The present invention relates to bioprinters and methods of operating bioprinters, especially bioprinters which extrude a bioink containing living cells through a conduit onto a workpiece. The invention also relates to the testing of bioinks, and testing conditions and parameters for printing with particular bioinks, as well as the testing and development of conduits and nozzles for such printing. The invention also more generally relates to the determination of properties of flow of one or more materials for example along a conduit.

Introduction

It is known to use a 3D bioprinters to build up a workpiece to form a bioengineered structure comprising living cells. Once incubated or grown, such bioengineered structures may be similar to natural tissues or even whole organs, and may find uses in application areas such as the testing of pharmaceuticals, biological research, and medical implantation.

A number of different techniques are known for depositing bioinks so as to build up a workpiece to form a bioengineered structure. For example, inkjet type techniques may use thermal, piezoelectric or electrostatic mechanisms to direct bioink droplets to a workpiece, but high heat and stress levels associated with such processes can disrupt cell membranes and affect cell viability.

Stereolithography and similar volumetric techniques can instead use highly directed and focussed UV light to selectively cure regions of a bioink layer, but cell damage due to the UV light, and the need for suitable photocurable materials and related difficulties with cytotoxicity are some of the difficulties which arise.

Another developing bioprinting technique is that of extrusion based bioprinting, in which the ink materials typically comprise living cells carried within a medium such as a hydrogel, and frequently include other components such as growth factors. The bioink is extruded under pressure from a nozzle, either before, during, or after cross-linking to cure the gel. The nozzle is typically provided by using a needle having an internal diameter of a few tens to a few hundreds of micrometers.

With extrusion based bioprinting, the rheological properties of the bioink are very important, both in terms of behaviour within conduits of the bioprinter including the needle or nozzle, and in terms of behaviour once the bioink leaves the nozzle and subsequently forms part of the developing workpiece. It may for example be important that once out of the nozzle the bioink forms consistent strands which retain their shape and which do not intermix within the workpiece.

At the same time, the bioink should pass at sufficient speed along conduits of the bioprinter to permit bioprinting to progress at a reasonable pace, while avoiding damage to the living cells and loss of cell viability, which can arise through excessive shear stress within the flowing bioink. For example, shear thinning and shear thickening of the bioink can lead to different volume flow rates within a conduit, and to different amounts of cell damage in different parts of the flow. It would therefore be desirable to be able to measure aspects of flow of bioink within a conduit, such as a conduit of a bioprinter. The invention addresses these and other issues and limitations of the related prior art.

It would also be desirable more generally to address related issues of how to determine properties of flow of materials such as fluids, liquids, gels, and other materials, for example such flow along a conduit or in other situations.

Summary of the invention

The invention provides methods and apparatus which can be used to observe the flow of a bioink within an extrusion bioprinter nozzle or other conduit, rather than just using numerical modelling or other predictive techniques.

More generally, the invention proposes methods and apparatus which can be used to observe the flow of one or more materials, for example flow within or along a conduit, or in other physical situations, including adjacent to a wall, or other boundary. To this end, the invention provides methods or apparatus in which a probe light sheet is directed through a flowing material, and an aspect of flow of the material is determined from imaging the material as illuminated by the light sheet. Typically, the probe light sheet is configured to extend in a direction of the flow of the material such that features of the material progress within the light sheet and can therefore be observed while being carried by the flow.

Methods and apparatus described herein use light sheet microscopy, for example light sheet fluorescence microscopy, to image the flow of material within a conduit, by forming a light sheet which extends across the conduit to illuminate the material, and observing movement of the material through the light sheet, the observed movement also being along the conduit.

Accordingly, the invention provides a method of detecting one or more properties of flow of a material along a conduit, comprising: directing a probe light sheet across the conduit through the material, such that the probe light sheet extends both across the conduit and in the direction of the flow of the material; acquiring at least one image frame of a particular layer of the material within the inside volume of the conduit, due to the layer being illuminated by the probe light sheet; and determining one or more properties of the flow of the material, in particular within that layer, from the at least one image frame.

In this way, the probe light sheet illuminates a single layer of the flowing material within the conduit, so that the one or more image frames of that layer can be acquired. The layer contains trajectories of flow of the material along the conduit, so that the at least one image frame can be used to measure that flow.

Although it may often be practical to determine such properties of flow from just a single image frame, for example by using a sufficiently long exposure to provide one or more tracks of features moving within the material flow within the image frame during that exposure, embodiments of the invention described in detail below largely operate by acquiring a plurality of successive such image frames corresponding to the particular layer of the conduit. In particular, the position of the light sheet and the image frame preferably remain fixed relative to the conduit while the material flows through the fixed light sheet position (although some movement of the light sheet and/or frame of reference of each image frame could be used if required, subject to such movement relative to the conduit being known).

The step of determining one or more properties of the flow of the material may then comprise tracking visible features of the material across the multiple image frames, and determining flow of the material from the tracked visible features.

The material flowing in the conduit may be any of a wide variety of materials. In the particular examples and embodiments described herein, the material is typically a bioink, for example for use or during use with an extrusion based bioprinter, but when considering the described techniques and apparatus it should be understood that these may be implemented to determine properties of flow of other materials in other contexts.

If the material under consideration is a bioink, then such a bioink will typically comprise a plurality of living cells, and each tracked visible feature may then correspond to such a living cell, as it flows along the layer within the conduit that is illuminated by the probe light sheet. However, other features of the bioink could be tracked, such as slight variations in structure and refractive index of the bioink medium, particles of features which have been deliberately introduced for the purposes of tracking the flow, and so forth.

Although the described embodiments focus on determining properties of flow of a material within a conduit, other properties may also be determined from the one or more image frames. For example, the image frames may also be used to detect visible changes to elements within the material, such as the elements which are observed as features for determining flow. Such changes may include modification or deformation of such elements within the flow, for example under flow conditions such as shear stress. By way of example, deformation, such as elongation or compression, of living cells within a bioink due to shear stress may be observed or measured. Other properties could include the measurement of concentrations or fluxes of particular particles, such as living cells in a bioink, within different parts of the flow.

The invention may typically function using fluorescence, and in particular light sheet fluorescence microscopy, such that the tracked features comprise fluorescent emissions from the living cells or other features, the fluorescence being stimulated by the light of the probe light sheet.

The step of determining one or more properties of the flow of the bioink (or other material) may further comprise determining velocities, along the layer of the light sheet and therefore along the conduit, of the tracked visible features. In this way a profile across the conduit of flow velocities of the bioink along the conduit can be determined.

Other properties of the flow of the bioink (or other material) which can be determined, either directly from the results of the tracking, of from a determined profile of flow velocities, may include one or more of: a shear stress profile across the conduit; a shear rate profile across the conduit; the flow behaviour index based on a power law flow model; detection or measurement of plug flow; detection or measurement of shear thinning flow; detection or measurement of Newtonian flow; detection or measurement of shear thickening flow; detection or measurement of biphasic flow; and a volumetric flow rate within the conduit.

Various flags and other metrics may also be generated based on such properties, for example flags which indicate that a particular property has fallen to one side or another of a threshold. Such flags could for example indicate that the volume flow rate is greater and/or less than a particular value.

The probe light sheet may typically be substantially planar in form across the inside of the conduit, but may deviate slightly from planar for example having a Gaussian form. The light sheet within the conduit may have a minimum thickness within the conduit of from 1 pm to 10 pm.

The probe light used to form the probe light sheet may typically be of a wavelength selected to trigger fluorescence in a suitable fluorescent dye for use on living cells, for example having a wavelength between about 300 nm to 650 nm. Typically, the light source used to generate the probe light will comprise one or more lasers, for one or more example semiconductor diode lasers, so that the light sheet is a sheet of laser light, although other light sources could be used such as one or more super luminescent diodes or other non laser light sources. Note that the probe light sheet could be formed by expanding a laser beam or other light beam to a required sheet form, or may be formed by scanning the laser beam or other light beam, for example in a scanning direction along the direction of flow of the bioink.

As described above, a series of successive image frames may be acquired which all image the same layer within the conduit so that trajectories of flow of the bioink (or other material) lie within the single layer. However, aspects of the invention may be used to effectively acquire three dimensional imagery of the conduit, and therefore rheological, flow velocity and related information for the whole conduit cross section. In particular, the described methods may be used for each of a plurality of layers within the conduit, so that flow is separately detected in each of these plurality of layers. The plurality of layers may preferably be non-overlapping. More particularly, the plurality of layers may be substantially parallel to each other. The layers may be described as z-slices within the conduit.

Applying the described techniques to multiple layers within the conduit allows a 3D measurement of the material flow to be constructed, and across a larger fraction of the cross sectional area of the conduit, for example effectively covering at least 50% or at least 90% of the cross sectional area. Two dimensional (in the area of the section of the conduit) or three dimensional properties of the material flow then may be generated from the multiple layers of detection and tracking of the flow, and/or separate properties of the flow may be generated from each layer.

In carrying out the described techniques on multiple layers within the conduit as mentioned above to determine properties of the flow in three dimensions and/or across a larger cross sectional area, sufficient image frames may be acquired sequentially for one layer to determine properties of flow of that layer before moving on to acquire sufficient image frames from each other layer in turn. Alternatively, a low number of image frames, for example just one image frame, may be acquired from each layer in turn, before repeating the cycle. In this way, the light sheet may be rapidly scanned through each layer in turn, and the whole scan repeated quickly enough to track features in each layer, so that the flow properties of all of the layer are effectively acquired at substantially the same time.

The conduit may be of any suitable profile, cross section, length and so forth, but for example may be of circular or elliptical internal cross section, and may have an internal diameter of between 50 pm and 1000 pm across a section where the light sheet is used to measure the flow of bioink or other material. The diameter may vary along the length of the conduit, or may vary at one or two points for example to provide a narrower nozzle structure at an end of the conduit or a wider input region at the start of the conduit. In some cases, the conduit may be referred to as a needle or nozzle, especially as the needle or nozzle of a bioprinter.

In order to permit the light sheet to be formed within the conduit, the entire conduit may be formed of a transparent material, or at least of a material which is transparent, or sufficiently transparent for example with at least 90% transmission, at the wavelength of the light sheet probe light and wavelengths of light to be imaged. For example, the conduit may be formed of a suitable glass or transparent plastic.

The described methods may be used for various purposes and in various scenarios. For example, in some scenarios the described methods may be used for testing particular bioinks and their flow, for under particular conditions such as selected pressures, flow rates, temperatures and so forth, along a conduit similar to a conduit expected to be used in bioprinting. In other scenarios the described methods may be used during operation of a bioprinter, as bioink is moved within the bioprinter within a conduit such as a connecting or delivery tube, or through a conduit such as a needle or nozzle which is being used to actively extrude the bioink onto a workpiece so as to form a bioengineered structure, or for testing the operation of such a needle or nozzle before or after use for extruding onto a workpiece.

The invention also provides apparatus arranged to carry out the described methods, such as apparatus comprising a conduit arranged to carry a flow of a material such as a bioink along the conduit, and light sheet optics arranged to form a probe light sheet across the conduit through the bioink or other material, such that the probe light sheet extends both across the conduit and in the direction of the flow of the bioink or other material.

Such apparatus may also comprise one or more of a microscope arranged to acquire at least one or a plurality of successive image frames of a layer within the conduit which is illuminated by the probe light sheet, for example a microscope including one or more of imaging optics and an imaging detector, and an analyser arranged to determine properties of the flow of the bioink (or other material) from the at least one or plurality of successive image frames of the illuminated layer.

To this end, the analyser may be arranged to determine one or more properties of the flow of the bioink (or other material) within the illuminated layer, by tracking visible features of the bioink (or other material) between the multiple image frames, and determining properties of the flow of the bioink or other material from the movement or trajectories of the tracked visible features. Aspects of the invention also provide a bioprinter comprising the above apparatus. For example, such a bioprinter may comprise one or more different bioinks, each such bioink being contained in a separate reservoir for delivery onto a workpiece through a separate conduit or needle. Each bioink may typically contain a plurality of living cells within a medium, and the tracked visible features may then correspond to fluorescence of said living cells under excitation by the probe light sheet.

The analyser or other elements arranged to carry out processing and analysis of the acquired images frames, including tracking of features within the image frames, and determination of properties of the flow of the bioink or other material, may for example comprise or be provided by one or more computer systems, with suitable computer program code being provided to implement such processes, analysis and determination. Such computer systems may typically be provided with suitable input and output facilities such as an image acquisition port, one or more data network connections, data storage facilities and so forth, as well as working computer memory for storing data and such computer program code, and one or more microprocessors for executing the computer program code.

The invention also provides one or more computer readable media carrying computer program code arranged to carry out the described data processing and analysis steps.

It was mentioned above that the described methods and apparatus may be used in a variety of different application areas and for different purposes in addition to the determination of flow properties of a bioink for example in a bioprinter. Some other such areas include: the determination of flow properties along a conduit of a medium used in cell or gene therapy to deliver cells such as stem cells to a patient, in which context such cells may also be vulnerable to the effects of shear flow within the conduit; extrusion printing of various other kinds of materials such as plastics or other materials, typically as filaments, in all kinds of 3D printing, including the printing of food, of medical devices (such as prosthetics, which may include materials that contain drugs or which dissolve), and of medical dosage formulations such as tablets and capsules or various kinds; the measuring of flows of materials within industrial, manufacturing, utility and similar contexts such as fluids, liquids, gases, and mixed phases or materials within pipelines, food stuffs and so forth; the determination of flow properties in medical contexts such as within patient, such as the flow of blood within a blood vessel, or air within passages leading to and including the lungs; properties of flows within passages of all sizes, ranging from microfluidic channels to large pipelines and similar.

In contexts such as those outlined above, properties of the flow may be determined from successive image frames by detection of various features within the image frames, which may correspond to a variety of different entities or structures within the medium, such as cells within blood, small particles within a gas such as air, slight variations in refractive index, variations and inconsistencies in mixing or constitution, and so forth.

Brief summary of the drawings

Embodiments of the invention will now be described, by way of example only, and with reference to the drawings of which:

Figure 1 illustrates schematically an extrusion type bioprinter which may be used for implementing aspects of the invention;

Figure 2a illustrates a light sheet microscope arrangement for detecting flow of bioink (or other material) in a conduit, for example an extrusion conduit of needle of the bioprinter of figure 1 ;

Figure 2b shows how a plurality of z-slice or conduit layers may be imaged by positioning the light sheet 46 at different planes within the conduit at different times;

Figures 3a to 3d show how features of a bioink (or other material), visible through illumination using a light sheet microscope such as that of figure 2a, can be tracked to determine a velocity profile of the material within the conduit;

Figure 4 shows how the light sheet microscope arrangement of figure 2a can be used to observe a plurality of layers or parallel planes Z1 , Z2, Z3 within the conduit, such that the tracking of figures 3a to 3d can be used to determine velocity profiles for a plurality of z-slices or layers through the conduit to build a 3D picture of material flow;

Figure 5 illustrates details of structure and operation of the analyser of figure 2a, for determining material flow and inferring and using properties of that flow;

Figure 6 illustrates various ways in which the light sheet microscope arrangement of figure 2a may be implemented within the bioprinter of figure 1 ;

Figure 7 shows some method steps which may be used for implementing the invention; and Figure 8 illustrates a more particular experimental implementation for measuring flow of bioink within a conduit.

Detailed description of embodiments

Referring now to figure 1 there is shown a bioprinter 10 which is arranged to deposit one or more different bioinks 12 onto a workpiece 14 to build up a bioengineered structure such as a synthetic tissue or organ. A multi-axis manipulator 16 carries or moves a conduit 18 which is used to extrude the bioink 12 received from a reservoir 20, such as a syringe, onto the workpiece 14. In practice, the reservoir 20 is usually closely coupled to the conduit 18 and also carried by the manipulator 16, but could instead be stationary and positioned elsewhere and connected by suitable flexible tubing to the conduit 18.

Such a bioprinter 10 will usually be arranged to extrude each of multiple different bioinks onto the workpiece in predetermined patterns so as to build up the desired bioengineered structure. To this end, the bioprinter may be provided with interchangeable reservoirs 20-1...n and/or conduits 18-1 ...n, each pair of reservoir and conduit being for containing and extruding a different bioink 12. In some bioprinters, these other reservoirs and/or conduits may be kept ready for collection by the manipulator 16 for example from a magazine 22, for use, before being placed back in the magazine 22 by the manipulator 16. Alternatively, some or all of the reservoirs and/or conduits may be retained on the manipulator ready for use.

Flow of the bioink 12 along the conduit 18 and onto the workpiece 14 may be achieved using gas pressure in the reservoir 20 currently in use, or in other ways. One or more valves may be provided between the reservoir and the conduit to provide control of flow of the bioink along the conduit. Each conduit 18 may typically comprise an elongate tube and, if desired, each such conduit may be provided with a terminating nozzle structure 24 such as a restriction or other form.

In order to provide fine scale structures and structural combinations of different bioinks within the workpiece, each conduit may typically have an internal diameter of the order of a few tens to a few hundreds of micrometers, although smaller and larger conduits may be used if required. Extrusion velocities of the bioink along a conduit and onto the workpiece may typically be of the order of a few hundred micrometers to a few millimetres per second.

Although figure 1 illustrates the extrusion of a single bioink through a single nozzle or conduit, it may sometimes be desirable to extrude more than one bioink, or a bioink with another material, along a single conduit at the same time. This could be used for example to provide increased printing resolution of each such bioink or material, to extrude different bioinks to be printed in close proximity, to induce a particular desired shear on living cells within the bioink to force them into developing along a particular cell lineage, or to induce a particular mixing between two or more bioinks during printing. When we describe the printing, extrusion, or flow of a bioink elsewhere in this document, this should be also taken to mean the printing, extrusion or flow or more than one bioink, or at least one bioink and at least one other material, along the conduit.

The bioprinter 10 may provide additional services such as a cleaning station 26 to which the manipulator may bring a conduit 18 (and associated reservoir if appropriate) for cleaning, and a testing station 28 to which the manipulator may bring a conduit 18 (and associated reservoir if appropriate) for testing or checking. In some arrangements, one or more of such additional services may be combined into the magazine 22 or another structure, or may be provided as separate structures.

The various parts of the bioprinter 10 may typically be operated using a bioprinter controller 30, for example in order to change the bioink 12 currently in use, by exchanging a bioink reservoir 20 and associated conduit 18 currently mounted on the manipulator with a different bioink reservoir and conduit from the magazine 22, controlling flow rate of a bioink though the conduit 18 and onto the workpiece for example through control of gas pressure in the associated bioink reservoir 20, precisely controlling movement of the manipulator 16 to lay bioink onto the workpiece, controlling cleaning of a conduit using cleaning station 26, receiving test results relating to a tested conduit and reservoir using testing station 28, monitoring bioink levels in each reservoir, and maintaining environmental temperature of the workpiece. Many other aspects of the bioprinter may also require control as understood by the person skilled in the art.

The bioinks 12 may be formulated in various ways to provide the required biological, chemical, and physical characteristics. Typically, each bioink comprises a medium in which are carried living cells. The medium may typically be a hydrogel, or a material which can be treated at a selected time to form a hydrogel. Gelatin based hydrogels such as gelatin methacrylates are commonly used. Other common options include alginate based materials and pluronics. Some examples of media and their use in formulating bioinks suitable for extrusions are given in Bertassoni L.E. et al., “Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels”, Biofabrication (2014) 6(2)

024105 and Ozbolat I.T. and Hospodiuk M., “Current advances and future perspectives in extrusion-based bioprinting”, Biomaterials (2016) 76:321-43, the contents of which are incorporated herein by reference to provide examples and details of different bioinks that may be used with the invention, and for all other purposes. The concentrations of living cells within bioinks using such media may vary widely, but concentrations in the region of 1 x 10 ⁵ — 1 x 10 ⁷ cells per ml may be typical.

In order for the bioink to extrude effectively from the conduit 18, including any nozzle 24 of the conduit, and to form integral and consistent strands while laying onto the workpiece 14, the physical and rheological properties of the bioink may need to be carefully formulated and controlled. In order to stabilize the bioink, hydrogel crosslinking is frequently used, for example through curing using a UV light source. This curing step may take place before, or while the bioink is present in the reservoir, or after the bioink has been extruded onto the workpiece, or even during the extrusion process for example in the conduit or immediately following exit from the conduit or nozzle. To this end, the bioprinter 10 of figure 1 may further comprise one or more bioink curing elements (not illustrated), for example provided using one or more suitable UK light sources, arranged to cure or cross link a hydrogel or other form of curable bioink, either within a reservoir, within a conduit, immediately on exit from a conduit, or once deposited on the workpiece 14.

However, during flow along the conduit 18, even slightly different rheological properties of a bioink may lead to significantly different flow regimes and properties, for example exhibiting different aspects of Newtonian and non-Newtonian flow, including shear thinning and shear thickening (most notably close to the walls of the conduit), with such flow regimes and properties depending with quite high sensitivity on parameters such as applied pressure, volumetric flow rate, and temperature, as well as whether the bioink has already been cured.

In turn, such flow regimes can have different impacts on the viability of the living cells within the bioink and therefore the success of the printed bioengineered structure, for example because high shear rates can lead to damage to the living cells, and low boundary flow rates can give rise to long residency times of some of the living cells within the conduit. According to some studies (for example see Guvendiren M. et al., “Designing Biomaterials for 3D Printing”, ACS Biomaterials Science & Engineering (2016) 2(10):1679-93, use of a shear thinning hydrogel enables improved extrusion through a liquid - like flow by applying pressure, with the bioink switching to a solid like behaviour upon the absence of pressure following extrusion, thus permitting improved bioprinting accuracy and shape fidelity.

T o this end, figure 2a illustrates schematically how a bioink conduit 18, such as a conduit of figure 1 , may be monitored to detect one or more properties of flow of a bioink 12 along the conduit using light sheet microscopy (LSM), or more particularly using light sheet fluorescence microscopy (LSFM). In figure 2a, the conduit 18 itself is shown in cross section, such that a bioink 12 will flow along the conduit in a direction which is either into or out of the page. A light source 40 provides probe light 42 which is then formed by light sheet optics 44 into a probe light sheet 46 which is directed into and across the inside of the conduit 18. In this way, the probe light sheet 46 forms a light sheet which extends through the bioink 12 both across the conduit and in the direction of flow of the bioink along the conduit, which typically will be parallel to a central axis of the conduit.

In figure 2a the conduit 18 is circular in cross section, but could take other forms. The conduit is preferably formed of a material such as a glass or plastic, so as to be reasonably transparent at least at the optical wavelength or some required wavelength range of the probe light 42 and optical wavelengths required for imaging the bioink as illuminated by the light sheet 46. However, one or more non-transparent materials could be used instead to form the conduit 18 with one or more transparent windows being provided to enable the light sheet 46 to enter the bioink and to enable the imaging described below to take place.

Imaging optics 50, for example implemented using a microscope objective, are provided so as to enable an imaging device 52 to acquire an image frame of a portion of the bioink 12 which is illuminated by the light sheet 46, and to this end the imaging optics 50 may have an optical axis 51 which is approximately normal to the major plane of the light sheet 46 so that the interaction between the light sheet and the bioink is imaged face- on, although more oblique viewing angles could be used. As discussed in more detail below, the imaging optics 50 are typically controlled to acquire a plurality of successive image frames of the illuminated bioink 12, i.e. a series of images spread over time, and an analyser 54 is then used to infer properties of the flow of the bioink along the conduit 18 from these successive image frames, although it may also be practical to infer such properties from a single such image frame. The imaging device 52 may typically be provided by a CMOS or CCD camera or imaging detector.

Although a fairly conventional arrangement of an imaging objective or similar imaging optics 50 and a pixelated imaging device 52 may be used to acquire the image frames, a variety of other techniques may additionally or alternatively be used, such as light scattering, shadow imaging, laser free confocal imaging, and multifocal / 3D projection imaging.

The light source 40 may typically comprise one or more pulsed or continuous wave lasers, for example semiconductor diode lasers, although other light source types may be used, such as one or more super luminescent diodes or other LED sources. The light sheet optics 44 may be constructed in various ways, for example by using a commercially available arrangement such as the Leica DLS module available for use with the Leica SP8 microscope (see https://www.leica-microsystems.com). With such a configuration the imaging optics 50 and imaging device 52 may also be provided as part of the same commercially available microscope arrangement.

However, for many implementations such as within a bioprinter, a more compact or flexible arrangement may be desirable, for example with laser light being delivered along optical fibres to a compact form of light sheet optics 44 such as a GRIN lens and/or other elements located adjacent to the conduit, and optical fibres also being used to collect light via a compact form of imaging optics 50 again located adjacent to the conduit, for delivery to an imaging device 52 spaced some distance from the conduit.

More generally speaking, the light sheet optics 44 may function in a variety of different ways, for example by shaping a laser beam output by the light source 40 using a cylindrical lens, or using a combination of a cylindrical lens and a microscope objective. In other arrangements the light sheet may be formed by scanning a laser beam to create a “digital light sheet”, and in this case the time to complete a full scan is preferably less than the acquisition time of a single image frame, and may be synchronized with such image acquisition. The light sheet may be directed into the conduit 18 from just one side, or from both sides of the conduit in opposite directions using a dual-sided arrangement which can help to improve consistency of light sheet intensity across the conduit.

Various techniques can also be used to help optimize the light sheet, typically to make it thinner or more penetrating, typically starting from use of a scanner or “digital” light sheet. For example, techniques of Bessel beam generation, Airy beams or lattice light sheets can be used, or various techniques that include the use of a phase mask or a modulation of the phase of the laser beam. A two photon light sheet technique may be used involving infrared femtosecond pulses to obtain two photon excitation to increase the penetration of the light sheet into and across the conduit. Another suitable technique includes use of infrared femtosecond pulses to obtain second harmonic generation from specific materials. Other suitable techniques include the use of digital micro-mirror devices or other deflectors, or holographic devices, and various combinations of the techniques mentioned herein.

In some arrangements, the light sheet may be formed by directing a scanned or cylindrically expanded laser beam through a condenser lens located on an opposite side of the conduit to that of the imaging optics, and from the condenser lens to a mirror located to one side of the conduit relative to the optical axis of the condenser lens and imaging optics. The mirror then redirects the light beam into the conduit to form the light sheet within the conduit. A symmetrical dual sided arrangement where a second mirror is located on the other side of the conduit, so as to form the light sheet within the conduit from both sides, may be used.

The light sheet optics 44 may include compensating optics to pre-adjust the direction and convergence of the light sheet beam for refractive effects which occur as the probe light passes through the transparent walls of the conduit 18. A description of various different schemes for forming suitable light sheets may be found in Olarte O.E. et al., “Light-sheet microscopy: a tutorial”, Advances in Optics and Photonics (2018) 10(1 ) 111- 179.

While extending across a full width of the conduit 18, the light sheet 46 may typically have a length along the conduit of the order of the diameter of the conduit for example from a few tens to the order of a thousand micrometers, and a thickness of around a few micrometers to perhaps as much as a few tens of micrometers, or from about 1 to 10 micrometers. Note that the thickness through the light sheet (approximately in the direction of the axis 51 of the imaging optics), will typically vary across the conduit, for example with an approximately Gaussian beam form in cross section. Preferably a waist of the light sheet, where the thickness is at a minimum, should be located about half way between the walls of the conduit 18, so that the variations in thickness across the conduit are minimized.

Note that in figure 2a, for clarity purposes, the light sheet 42 is not shown to scale.

In practice, the light sheet 42 is likely to be smaller, and often many times smaller, in thickness, than seen in figure 2a in comparison to the size of the conduit 18.

A z-drive arrangement 54 may be provided which permits the light sheet to be directed across the conduit 18 at different depths within the conduit 18, that is at different distances from the imaging optics, so that different but preferably parallel z-slices of the bioink within the conduit can be illuminated by the light sheet 46, and therefore imaged, at different times. The z-drive 54 may achieve this by providing relative movement between the light sheet optics (and if necessary the light source) on the one hand, and the conduit 18 on the other, by moving one or both of the light sheet optics and conduit. However, the same effect may also or instead involve adjusting the configuration of the light sheet optics themselves, or in other ways. Figure 2b shows the conduit of figure 2a, but showing such a plurality of different light sheet positions, which are formed at different times so as to image the corresponding different layers within the conduit. Although five different light sheet positions are shown in figure 2b, as few as one or two such positions may be used, or a much larger number such as at least 10, or at least 100. The elements of figure 2a which provide the light sheet 46 within the conduit 18, and permit imaging of the bioink as illuminated by the light sheet may together comprise a light sheet microscope generally indicated as 60, although the conduit 18 would usually be considered to be a sample rather than forming part of the light sheet microscope 60 itself. Aspects of the light sheet microscope 60 may then be controlled using a light sheet microscope controller 62. Such aspects may for example include timing and/or wavelength control of the light source 40, z-slice control of the position of the light sheet within the conduit 18 through control of the z-drive 54 and/or the light sheet optics 44, timing control of the light sheet optics for example if a scanning operation is used in forming the light sheet, control of the imaging device for example to coordinate image frames with respect to timing aspects of the light source and/or light sheet optics, and so forth. The light sheet microscope controller may in turn exchange control data with the bioprinter controller 30 already discussed in respect of figure 1 , or the bioprinter controller 30 and light sheet microscope controller may be implemented in a single control function or unit.

Image frames I of the bioink as illuminated by the light sheet 46, which are output by the imaging device 52 along with any required timing or other metadata, are passed to an analyser 54 which is arranged to infer properties P of the flow of the bioink 12, or of the bioink itself within the conduit 18 from the image frames. Inferred properties P may be stored, displayed or further processed at the analyser itself, or may be passed on to other computer elements such as personal computer 56 shown in figure 2a, for further analysis, monitoring of the bioprinter, and so forth. Such properties and related data may be transmitted to such other computer elements in a variety of ways such as over a wired or wireless network or data collection.

The inferred properties P may also or instead be passed to the bioprinter controller 30 to enable the bioprinter to take suitable actions based on the properties. For example, adverse or undesirable flow properties of the bioink may be addressed by actions such as adjusting a temperature of the bioink in a reservoir or conduit, changing a bioink flow rate through a conduit, cleaning a conduit for example using a cleaning station as illustrated in figure 1 , or in other ways.

Figures 3a to 3c illustrate a plurality of successive image frames 72 of the bioink 12 as illuminated by a light sheet 46 within a conduit 18, acquired using the imaging device of figure 2a or in a similar manner, and passed to the analyser 54 for determining properties of the flow of the bioink 12. Each image frame 72 represents a plan view of the light sheet 46 as projected across the conduit 18, where the position of the light sheet, in terms of distance from the imaging optics 50 or “z-slice” is unchanged between the image frames. The direction of flow of bioink along the conduit 18, and therefore also substantially in the plane of each image frame, is shown by the “y-axis” arrow in the bottom left hand corner of figure 3a.

The shorter dimension of each image frame in figures 3a to 3c then corresponds to the extent of the light sheet across the conduit 18, which is labelled using the x-axis arrow in the bottom left hand corner of figure 3a, and this dimension preferably extends at least to each wall of the conduit at the current z-slice level of the light sheet, so that the bioink close to the conduit wall is imaged. Boundaries of the conduit are visible and shown as broken lines 18’ in these figures, and are shown for reference in the graph panel of figure 3d. If the light sheet is projected across a narrower, lower or upper part of the conduit than through the widest point, then the walls of the conduit may increasingly encroach in from the sides of the image frame, as seen in some of the image frames of figure 4.

The three image frames of figures 3a to 3c are successive in the sense that they are spaced apart in time, which progresses through the sequence of figures. Other image frames may have been acquired between those shown, as well as before and after those shown. A typical spacing in time between each image frame, and therefore the effective frame rate, may be of the order to 10 to 100 milliseconds or 100 to 10 Hz. An optimum choice of frame rate may be made depending on expected flow rate of the bioink, available signal to noise ratio at different frame rates, and so forth.

Although not necessarily shown to scale, three living cells 70 contained within the bioink are shown within each image frame. Of course, fewer or more cells might be visible in any actual such image frame, with a typical number be determined by the thickness of the light sheet 46, the number density of cells 70 within the bioink, and the dimensions of the imaged light sheet portion. In figures 3b and 3c previous positions of each cell are shown using broken lines.

These living cells 70 may be visible in each image frame through fluorescence of the cells in response to the probe light of the light sheet, either due to inherent fluorescence or through suitable staining of the cells, in which case the technique used to image the cells may be referred to as light sheet fluorescence microscopy. Alternatively, or additionally, the cells may be visible in each image frame through elastic scattering of the probe light of the light sheet from the living cells, or in other ways.

Cells which in principle are within the lateral boundaries of an image frame but which are not illuminated by the light sheet in its current z-slice position do not fluoresce or scatter the probe light, so are not visible in the image frames. In this way, the light sheet is used to geometrically select for a small number of cells which fall within the light sheet and therefore a current conduit layer at any one time. Because the light sheet and therefore conduit layer extends parallel to the direction of flow of the bioink along the conduit 18, each cell can be seen in successive image frames, as long as time between frames is not too long.

The analyser 54 shown in figure 2a is then arranged to process the image frames to track cells which become visible in the series of image frames, and to infer properties of the flow from the tracked cells. In particular, the velocity of the flow at any particular lateral position across the conduit may be determined by measuring the distance travelled by a particular cell between each of multiple image frames.

By acquiring a sufficient number of successive image frames, a sufficiently large number of cells can be tracked so as to determine a reasonably accurate profile of flow velocity across the conduit. A result of this determination is illustrated in figure 3d, which provides a graph of inferred flow velocity v as a function of position across the conduit x, with the velocity in this case being relatively high in the centre of the conduit and falling off to approximately zero at each side wall. Other properties of the flow can then be inferred from the profile of flow velocity as required, for example profiles of shear stress and/or shear rate, and regions or particular flow types such as shear thinning, shear thickening, and Newtonian flow.

Although figures 3a to 3c illustrate how properties of the flow of the bioink may be inferred by tracking a number of living cells contained within the bioink across a number of successive frames, more generally any suitable features of the bioink may be tracked. For example, instead as or as well as tracking the movement of living cells, the analyser may track the movement of non-cell markers which have been introduced into the bioink for this and/or for other purposes. Other features which may be sufficiently visible to permit such tracking may include imperfections and variations within the bioink itself, for example due to changes in density, medium properties and so forth, boundaries which develop between different portions of the medium due to complex flow regimes, and so forth.

The described methods and apparatus may also be implemented to determine properties of flow of a material which is not a bioink, as discussed elsewhere in this document. In such cases the tracked features may be any suitable features visible in the image frames which are suitable for tracking. The invention may also be carried out by determining properties of flow from a single image frame, for example where the exposure time for the frame is of a suitable length such that one or more features of the flowing material are seen as tracks within the image frame, whereby the length of each track corresponds to distance travelled by that feature within the exposure time, and therefore velocity of flow of that feature. Such techniques may require the exposure time to be adapted manually or automatically in response to the apparent flow rate to optimise the number of usable tracks seen within an image.

In other embodiments, a plurality of successive images may be acquired of the same layer within the conduit, but instead of tracking the movement of features between successive frames, the length of tracks in each image frame due to a long exposure time are used to determine velocity of flow corresponding to the features causing those tracks.

Whereas figures 3a-3d illustrate successive image frames and a corresponding determined velocity profile for a single z-slice or layer through the conduit, corresponding to a single distance of the light sheet 46 from the imaging optics, figure 4 shows how the apparatus of figure 2a may be used to determine profiles of bioink flow properties at multiple z-slice positions, for example as illustrated in figure 2b which shows five such positions, thereby determining a three dimensional view of bioink flow within the conduit. In this figure, the successive image frames labelled Z2-1 to Z2-3 are taken when the light sheet is being formed at a central, diametric plane across the conduit at depth Z1 , and correspond to figures 3a to 3c. The corresponding velocity profile determined from tracking features such as living cells across these and further frames is shown at panel Z1-P.

The three successive images Z1-1 to Z1-3 are taken when the light sheet is being formed below the diametric plane of Z2, at a depth Z1 which is further from the imaging optics than Z2. Because the light sheet is not being formed across a full diameter of the conduit, boundaries of the conduit are visible and shown as broken lines 18’, shown also for reference in the graph panel Z1-P.

Similarly, the three successive image frames Z3-1 to Z3-3 are taken when the light sheet is being formed above the diametric plane of Z2, at a depth Z3 which is closer to the imaging optics than Z2. Because the light sheet is not being formed across a full diameter of the conduit, boundaries of the conduit are again visible and shown as broken lines 18’, shown also for reference in the graph panel Z3-P.

In some embodiments, a series of sufficient successive image frames are recorded at one particular z-slice, for example Z1 , in order to make a determination of properties of bioink flow in that layer, before the position of the light sheet is shifted in order to make a determination of properties of bioink flow at each of a second and subsequent positions Z2, Z3. The number of successive frames required at a particular z-slice to make such a determination may for example be around 10 to 100. Once all required positions of the light sheet have been used to probe all conduit layers, the sequence of positions may be repeated. If twenty different positions are used, each with 100 frames at 40 Hz frame rate, this would lead to a full three dimensional scan of the conduit for determining flow properties which took about 50 seconds. However, different frame rates, numbers of different z-slice positions, and number of successive frames at each position can be used.

If the imaging device can operate at a sufficiently high frame rate with adequate image quality to track features as required, then interleaving of successive image frames between different z-slice positions could be used. For example, the light sheet could be formed successively at each of four z-slice positions to take a single image frame at each, and this cycle repeated multiple times to enable the analyser to track features at each z- slice position essentially simultaneously, subject to the repeat frame rate at each such position being high enough to be able to track any one feature accurately before it moves out of view.

Figure 5 illustrates in more detail how the analyser 54 may be arranged in order to provide the required functionality of tracking features in the bioink such as living cells, and thereby inferring properties of the flow of the bioink. The analyser 54 may typically be provided using a suitable computer system, including memory, one or more microprocessors, and suitable input and output mechanisms, such as network connections and a data bus connection to the imaging device 52, LSM controller 62, or other element of the system which provides the required image frames generated by the imaging device 52. The functionality described herein may then be typically implemented using suitable computer software executing on the one or more microprocessors. If desired, the analyser 54 could be incorporated as part of the bioprinter controller 30, and/or the LSM controller 62, and/or a further computing device such as personal computer 56 as shown in figure 2a. The analyser 54 may carry out some or all of the described analysis as the image frames become available, shortly thereafter, or some longer time afterwards.

The analyser 54 comprises, or has access to, an image store 80 in which a series of successive image frames 72 of the bioink, as illuminated by light sheets as discussed above, are stored. A plurality of such series, for example at least one series for each of a plurality of different z-slices or conduit layers as discussed above, may be stored if required, and in figure 5 three such series are shown as S1 , S2 and S3. In principle, the processes of image analysis carried out by the analyser 54 in order to infer properties of bioink flow could be carried out without storing such series, instead processing and then discarding each subsequent image frame 72 as it is received from the imaging device 52, but this is likely to lead to a more complex implementation.

The supply of image frames 72 to the image store 80 is generally indicated in this figure by input /. A second data stream indicated as m provides metadata relating to the image frames, for example timing and z-slice position data describing the image frames, which may for example be received from the LSM controller 62. Meta-data for each of the image frame series S1, S2, S3 held in image store 80 is shown as meta data ml, m2, m3 respectively.

The analyser also comprises a feature tracker 82 which is arranged to receive a series of image frames such as S1, S2 or S3, and to track features seen in some or all of the successive frames of the series, so as to determine movement of each such feature across two or more of the images. These features may correspond to living cells 70 illuminated by the light sheet within the bioink, or other features illuminated by the light sheet as discussed above. An output of the feature tracker 82 may then be a set of velocity profiles 84, each velocity profile representing a profile of bioink flow velocity across a plane within the conduit illuminated by the light sheet for a particular z-slice position. In figure 5 these velocity profiles are shown as V1, V2 and V3, corresponding to image frame series S1, S2 and S3 respectively.

The feature tracker 82 may be implemented, for example, using feature tracking software available in the public domain, such as the “TrackMate” software implemented within the ImageJ project. This is available from https://imaqei.netn rackMate, and is discussed in Tinevez et al., " TrackMate: An open and extensible platform for single-particle tracking", Methods 115: 80-90 (2017), PMID 27713081 . Using this software, a Laplacian of Gaussian spot detection algorithm can be used for detection of the image frame features such as living cells to be tracked, with a suitably selected target feature diameter in terms of image pixels and an appropriate contrast constraint. An LAP (linear assignment problem) tracker algorithm available within the TrackMate software can then be used for relatively slow flow speeds, or a Kalman filter implementation which is also available, for higher flow speeds.

The velocity profiles 84 are then received by a flow behaviour unit 86 which infers particular target properties of the flow of bioink from the velocity profiles 84. Some such inferred properties are indicated in figure 5 as comprised in properties data store 88, and may be output by the analyser as properties P shown in figure 2a, in various ways and for various purposes. However, the determined properties of the bioink flow may also include flow velocities themselves, such as one or more flow velocity profiles, as well as profiles related to the velocity profiles such as shear rates, shear stresses, profiles of shear rate and/or shear stress, and so forth. At least some target properties can be inferred by the flow behaviour unit 86 by fitting the velocity profiles 84 with a suitable velocity equation for flow in conduits, such as the power law equation often referred to as the Ostwald de Waele model:

(1 ) where v(r) is the flow velocity at radius r from the conduit central axis, P _f is the frictional pressure in the conduit, L is the conduit length, R is the conduit radius, and K and n are the flow consistency index and the flow behaviour index respectively. Based on the determined value of n, the flow can be described as Newtonian if n=1, shear thickening if n>1, shear thinning if n<1, or plug flow as n approaches zero.

As well as properties such as K and n, the volumetric flow rate Q of the bioink 12 through the conduit can also be calculated as an integral of flow velocity over cross sectional area of the conduit. An average residency time T _a of cells of the bioink within the conduit can be calculated as a product of average flow velocity and conduit length, and other residency times such as an upper quartile residency time T _u can similarly be calculated for example from an estimate of the flow rate for the slowest quartile of the cross sectional area. From parameters such as the flow behaviour index n or more directly from the velocity profile v(r) or related parameters such as the shear rate profile, parameters such as an expected cell death rate or attrition rate D could also be calculated. Other properties of the bioink that may be automatically observed, recorded, or used as discussed below, could be for example a degree of deformation of living cells, or a distribution of concentration or flux of living cells within different parts of the flow. The cells within the bioink may for example be observed to deform or elongate, in particular under conditions of higher shear stress, and a measure of such deformation E may be derived for example as a function or position across the conduit E(r).

Thresholds can be applied to some inferred properties of the flow, such as the values of n, Q, T _a , T _u , D mentioned above, to provide further inferred properties in the form of Boolean or logical indicators of different flow conditions can then also be derived. For example, inferred properties element 88 as illustrated in figure 5 also includes a flow type flag 90 indicating whether the present flow is determined to a = strongly shear thickening, b = shear thickening, c = Newtonian, d = shear thinning, or e = plug flow, depending on the fitted value of the flow behaviour index n compared to pre-defined or calculated boundaries between these regimes. A volume flow flag 92 is also illustrated which indicates whether flow is 1 = too low, 2 = acceptable, or 3 = too high, depending on the volumetric flow rate Q and corresponding pre-defined or calculated boundaries between these regimes.

Other aspects related to flow of the bioink or other medium which may be determined from the acquired images may be: a determination or measure of non-laminar or turbulent flow, which may for example be apparent from a spread of determined flow velocities along a particular flow line within the conduit, or from apparent erratic velocity of particular features; a detection of one or more blockages or obstacles within the conduit, which may for example be apparent from flow which appears laminar, but which deviates in direction from an expected path; damage to the conduit, for example in the form of obstacles or leakage which may be evident again from irregularities in the observed flow, or deviations from the expected flow directions.

Based on inferred properties of the flow, control logic 94 may then send corresponding data to other elements of the system such as the bioprinter controller 30 and/or the LSM controller so that these control elements can take suitable consequent actions, and one or more alarms 96 may be triggered to bring particular situations to the attention of a user of the apparatus or bioprinter. Suitable consequent actions may include for example, performing a nozzle or conduit cleaning operation if volumetric flow rate has fallen too low, a halting bioprinting if the flow behaviour index indicates strongly shear thinning or strongly shear thickening behaviour, or adjusting the nozzle movement speed in accordance with determined volumetric flow rate so that the correct amount of bioink is extruded at any particular location. Alarms, which could be audible, visible, or through data messaging to a control screen or hand held device, could for example indicate that the flow behaviour index indicates strongly shear thinning or strongly shear thickening behaviour, that the volumetric flow rate is too low or too high, or an estimated cell death rate exceeds a threshold.

As well as or instead of using the described apparatus and methods for monitoring flow of bioink in a conduit actively being used within a bioprinter 10 to deposit bioink onto a workpiece, the described apparatus and methods may also or instead be used to test the flow of bioink in a conduit of a bioprinter for testing or monitoring purposes when the conduit is not actively being used to deposit bioink onto a workpiece. Figure 6 is similar to figure 1 , but also shows some locations in which elements of the light sheet microscope 60 such as the imaging optics 50, light sheet optics 44, and light source 40, may be located in order to fulfil various roles.

For example, LSM unit 110-1 in figure 6 may be provided as part of, or fixedly coupled to the multi-axis manipulator 16 so as to be available to actively monitor bioink flow within any conduit currently being carried by the manipulator. Some components of the light sheet microscope arrangement being used here may be located elsewhere within the bioprinter and connected to the LSM unit 110-1 for example using a flexible umbilical 112. For example, a separate LSM base unit 112 may house the required light source, optionally some or all of the light sheet optics, optically some or all of the imaging optics, and the imaging device, with umbilical 112 comprising optical fibres to carry probe light to the LSM unit 110-1 where final formation of the light sheet into the conduit takes place, and to carry imaging light from the conduit to the imaging device 52.

In other arrangements, a separate LSM unit 110-1 may be provided for, and fixed to or in relation to each conduit and or bioink reservoir.

In other arrangements, an LSM unit 110-2 may provide the required functionality to project a light sheet into, and acquire associated images of a conduit currently disposed at and being tested by a testing station 28. In yet other arrangements, an LSM unit 110-3 may provide the required functionality to project a light sheet into, and acquire associated image frames of a conduit currently disposed within a magazine 22 or other storage facility for bioink conduits and/or reservoirs.

The invention may also be used to infer properties of a bioink flowing within a conduit for the purposes of testing the properties and behaviour of different bioink formulations, different conduits, and different conditions such as extrusion pressure, temperature and so forth. In such situations apparatus and methods as described herein can still be used but outside of the context of a functional bioprinter. Results of such testing, including for example the inferred properties shown in the properties data store 88 of figure 5, may then be used to assist in preparing improved bioink formulations, for providing optimum parameters for operating a bioprinter using such formulations, developing improved conduits for bioink extrusion, or conduits more particular adapted to exhibit required properties and impose require behaviours on particular bioinks and so forth. For example, the invention may be used to help design conduits or nozzles for printing which can reduce overall shear stress on living cells in a bioink, to permit, enhance or control mixing between two bioinks, or more generally between two materials which have been introduced into the conduit, and so forth.

Figure 7 summarises in method steps how the described apparatus may be used to determine or infer parameters of the flow of bioink (or other material) within a conduit, including bioinks, conduits, and situations such as in bioink testing and bioprinter use as described above. At step 210 a light sheet 46 is formed, using a light sheet microscopy arrangement, through bioink 12 which is flowing within a conduit such as a conduit being used to extrude the bioink onto a workpiece by a bioprinter, or within a conduit being used to test the bioink. The light sheet 46 extends across the conduit, either across a full diameter through the central axis of the conduit, or across a narrower width of the conduit. The light sheet 46 ensures that only a single layer of the bioink is illuminated at any one time, and extends along the conduit in the direction of flow of the bioink so that a feature such as a living cell within the bioink remains within the illuminated layer to enable tracking of that feature to be carried out.

At step 220 a series of successive image frames of the layer of bioink which is being illuminated by the light sheet are acquired, for example using a microscope objective and imaging device. An optical axis of the microscope objective is preferably approximately normal to the illuminated layer of bioink being imaged. The successive image frames have a frame rate which permits features of the bioink such as living cells to be tracked across multiple image frames to enable their velocity to be determined. The features may be visible in the image frames through fluorescence which is stimulated by the probe light of the light sheet, for example through using a fluorescent dye on the living cells, and/or the features may be visible through elastic scattering.

The acquired image frames are then used in step 214 by tracking multiple different features visible in the bioink across multiple frames, to derive estimates of velocity of the bioink at different positions across the conduit cross section within the illuminated layer. In this way a velocity profile across the conduit is derived. In many situations, the flow will be essentially laminar, and the velocity profile will then represent the instantaneous and ongoing flow within the conduit. If the flow is turbulent or otherwise structured in some way, then the velocity profile may represent a profile of average velocity. Visible features may be tracked over several tens or several hundreds of frames or more to acquire a sufficiently accurate velocity profile with an adequate number of different tracked features across the full conduit width illuminated by the light sheet.

The velocity profile determined in step 214 is then used at step 216 to infer properties of the flow of the bioink within the conduit. Power law flow properties such as the flow behaviour index n and the flow consistency index K may be determined for example by fitting the velocity profile to a power law model such as the Ostwald de Waele model. Other properties such as volumetric flow rate, residency times of cells within the bioink and so forth may be derived or inferred, for example as already described. Boolean or logical indicators of different flow conditions may be derived by using predetermined or otherwise calculated thresholds based on derived properties. Although figure 7 proposes to first calculate a velocity profile across the conduit, and to then derive other properties of the flow from this velocity profile, embodiments of the invention may be used to derive a velocity profile or other velocity data relating to flow without proceeding to then also use such velocity date to derive further flow properties, and in this context any such velocity data should therefore also be considered to be properties of the flow which are inferred from the image frames.

The described arrangements and methods may also avoid the intermediate calculation of velocities or velocity profiles which are then used to infer other properties, for example by calculating various properties of the flow, other than velocity profiles, directly from results of the feature tracking.

Steps 210 - 216 of figure 7 show how properties of bioink flow can be found using a light sheet across the conduit at a particular position. At an additional optional step 218, the position of the light sheet is shifted to a configuration which is preferably parallel to, but spaced, from the initial configuration, and a further series of successive image frames is acquired at this new position. In this way, a plurality of series of image frames are acquired, each being used for feature tracking to derive a velocity profile at a different slice through the conduit. In this way a three dimensional profile of flow velocity can be built up so as to derive a more complete picture of flow properties within the conduit.

As illustrated by the broken line connections in figure 7, the further series of image frames using different light sheet positions may be acquired before feature tracking of any of the series is carried out, after feature tracking of an already acquired series, or even after a derivation of flow properties using an already acquired series, although this final option may prove unsatisfactory in that a more complete calculation of flow properties can be carried out using multiple acquired image frame series from different conduit cross sections.

A further optional step is illustrated at step 220, in which various actions may be taken on the basis of the inferred flow properties, such as the triggering of various alarms, or bioprinter control actions as already discussed above.

A particular experimental implementation of the invention is depicted in figure 8. A bioink reservoir was provided using an infusion syringe pump 310 (Cole-Parmer Single- Syringe) to drive water from a glass syringe 312 (Hamilton syringe 1001 ) along a 3mm tube 314 (Adtech FEP tube) to drive a gel extrusion syringe 316 (disposable luer-Lock syringe) attached to a 400 pm internal-diameter tube 318 (Adtech FEP tube) which provides the conduit 18 along which the bioink 12 flows. The tube 318 is fixed to a petri dish 320 (35 mm glass bottom petri dish) for holding it in the light sheet microscope discussed below. For the four bioink medium materials extruded along the tube 318 using this experimental set up, the flow rates used varied from 0.22ml/h to 8 ml/h, with corresponding central conduit flow velocities of up to about 50 mm/s

The four different bioink media used in the experiments were a gelatin methacrylate (GelMA) solution, a GelMA hydrogel preparation, a pluronic preparation, and an agar. Samples of a human osteosarcoma cell line were prepared and stained with CellTracker Violet (2, 3, 6, 7- tetrahyfro-9-bromomethyl-1 H, 5H-quinolozino-(9, 1-gh) coumarin, ThermoFisher). Cell samples were added to the bioink media at a concentration of 1 x 10 ⁶ ml ¹ . The viabilities of the cells were assessed before and after extrusion using the LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells, (ThermoFisher).

To acquire quasi-real time image frames of the bioink media flowing through the tube 318, a commercial light sheet fluorescence microscope, the Leica SP8 with DLS module was used. In the Leica light-sheet microscope the illumination objective 330 (FIC PL FLUOTAR 2.5x/0.07 DRY) sends a 405 nm wavelength laser beam vertically to two mirrors 332 screwed around the detection objective 334 (FIC APOL 10x/0.30 water). By scanning the laser at a frequency of 1400 Hz, a digital light sheet of 3.6 pm thickness was created in between the two mirrors horizontally. The 400 pm internal diameter tube 318 was positioned in between the two mirrors, immersed in water. The detection objective 334 was held vertically, and the collected image light passed through a 455-495 nm band pass filter 336 to select for fluorescence from the bioink material for collection with a 2048x2048 pixel camera 338 (Hamamatsu Flash 4 V2). The selected field of view of the layer of bioink material illuminated by the light sheet had dimensions of 735.76 pm x 420 pm and the effective pixel size at the light sheet layer was therefore 0.359 nm.

Two types of image frame data were acquired, one two-dimensional and one three- dimensional. In the first type, the central plane of the 400 pm-diameter tube 318 was imaged to obtain the maximum acquisition speed. The image frames were recorded every 28 ms, with an exposure time of 10.57 ms and a light time (time the camera shutter is opened) of 6.33 ms. 5000 frames were collected per series of image frames, which amounts to 25.4 GB of data each, with about 20 such image frame series acquired per bioink medium tested, while changing the speed of the flow.

In the 3D experiment, series of ten image frames at each of 266 light sheet planar positions, spaced at 2 pm, were acquired, each image frame having an exposure time of 7 ms and a light time of 4.2 ms, leading to the acquisition of one complete volume of image data every 7.68 s. The number of image frames per series was limited to ten so as to reduce the dataset size to more manageable levels, at about 13.2 GB. The TrackMate software discussed above was used for cell detection and tracking. In brief, the LoG (Laplacian of Gaussian) a spot detection algorithms were chosen based on the size (5-20 pixels) of the blob (cells) needed to be detected. The blob diameter was adjusted between 10-20 pixels and the threshold was set based on the contrast between the blob and the background. The LAP Tracker module of the TrackMate software, which relies on the Linear Assignment Problem (LAP) mathematical formulation, was used for low flow speeds, and the Linear Motion LAP Tracker which relies on a Kalman filter was used for high flow speed. Finally, erroneous tracks were filtered out or manually cured after morphological inspection.

The fluid behaviour was determined by fitting the experimental velocity profiles with the Ostwald de Waele model shown in equation (1) above, and the shear rate profile g was obtained using the equation: where the symbols are explained earlier in respect of equation (1). The volumetric flow rate of the extruded material Q through the tube 318 was calculated as the product of the average flow velocity and the cross-sectional area of the tube. The time spent by the cells in the tube before extrusion from the open end is known as the residence time and was calculated as the product of the tube length and the average flow velocity.

The high-speed imaging of the middle plane of the tube 318 made the tracking of the cells flowing through the tube more straightforward. Before gelling using UV light, the GelMA medium flowed at a high speed in the centre of the tube, with the distance between the same cell at subsequent frames being quite large, whereas going towards the tube walls the flow slows down. In the centre, the imaged cell shapes were distorted because of the high speed of the flow, but at the tube edges they were well resolved it was observed that they rolled on the surface of the tube, clockwise on the right side and counter clockwise on the left side. From 3D reconstructions it was seen that the flow was consistent through the whole tube volume.

The pluronic medium was observed to flow with a velocity that was almost constant throughout the tube, but close to the tube wall the flow became rapidly slower, with cells slipping on the tube surface. When gelled using UV light, the GelMA medium flowed with a constant velocity across the whole tube, behaviour also observed using the gelled agar medium. However, when gelled, GelMA is at least a two-phase medium, with solid gel chunks and liquid media flowing present at the same time. The cells in the liquid phase are believed to experience higher shear rate than those trapped in the gel chunks: in fact, the cells flowing in the liquid phase were observed to have a disturbed flow behaviour, which may introduce shear to those cells affecting their viability. Additional experiments involved curing the gel within the conduit using a UV light, so that the GelMA medium studied without curing, flowing along the conduit after curing using UV light, and flowing along the conduit while being cured using the UV light.

Cell detection and tracking with the TrackMate software lead to the estimation of the velocity of the cells passing through the tube. Non-gelled GelMA medium showed a gradient in the mean velocities, from the slowest cells near the tube wall to the fastest at the tube centre. The fitting of the power law flow model (equation 1) confirmed that the material exhibited Newtonian behaviour (n = 1) at all tested concentrations and velocities of flow. However, for the pluronic medium, the flow behaviour index n indicated a high shear thinning flow type, with n<0.08 at the three different flow speeds used.

In the case of gelled agar and GelMa media, the flow profile was largely consistent between different gel concentrations and at different velocities tested. In these case, n = 0 which means that these media were infinitely shear-thinning. Knowing that this is only an ideal condition, the movement of the gel can be attributed to “shear banding” along the tube, in which a layer of shear-thinned hydrogel near the walls acts as a lubricant allowing the rest of the solid-like hydrogel to slip through the tube in a process known as “plug flow”, which may protect the cells within the plug flow from shear and extensional forces.

Cell viability was tested only for the GelMA medium. At both 3 hours and 24 hours after extrusion, GelMA medium extruded in a gelled form showed better cell viability than the GelMA extruded in a non-gelled form; however, in both cases cell viability was higher at 3 hours that at 24 hours after extrusion. It was noticeable that in both cases the extrusion speed and the subsequently tested cell viability were inversely proportional, with a drastic decrease in viability when extruding at volume flow rate of 10 ml/h compared to a volume flow rate of 5ml/h. These findings are in agreement with our flow mechanics analysis, in which the non-gelled GelMA material experienced high shear rates flowing as a Newtonian fluid, while gelled GelMA experienced zero shear through plug flow behaviour.

Although particular embodiments and applications of the invention have been described, it will be apparent to the skilled person that various modifications and alterations can be made without departing from the scope of the invention. For example, although the described embodiments describe primarily the determination of properties of flow of bioinks, properties of flow of other materials may be determined, such as the flow of extrusion materials in other 3D printing applications and areas (including structural 3D printing of plastics, metals and other materials, of food material, and so forth), as well as the flow of materials in other areas of industry, manufacturing, medicine and so on. Similarly, although the described embodiments describe primarily the determination of properties of flow of materials by the tracking of visible features across multiple successive image frames corresponding to a flow layer illuminated by a light sheet, in some embodiments other techniques besides or as well as feature tracking may be used such as by measuring the lengths of tracks seen in a single image frame due to features moving across the image frame during an extended exposure time. Such techniques may for example be more attractive than tracking techniques in situations of high flow velocity relative to practical exposure times.