.

[0054] Features of any aspect or embodiment described herein may, wherever appropriate, be applied to any other aspect or embodiment described herein. Where reference is made to different embodiments, it should be understood that these are not necessarily distinct but may overlap. It will be appreciated that all of the preferred features of the optical reference target according to the first aspect described above may also apply to the other aspects of the invention.

[0055] BRIEF DESCRIPTION OF THE DRAWINGS

[0056] One or more non-limiting examples will now be described, by way of example only, and with reference to the accompanying figures in which:

[0057] Figure 1 is a schematic diagram of an optical reference target according to an embodiment of the present invention;

[0058] Figure 2 is another schematic diagram of the optical reference target;

[0059] Figure 3 is a schematic diagram of another optical reference target according to an embodiment of the present invention;

[0060] Figure 4 is a schematic diagram of a microscope system being calibrated using a optical reference target according to an embodiment of the present invention;

[0061] Figures 5-7 are schematic diagrams of various steps in a method of manufacturing an optical reference target according to an embodiment of the present invention; and

[0062] Figure 8 is a schematic diagram of another optical reference target according to an embodiment of the present invention.

[0063] DETAILED DESCRIPTION

[0064] Figure 1 shows an optical reference target 2 according to an embodiment of the present invention. The optical reference target 2 comprises a glass slide 4 and a plurality of polymer fibres 6 disposed on the glass slide 4. In this embodiment the optical reference target 2 is a microscopy resolution and refractive index target.

[0065] Figure 2 shows in more detail a section of the optical reference target 2 indicated by the label F2 in Figure 2. The polymer fibres 6 have a consistent width W, typically between 100 nm and 5 mm (e.g. between 100 nm and 20 pm). The polymer fibres 6 also have a consistent refractive index.

[0066] Figure 3 shows in detail another optical reference target 52. The optical reference target 52 comprises two types of polymer fibres 56, 58. A first type of polymer fibre 56 has a first width Wi and a first refractive index Rh, and the second type of polymer fibre 58 has a second, larger width W2 and a second refractive index RI2.

[0067] The properties of the optical reference targets 2, 52 (e.g. the widths and RIs of the polymer fibres) are known (e.g. determined during manufacture as described below), and provided with the optical reference targets 2, 52.

[0068] Figure 4 shows a process of characterising a microscope system 100 using the optical reference target 52. The microscope system 100 comprises a light source 102, a transmission microscope 104, a sample stage 106 and a controller 108. The optical reference target 52 is positioned on the sample stage 106.

[0069] The controller 108 controls the light source 102, the microscope 104 and the sample stage 106 to capture images of the polymer fibres 56, 58 in the optical reference target 52.

[0070] Because the polymer fibres 56, 58 have consistent and known widths and RIs, they provide reference structures for calibrating and characterising a microscope.

[0071] For instance, the controller 108 (or an operator thereof) may identify the apparent width of polymer fibres 56, 58 in the images, and use this to determine a magnification delivered by the microscope 104. Additionally or alternatively a resolution of the microscope 104 may be estimated by identifying the smallest polymer fibre width that can be resolved. For instance, if the second polymer fibre 58 can be resolved by the first polymer fibre 56 cannot, the resolution may be estimated to be between Wi and W2.

[0072] The controller 108 may identify aberrations produced by the microscope 104 by assessing the apparent width of the polymer fibres 56, 58 at different locations in the images. Any variation in apparent width may indicate optical aberrations.

[0073] The controller 108 may calibrate refractive index measurements using the known refractive indices of the polymer fibres 56, 58.

[0074] A method of manufacturing an optical reference target such as the target 2 shown in Figures 1 and 2, will now be explained in more detail with reference to Figures 5- 7

[0075] In a first step, illustrated in Figure 5, a bulk quantity of liquid polymer 200 (e.g. polymer in solution) is prepared. The polymer 200 is chosen to have a desired viscosity and refractive index, e.g. by referring to published properties of different polymers.

[0076] In a second step, illustrated in Figure 6, one or more additives 202 are added to the polymer 200 to add desired properties to the polymer 200. For instance, a fluorescent dye may be added.

[0077] The refractive index of the polymer 200 is then precisely measured, as this may vary slightly from the nominal Rl of the polymer 200. It is relatively straightforward to precisely measure the refractive index of the bulk quantity of polymer 200.

[0078] Next, the liquid polymer 200 is transferred to an electrospinning apparatus 204, shown in Figure 7. The electrospinning apparatus 204 comprises a needle 206, a collector plate 208 and a high-voltage power supply 210.

[0079] In use, the power supply 210 applies a large voltage between the needle 206 and the collector plate 208. Liquid polymer 200 is extruded from the needle 206 and becomes highly charged due to the electric field. A jet of polymer 200 accelerates towards the collector plate 208. Electrostatic forces between different parts of the jet causes the polymer jet to spin and elongate as it dries to form long polymer fibres 212 with predictable and consistent widths. These fibres 212 are collected on the collector plate 208 to form the optical reference target.

[0080] Figure 8 shows another optical reference target 302 according to an embodiment of the present invention. The optical reference target 302 comprises a plurality of polymer fibres 306. In this embodiment, several of the plurality of polymer fibres 306 comprise integral spindle-shaped beads 308, and several of the polymer fibres 306 comprise flat polymer ribbons 310.

[0081] While the invention has been described in detail in connection with only a limited number of embodiments, it should be readily understood that the invention is not limited to such disclosed embodiments. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.

Claims

Claims1. An optical reference target comprising a reference structure provided by a polymer fibre.

2. The optical reference target of claim 1, comprising a microscopy reference target.

3. The optical reference target of claim 1 or 2, comprising a resolution target and / or a refractive index target.

4. The optical reference target of any preceding claim, comprising a plurality of reference structures provided by a corresponding plurality of polymer fibres.

5. The optical reference target of claim 4, comprising reference structures with different properties.

6. The optical reference target of claim 4 or 5, wherein some or all of the polymer fibres share one or more common structural or material properties.

7. The optical reference target of any of claims 4-6, comprising a network of polymer fibres.

8. The optical reference target of any of claims 4-7, wherein some or all of the polymer fibres are aligned.

9. The optical reference target of any of claims 4-8, wherein some or all of the polymer fibres extend in different directions10. The optical reference target of any of claims 4-9, wherein some or all of the polymer fibres are distributed randomly.

11. The optical reference target of any preceding claim, wherein the polymer fibre comprises one or more integral bead structures.

12. The optical reference target of any preceding claim, wherein the polymer fibre comprises a ribbon.

13. The optical reference target of any preceding claim, wherein the polymer fibre is a microfiber or a nanofiber.

14. The optical reference target of any preceding claim, wherein the polymer fibre is formed of a homogeneous polymer.

15. The optical reference target of any preceding claim, wherein the polymer fibre is substantially suspended in a fluid medium along its length.

16. The optical reference target of any preceding claim, wherein the polymer fibre comprises a fluorescent additive and a reflective additive.

17. A method of manufacturing an optical reference target comprising: forming a polymer fibre using a physical manufacturing process; and arranging the polymer fibre to provide a reference structure of an optical reference target.

18. The method of claim 17, wherein forming the fibre comprises an electrospinning process, a melt-blowing process or a drawing process.

19. The method of claim 17 or 18, comprising selecting one or more desired properties for the optical reference target and then forming the polymer fibre to have said one or more desired properties.

20. The method of any of claims 17-19, comprising: providing a bulk quantity of polymer material; determining one or more properties of the bulk quantity of polymer material; and forming the polymer fibre from the bulk quantity of polymer material.21 . The method of claim 20, comprising determining a refractive index of the bulk quantity of polymer material.

22. The method of claim 20 or 21 , comprising adding one or more additives to the bulk quantity of polymer material.

23. A method of calibrating an imaging system using an optical reference target comprising a reference structure provided by a polymer fibre.

24. The method of claim 23, wherein the imaging system comprises a microscope system.

25. The method of claim 24, wherein the microscope system comprises a refractive-index-based microscope.

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