Sample holder, and systems and methods for imaging and illuminating samples in a sample holder

Abstract

Sample holder, and systems and methods for imaging and illuminating samples in a sample holder A sample holder (310, 310', 310'', 710) comprises: one or more recesses (312) for receiving a respective sample (350) for imaging, each recess comprising: sidewall portions (316) extending from a first end of the recess to a second end of the respective recess, and a base portion (314) located at one of said first end or said second end. The respective sidewall portions of each recess are inclined towards the respective sidewall portions of the other recesses of the plurality of recesses. A method comprises: providing a sample holder (310) comprising at least one recess (312) for receiving the sample (350); and imaging the sample using a camera (302); wherein each recess comprises: sidewall portions (316) extending from a first end to a second end of the recess, and a base portion (314) located at one of said first end or said second end of the recess, wherein respective central axes of each recess are aligned with lines of sight from the camera. The sidewall portions of each recess may be aligned with lines of sight from the camera. The recesses may be illuminated from the first end with convergent light. The convergent light may converge towards a point located at an effective aperture of the camera.

Description

Sample holder, and systems and methods for imaging and illuminating samples in a sample holderFIELD

[0001] The present disclosure relates to a sample holder and to systems and methods for imaging and illuminating samples in a sample holder.BACKGROUND

[0002] Sample holders such as petri dishes and well plates are typically used for imaging biological samples and small organisms, such as Caenorhabditis (C. elegans). Such sample holders generally have a flat base on which the sample or organism is supported, optionally on a layer of material such as agar, and side walls to retain the organisms. The sample holder may be subdivided, as is the case for a well plate or a compartmented petri dish, to provide multiple compartments or recesses for studying multiple samples or organisms. A camera may be used to image the samples or organisms from above. However, organisms move around their respective compartment and it can be difficult to image an organism close to a sidewall of a compartment due to occlusion of the image by the sidewall.SUMMARY

[0003] Features of the invention are set out in the appended claims.

[0004] According to a first aspect of the present disclosure, there is provided a sample holder comprising: a plurality of recesses for receiving a respective sample to be imaged, each recess comprising: sidewall portions extending from a first end of the recess to a second end of the respective recess; and a base portion located at one of said first end or said second end; wherein the respective sidewall portions of each recess are inclined towards the respective sidewall portions of the other recesses of the plurality of recesses.

[0005] In some embodiments, the sidewall portions are inclined along straight paths.

[0006] In some embodiments, each recess of the plurality of recesses defines a respective central axis extending from the first end to the second end and passing through a centre of the base portion; wherein the central axes of the recesses of the plurality of recesses converge towards each other in a direction from the first ends to the second ends of the recesses.

[0007] In some embodiments, the central axes converge along paths corresponding to lines of sight from a point. The point may be located at a distance beyond the second end of the recesses.

[0008] In some embodiments, said plurality of recesses is a first plurality of recesses, wherein the respective central axes of the recesses of said first plurality of recesses convergetowards a first point; wherein the sample holder further comprises a second plurality of said recesses, wherein respective central axes of the recesses of said second plurality of recesses converge towards a second point.

[0009] In some embodiments, the sample holder may comprise one or more further pluralities of recesses, wherein respective central axes of the recesses of the or each further plurality of recesses converge towards a respective further point.

[0010] According to a second aspect of the present disclosure, there is provided a sample holder comprising: one or more recesses for receiving a respective sample to be imaged, each recess comprising: sidewall portions extending from a first end of the recess to a second end of the recess; and a base portion located at one of the first end or the second end; wherein said sidewall portions converge towards each other in a direction from the first end to the second end of the recess.

[0011] In some embodiments, the sidewall portions converge along straight paths.

[0012] In some embodiments, the straight paths correspond to lines of sight from a point. The point may be located at a distance beyond the second end of the recesses.

[0013] In some embodiments, the sample holder comprises a plurality of said recesses, including at least a first recess and a second recess; wherein respective sidewall portions of the first recess converge towards respective sidewall portions of the second recess.

[0014] In some embodiments, said plurality of said recesses is a first plurality of said recesses, wherein respective sidewall portions of the recesses of said first plurality of recesses converge along straight paths towards a first point; wherein the sample holder further comprises a second plurality of said recesses, wherein respective sidewall portions of the recesses of said second plurality of recesses converge along straight paths towards a second point.

[0015] In some embodiments, the sample holder may comprise one or more further pluralities of recesses, each plurality of recesses converging towards a respective further point.

[0016] In some embodiments, said first plurality of recesses is positioned adjacent or spaced apart from said second plurality of recesses.

[0017] In some embodiments, said first plurality of recesses is at least partly interleaved with said second plurality of recesses.

[0018] In some embodiments of the first or second aspects, the sidewall portions at least partially define a periphery of the respective recess.

[0019] In some embodiments, said periphery is at least one of: circular, elliptical, rectangular, square, hexagonal, T-shaped, Y-shaped, a maze structure, or an n-sided polygon.

[0020] In some embodiments, said sidewall portions are opaque.

[0021] According to a third aspect of the present disclosure, there is provided a system for imaging a sample in a sample holder, the system comprising: a sample holder according to one of the first or second aspect of this disclosure, as defined above; and a camera, arranged for imaging samples received in respective recesses of said sample holder; wherein a central axis and / or the sidewall portions of the or each recess are aligned along lines of sight from the camera.

[0022] In some embodiments, the sidewall portions of the or each recess converge towards a point located at an effective aperture of the camera.

[0023] In some embodiments, the camera is an entocentric camera.

[0024] According to a fourth aspect of the present disclosure, there is provided a system for illuminating a sample in a sample holder, the system comprising: a sample holder according to one of the first or second aspects defined above; and at least one beam shaping element for transmitting light, in use, from a light source to the or each recess of the sample holder, wherein the light transmitted to the recess or recesses of the sample holder is substantially convergent.

[0025] In some embodiments, respective central axes of the recesses and / or said sidewall portions of the or each recess converge towards a point; and said light transmitted by the beam shaping element converges towards said point.

[0026] In some embodiments, the sample holder comprises a first plurality of said recesses, wherein respective central axes and / or the respective sidewall portions of the recesses of said first plurality of recesses converge along straight paths towards a first point; wherein the sample holder further comprises a second plurality of said recesses, wherein respective central axes and / or the respective sidewall portions of the recesses of said second plurality of recesses converge along straight paths towards a second point; wherein said beam shaping element is a first beam shaping element, configured to transmit light, in use, from a first light source to the first plurality of recesses, wherein said light transmitted by the first beam shaping element converges towards said first point; the system further comprising a second beam shaping element, configured to transmit light, in use, from a second light source to the second plurality of recesses, wherein said light transmitted by the second beam shaping element converges towards said second point. In some embodiments, said first and second light sources may be provided by the same illumination module.

[0027] In some embodiments, said beam shaping element comprises a baffle defining at least one channel for transmitting light, in use, from the light source to a respective recess of the sample holder.

[0028] In some embodiments, the or each channel converges in a direction towards the sample holder.

[0029] In some embodiments, said sidewall portions of the or each recess of the sample holder converge towards a point; and said at least one channel of the baffle converges towards said point.

[0030] In some embodiments, said sample holder is a sample holder comprising a plurality of said recesses, wherein the baffle defines a plurality of channels for transmitting light from the light source to a respective recess of said plurality of recesses of the sample holder; wherein respective central axes of the channels converge in a direction towards the sample holder.

[0031] In some embodiments, respective central axes of the recesses and / or said sidewall portions of each recess of the plurality of recesses converge towards a point; and the respective central axes of the channels of the baffle converge towards said point.

[0032] In some embodiments, the or each channel is defined by respective channel sidewalls.

[0033] In some embodiments, the or each channel is defined by a sequence of apertures.

[0034] In some embodiments, the or each beam shaping element comprises at least one focusing element.

[0035] In some embodiments: the system further comprises said light source; respective central axes of said recesses of the sample holder and / or said sidewall portions of the or each recess of the sample holder converge towards a point; and the or each focusing element is configured to form an image of at least part of the light source at said point. In some embodiments, the or each focusing element is configured to form an image of at least part of the light source at said point.

[0036] In some embodiments, the system further comprises: said light source; and a camera, arranged for imaging samples received in respective recesses of said sample holder; wherein the or each focusing element is configured to form an image of at least part of the light source at an effective aperture of the camera.

[0037] In some embodiments, the light source comprises an extended light source having a dark spot at its centre; and the or each focusing element is configured to form an image of said dark spot at an effective aperture of the camera.

[0038] In some embodiments, the focusing element has a focal length f; said light source is positioned a distance 2f from the focusing element; and said effective aperture of said camera is positioned a distance 2f from the focusing element.

[0039] In some embodiments, said at least one focusing element comprises a plurality of said focusing elements; wherein each focusing element is configured to converge light through a respective recess of said sample holder.

[0040] In some embodiments, the or each focusing element comprises at least one of: a lens, a Fresnel lens, an axicon, a Fresnel axicon, a concave mirror and / or a hologram plate.

[0041] According to a fifth aspect of the present disclosure, there is provided a baffle structure for transmitting light, in use, from a light source to a sample holder, such that the light transmitted to the sample holder is substantially convergent; wherein said baffle structure defines at least one channel, extending from a first end of the baffle structure to a second end of the baffle structure, for transmitting light from the light source to the sample holder.

[0042] In some embodiments, the baffle structure defines a plurality of channels, wherein respective central axes of the channels converge in a direction from the first end to the second end of the baffle structure.

[0043] In some embodiments, the or each channel converges in a direction from the first end of the baffle structure to the second end of the baffle structure.

[0044] In some embodiments, the or each channel is defined by respective channel sidewalls in the baffle structure.

[0045] In some embodiments, the channel sidewalls converge along straight paths corresponding to lines of sight from a point.

[0046] In some embodiments, the or each channel may be defined by a sequence of apertures. The apertures may be defined in a corresponding sequence of planar elements.

[0047] According to a sixth aspect of the present disclosure, there is provided a method for imaging a sample, the method comprising: providing a sample holder comprising a plurality of recesses for receiving respective samples; and imaging the samples using a camera; wherein each recess of the plurality of recesses comprises: sidewall portions extending from a first end of the recess to a second end of the recess, and a base portion located at one of the first end or the second end of the recess; wherein respective central axes of each recess are aligned with lines of sight from the camera.

[0048] According to a seventh aspect of the present disclosure, there is provided a method for imaging a sample, the method comprising: providing a sample holder comprising at least one recess for receiving the sample; and imaging the sample using a camera; wherein the or each recess comprises: sidewall portions extending from a first end of the recess to a second end of the recess, and a base portion located at one of the first end or the second end of the recess; wherein the sidewall portions of the or each recess are aligned with lines of sight from the camera.

[0049] In some embodiments, the method further comprises: illuminating the or each recess from the first end with convergent light.

[0050] In some embodiments, the convergent light converges towards a point located at an effective aperture of the camera.

[0051] In some embodiments, the sample holder is a sample holder according to one of the first or second aspects defined above.

[0052] In some embodiments, the method is implemented using the system according to one of the third or fourth aspects defined above.

[0053] In some embodiments, the method includes illuminating the or each recess of the sample holder using light transmitted from a light source through a beam shaping element according to the fifth aspect defined above.BRIEF DESCRIPTION OF DRAWINGS

[0054] A more complete understanding of the subject matter may be derived by referring to the detailed description and claims when considered in conjunction with the following figures, wherein like reference numbers refer to similar elements throughout the figures:

[0055] Figures 1A and 1 B schematically illustrate an example system for imaging a sample in a sample holder;

[0056] Figure 1C illustrates an image obtained by the camera of the sample holder using the system illustrated in Figures 1A and 1 B;

[0057] Figures 2A to 2C schematically illustrate further example systems for imaging a sample in a sample holder;

[0058] Figure 3A schematically illustrates an imaging system including a sample holder according to an example embodiment of the present disclosure;

[0059] Figure 3B schematically illustrates an imaging system including a sample holder according to another example embodiment of the present disclosure;

[0060] Figure 3C illustrates an image obtained by the camera of the sample holder using the system illustrated in Figure 3A;

[0061] Figure 3D schematically represents a sample holder comprising four pluralities of recesses, each plurality of recesses being configured for imaging by a camera positioned at a respective one of four different locations;

[0062] Figure 4 schematically illustrates the imaging system of Figure 3A in use for brightfield imaging using transmissive illumination according to an example embodiment of the present disclosure;

[0063] Figures 5A schematically illustrates a system for illuminating and imaging samples in a sample holder according to an example embodiment of the present disclosure;

[0064] Figure 5B schematically illustrates another system for illuminating and imaging samples in a sample holder according to an example embodiment of the present disclosure;

[0065] Figure 6 schematically illustrates another system for illuminating and imaging samples in a sample holder according to an example embodiment of the present disclosure;

[0066] Figure 7A schematically illustrates another system for illuminating and imaging samples in a sample holder according to an example embodiment of the present disclosure; and

[0067] Figure 7B schematically illustrates another system for illuminating and imaging samples in a sample holder according to an example embodiment of the present disclosure.DETAILED DESCRIPTION

[0068] The following detailed description is merely illustrative in nature and is not intended to limit the embodiments of the subject matter or the application and uses of such embodiments. As used herein, the words “exemplary” and “example” mean “serving as an example, instance, or illustration.” Any implementation described herein as exemplary or an example is not necessarily to be construed as preferred or advantageous over other implementations. Furthermore, there is no intention to be bound by any expressed or implied theory presented in the preceding technical field, background, or the following detailed description.

[0069] It will be readily understood that the components of the embodiments as generally described herein and illustrated in the appended figures could be arranged and designed in a wide variety of different configurations. Thus, the following more detailed description of various embodiments, as represented in the figures, is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. While the various aspects of the embodiments are presented in drawings, the drawings are not necessarily drawn to scale.

[0070] Figures 1 A and 1 B schematically illustrate an example system 100 for imaging a sample in a sample holder 110 using a camera 102. In the Figures, the camera is represented by its effective aperture. The sample holder 110 is provided in the form of a well plate 110 and comprises a plurality of wells or recesses 112. Each recess 112 comprises a base portion 114 at a first, or lower, end of the recess 112, and sidewall portions 116. The sidewall portions 116 extend away from the base portion 114 towards a second, or upper, end of the respective recess 112 and define a periphery of the respective recess 112. In this example, the base portions 114 of the individual recesses 112 are provided by an upper surface of a base plate 118 of the well plate 110, and the sidewall portions 116 of the recesses 112 are provided by surfaces of a peripheral wall 120 and / or dividing walls 122 of the well plate 110 which subdivide the well plate 110 into the individual recesses 112.

[0071] In a conventional well plate, the sidewall portions 116 of the recesses 112 are parallel to a central axis of the well plate 110, orthogonal to the base plate 118 of the well plate 110, as shown in Figures 1A and 1 B.

[0072] A limitation arises when using a typical optics (e.g., an entocentric camera) to image the bottom of the recesses, due to lines of sight from the camera 102 to the base of recesses being occluded by the peripheral 120 and / or dividing 122 walls of the well plate 110. Although these walls 120, 122 are typically perpendicular to the base plate 118 of the well plate 110, they do not appear to be perpendicular when viewed by the camera 102 due to the geometry of the system. This is illustrated in Figure 1A, in which the optical axis 104 of the camera 102 is aligned with the centre of the well plate 110 and parallel to the side wall portions 116 of the recesses. The image plane 106 of the camera 102 is aligned with the upper surface of the base plate 118 of the well plate 110, that is, with the base portions 114 of the recesses 112. In this arrangement, when imaging the bottom of the recesses 112 away from the centre of the well plate 110, the dividing walls 122 obscure the camera’s view of some parts of the recesses. In Figure 1A, imaging rays 130, 132 are shown from the inner and outer edges of the base portion 114 of one of the recesses 112a to the aperture of the camera 102. These imaging rays 130, 132, can alternatively be considered as lines of sight from the camera 102 to the recess 112a. While the camera 102 has a clear view of the outer edge of the recess 112a along the ray 132, it can be seen that the ray 130 from the inner edge of the recess 112a is obscured the dividing wall 122a before reaching the camera 102.

[0073] The peripheral and dividing walls 122 of the well plate 110 may be opaque or transparent. Although transparent walls do not fully obscure the rays, the refractive, absorptive and absorptive properties of the walls still degrade the image quality and complicate the image, challenging analysis methods.

[0074] Figure 1 B schematically illustrates the field dependence of the occlusion of the recesses 112 by the dividing walls 122 of the well plate 110, for the same system 100 as Figure 1 A. In Figure 1 B, the optical axis 104 of the camera 102 is again aligned with the centre of the well plate 110. For each recess 112, Figure 1 B indicates a ray 140 from the respective recess 112 to the camera 102 corresponding to the limit of the camera’s unoccluded view of the base portion of the recess 112. The shaded areas 142 in Figure 1 B indicate occluded regions of each recess 112. It can be seen that the occluded portion of the image plane 106 within each recess 112 depends on the radial distance r from the optical axis 104 of the camera and becomes worse with increasing r.

[0075] Figure 1 C illustrates an image 190 obtained by the camera 102 of the well plate 110 using the system 100 of Figures 1A and 1 B. It can be seen that portions of the sidewalls 116 are visible in each of the wells 112, to an extent that increases with radial distance from the centre of the array of wells or recesses 112 of the well plate 110, which is aligned with the optical axis 104 of the camera 102. Similarly, a portion of each well or recess 112 is occluded by sidewall portions 116 of the respective recess 112, to an extent that increases with radial distance from the optical axis 104 of the camera 102.

[0076] To avoid the above limitation, a camera with a telecentric lens may be used for imaging samples in a conventional sample holder. Telecentric lenses are a special design of lens that has a larger aperture than the sample being imaged, and views the sample along ray bundles perpendicular to the sample at every point in the aperture of the lens. As a result, all structures (walls, compartment dividers, etc.) perpendicular to the plane of the sample being imaged appear perpendicular to the camera and do not occlude the sample. However, this is not a scalable solution because telecentric lenses are very expensive, particularly for the scale required to image a whole well plate in a single field of view, that is, from a single camera position. Telecentric lenses also have a low numerical aperture (i.e., a low light collection ability), which limits their suitability for fluorescence and bioluminescence imaging.

[0077] Another approach is to use a patrolling camera, whereby a single camera and light source move from well to well, imaging each well sequentially. However, this does not allow for synchronous or continuous (e.g., high speed or video frame rate) imaging of the wells (important for dynamic assays over cell cultures, e.g., dynamic heart tissue models, or for imaging motile micro-animals such as nematode worms), significantly reduces throughput, and increases the size, complexity and cost of the imaging system. In addition, vibration and noise from the motion of the camera and light source can introduce an inconsistent stimulus between samples in different compartments of the sample holder when imaged sequentially over an extended period of time.

[0078] Figure 2A schematically illustrates another example system 200 for imaging a sample in the conventional sample holder 110 using the camera 102, in which the samples 250 to be imaged are located higher up in each recess 112. In this example, the samples 250 are located above the base portion 114 of the respective recess 112 because they are supported on the surface 253 of a medium 252 (e.g., agar) contained in the recess 112. However, similar considerations apply to samples located within the volume of a liquid or other media, away from the base of the recesses 112. The camera 102 is positioned and / or configured such that the image plane 206 of the camera 102 is substantially aligned with a plane containing the samples 250. The samples 250 are illuminated by an extended light source in the form of an illumination module 260 having a planar light emitting surface 262. The illumination module 260 is located below the well plate 110 such that a sample 250 in a recess is illuminated by light from the illumination module 260 which has passed through the base portions 114 of the respective recess 112. Some light rays from the illumination module 260 are obscured by the peripheral and / or dividing walls 120, 122 of the well plate 110. Figure 2A indicates example rays 264a-c from the illumination module 260 passing through a respective sample 250 to the camera 102. Rays 264a and 264b pass through or are obscured by the dividing walls 122, impairing the image of the samples 250 at the camera 102. Unlike the problem discussed with reference to Figures 1A and 1 B above, this problem may alsoimpact samples located in central recesses 112 and can include obscuration from both the peripheral wall 122 and the dividing walls 122 of the well plate 112.

[0079] Figure 2B schematically illustrates another example system 200’, similar to the system 200 shown in Figure 2A. The system 200’ differs from the system 200 in that the well plate 110 shown in Figure 2A is substituted by a well plate 110’ having a greater density of compartmentalization. That is, for the same area, the well plate 110’ comprises a larger number of compartments or recesses 112’ than the well plate 110. As shown in Figure 2B, for a sufficiently high density of compartmentalization, some light rays from the illumination module 260, for example light ray 266, travel through more than one recess 112’. As a result, when imaging samples 250’ distributed throughout the volume of the recesses 112’, it may not be possible to determine within which recess 112’ the sample 250’ is contained. Even when samples are constrained to the surface 253 of the support medium 252, this further complicates the image analysis.

[0080] Figure 2C schematically illustrates another example system 200”, similar to the system 200 shown in Figure 2A. The system 200” differs from the system 200 in that the well plate 110 shown in Figure 2A is substituted by a sample holder 110” having a single recess 112” defined by a base portion 114” and sidewall portions 116”. For example, the sample holder 110” may be a conventional petri dish 110” having a baseplate 118 and peripheral wall 120. As illustrated by Figure 2C, the problem described above with reference to Figure 2A is applicable to the sample holder 110” having a single recess 112”, because some of the light rays 268a, 268b from the illumination module 260 which would otherwise be transmitted to the camera 102 via a respective sample 250” in the recess 112” may be obscured by the peripheral wall 220. For example, light ray 268a is transmitted to the camera, while light ray 268b is obscured by the peripheral wall 200.

[0081] Figure 3A schematically illustrates an imaging system 300 including a sample holder 310 according to an example embodiment of the present disclosure. The system 300 includes a camera 302 for imaging samples in the sample holder 310. In the Figures, the camera 302 is represented by its effective aperture. In this embodiment, the sample holder 310 is provided in the form of a well plate and comprises a plurality of wells or recesses 312. Each recess 312 comprises a base portion 314 at a first, or lower, end of the respective recess 312, and sidewall portions 316. The sidewall portions 316 of each recess 312 extend away from the respective base portion 314 towards a second, or upper, end of the respective recess 312. Together the sidewall portions 316 of an individual recess 312 define a periphery of the respective recess 312. In this example, the base portions 314 of the individual recesses 312 are provided by an upper (planar) surface of a base plate 318 of the well plate 310, while the sidewall portions 316 of the recesses 312 are provided by surfaces of the dividing walls 322 which subdivide the well plate 310 into the individual recesses 312 and, in some instances,the peripheral wall 320 of the well plate 310. The open ends of the recesses 312 terminate at a common plane. In contrast to a conventional well plate, such as that shown in Figures 1A and 1 B, the central axes 332 of the recesses 312 converge towards each other in a direction from the first end to the second end of the recesses, i.e. , in a direction towards the camera 302. (Here, the central axis 332 of a recess 312 passes through the centre of the respective base portion 314 and the centre of the respective opening of the recess 312.) In particular, the central axes 332 of the recesses 312 converge towards a point 340 along lines of sight from the point 340. As a result, the respective sidewall portions 316 of each recess 312 of the sample holder 310 are inclined towards the respective sidewall portions 316 of the other recesses 312 of the plurality of recesses. In addition, the sidewall portions 316 of each respective recess 312 of the sample holder 310 converge towards each other from the first end to the second end of the respective recess 312. That is, a cross-section of a recess 312 is smaller at the second end of the recess 312 (i.e, the upper or open end in Figure 3A), than at the first end of the recess 312 (i.e., the lower or closed end in Figure 3B).

[0082] When viewed from above, each recess 312 may be circular, elliptical, square, rectangular, hexagonal, or any other suitable shape. However, the sidewall portions 316 are inclined along (i.e., are aligned with) straight-line paths 330 as shown in the Figure 3A, which shows a vertical-plane cross-section. The sidewall portions 316 of each individual recess 312 also converge towards the point 340. That is, the sidewall portions 316 of each individual recess are aligned with lines of sight from a point 340. Furthermore, the sidewall portions 316of the plurality of recesses 312 all converge to the same point 340. As a result, the dividing walls 322 taper and are narrower at an upper end (open end) than at the lower end (closed end).

[0083] The camera 302, for example an entocentric camera, is arranged for imaging samples in the well plate 310, and is configured such that the image plane 306 of the camera 302 is aligned with the base portions of the recesses of the well plate 310. The optical axis 304 of the camera 302 passes through the centre of the well plate 310 and is orthogonal to the base plate 318 of the well plate 310, and thus to the base portions 316 of the recesses 312. The camera 302 is positioned relative to the well plate 310 such that the central axes 332 and the sidewall portions 314 of each recess 312 are aligned along lines of sight from the camera 302. To achieve this, the camera 302 is positioned such that the centre of the centre of its effective aperture coincides with the point 340 at which the central axes 332 and sidewall portions 316 of the recesses 312 converge. The effective aperture of the camera may be the entrance pupil of the camera. Note that, depending on the design of the camera lens, the entrance pupil may be located at a different position from the physical aperture stop of the lens. Angling the sidewalls 316 of the recesses 312 to align with lines of sight of the camera, as shown in Figure 3A, ensures that the sidewall portions 316 of the sample holder 312(dividing walls 322 and peripheral wall 320) do not obscure any lines of sight from the camera 302 for samples at any depth in the recesses 312. In images recorded by the camera 302 (for example see Figure 3C, discussed below), the sidewall portions 316 of a recess 312 appear to be perpendicular, that is, the apparent magnification (i.e., the apparent size) of the recesses is the same for the top and the bottom of the recess 312. Thus the sample holder of the present disclosure may enable all the recesses of a sample holder, such as a well-plate, to be imaged using a single, stationary entocentric camera, thereby reducing the cost and complexity of an imaging system, and enabling video rate imaging of an entire well plate or other compartmented sample holder.

[0084] In addition, angling the sidewalls 316 of the recesses 312 to align with lines of sight of the camera also ensures that the sidewall portions 316 do not obscure light rays from a light source located below the sample holder 312 (that is, on the opposite side of the sample holder 310 from the camera 302) from being transmitted via a sample located in a recess to the camera.

[0085] It may be helpful to consider the sidewall portions 316 of each recess 112 as being aligned with “view frustums” from the camera 302 to the base portion 314 of the respective recess 312.

[0086] The sample holder 310 of Figure 3A may be formed as a single part. Alternatively, the sample holder 310 may be assembled from a base plate and a separate structure (which may itself be of unitary or compound construction) comprising the peripheral and dividing walls 320, 322. The sample holder 310 may comprise a transparent base plate 318, to allow for transmissive illumination of the samples. The dividing walls 322 and / or peripheral wall 320 of the sample holder 310 may be transparent or opaque, for example, light absorbent. In the sample holder 310 shown in Figure 3A, the base portions 314 of the recesses 312 are flat, which is important for uniform transmissive illumination. However, for other applications, such as imaging bioluminescence, this may not be necessary. In some alternative embodiments, the upper end of the recesses 312 may be closed.

[0087] Figure 3B schematically illustrates an imaging system 300’ including a sample holder 310’ according to another example embodiment of the present disclosure. The system 300’ includes a camera 302 for imaging samples in the sample holder 310’. The sample holder 310’ is similar to the sample holder 310 of Figure 3A in that is provided in the form of a well plate having a base plate 318’ and comprises a plurality of wells or recesses 312’. Each recess 312’ comprises a base portion 314’ and sidewall portions 316’, the base portions 314’ of the individual recesses 312’ being provided by a surface of a base plate 318’ of the well plate 310’, and the sidewall portions 316’ of the recesses 312’ being provided by surfaces of the dividing walls 322’ and, in some instances, the peripheral wall 320’ of the well plate 310’.

[0088] However, the system 300’ differs from the system 300 of Figure 3A in that the camera 302 is arranged to image samples in the sample holder 300’ through the base plate 318’. Note that, although the system 300’ is illustrated in Figure 3B with the camera 302 above the sample holder 310’ and the recesses 312’ open at their lower ends, the entire system 300’ can be rotated to any orientation. As a result, the sample holder 300’ differs from the sample holder 300 of Figure 3A in that the base portion 314’ of each recess 312’ is located at the second end of the recesses 312’, and the open end of each recess 312’ is located at the first end of the recesses 312’. The central axes 332’ and the sidewall portions 316’ of the recesses 312’ of the sample holder 310’ converge towards each other in a direction from the first end to the second end of the recesses, i.e., in a direction towards the camera 302, in the same way as for the sample holder 300. However, because the base portions 314’ are located at the second end of the recesses in the sample holder 312’ of Figure 3B, the central axes 332’ and the sidewall portions 316’ of the recesses 312’ diverge from the base portions 314’ towards the open ends. This is different from the sample holder 300 of Figure 3A, in which the base portions 314 are located at the first end of the recesses 312, and the central axes 332 and the sidewall portions 316 of the recesses 312 converge from the base portions 314 towards the open ends.

[0089] Figure 3C illustrates an image 390 obtained by the camera 302 of the sample holder 310 using the system 300 illustrated in Figure 3A. (A similar image would be obtained using the system 300’ illustrated in Figure 3B.) The entire base portion 314 of each recess 312 is visible, without any occlusion by the sidewall portions 316 of the wells 312. Due to the sidewalls 316 being angled as described above with reference to Figures 3A and 3B, the apparent magnification of each recess 312 is constant from the first end to the second end of the recess.

[0090] In alternative embodiments of the sample holder of the present disclosure in which the sample holder comprises a plurality of recesses, in particular sample holders comprising a high density of recesses, the sidewalls of an individual recess may not necessarily converge towards each other. For example, the sidewall portions of each recess may be parallel to a central axis of the respective recess. Nonetheless, the sidewalls portions of each recess of the plurality of recesses are still inclined towards the respective sidewall portions of the other recesses of the sample holder. For example, central axes of the plurality of recesses may converge towards each other, for example towards a point located, in use, at the effective aperture of the camera.

[0091] While the sample holders 310, 310’ of Figures 3A and 3B are both shown in the form of a well plate, the sample holder of the present disclosure may have other configurations comprising a single or multiple recesses. For example, the sample holder may be a petri dish, a compartmented petri dish (e.g., 2-, 3-, 4-compartments), a well plate havingany number of wells or recesses (e.g., 6-, 36-, 96-, 384-, 1536-well plates), or any other geometry of sample holder having one or more recesses. The shape of the recess(es) (i.e. , the shape of the base portion 314, 314’) may be any shape, for example, circular, elliptical, rectangular, square, hexagonal, any other n-sided polygon, T-maze, or other maze configuration, depending on the application. In other embodiments, the recesses may be nested within each other, for example the recesses may be provided in the form of nested circles or ellipses. The size or area of the recesses will depend on the application. For example, the transverse dimensions of the recesses may range from of the order of a millimetre to of the order of a centimetre or more. The transverse size and pitch of the recesses may be configured to match standard devices, for example the open ends of the recesses may have a size and pitch compatible with a multi-channel pipette. The height of the peripheral and dividing walls 320, 320’, 322, 322’ may be low enough that they remain close to the focal plane 306 of the camera. For example, for a camera having a focal distance of around 20cm, the height of the peripheral and dividing walls 320, 322 may be up to a few centimetres.

[0092] In use, samples may be located on the base portions 314, 314’ of recesses 312, 312’, or may be located anywhere within the volume of the recesses 312, 312’. For example, a sample may be provided on or in another substance in the well, e.g., in a liquid or on a solid substance such as agar. Accordingly, the image plane 306, 306’ on which the camera is focussed is not necessarily aligned with the base portions 314, 314’ of the recesses 312, 312’, but may be located higher up in the recesses.

[0093] Although the camera 302 is shown having an optical axis 104 centred on and perpendicular to the plane of the base 318, 318’ of the sample holder 310, 310’ in Figures 3A and 3B, other embodiments of sample holder may be configured for use with a camera 302 located off-centre and / or at a different angle with respect to the sample holder, by configuring the sample holder such that the sidewall portions converge to a point at the intended camera location.

[0094] In yet further embodiments of the sample holder, the sample holder may include more than one plurality or group of recesses, wherein the recesses belonging to a first group of recesses converge towards a first point, while the recesses belonging to a second group of recesses converge towards a second point, and so on. A further camera may be provided to image samples in each further group of recesses, located at the respective further point of convergence. Alternatively, a single camera may be used to sequentially image each group of recesses from its respective point of convergence, with the camera or sample holder being moved relative to the other to image each group. The different groups of recesses may be arranged adjacent to each other, for example in a row or array. For example, a sample holder (or a portion of a sample holder) may be divided into four quadrants, with a camera perquadrant, with the respective sidewalls of the recesses in each quadrant conforming to lines of sight from the respective camera.

[0095] Figure 3D represents such a sample holder and system. In Figure 3D, an array of circles represents the upper ends or openings of an array of recesses provided in a sample holder 310”. The array of recesses is divided into quadrants. The bundles of central axes 332a, 332b, 332c, 332d of the respective plurality of recesses of each quadrant (and optionally the sidewall portions of the recesses of the respective plurality of recesses) converge towards a respective point 340a, 340b, 340c, 340d. Note that this arrangement could alternatively be considered as being equivalent to multiple copies of the system shown in Figure 3A, arranged adjacent to each other, wherein the multiple sample holders 310 of Figure 3A are joined to form a single sample holder 310”. Embodiments such as that shown in Figure 3D may allow cameras having smaller lenses and / or image sensors to be used to obtain the desired image resolution. In turn, this may allow the camera to be positioned closer to the sample holder by reducing the focal length required, resulting in a more compact system which may be more amenable to scaling up. Alternatively, a single camera could be moved between each of the positions to image each plurality of recesses sequentially.

[0096] Other advantages of dividing the sample holder into multiple regions, each configured to be imaged from a different camera position, are that this may prevent the angles of inclination of the recesses from becoming too extreme. Smaller angles make it easier to access and view (by eye) samples in the recesses, aiding in manual handling and sample preparation, and may result in better image quality due to the angles being closer to the optical axis of the camera. In yet other further embodiments, the recesses belonging to one group may be interleaved with or nested within recesses belonging to another group. In yet further embodiments, the point of convergence of the central axes and / or sidewalls of each individual recess may be optimised to account for the non-ideal behaviour of a specific camera lens. For example, the point of convergence of the sidewalls may vary with the radial distance of the recesses from a particular location of the sample holder, corresponding in use to a radial distance from the optical axis of the camera. This may be helpful if needed to take account of field effects due to the camera lens.

[0097] The sample holder described above may be used with all forms of imaging, including, but not limited to brightfield, darkfield, fluorescence, and bioluminescence.

[0098] Figure 4 schematically illustrates the system 300 of Figure 3A in use for brightfield imaging using transmissive illumination from an extended light source 460 located below the well plate 310. The extended light source 460 is provided in the form of an illumination module 460 comprising a planar light emitting surface 462 that emits light over a wide range of angles towards the well plate 310. A sample 350 in a recess 312 of the well plate 310 may be illuminated by light rays originating from various locations of the extendedlight source 460 (e.g., light rays 464a, 464b, 464c). However, only rays that reach the aperture of the camera (e.g., light ray 464a, shown as a solid line) contribute to the image (or signal). For example, ray 464a is transmitted directly through a sample 350 to the camera 302 and contributes to the image of the sample 350 at the camera 302. Other rays (e.g., light rays 464b, 464c, shown as dashed lines), which do not propagate directly to the camera 302, may nonetheless be scattered from turbid or scattering media. Some of this scattered light may reach the camera 302 resulting in noise on the image, thereby reducing image quality and signal-to-noise ratio. The example embodiments shown in Figures 5A, 5B, 6, 7A and 7B include alternative arrangements for illuminating the sample holder 310 to further improve image quality.

[0099] Figure 5A schematically illustrates a system 500 for illuminating and imaging samples in the sample holder 310 according to an example embodiment of the present disclosure. The system 500 includes the sample holder 310 and camera 302 arranged as previously shown in Figure 3A, and further includes an extended light source 560 arranged to illuminate the sample holder 310 through the respective base portions 314 of the recesses 312, and a beam shaping element 570 configured to transmit light from the light source 560 to the recesses 312 of the sample holder 310, such that the light 564 arriving at the sample holder 310 is convergent. In this embodiment, the beam shaping element 570 is provided by a focusing element, in the form of a converging lens 570 of focal length f, positioned below the sample holder 310. In this example, the light 564 arriving at each respective recess 312 of the sample holder 310 is convergent. In particular, the illumination light arriving at each recess 312 converges towards the same point 340 to which the central axes 330 of the recesses 312 and / or the sidewall portions 316 of the recess 312 converge.

[0100] Using cameras equipped with sufficiently high-quality image sensors and imaging lenses, microscopic resolution can be achieved by using a small, high resolution image sensor far from the sample (far meaning that the sample-to-camera distance is large compared to the diameter of the camera’s lens). To realise this resolution in the images obtained, light scattered by liquid or solid media in the sample holder must be minimized as this reduces contrast on the image, as discussed above with reference to Figure 4. One way to reduce scattering is to match the light cone of the illumination light reaching the camera to the view frustrum or angle of view of the camera. This way, only light that is going to hit the image sensor of the camera and contribute to the signal recorded in the image passes through the sample. This eliminates the much larger amount (in terms of flux) of light from a uniform or point source that would otherwise hit the sample but not the image sensor, which would produce a large quantity of scattered light arriving at the camera. In addition, this helps to minimise the light impingement on the sample which can be important for some biologicalapplications where chemicals being tested could photodegrade or where the sample is light sensitive.

[0101] In Figure 5A, the light source 560 is positioned a distance 2f below the lens 570, where f is the focal length of the lens 570. The sample holder 310 is positioned above the lens, and the camera 302 is positioned above the sample holder 310, at a distance of 2f from the lens 570. By distance, we mean a distance along the optical axis of the system, since in practice the optical path from sample holder to camera may include one or more mirrors. The lens 570 casts an image of the light source 560 onto the effective aperture or entrance pupil of the camera 302. A uniform extended light source 560 may be used such that the image of the light source 560 is cast over an extent equal to or slightly larger than the desired or selected camera aperture. In some embodiments, the image of the light source 560 itself may define the effective aperture. That is, rather than using a mechanical stop to reduce the effective aperture of the camera, the same effective aperture stop could be achieved by casting an image of the light source having an extent equal to the desired camera aperture. The lens 570 has a diameter slightly larger than the transverse dimensions of the sample holder 310 and is positioned close enough to the sample holder 310 that the entirety of the base of the sample holder 310 is illuminated. The lens 570 focuses the illumination light from the light source 560 on to the effective aperture of the camera 302, ensuring that only light contributing to the image formed at the image sensor of the camera passes through each sample. Since all the light passing through the recesses 312 of the sample holder 310 converges on the aperture of the camera 302, the ratio of scattered light generated by a sample to light contributing to its image is minimized, thereby maximizing image quality. A 2f:2f arrangement as shown in Figure 5A may be advantageous in that it is the most compact along the optical axis. However, the skilled person will appreciate that other camera-lens and lens-light source distances may be used to image the light source onto the camera.

[0102] In the embodiment shown in Figure 5A, the base plate 318 of the sample holder 310 is transparent but the dividing walls 322 and peripheral walls 320 are absorbent at illumination wavelengths. Light-absorbing (e.g., black) walls between the recesses 312 further improve image quality by eliminating any spread of scattered light between the recesses 312.

[0103] In embodiments in which the sample holder includes more than one plurality or group of recesses (for example a first group of recesses converging towards a first point, a second group of recesses converging towards a second point, and so on), additional focusing elements (e.g., additional lenses ) may be provided, each one configured to converge light from a respective light source through a respective group of recesses to the respective point of convergence. This may be useful as it may be less expensive to provide multiple smaller lenses rather than a single larger lens. Alternatively, if imaging different groups of recesses sequentially using a single camera by moving the sample holder, a single lens slightly largerthan the area occupied by one group may be used for illumination, so that each group is illuminated in turn.

[0104] In yet other embodiments, the lens 570 may be replaced by an array of lenses (or other focusing elements), for example one lens may be provided for each recess of the sample holder. This may be particularly useful in embodiments in which the sample holder comprises a plurality of recesses having a regular shape and distribution, for example a well plate having circular wells arranged in an array. Additionally, a separate light source (e.g., a point light source) may be provide per lens. Small, relatively low quality lenses can be used to converge the rays of light passing through each recess to be convergent on the camera aperture. An advantage of using an array of lenses, rather than a single large lens, is that it can be readily scaled up to larger sample holders. Another advantage of providing a separate focusing element and light source per recess is that the individual light sources may be configured to have different intensities, for example to ensure uniform illumination of each recess for all view angles of the camera.

[0105] It should be noted that focussing of the illumination light onto the camera does not require particularly high-quality imaging. More generally, it is sufficient that most of the illumination light is “pointing” towards the camera aperture. Therefore, although the beam shaping element 570 of the present embodiment is provided in the form of a converging lens 570, the skilled person will appreciate that other embodiments may use different focusing or converging elements. For example, the converging lens 570 could be replaced by a Fresnel lens, an axicon, a Fresnel axicon (“fraxicon”), a hologram plate or any other focusing device, or by an array of such focusing or converging elements. Here, we use the term “focusing” loosely, as, for example, an axicon would direct all the light to the point of the camera but would not produce a faithful image of the light source 570.

[0106] Figure 5B schematically illustrates a system 500’ for illuminating and imaging samples in the sample holder 310 according to an example embodiment of the present disclosure. The system 500’ differs from the system 500 in that the extended light source 560 of the system 500 is replaced by an annular light source 560’, for dark field imaging of samples in the sample holder 310. The annular light source 560’ may, for example, be provided by an extended light source with a dark spot 56T placed at its centre. The annular light source 560’ is imaged onto the plane of the effective camera aperture by the lens 570 using the same optical arrangement as in Figure 5A. The dark centre 56T of the annular light source 570’ is slightly larger than the camera aperture. Therefore, in the absence of any samples in the sample holder 310, a dark image would be obtained at the camera 302. Only light scattered by samples in the recesses 312 of the sample holder 310 is collected by the camera, producing a darkfield image of the samples. In this embodiment, a benefit of using the lens 570 to image the light source 560’ on to the camera aperture is that the dark field illumination can beprovided for each one of the recesses using a single light source 560’. Moreover, the convergent illumination light is travelling as close as possible towards the camera without reaching the image sensor, which maximizes the darkfield signal given the preference for forwards scattering.

[0107] Figure 6 schematically illustrates a system 600 for illuminating and imaging samples in the sample holder 310 according to an example embodiment of the present disclosure. The system 600 includes the sample holder 310 and camera 302 arranged as previously shown in Figure 3A, and further includes a light source 660 arranged to illuminate the sample holder 310 through the respective base portions 314 of the recesses 312, and a beam shaping element 670 in the form of a baffle structure, according to an example embodiment of the present disclosure. The extended light source 660 is provided in the form of an illumination module 660 comprising a planar light emitting surface 662 that emits diffuse light over a wide range of angles towards the sample holder 310. The beam shaping element 670 is positioned between the light emitting surface 662 of the light source 660 and the sample holder 310 and is configured to transmit light from the light source 660 to the recesses 312 of the sample holder 310 such that the light arriving at the sample holder 310 is substantially convergent. In particular, the light arriving at each recess 312 of the sample holder 310 may be substantially convergent.

[0108] In this embodiment the beam shaping element 670 is provided in the form of a baffle structure comprising channels 672 for transmitting light from the light source 660 to the recesses 312, such that the transmitted light converges as it propagates towards the recesses 312. Each channel transmits light to a respective recess 312 of the sample holder 310. Each channel 672 is defined by respective channel central axis 632 and channel sidewall portions 674 that converge in a direction towards the sample holder 310, in particular towards a point 340. The sidewall portions are preferably optically absorbent for the light emitted from the illumination module 660. Accordingly, each channel 672 substantially restricts the light rays transmitted through the channel 672 to a beam of light convergent on the point 340, that is, at the centre of the effective aperture of the camera 302. In use, the baffle structure 670 and the sample holder 310 are arranged so that the channel sidewall portions 674 and the sidewall portions 314 of the recesses 312 of the sample holder 310 converge towards the same point 340.

[0109] Figure 6 illustrates examples of light rays 664a-c and 665a-c from the illumination module 660 entering respective channels 672a and 672b of the baffle structure 670. The rays 664a-c are transmitted through the centre of the entrance aperture (the lower opening) of the channel 672a. The ray 664a is aligned with a central axis 632 of the channel 672a (and thus with a line of sight from the camera 302) and therefore passes unobstructed through the centre of the exit aperture (the upper opening) of the channel 672a beforepropagating through the corresponding recess 312a of the sample holder 310 to the camera 302. Similarly, ray 665a is aligned with a central axis 632 of the channel 672b (and thus with a line of sight from the camera 302) and therefore passes unobstructed through the centre of the upper opening of the channel 672b before propagating through the corresponding recess 312b of the sample holder 310 to the camera 302. In contrast, the rays 664b and 664c are incident at an angle relative to the ray 664a and are absorbed by the channel sidewall portions 674 and are therefore prevented from reaching the sample holder 310 and causing problematic scattering. Likewise, the rays 665b and 665c are incident at an angle relative to the ray 665a and are also absorbed by the channel sidewall portions 674. Accordingly, only a tight bundle of rays (represented by rays 664a, 665a) defined by the entrance and exit apertures of the respective channel 672 of the baffle structure 670 reach the respective recess 312 of the sample holder 310. Due to the geometry of the system 600, these rays are travelling towards the camera 302. In this way, the baffle structure 670 prevents rays that would not otherwise reach the camera 302 from hitting a sample in the sample holder 310. As a result, scattering is reduced. Another advantage of removing this unwanted light, is that it is prevented from causing any harmful effect on the samples.

[0110] In alternative embodiments of the baffle structure, the channel sidewalls of each individual channel may be non-convergent, provided that the central axes 632 of the plurality of channels are convergent. For example, the channel sidewalls of a channel may be parallel to the central axis of that channel.

[0111] A particular advantage of the baffle structure 670 of this embodiment is that it allows use of a diffuse light source such as the planar or flat-panel illumination module 660 which may significantly reduce the depth of the overall system because, unlike the lens system described above, it does not require sufficient depth for a point source of light to expand to fill the illumination lens 570. A further advantage is that it can be readily scaled up to larger sample holders as it does not require an expensive large diameter lens.

[0112] The baffle structure 670 is preferably made of an optically absorbing material. It may be a single structure with open channels 672 through it. The transverse shape of each channel 672 may be, for example, circular, elliptical, rectangular, square, hexagonal, T-maze, or any other shape, depending on the application. The transverse shape of each channel may match the shape of the corresponding recess 312 of the sample holder 310. However, the channels 472 and recesses 312 may have different shapes. For example, it may be preferable for the recesses 312 to be circular in transverse cross-section (e.g., to present a uniform peripheral wall around the sample which may be beneficial for samples in the form of small animals), whereas it may be preferable for the channels 472 to have a cross-section corresponding to a hexagonal or another tessellating shape (e.g., to reduce the amount of material used to form the dividing walls between the channels 472.)

[0113] In the system 600 shown in Figure 6, the transverse size or area of the channels 672 is matched to the transverse size or area of the corresponding recesses 312 of the sample holder 310, so that the channels 472 can be considered to be continuations or extensions of the recesses 312. That is, the sidewall portions 316 of the recesses and the channel sidewall portions 674 conform to the same lines of sight or view frustum from the point 340. However, in some embodiments, the channels 672 may be slightly enlarged compared with the recesses 312 to improve illumination at the edges of the recesses 312. That is, the angle subtended by each of the channels 672 at the point 340 may be slightly larger than that subtended by the respective recesses.

[0114] Although the system 600 shown in Figure 6 includes a gap 678 between the sample holder 310 and the baffle structure 670, in other embodiments the baffle structure may abut or be joined to the base of the sample holder 310.

[0115] In some alternative embodiments, the light source 660 of system 600 may be replaced by a number of independent light sources, one for each channel 672 of the baffle structure 670. A diffuser may be provided between each individual light source (e.g., an LED) and the corresponding channel 672, for example by positioning a diffuser(s) across the lower ends of the channels 672. The intensity of each light source may be set or adjusted independently. The separate light sources may be provided by an illumination module with locally-adjustable brightness, for example an array of LEDs or a display panel. An advantage of this arrangement is a reduction in waste of electrical and optical power by not illuminating the opaque parts of the baffle structure 670. Another advantage is that it enables the intensity of each light source to be independently adjusted to give uniform intensity on the camera to compensate for the cosine dependence (Lambert’s law) of the irradiance of typical light source(s), which can lead to a fall-off in intensity as seen by the camera for wider field angles. A uniform illumination intensity at the camera makes best use of the dynamic range of the camera, by avoiding the longer exposure that would be required if the outer recesses were darker than the central ones.

[0116] In an alternative embodiment, the “channels” of the baffle structure may effectively be defined by a sequence of two or more apertures. For example, the beam shaping element may be provided by a sequence of two or more planar layers with apertures cut in them. For example, the beam shaping element 670 of Figure 6 could be replaced by two planar, light absorbing layers, including a lower planar layer comprising apertures (effectively, entrance apertures) coincident with the entrance apertures of the beam shaping element 670, and an upper planar layer comprising apertures (effectively, exit apertures) coincident with the exit apertures of the beam shaping element 670. In use, light transmitted from the light source 660 to each respective recess 312 of the sample holder 310 would pass through a respective first aperture before passing through a respective said second apertureto the respective recess 312. The exit apertures may have a smaller area than the entrance apertures.

[0117] Figure 7A schematically illustrates a illustrates a further system 700 for illuminating and imaging samples in a sample holder, including an alternative baffle structure 770 for the beam shaping element, according to an example embodiment of the present disclosure. The system 700 includes a sample holder 710, a camera 302 arranged for imaging samples in the sample holder 710, a light source 660 arranged to illuminate the sample holder 710 through the respective base portions 314 of the recesses 312, and the baffle structure 770. The sample holder 710 is similar to the sample holder 310 of Figure 3A, but has a smaller number of recesses to improve clarity of the Figure. The extended light source 660 is provided in the form of an illumination module 660 comprising a planar light emitting surface 662 that emits diffuse light over a wide range of angles towards the sample holder 710. In this embodiment, the baffle structure 770 comprises a series of light blocking, planar layers 771a, 771 b, 771c, positioned between the sample holder 710 and the light source 660. The layers 771a, 771b, 771c have clear apertures 773 in them, arranged to define “channels” 772 for transmitting light from the light source 660 to corresponding recesses 312 of the sample holder 710.

[0118] Each “channel” 772 defines a channel central axis 732, which passes through the centre of each aperture 773 of the respective channel 772. The channel central axes 732 converge towards each other in a direction as they approach the sample holder 310. In particular, the central axes 732 converge at a point 340 at which the effective aperture of the camera 302 is located. The central axis 732 of each channel 772 is aligned with the central axis of a respective recess 312 of the sample holder 710. Accordingly, the light transmitted by the baffle structure 770 to the recesses of the sample holder 710 is convergent on the point 340, that is, at the effective aperture of the camera 302. The size of the apertures 773 in different layers 771a, 771b, 771c may also decrease with decreasing distance from the sample holder and camera. Accordingly, the apertures 773 may define the “sides” of the channels 772, which may also converge towards the point 340 at which the effective aperture of the camera 302 is located. That is, the “sides” of the channels 772 may be aligned along lines of sight 730 from the camera 302 as shown in Figure 7. Therefore, the beam of light transmitted by an individual channel 770 may itself be substantially convergent at the effective aperture of the camera 302.

[0119] The apertures 773 are configured such that light rays entering one “channel” may propagate to the corresponding recess of the sample holder, but are blocked from reaching another recess. This ensures that only light from the illuminator that passes through a given recess 312 to the camera 302 is incident on a sample in the recess 312. Light rays at other angles that would cause scattering in the sample but not contribute to an image of thesample are blocked. Figure 7A illustrates light rays 764a, 764b emerging from part of the light emitting surface 662 of the illumination module 660 and entering a channel 772b of the baffle structure 770. The ray 764a is aligned with a central axis 732 of the channel 772b (and thus with a line of sight from the camera 302) and therefore passes unobstructed through the centre of each aperture 773 of the respective channel 772b before propagating through the corresponding recess 312b of the sample holder 310 to the camera 302. In contrast, the ray 764b, which is propagating towards a different recess 772 of the sample holder, is blocked by the light-blocking layer 771b. In this way, the baffle structure 770 prevents this ray 764b, that would not otherwise reach the camera 302, from hitting a sample in the sample holder 710 and causing unwanted scattering.

[0120] Figure 7B schematically illustrates a system 700’ according to another example embodiment. The system 700’ includes a sample holder 710’, two cameras 302a, 302b arranged for imaging samples in the sample holder 710’, an illumination module 660 arranged to illuminate the sample holder 710’ through the respective base portions 314 of the recesses 312, and a baffle structure 770’ located between the light source 660 and the sample holder 710’. The sample holder 710’ includes two sets of recesses 312a, 312b, each having sidewalls 316 convergent along lines of sight 730a, 730b from a respective point 340a, 340b. The sample holder 710’ effectively corresponds to two copies of the sample holder 710 of Figure 7A fixed together side-by-side. The baffle structure 770’ differs from the baffle structure 770 of Figure 7A in that it comprises two sets of “channels” 772a, 772b, the channels being defined by apertures 773 in a series of light blocking, planar layers 771a’, 771 b’, 771c’. The baffle structure 770’ effectively corresponds to two copies of the baffle structure 770 of Figure 7A fixed together side-by-side. Samples in recesses 312a of the first set of recesses may be imaged using a first camera 302a, while samples in recesses 312b of the second set of recesses may be imaged using a second camera 302b.

[0121] At the centre of the sample holder 710’, adjacent recesses 712 converge on different points 340a, 340b. The planar, light blocking layers 771a’, 771 b’, 771c’ and apertures 773 of the baffle structure 770’ are carefully positioned, such that one particular area 664 of the illumination module 660 illuminates one recess in each of the first and second sets 312a, 312b of recesses, whilst blocking that area of the illumination module 660 from illuminating other recesses in each of the first and second sets of recesses 312a, 312b. The “channels” 772a, 772b transmitting light to adjacent recesses convergent on different points 340a, 340b may cross each other and can share one aperture 773 as shown in Figure 7B. For example, the area 664 of the illumination module 660 illuminates one recess in each of the first and second sets of recesses (dashed light rays 780a, 780b) whilst the baffle structure 770’ blocks that area of the illumination module 660 from illuminating other recesses in each of the first and second sets of recesses (dotted light rays 782a, 782b). In Figure 7B, parts of the light-blocking layers 771a’, 771b’, 771c’ that are positioned to baffle light from parts of the illumination module 660 from illuminating multiple recesses of the sample holder 710’ are shown with a hatched pattern.

[0122] The arrangement shown in Figure 7B is particularly useful for illuminating multiple zones of a sample holder simultaneously so that they can be imaged simultaneously. It would be difficult to achieve this using the baffle structure 670 of Figure 6, since it is not possible for solid walled channels 672 to cross each other, as would be required for illuminating adjacent closely-spaced recesses belonging to different zones.

[0123] Each of the systems 500, 500’, 600, 700, 700’, described above with reference to Figures 5A-B, 6 and 7A-B, may be implemented using an inverted sample holder like the sample holder 310’ shown in Figure 3B. For example, the sample holder 310 of Figures 5A- B may be substituted by the sample holder 310’ of Figure 3B.

[0124] Features of the embodiments described with respect to Figure 5A-B, 6 and 7A- B may be combined. For example, the systems 600, 700, 700’ may be modified to include a focussing or converging element (e.g., the lens 570 of Figures 5A-B) between the light source and the baffle structure 670, 770, 770’. In particular, the system 600, 700, 700’ may be modified to include an array of lenses between the light source and the baffle structure 670, 770. 770’, together with a point light source per lens, such that each lens of the array converges light from the respective light source through a respective recess of the sample holder.

[0125] In the embodiments described above with respect to Figures 3 to 7, the recesses of the sample holder 310, 310’, 310”, 710, 710’ (and also the channels 672, 772, 772b of the baffle structure 670, 770, 770’) converge in both transverse dimensions towards a point 340. (Because Figures 3 to 7 show a longitudinal cross-section through the recesses and channels, the convergence in only one of these transverse dimensions is shown.) However, in some embodiments, the recesses (and channels, if present) may converge in only one transverse dimension (i.e. , towards a line). For example, such a configuration may be used if the recesses are elongated rectangles (e.g., to provide a “racetrack” for motile micro-animals), in which case it may be sufficient for only the sidewall portions forming the long sides of the rectangular recess to be convergent. The illumination light may similarly be converged in only one transverse dimension, for example by using a cylindrical lens.

[0126] Although the camera 302 in the example embodiments shown in Figures 3 to 7 is provided in the form of an entocentric camera, the skilled person will appreciate that the invention may be implemented using other configurations of camera.

[0127] While the sample holder, and systems for illuminating and imaging samples in the sample holder have been described with the camera located above the sample holder, the sample holder being optionally illuminated from below, it will be appreciated that the sampleholder and systems can be used in any orientation. That is, the entire system (e.g., system 300, 300’, 500, 500’, 600, 700, 700’) may be rotated to any angle. Furthermore, one or more mirrors may be used to rotate the direction of the optical axis of the system between various elements of the system, for example between the light source and sample holder, and / or between the sample holder and camera. In addition, using an alternative embodiment of the sample holder such as that shown in Figure 3B, in which the respective base portion of each recess is located at the second end of the respective recess (rather than at the first end as shown in Figures 3A and 4 to 7), the camera may be used to image samples through the respective base portions, with illumination being provided through the first (optionally, open) end of the recesses.

[0128] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by this detailed description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0129] Reference throughout this specification to features, advantages, or similar language does not imply that all of the features and advantages that may be realized with the present invention should be or are in any single embodiment of the invention. Rather, language referring to the features and advantages is understood to mean that a specific feature, advantage, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, discussions of the features and advantages, and similar language, throughout this specification may, but do not necessarily, refer to the same embodiment.

[0130] Furthermore, the described features, advantages, and characteristics of the invention may be combined in any suitable manner in one or more embodiments. One skilled in the relevant art will recognize, in light of the description herein, that the invention can be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments of the invention.

Claims

CLAIMS1 . A sample holder comprising: a plurality of recesses for receiving a respective sample to be imaged, each recess comprising: sidewall portions extending from a first end of the recess to a second end of the respective recess; and a base portion located at one of said first end or said second end; wherein the respective sidewall portions of each recess are inclined towards the respective sidewall portions of the other recesses of the plurality of recesses; wherein each recess of the plurality of recesses defines a respective central axis extending from the first end to the second end and passing through a centre of the base portion; wherein the central axes of the recesses of the plurality of recesses converge towards each other from the first ends to the second ends of the recesses.

2. The sample holder of claim 1 , wherein said central axes converge along paths corresponding to lines of sight from a point.

3. The sample holder of claim 1 or claim 2, wherein the sidewall portions of each recess are parallel to the central axis of the respective recess.

4. The sample holder of claim 2, wherein the sidewall portions are inclined along straight paths corresponding to lines of sight from said point.

5. The sample holder of any one of the preceding claims, wherein said plurality of recesses is a first plurality of recesses, wherein the respective central axes of the recesses of said first plurality of recesses converge towards a first point, wherein the sample holder further comprises a second plurality of said recesses, wherein the respective central axes of the recesses of said second plurality of recesses converge towards a second point.

6. A sample holder comprising:one or more recesses for receiving a respective sample to be imaged, each recess comprising: sidewall portions extending from a first end of the recess to a second end of the recess; and a base portion located at one of said first end or said second end; wherein said sidewall portions converge towards each other from the first end to the second end of the recess.

7. The sample holder of claims 6, wherein said base portion is located at said first end.

8. The sample holder of claim 6 or claim 7, wherein said sidewall portions converge along straight paths.

9. The sample holder of any one of claims 6 to 8, wherein said straight paths correspond to lines of sight from a point.

10. The sample holder of any one of claims 6 to 9, comprising: a plurality of said recesses, including at least a first recess and a second recess; wherein respective sidewall portions of the first recess converge towards respective sidewall portions of the second recess.

11. The sample holder of claim 10, wherein said plurality of recesses is a first plurality of recesses, wherein respective sidewall portions of the recesses of said first plurality of recesses converge towards a first point, wherein the sample holder further comprises a second plurality of said recesses, wherein respective sidewall portions of the recesses of said second plurality of recesses converge towards a second point.

12. The sample holder of any one of the preceding claims, wherein the sidewall portions at least partially define a periphery of the respective recess.

13. The sample holder of claim 12, wherein said periphery is at least one of: circular, elliptical, rectangular, square, hexagonal, T-shaped, Y-shaped, a maze structure, or n-sided polygon.

14. The sample holder of any one of the preceding claims, wherein said sidewall portions are opaque.

15. A system for imaging a sample in a sample holder, comprising: a sample holder according to any one of the preceding claims; and a camera, arranged for imaging samples received in respective recesses of said sample holder; wherein a central axis and / or the sidewall portions of the or each recess are aligned along lines of sight from the camera.

16. A system according to claim 15, wherein the sidewall portions of the or each recess converge towards a point located at an effective aperture of the camera.

17. A system for illuminating a sample in a sample holder, comprising: a sample holder according to any one of claims 1 to 13; and at least one beam shaping element configured for transmitting light, in use, from a light source to the or each recess of the sample holder, wherein the light transmitted to the recess or recesses of the sample holder is substantially convergent.

18. A system according to claim 17, wherein: respective central axes of the recesses and / or said sidewall portions of the or each recess converge towards a point; and said light transmitted by the beam shaping element converges towards said point.

19. A system according to claim 17, wherein: said sample holder is a sample holder according to claim 5 or claim 11 ; wherein said beam shaping element is a first beam shaping element, configured to transmit light, in use, from a first light source to the first plurality of recesses, wherein said light transmitted by the first beam shaping element converges towards said first point;further comprising a second beam shaping element, configured to transmit light, in use, from a second light source to the second plurality of recesses, wherein said light transmitted by the second beam shaping element converges towards said second point.

20. A system according to any one of claims 17 to 19, wherein said beam shaping element comprises a baffle defining at least one channel for transmitting light, in use, from the light source to a respective recess of the sample holder.

21. A system according to claim 20, wherein the or each channel converges towards the sample holder.

22. A system according to claim 21 , wherein: said sidewall portions of the or each recess of the sample holder converge towards a point; and the or each channel of the baffle converges towards said point.

23. A system according to any one of claims 20 to 22, wherein said sample holder is a sample holder according to claim 1 or 10, wherein the baffle defines a plurality of channels for transmitting light from the light source to a respective recess of said plurality of recesses of the sample holder; wherein respective central axes of the channels converge in a direction towards the sample holder.

24. A system according to claim 23, wherein: respective central axes of the recesses and / or said sidewall portions of each recess of said plurality of recesses of the sample holder converge towards a point; and the respective central axes of the channels of the baffle converge towards said point.

25. A system according to any one of claims 20 to 24, wherein: the or each channel is defined by respective channel sidewalls.

26. A system according to any one of claims 20 to 24, wherein:the or each channel is defined by a sequence of apertures.

27. A system according to any one of claims 17 to 26, wherein said or each beam shaping element comprises at least one focusing element.

28. A system according to claim 27, wherein: the system further comprises said light source; respective central axes of said recesses of said sample holder and / or said sidewall portions of the or each recess of said sample holder converge towards a point; and the or each focusing element is configured to form an image of at least part of the light source at said point.

29. A system according to claim 27, wherein the system further comprises: said light source; and a camera, arranged for imaging samples received in respective recesses of said sample holder; wherein the or each focusing element is configured to form an image of at least part of the light source at an effective aperture of the camera.

30. A system according to claim 29, wherein said light source comprises an extended light source having a dark spot at its centre; and wherein the or each focusing element is configured to form an image of said dark spot at an effective aperture of the camera.

31. A system according to any one of claims 27 to 30, wherein said at least one focusing element comprises a plurality of said focusing elements; wherein each focusing element is configured to converge light through a respective recess of said sample holder.

32. A system according to any one of claims 27 to 31 , wherein said or each focusing element comprises at least one of: a lens, a Fresnel lens, an axicon, a Fresnel axicon, a concave mirror and a hologram plate.

33. A method for imaging a sample, the method comprising: providing a sample holder comprising a plurality of recesses for receiving respective samples; and imaging the samples using a camera; wherein each recess of the plurality of recesses comprises: sidewall portions extending from a first end to a second end of the recess, and a base portion located at one of said first end or said second end of the recess, wherein respective central axes of each recess are aligned with lines of sight from the camera.

34. A method for imaging a sample, the method comprising: providing a sample holder comprising at least one recess for receiving the sample; and imaging the sample using a camera; wherein the or each recess comprises: sidewall portions extending from a first end to a second end of the recess, and a base portion located at one of said first end or said second end of the recess, wherein the sidewall portions of the or each recess are aligned with lines of sight from the camera.

35. The method according to claim 33 or claim 34, further comprising: illuminating the or each recess from the first end with convergent light.

36. The method according to any one of claims 33 to 35, wherein the convergent light converges at an effective aperture of the camera.

37. The method according to any one of clams 33 to 36, wherein the sample holder is a sample holder according to any one of claims 1 to 14.

38. The method according to any one of clams 33 to 37, wherein the method is implemented using the system according to any one of claims 15 to 32.

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