GB2519961A

GB2519961A - Particle identification apparatus, system, and method

Info

Publication number: GB2519961A
Application number: GB1319342.0A
Authority: GB
Inventors: John A Hunt; Susan Wilson; Stephen Walker; Michael John Loughran
Original assignee: Cellect Biotech Ltd
Current assignee: Cellect Biotech Ltd
Priority date: 2013-11-01
Filing date: 2013-11-01
Publication date: 2015-05-13
Also published as: GB201319342D0

Abstract

A particle identification apparatus for particle identification via luminescence comprises a slide assembly comprising a slide and slide holder. At least one surface of the slide has a photosensitive coating and the assembly has a removable cover such that prior to removal the cover prevents irradiation of the slide surface by ambient light and in use the cover is removable to expose said surface for application of particles thereto. A system (figure 2) includes the slide assembly and a light tight enclosure 16, 18 with fluid inlet and slide retaining means. The enclosure allows the slide assembly to be secured therein and the removable cover to be removed from the slide assembly such that fluid may be applied to the slide in the enclosure without exposing the slide to ambient light. The slide may be obliquely angled in the enclosure relative to the support or fluid inlet.

Description

Title -Particle Identification Apparatus, System, and Method The present invention relates to particle identification, and more particularly to apparatus, systems, and methods for particle identification via luminescence.

In medical, pharmaceutical, diagnostic and scientific applications it is desirable to be able to clearly identify, and separate, individual particles, for example cells.

There any many well-known methods of performing such separation, including, but not limited to, centrifugation methods, density gradient separation methods, magnetic-based separation methods, flow cytometry methods, and fluorescence assisted cell sorting methods. More advanced methods include, for example, the use of optical tweezers and the like.

A particular method of particle-identification and separation is disclosed in WO 02/078906. The method of WO 02/078906 is based on the selective adherence or attachment of particles to metal grains formed in a light sensitive substrate containing a photosensitizable metal compound by irradiation of the substrate and development of the photosensitized substrate or layer.

It is recognised that the method disclosed by WO 02/078906 provides a particularly effective approach in separating particles, and that such a method has the potential to be widely utilised in diagnostic tests and screening for medical conditions which are readily identifiable by particular characteristics or types of cells, for example cancer, amongst many other possible uses.

However WO 02/078906 focuses on the provision of a scientifically viable method only and does not contemplate the implementation of that method in a practical sense. In general, diagnostic tests and screening may require non-standard, dedicated equipment, which is often only found in specialised laboratories, and is therefore not available for widespread use. The need for such equipment may severely limit the extent to which diagnostic tests are available, and may also result in unreasonable delays in the processing of the results of any such tests.

It is therefore an objective of the present invention to provide a system which facilitates the utilisation of the methods that are disclosed by WO 02/078906 in a practical manner, for example as a diagnostic test.

According to a first aspect of the present invention there is provided a system for the identification of particles, the system comprising: a slide assembly having a slide, and a slide holder, the slide being supported by the slide holder; wherein a surface of the slide has a photosensitive coating and the slide assembly has a removable cover to protect the coating from exposure to light; and, a light tight enclosure having at least one fluid inlet and a slide retaining formation for releasably retaining a slide within the enclosure relative to the fluid inlet for application of particles-and one or more fluid reagent to the slide in use, wherein the enclosure is configured to allow removal of the cover from the slide assembly once within the light tight enclosure.

According to a second aspect of the present invention there is provided a particle identification slide assembly, the slide assembly comprising: a slide; and a slide holder, the slide being supported in the slide holder; wherein at least one surface of the slide is provided with a photosensitive coating; and wherein the slide assembly is provided with a removable cover, such that prior to removal the cover prevents irradiation of the at least one surface of the slide by ambient light and in use the cover is removable to expose said at least one surface for application of particles-thereto.

According to a third aspect of the present invention there is provided apparatus for the identification of particles, the apparatus comprising a light tight enclosure for use within the system according to the first aspect of the invention.

According to a fourth aspect of the invention, there is provided apparatus for the identification of particles, the apparatus comprising a light tight enclosure having at least one fluid inlet and a slide retaining formation for releasably retaining a slide within the enclosure relative to the fluid inlet for fluid application of particles-to an exposed surface of the slide in use, wherein the enclosure is configured to be positioned relative to a support surface in use and to allow the slide to be obliquely angled relative to the fluid inlet and/or support surface when held by the slide retaining formation to promote wetting of the slide by flow of a fluid applied via the fluid inlet along a surface of the slide.

Thus the flow of applied fluid along the slide may advantageously occur at least in part, or else substantially, under the action of gravity.

Any of the features that are discussed below may be applied in relation to any of the aspects of the present invention.

The system and/or enclosure may be portable, e.g. in one hand. The apparatus and/or enclosure may be generally pocket-sized. The system may be entirely manually operated, such that no power source is required.

The light tight enclosure may be reversibly actuable between an open configuration and a closed configuration, for example to allow for insertion and removal of a slide in use.

The light tight enclosure may comprise first and second body portions, which may allow relative actuation between the open and closed conditions. The first and second body portions may or may not be joined by a hinge. The first and second body portions may each comprise substantially concave and/or hollow portions.

The hollow portions may form an internal chamber of the light tight enclosure, for example when peripheral edges of the first and second body portions are brought together.

The first and/or second body portion may be shaped to define an opening in the enclosure in the closed condition. The first and/or second body portion may comprise a light seal member covering the opening, for example mounted in the interior of the enclosure. The light seal may be flexible. The opening and/or light seal may be provided at an interface between the first and second body portions.

The light seal may be mounted to one of the first or second body portions and may contact the other body portion when the enclosure is closed. The light seal may act as a wiper seal in use. The opening may be a slot or mouth and the light seal may be rectangular in form. The opening may allow removal of the slide holder cover in use, e.g. when the slide holder is mounted in the enclosure.

During removal of the slide holder cover the seal may act so as to maintain the light tight nature of the enclosure.

The light tight enclosure may comprise at least one projection, such as a tab. The at least one projection may be used to open the enclosure and may depend outwardly from a peripheral edge or wall of the enclosure. The light tight enclosure may comprise a plurality of projections. A projection may be provided on each of the first and second body portions, for example on a corresponding wall or side thereof. The peripheral edge or tip of the projection on the first body portion may be offset or recessed/set-back relative to the tab on the second body portion. This feature may help to prevent accidental opening of the light tight enclosure during use, for example by one (i.e. the upper) projection in use being within the periphery of the other projection. Each of the tabs may be provided with a grip formation.

The light tight enclosure may comprise a plurality of fluid inlets. At least one of the fluid inlets may be angled relative to the light tight enclosure. At least one of the fluid inlets may be provided at an angle in the range of 25-65 degrees. Most preferably, at least one of the fluid inlets is angled at 45 degrees relative to the light tight enclosure.

The angle of the fluid inlet facilitates effective wetting, e.g. directional wetting of a slide contained within the light tight enclosure. Further, the angled fluid inlet may be arranged such that fluid may be applied towards one end of a slide in use such that it can run over the slide towards its other end to ensure consistent wetting.

The enclosure may comprise a plurality of fluid inlets, for example two or three fluid inlets. One or more further fluid inlets may be angularly oriented relative to a first fluid inlet. The one or more further inlets may be perpendicular to the enclosure and/or slide mounted therein. The first inlet may be for wetting the slide.

The further inlet may be for applying cells to the slide once wet. Once the slide is evenly wetted by the fluid from the first inlet, it is found that fluid from the additional inlets will spread evenly over the slide and thus any additional inlets do not need to be angled or arranged towards one end of the slide.

Each of the plurality of fluid inlets may be substantially tubular in form. Each of the plurality of fluid inlets may comprise an inlet adapter. The inlet adapter(s) may be removably attached to the enclosure and may be disposable/replaceable or reusable. Each inlet adapter may have a bayonet fitting arrangement. Each adapter may extend through a corresponding fluid inlet port in the enclosure into the interior of the light tight enclosure.

Each fluid inlet may comprise a closure, for example a light closure within an internal duct of the inlet. The closure may take the form of a valve. The closure may be substantially light tight when closed. The closure may comprise a resiliently deformable closure material extending across an inlet opening. The closure may have one or more slit therein, thereby defining opposing and/or adjacent closure portions, which portions may co-apt when closed. A fluid delivery nozzle may be insertable through the slit to resiliently bias the closure into an open condition. The closure may comprise a split valve membrane.

The fluid inlet may be adapted so as to allow use/insertion of a variety of fluid delivery devices, e.g. nozzles, therewith. The inlet may comprise an abutment or restriction formation, for example so as to prevent the over insertion of a fluid delivery device, e.g. so as to maintain a desired spacing/orientation between the fluid delivery device and a slide within the enclosure.

The slide retaining formation may be provided on an interior of the light tight enclosure. The slide retaining formation may be upstanding from an interior surface or slide-supporting member of the light tight enclosure. The slide retaining formation may comprise a projection and may be shaped so as to correspond to a receiving portion of the slide holder. The retaining formation may be a male formation and may be shaped to engage with a correspondingly shaped formation, e.g. a female portion or opening, of the slide holder. The slide retaining arrangement may be arranged to prevent translation of the slide holder in a plane (i.e. in two orthogonal) directions and rotation of slide holder about two or three axes. The retaining formation may have a spine/spigot portion and one or more fin depending therefrom, e.g. in the direction of insertion of the slide in use.

The enclosure may comprise one or more slide supporting member in its interior, for example such that a slide is supported in an elevated position above a base of the enclosure in use. The slide supporting members may comprise a plurality of projections, e.g. such as upstanding internal walls or ridges, having free ends/edges on which the slide holder sits in use. The upper ends/edges may be aligned in a substantially horizontal plane. The plurality of projections may be spaced apart from each other and may extend in a direction that is substantially parallel with the direction of insertion of a slide in use. The projections may have a rounded/curved end.

One of the slide supporting members may bear the slide retaining formation. A central slide supporting member may be of a greater height than the remainder of the supporting members so as to define the slide retaining formation.

The slide support formation may support the slide at a desired orientation relative to the fluid inlet(s) and may allow excess liquid to run off the slide and be held within the light tight enclosure without immersing the slide in the run-off.

The system may comprise a base for supporting the enclosure relative to a support surface in use. The base may comprise an enclosure receiving portion such as a tray or planar support surface. The base may comprise an orientation adjustment mechanism for adjusting/setting the orientation of the enclosure relative to the support surface in use.

The base may comprise a plurality of legs and/or feet, typically on an underside of the base and/or enclosure receiving portion, for example in proximity to one or more corners thereof. The plurality of legs and/or feet may be adjustable, e.g. individually adjustable relative to the enclosure receiving portion. The plurality of legs and/or feet may comprise a threaded formation. Thus the position of the plurality of feet may be adjusted relative to the corresponding legs, thereby adjusting the height/orientation of the base relative to the support surface. This control is particularly advantageous for facilitating correct wetting of a slide within the light tight enclosure.

The plurality of feet may comprise a slip-resistant material or grip formations.

The base may comprise one or more orientation/level indicator, such as a spirit level.

The light tight enclosure and base may comprise corresponding engagement formations. The enclosure may be adjustably and/or removably engageable with the base. The enclosure may be pivotably engageable with the base. One of the enclosure and base may comprise a pivot projection and the other may comprise a pivot recess for receiving said projection. A slot or channel may lead to the pivot recess in order to allow location of the pivot projection in the recess. An exterior light tight enclosure may comprise at least one recess and/or slot formation. This may advantageously allow the light tight enclosure to be tilted on the base for directional wetting of a slide. Any of the pivot projection, recess and/or slot may be rounded. A plurality of opposing pivot projections/recesses may be provided, e.g. on opposing sides of the enclosure/base.

The slide may be received within the slide holder. The slide holder may be open-sided. The slide holder may comprise a frame portion and a base portion. The frame portion may surround the peripheral edges of the slide in use and may provide an upstanding peripheral wall of depth greater than or equal to the depth of the slide. The frame portion may be upstanding from the such that a recess is formed. One or more dimension of the recess may substantially match a corresponding dimension of the slide to be held. The slide may be received within the recess with a friction fit.

When a slide is held within the slide holder, an upper surface of the slide may be substantially flush with an upper/open face of the frame.

The base portion of the slide holder may comprise one or more apertures. The plurality of apertures may be arranged so as to allow run-off and/or drainage of fluid during the wetting of a slide.

The slide holder may comprise one or more grip formation, for example depending outwardly from the frame portion, such as a plurality of projections for manipulation of the slide holder during use.

The slide holder may comprise a locating formation, which may be shaped so as to engage with the slide retaining formation of the light tight enclosure, e.g. via a friction fit, snap fit, or any other such suitable engagement. The locating and/or retaining formation may comprise a plurality of spaced formations. The plurality of locating formations may be received in the spaces between the plurality of retaining formations.

The slide may be formed of any suitable substrate material that is sufficiently rigid, for example glass, plastic, tile, metal, or the like. A transparent material such as glass or plastic is preferred and a glass slide such as a microscope slide or the like is especially preferred. The slide may be provided with a photosensitive surface layer over a major surface thereof.

The photosensitive surface may be a light sensitive substrate. The light sensitive substrate may comprise at least one photosensitizable metal compound, e.g. as a single crystal layer. In one preferred embodiment of the invention the light sensitive substrate may comprise a photographic emulsion, e.g. comprising a photosensitisable metal compound, such as silver halide.

The removable cover of the slide holder may comprise a layer of metallic foil. The removable cover may comprise a light reflecting or impervious material so as to provide a light tight cover which prevents irradiation of the slide by ambient light.

The removable cover may be attached to the slide holder and/or the slide, for example over the photosensitive surface of the slide.

The removable cover may be adhesively attached to the slide assembly. The removable cover may comprise a tab extending beyond the perimeter of the slide holder. The tab portion may be of length greater than the length of the slide holder. The tab may fold back over itself at a first end of the slide holder and may extend the full length of the slide holder to define an overhang at the other end of the slide holder. The removable cover may be removed by pulling the tab at its free end to cause peeling the tab from the first end. The removable cover may be attached to the slide holder and/or the slide.

According to a fifth aspect of the present invention there is provided a method of particle identification, the method comprising: i. providing a slide having a photosensitive surface; ii. protecting the photosensitive surface from irradiation by light; iii. inserting the slide into a light tight enclosure; iv. exposing the photosensitive surface to the internal environment of the enclosure; and v. applying particles and one or more reagents to the photosensitive surface, such that the particles to be identified luminesce as a result of the interaction between the particles and the reagent.

The method may comprise pre-wetting the photosensitive surface of the slide prior to applying the particles and one or more reagents to the photosensitive surface.

The pre-wetting may comprise application of a fluid at an oblique angle towards one end of the slide surface. The method may comprise the step of tilting and/or pivoting the slide and/or enclosure, so as to cause or allow the pre-wetting fluid to flow down the surface of the slide and promote even wetting of the photosensitive surface. The pre-wetting of the slide may beneficially allow the particles and reagent to spread evenly across, and substantially cover, the entirety of the photosensitive surface of the slide. A substantially single layer of particles may be applied using this technique without the need for expensive or complex apparatus.

The particles and the one or more reagents may be applied at the same time or in succession. It may be advantageous in some embodiments to allow the particles a short time to settle on to the substrate and then apply the other reagents somewhat later. The particles and one or more reagents may be applied through the same or different fluid inlets.

The method may comprise the step of applying a further/developing reagent to the photosensitive reagent, such that the photosensitive layer is developed and traps the desired cells in place. The developing reagent may comprise a photographic developer.

The particles and one or more reagents may be applied by a fluid delivery device, such as a pipette or syringe, which may have a dispensing nozzle.

The pre-wetting liquid may comprise water and may be a saline solution or another sterile, typically non-toxic fluid.

The particles may be any particles that are capable of being labelled to luminesce, but the invention is particularly applicable to biological particles, such as cells, most especially living cells. The one or more reagents may be selected so as to chemically react with a desired sub-class of cells or individual cell type.

Any of the method steps may be performed using features of the apparatus or system defined in other aspects of the invention.

Practicable embodiments of the invention are described in further detail below with reference to the accompanying drawings, of which: Figure 1 shows a three-dimensional view of an enclosure according to one example of the present invention in its closed configuration; Figure 2 shows a three-dimensional view of a system according to an example of the present invention, when assembled prior to use; Figure 3 shows a longitudinal section through a system according to the present invention, in use; Figure 4 shows a three-dimensional view of an empty slide holder according to an example the present invention; Figure 5 shows a longitudinal section through a slide assembly according to an example of the invention; and, Figure 6 shows a three-dimensional view of a base for use in conjunction with the present invention.

The system, apparatus, and method described herein provide a way of facilitating the utilisation of the methods that are disclosed by WO 02/078906 in a practical manner, for example as a diagnostic test. The system of the present invention is advantageous in that it is portable and robust and can be used to identify and isolate particles, such as cells, in the field as well as in a more controlled laboratory environment. Furthermore, the system requires no electricity, and can be operated entirely manually. The system, apparatus, and method are further beneficial in that they do not require a skilled operator for use.

With reference to Figures 1 to 3, there is shown an enclosure, generally designated 10, according to the present invention, in the form of a housing having a light tight interior and a plurality of fluid inlet adapters 12, 14 and 15. The enclosure 10 has a main body which comprises first 16 and second 18 portions, each being generally concave in shape such that when brought together at their respective peripheral edges, the opposing portions form a substantially complete enclosure.

The first 16 and second 18 portions of the main body are joined by a hinge 20.

When closed, the body portions are releasably connected together by catch formations 21 as shown in Figure 2. In other embodiments, the hinge could be omitted in favour of further releasable connection formations. The catch formations could be swapped for other conventional releasable fastening arrangements, typically of quick-release design, such as one or more latch, clip or similar.

The enclosure 10 has a substantially rectangular cross-section, such that the housing is generally cuboid in form. First 22 and second 24 ends of the enclosure may be provided with rounded/curved edges. The hinge 20 is located at the second 24 end of the enclosure 10.

As shown in Figure 2, one of the first and the second body portions 16, 18 comprises an upstanding lip formation 19 about its peripheral edge. The lip creates an overlap (i.e. an interference fit) with the opposing peripheral edge of the other body portion so as to create a substantially light-tight interface therebetween when closed.

The enclosure 10 further comprises a pivot engaging recess 23 and an associated slot or channel 25. A plurality of pivot engaging recesses 23 and slots 25 are arranged such that there is one recess 23 and one slot 25 located on each side of the enclosure 10. The recesses 23 and slots 25 are located towards the second end 24 of the enclosure 10. Each slot 25 extends from the corresponding recess 23 and is arranged so as to locate/guide a pivot point (to be described below) into the pivot engaging recesses 23 in use. The slots 15 extend from a floor towards a top surface of the enclosure 10, e.g. spanning substantially the entire height of the enclosure 10.

The plurality of pivot engaging recesses 23 are substantially circular in plan and may be rounded in section. The depth of each slot 25 may be variable i.e. with distance towards the recess 23 or else laterally, e.g. having a curved cross sectional profile.

The first 16 and second 18 portions of the main enclosure body have substantially hollow sections, such that when the enclosure 10 is in its closed configuration the enclosure 10 has an internal chamber 26 as shown in Figure 3. However each body portion has a number of feature/formations extending into the internal chamber 26 as will be described below.

The first portion 16 has a plurality of upstanding spigots 30,32,34 arranged to receive the corresponding adapters 12, 14, 15. The first portion 16 further comprises an external recess 36 and an actuation projection in the form of a tab 38.

The recess 36 is provided in an upper surface of the first portion 16, at the first end 22. The tab 38 is located within, and extends from, the recess 36. The tab 38 may be provided with a grip formation 39, which in this example takes the form of an indentation in the external profile or edge of the tab.

A first spigot 30 is angled relative to the upper surface/wall of the first portion 16.

Second 32 and third 34 spigots are substantially perpendicular to the upper surface/wall of the first portion 16. Each of the spigots 30,32,34 is adapted to receive corresponding adapters 12, 14, 15 via a bayonet fining, although other fitting arrangements that allow selective attachment of the adapters may be used.

Each of the spigots 30,32,34 have a substantially circular cross section and internal passageway. Each of the spigots 30,32,34 act as a fluid inlet port into the enclosure of internal diameter to accommodate a fluid insertion nozzle. Each of the spigots 30,32,34 provide a female connection for the adapters.

Each of the adapters 12, 14, 15 have an upper portion 46 and a lower portion 48.

Both the upper 46 and lower 48 portions are substantially cylindrical in form. The upper portion 46 has a larger diameter, e.g. which substantially corresponds to the external diameter of the corresponding spigot 30,32,34, and the lower portion 48 has a smaller diameter, e.g. which substantially corresponds to the internal diameter of the corresponding spigot. Each of the adapters therefore take the form of a hollow peg or the like.

The change in diameter between the upper 46 and lower 48 portions of the adapters creates an internal abutment formation, against which a fluid delivery device, such as a pipette or syringe nozzle (not shown) can be located in use. This advantageously prevents over-insertion of the fluid delivery device and ensures adequate spacing between an end opening of the fluid delivery device and the slide surface in use, i.e. so as to prevent contact there-between. The abutment formation is tapered. This may allow a varying number of standard conventional fluid delivery devices to be accommodated.

Each of the adapters 12, 14, 15 has an internal closure 50, which is light tight, when closed. In this example, the closure 50 takes the form of an elastomeric membrane spanning the internal diameter of the adapter, typically towards its lower portion 48. The membrane has one or more discontinuity or slit which is closed in an undeformed condition, i.e. by the opposing edges of either side of the slit being in contact or overlapping. The closure may have two slits in a X-arrangement or may be akin to a duckbill valve, umbrella valve or similar.

The first spigot 30 and adapter 12 are disposed at an angle of substantially 45 degrees relative to the surface of the light tight enclosure 10, although an oblique angle of between 200 and 70° may be usable. Furtherthe first spigot 30 and adapter 12 may be disposed such that fluid injected therethrough contacts the slide at or towards one end of the surface thereof.

The second portion 18 of the main body provides a floor of the enclosure 10. The hollow section of the second portion 18 of the main body 14 comprises a plurality of upstanding formations for positioning/alignment of a slide holder in the enclosure interior in use. A first kind of formation comprises internal walls or ridges 52. A second kind of formation comprises a plurality of abutment formations 54.

Each of the upstanding ridges 52 is elongate, and extends along approximately two thirds of the length hollow section of the second portion 18. The ridges are substantially identical in form and aligned in parallel, side-by-side. The upper edges of the upstanding ridges 52 form a substantially level support structure relative to the enclosure floor, atop which a slide holder can sit in use as shown in Figures 2 and 3.

The plurality of upstanding formations 54 also take the form of walls, having a flat abutment edge, and are spaced apart from each other such that they may receive corresponding projections 74 on a slide holder 66 (to be described below) therebetween during use. A central upstanding formation 55 is a retaining formation which has a greater height than the remainder of the upstanding spigot formations 54. The central upstanding spigot formation 55 is shaped so as to be received by a corresponding retaining formation 76, in this example, a female formation, on the slide holder 66 during use. The central upstanding spigot formation 54 has a substantially cylindrical main body and a fin-like secondary body depending therefrom, such that the upstanding spigot formation 54 resembles a key or the like in section.

At the first end 22 of the second body portion 18, there is provided a recess in the peripheral edge, thereby forming an opening 56 at the first end of the closure when closed. Behind the opening 56, within the interior of the enclosure, a flexible sealing formation 58 is mounted to the floor of the second body portion 18. The formation 58 serves as a light seal and extends across the entire width of the recess in the second portion 18. The seal 58 is substantially elongate/rectangular in form and is mounted to the enclosure floor by a rigid upstanding receiving portion 60.

The sealing formation 58 is made of a suitable elastomeric material. Silicone rubber or PTFE blend Elastomers are especially suitable as they have heat resistance characteristics adequate to resist sterilisation over the lifetime of the apparatus, for example at 130°C in an Autoclave.

The seal 58 is held in contact or close proximity with an opposing internal surface of the upper portion 16 of the main enclosure body when the enclosure 10 is in its closed configuration. In this example, the surface is the underside of the recess 36 at the first end of the enclosure.

The second portion 18 of the main body further comprises a projection at the first end 22 in the form of tab 62. The tab is directly beneath and/or opposing the tab 38 in the upper body portion 16. The respective tabs are spaced by a short distance to allow the tabs to be prised apart to open the enclosure. In this example the tabs are spaced by the opening 56. The tab 62 extends outwardly from the enclosure body to a greater extend (i.e. beyond) the perimeter of the tab 38. This helps to prevent unwanted opening of the tabs, for example by accidentally catching the tab 38, and thus requires a purposeful opening action to be force the upper and lower body portions apart.

The enclosure has a plurality of support feet 64 on its underside, e.g. in the form of rounded nodes, typically located towards each corner of the enclosure.

A slide holder 66 according to the present invention is shown in Figures 4 and 5, of which Figure 4 shows an empty slide holder as viewed from the back, that is to say the underside in normal use, and Figure 5 shows a longitudinal section through a slide assembly including the slide.

The slide holder 66 comprises a base 68 and a peripheral frame portion 70.

The frame portion 70 is rectangular in form and is upstanding from the base 68.

The frame 70 has a hollow central portion within which the base portion 68 is located. The frame portion 70 has a depth which is greater than the depth of the base portion 68, thereby defining an open recess or depression spanning the majority of the open face area of the slide holder 66. The slide holder is thus generally tray-like in form.

The frame portion comprises a plurality of projections 72, which in this example are semi-circular in form, and are located on opposing sides of the frame 70. The plurality of projections 72 provide grips to facilitate the manipulation of the slide holder 66 during use, i.e. without touching the slide therein.

The slide holder 66 comprises locating formations which depend outwardly from the frame. The locating formations comprise a plurality of projections 74, and a retaining formation 76. The projections 74 are dimensioned so as to fit between adjacent upstanding walls 54 in the enclosure. The retaining formation 76 has an opening to receive the correspondingly shaped retaining formation 55 in the second body portion 18. The retaining formation is a female connector and in this example is substantially the shape of a keyhole, so as to provide for ease of location as well as inhibiting the freedom of rotational movement of the slide holder once engaged with the retaining formation 55.

The base portion 68 comprises a plurality of apertures 78. The apertures 78 take the form of windows and are elongate in form, such that a significant area of the base is open. The slide holder in this example has a trough or channel 80 at opposing sides thereof, i.e. at the inner edge of the frame. The openings 78 and channels 80 allow fluid run-off from a slide in the holder 66 in use.

A slide assembly is shown in Figure 5 in which a substrate 82 is provided within the slide holder 66. The substrate 82 has a photosensitive surface, typically provided as an outer surface layer on a major face of the substrate. The photosensitive surface may be in accordance with W002/078906 and may comprise photosensitive crystals at its surface, for example comprising silver halide. However the precise material makeup of the surface is not critical to the invention provided it can function to allow identification/isolation of particles at the substrate surface.

Substrate 82 is referred to herein as a slide, having a generally planar outer surface and encompasses any suitable substrate material that is sufficiently rigid, for example glass, plastic, tile, metal, or the like. The substrate may otherwise be referred to as a a tile, slate or other similar, generally planar, substrate. However it is generally preferred that a transparent substrate/slide is provided to allow inspection/lighting of the slide by way of a microscope or other imaging device after use in conjunction with the invention.

The slide assembly comprises a removable cover 84 extending across the entire open side of the slide holder 66, i.e. opposing the base. The removable cover 84 is attached around its perimeter to the upper edge of the frame 70. The cover 84 has a length dimension that is greater than the slide holder length so as to form an overhang. The removable cover 84 is doubled back on itself, e.g. at one end of the slide holder, and overhangs the opposing end of the slide holder so as to provide a tab portion 86. The tab portion 86 extends away from the end of the slide holder 66 so as to provide a graspable formation for peeling the cover away from the slide and holder when required. Thus it will be appreciated that the cover is more than double the length of the slide holder in this example of the invention.

The cover 84 comprises a light reflecting/impervious material layer, such as a metallic layer, and, in this example, an aluminium foil. The light reflecting layer may have one or more polymer layers, such as a lacquer, applied thereto, e.g. to protect the light reflecting layer and/or to promote adherence with the slide holder.

The removable cover 84 is sealed to the slide holder 70, and the seal formed is light tight. The cover may be sealed to the slide holder by use of an adhesive or may be heat sealed. This seal is broken when the cover is removed by pulling the extended tab.

Further, and especially if the slide is made from a transparent material, there may also be a light reflecting/impervious cover arranged on the back of the slide holder (not shown in the Figures). The light reflecting/impervious layer on the back of the slide holder may be the same as that on the front, such as a metallic layer, or may be different. The light reflecting/impervious layer may be sealed to the back of the slide holder in the same fashion as the cover on the front of the holder to provide a light tight seal, and may be removable the same fashion as that on the front of the holder, i.e. by pulling an extended tab. The tabs of the two covers may be genrally aligned at their free ends, and/or may be connected, such that both covers can be removed at once if desired.

The slide surface bearing the photosensitive material faces the cover 84 in the slide assembly. The cover 84 protects the photosensitive surface of the slide from contact and from dust and other extraneous matter. The slide is typically a close fit within the slide holder frame. The photosensitive surface of the slide may be spaced a small distance from the cover when assembled.

The slide holder is typically formed of a conventional plastic material in the medical device field, for example polypropylene. The slide holder may be injection moulded and may be disposable or recyclable after use.

The slide holder may be formed of a dark coloured material such that the holder is light tight, and thus in conjunction with the light reflecting/impervious cover layers sealed to the front and back of the holder ensures the photosensitive slide within the holder is not exposed to light until the cover layers are removed for use. A suitable means of sealing and choice of material for the holder and for the light reflecting cover layers ensures that the cover layers are sealed to the holder to form a light tight seal, but that this seal is sufficiently weak that the cover layers may readily be removed by pulling the tabs. For example, a suitable cover layer comprises an aluminium foil treated with a PET lacquer that may be heat-sealed to the polypropylene holder to provide a light-tight seal that may readily be removed by pulling the tab.

A base 88 for use with the light tight enclosure 10 of the present invention is shown in Figures 2, 3 and 6. The base comprises a receiving portion 90 a plurality of legs 92 and a plurality of feet 94, and a plurality of level indicators 95.

The receiving portion 90 is substantially rectangular in plan, and is dimensioned so as to receive a light tight enclosure 10. The receiving portion 90 takes the form of a tray, having a planar receiving surface on which the enclosure 10 sits in use.

The receiving portion 90 also comprises a plurality of upstanding walls 96 on opposing sides of the base 88, between which the enclosure can be received with a close fit.

A pivot projection 98 is provided on each upstanding wall 96, i.e. on opposing/inwardly-facing faces thereof. The pivot projections 98 are shaped and dimensioned so as to fit within the plurality of pivot engaging recesses 23 disposed on the enclosure 10, e.g. such that the enclosure can be releasably held thereon in use. The plurality of projections 98 thus act as pivot points and may be rounded in form, for example dome-shaped.

The legs 92 may be substantially cylindrical in form, and depend downwardly from an underside of the receiving portion 90. The legs 92 may each comprise a threaded portion along at least part of their length.

The plurality of feet 94 may each comprise a hollow central portion shaped to receive the lower end of a corresponding leg 92. In one embodiment, the feet 94 have threaded formations corresponding to the threaded portions of the legs 92, such that the feet may be attached to the legs via a screw fit. The screw fit nature of the attachment means that the length of each combined leg and foot may be selectively adjusted by rotation of the feet, thereby allowing the height and angle of the base 88 to be selectively adjusted. Alternatively the legs may be received in threaded formations in the receiving portion 90 of the base, such that rotation of the leg and foot in unison may adjust the height/angle of the receiving portion 90. Other types of height adjustment mechanism may be used provided they allow selective and accurate adjustment at each leg independently.

The feet 94 may each have a non-slip coating or surface on their underside.

The plurality of level indicators 95 are located on the receiving portion 90.

Preferably an indication of the level of the base 88 relative to horizontal is provided relative to two horizontal axes, i.e. in two directions. Typically two level indicators are provided at perpendicular orientations, e.g. aligned with a side and an adjacent end of the base 88 respectively. Spirit levels are used as level indicators in this

example.

The enclosure 10 and base 88 are intended to be reusable and may be formed of a material that is resistant to autoclaving or other conventional cleaning/sterilisation processes. A high temperature ABS or other resin/plastic is preferably used to ensure the surface quality of the enclosure is not warped or otherwise degraded by autoclaving.

Prior to use, the adapters 12, 14, 15 are located within the fluid inlets 30,32,34, of the enclosure 10 such that a light tight seal is formed.

The enclosure 10 is opened about its hinge 20 to allow insertion of a slide assembly of the kind shown in Figure 5. When in use, the grip projection 38 on the first body portion 16 of the enclosure 10 is used to move the enclosure 12 from its closed configuration to its open configuration. A slide assembly according to the present invention, as discussed above, is placed into the enclosure 10 such that the slide holder is seated atop walls 52 in the enclosure and the frame is located against formations 54. The slide holder retaining formation 76 is received by the upstanding enclosure retaining formation 55 such that the slide holder is held in place at its end which opposes the enclosure opening 56.

The first body portion 16 is closed and engages with the second body portion 18 by clips 21 so as to form a light-sealed interior. The graspable tab 86 of the removable cover 84 is located such that it extends outwardly from the first end 22 of the enclosure via opening 56 when closed. The fold in the cover is provided at the far end of the slide holder relative to the opening 56.

The graspable tab end 86 is then gently pulled so as to retract the removable cover 84 from the surface of the slide assembly, and expose the photosensitive surface of the slide to the interior of the light tight enclosure. As the graspable tab 86 is pulled, and the cover 84 is removed from the interior of the enclosure, the sealing formation 58 acts so as to retain the light tight nature of the enclosure 10, by resiliently pressing the cover against the opposing wall 60 as it passes through the seal. Further, if there is a removable cover on the lower surface of the slide holder this may be removed at the same time as the cover on the top surface of the slide holder.

Either before or after slide insertion, the enclosure 10 may be located on the base 88 such that the pivot projections 98 engage the pivot recesses 23 on the enclosure. The guiding channels 25 on the enclosure ease the mounting action.

The angular orientation of the base can be adjusted as necessary to ensure the base is horizontal.

A fluid delivery device containing a wetting solution, typically comprising water, such as phosphate buffered saline, is inserted into the first fluid inlet defined by adapter 12 and/or spigot 30, and is used to pre-wet the photosensitive surface of the slide. The angle of application to the slide significantly helps wetting along the length of the slide surface. The enclosure 10 may then be tilted manually, for example to an angle of approximately 45°, about pkot points 98 so as to ensure that the entirety of the photosensitive surface is covered by the wetting agent.

This controlled pivoting action, relative to the direction of insertion of the fluid is beneficial in ensuring correct wetting. The slide holder construction, including the openings and /or troughs therein allows free runoff of the wetting fluid, thereby preventing wicking at the slide edges and ensuring a thin and even fluid layer remains on the slide. The light tight enclosure 10 is then returned to a horizontal position. The manner in which the slide holder is retained ensures the slide holder does not move relative to the enclosure during agitation.

The pre-wetting of the slide is beneficial as it enables the particles and reagent applied there-after to spread across, and substantially cover, the entirety of the photosensitive surface of the slide. It is particularly beneficial to achieve as close as possible to a single layer of particles (i.e. a monolayer) over the slide surface.

Pre-wetting of the slide surface thus promotes fluid movement at the slide surface to achieve as close to a monolayer as possible in a field-ready apparatus.

A second fluid delivery device containing the particles and one or more light activation reagent, is inserted into the second fluid inlet (i.e. adapter 14 and/or spigot 32), and the particles and reagents are dispensed onto the slide. The desired particles to be identified then luminesce as a result of the reaction with the reagent. This process is allowed to happen for a pre-determined period of time such as for example 10-20 minutes. This time allows settling of the particles to be identified and exposes the local area of the slide surface (i.e. the photosensitive crystals therein) to the emitted light.

A third fluid delivery device containing a developing agent is then inserted into the third fluid inlet (i.e. adapter 15 and spigot 34), and the developing agent is dispensed onto the slide. The developing agent acts to develop the photosensitive surface of the slide, thereby fixing the particles that caused luminescence. Thus the identified particles can be held in place on the slide surface. In this example the photographic developer converts the exposed photo crystals from silver halide into silver, which traps the cells. Optionally a silver halide fixing solution, for example ILFOFID Hypam diluted 1+4, can be applied through the third inlet.

Once fixed, the slide can be flushed/washed with fluid to remove any particles that have not been fixed to the slide. This can be achieved initially by application of fluid via one of the fluid inlets, typically the first inlet. Additionally or alternatively, the slide holder and slide can be removed and rinsed.

The fluid delivery devices used may be pipettes or syringes or similar. The dispensing portion, i.e. the nozzle end, can be inserted into, and retracted from, the fluid inlets without breaking the light tight seal due to the requirement to resiliently deform the membrane 50 within the inlet.

Use of a plurality of fluid inlets and/or associated adapters for injecting the various fluids is advantageous in avoiding cross-contamination of the delivery devices. In view of the sensitivity of the method it is particularly desirable to avoid contaminating other fluids or equipment with the particles of interest for detection.

When the procedure has been completed, the projection 38 on the first portion 16 of the main body of the enclosure 10 is used to open the enclosure. The slide assembly can thus be removed for analysis.

The enclosure and base can be washed/sterilised for re-use. This apparatus provides a robust kit for use in the field, for example such that particle identification can be performed on site on any suitable, generally level surface. This is beneficial particularly for identification of living cells. The apparatus is particularly well suited for table top use and may also be used in a controlled lab environment.

Furthermore the apparatus does not require a power source, is cost-effective and can provide viable isolated particles for analysis, storage, culture or therapy immediately after separation.

In view of the above process, it will be appreciated that the apparatus and methods described above allow any or any combination of identification, isolation and/or fixing of particular particles from within a mixture of particles according to a selective reaction with a chemical/biochemical reagent. The basic technique for particle identification involves: conjugating luminophoric (light emitting) agents with specific antibodies and adding them to a particle mixture. They then attach to specific antigens on the target particles in the mixture. The selective labelling of target particles is achieved with a photophoric probe that can be activated to emit light. The method then involves applying the sample to a photosensitive substrate and activating the photophoric probe to emit light which photosensitises the substrate and, on development, forms metal, particularly silver, grains to which the labelled particles will attach, leaving the others free to be washed off.

The process and apparatus have been tested using particles in the form of polystyrene beads as follows: a proportion of beads was tagged by labelling with a photophoric probe on the surface, and also coloured by staining such that these particles can be identified visually. The tagged particles were then mixed with a considerable excess of unlabelled particles stained a different colour. When run through the process, it has been found that the target beads can be easily separated by being fixed on the surface of the photosensitive plate and identified by their colour even when the ratio of target particles to unlabelled particles is as low as 1:1,000,000 to 1:8,000,000. Further, where single figures or double figures of target particles are provided in such concentrations it is found that 50-100% of the target particles within a mixture of particles are fixed and selectively recovered after rinsing the plate.

Any of the details of the process described in WO 02/078906 may be used with the apparatus/process of the present invention and so the contents of WO 02/078906 is incorporated herein by reference.

The terms light tight', light impervious' or light seal' as used herein are intended to mean substantially impenetrable to wavelengths of light to which the substrate/slide is sensitive (i.e. to a degree that prevents a light-sensitive reaction to occur). Accordingly it may not be essential that the enclosure is completely impenetrable to all ambient light.

Among particles that can be identified/separated using the methods and apparatus described herein include in particular particles of biological origin, such as living cells, dead cells, sub-cellular components, and organelles and other macromolecules or molecular complexes or aggregates.

Claims

Claims: 1. A system for the identification of particles, the system comprising: a slide assembly having a slide; and a slide holder, the slide being supported by the slide holder; wherein a surface of the slide has a photosensitive coating and the slide assembly has a removable cover to protect the coating from exposure to light; and a light tight enclosure having at least one fluid inlet and a slide retaining formation for releasably retaining a slide within the enclosure relative to the fluid inlet for application of particles and one or more fluid reagent to the slide in use; and wherein the enclosure is configured to allow removal of the cover from the slide assembly once within the light tight enclosure.
2. A system according to claim 1, wherein the light tight enclosure is portable.
3. A system according to claim 1 or 2, wherein the light tight enclosure comprises first and second body portions, which are shaped to define a generally hollow interior of the enclosure in use, the first and second body portions being selectively actuable between the open and closed conditions to allow for insertion and removal of the slide assembly therein.
4. A system according to claim 3, wherein the first and/or second body portion is shaped to define an opening in the enclosure in the closed condition.
5. A system according to claim 4, wherein the first and/or second body portion comprises a light seal member covering the opening to prevent ingress of ambient light into the enclosure.
6. A system according to claim 5, wherein the light seal is flexible so as to allow removal of the removable cover from the slide assembly when mounted in the enclosure in the closed condition.
7. A system according to any preceding claim, wherein the fluid inlet is obliquely angled relative to a slide when supported within the enclosure.
8. A system according to claim 7, wherein said fluid inlet is arranged towards an end of a slide when supported within the enclosure.
9. A system according to any preceding claim, wherein the light tight enclosure comprises a plurality of fluid inlets arranged to deliver fluid to the slide surface in use.
10. A system according to any preceding claim, wherein the, or each, fluid inlet comprises a light tight closure, said closure being openable by insertion of a fluid delivery device into the inlet.
11. A system according to claim 10, wherein the closure comprises a resiliently deformable material spanning an internal duct of the inlet.
12. A system according to any preceding claim, wherein the, or each, fluid inlet comprises an inlet adapter member which is removably attachable to the enclosure.
13. A system according to any preceding claim, wherein the, or each, fluid inlet comprises an abutment formation for limiting the depth of insertion of a fluid delivery device therein.
14. A system according to any preceding claim, wherein the slide retaining formation is arranged to cooperate with a corresponding formation on the slide holder so as to prevent movement of the slide within the enclosure.
15. A system according to any preceding claim, wherein the enclosure comprises a slide supporting structure within its interior comprising a plurality of projections which are aligned in height relative to a floor of the enclosure so as to suspend the slide above the floor in use.
16. A system according to any preceding claim, further comprising a base for supporting the enclosure relative to a support surface in use, the base may comprising an orientation adjustment mechanism for setting the orientation of the enclosure relative to the support surface in use.
17. A system according to claim 16, wherein the base comprises a plurality of legs for supporting the enclosure above the support surface, wherein the height of each of the legs in individually adjustable.
18. A system according to claim 16 or 17, wherein the base or enclosure comprises an orientation sensor or indicator.
19. A system according to any one of claims 16 to 18, wherein the light tight enclosure is pivotably engageable with the base.
20. A system according to any preceding claim, wherein the slide holder comprises a peripheral frame portion and a base portion, the base portion comprising one or more apertures.
21. A system according to any preceding claim, wherein the slide holder comprises a locating formation shaped to engage with a slide retaining formation of the light tight enclosure.
22. A system according to any preceding claim, wherein the removable cover comprises a tab extending beyond the perimeter of the slide holder.
23. A system according to claim 22, wherein the tab portion is of length greater than the length of the slide holder and is folded back upon itself at a first end of the slide holder so as to define an overhang at the other end of the slide holder.
24. A system according to claim 23, wherein the overhang passes through an opening in the enclosure in use to allow removal thereof when the enclosure is closed.
25. A slide assembly for identification of particles, the slide assembly comprising: a slide; and a slide holder, the slide being supported in the slide holder; wherein at least one surface of the slide is provided with a photosensitive coating, and wherein the slide assembly is provided with a removable cover, such that prior to removal the removable cover prevents irradiation of the surface of the slide by ambient light and in use the cover is removable to expose said at least one surface for application of particles thereto.
26. Apparatus for the identification of particles, the apparatus comprising a light tight enclosure having at least one fluid inlet and a slide retaining formation for releasably retaining a slide within the enclosure relative to the fluid inlet for fluid application of particles to an exposed surface of the slide in use, wherein the enclosure is configured to be positioned relative to a support surface in use and to allow the slide to be obliquely angled relative to the fluid inlet and/or support surface when held by the slide retaining formation to promote wetting of the slide by flow of a fluid applied via the fluid inlet along a surface of the slide.
27. A method of particle identification, the method comprising: providing a slide having a photosensitive surface; protecting the photosensitive surface from irradiation by light; i. inserting the slide into a light tight enclosure; ii. exposing the photosensitive surface to the internal environment of the enclosure; and iii. applying particles and one or more reagents to the photosensitive surface, such that the particles to be identified luminesce as a result of the interaction between the particles and the reagent.
28. The use of a system, apparatus or method according to any preceding claim for identification of biological particles, such as cells.
29. A system, apparatus or method substantially as hereinbefore described with reference to the accompanying drawings.