Imaging apparatus and method

Abstract

An imaging apparatus for collecting and imaging a sample inside a vessel comprising: a first surface comprising or configured to receive a first optical element; a light source housing for a light source; a moveable spacer secured at a distal end of the light source housing by a resilient membrane and moveable relative to the distal end of the light source housing, wherein the moveable spacer comprises a second surface comprising or configured to receive a second optical element; a mechanism for controllably moving said moveable spacer from a retracted position to a protruding position relative to the distal end of the light source housing, wherein the movable spacer is biased against movement into the protruding position by the resilient membrane, wherein the first surface and / or the second surface of the moveable spacer comprise one or more interfacing features configured to form an at least partially closed sample chamber for imaging, wherein the at least partially closed sample chamber is defined, at least in part, by the first and second optical elements and / or at least part of the interfacing features.

Description

[0001] Imaging Apparatus and Method

[0002] Field

[0003] The present invention relates to an imaging apparatus and method, for example, an in line microbial cell imaging apparatus.

[0004] Background

[0005] Cell counting technology and high-throughput imaging is currently focused mainly on mammalian cells. The task of imaging (and counting via imaging) of microbial, and in particular bacterial cells, is non-trivial due to their small size, extensive size changes (up to 100 times), the fact that bacteria can swim, as well as by the fact that biotechnology relevant media is turbid and often viscous. A need for in-line imaging devices that are capable of offering high resolution imaging of microbial cells has been identified.

[0006] Summary

[0007] In accordance with a first aspect, there is provided an imaging apparatus for collecting and imaging a sample comprising: a first surface comprising or configured to receive a first optical element; a light source housing for a light source; a moveable spacer secured at a distal end of the light source housing by a resilient membrane and moveable relative to the distal end of the light source housing, wherein the moveable spacer comprises a second surface comprising or configured to receive a second optical element; a mechanism for controllably moving said moveable spacer from a retracted position to a protruding position relative to the distal end of the light source housing, wherein the movable spacer is biased against movement into the protruding position by the resilient membrane, wherein the first surface and / or the end surface of the moveable spacer comprise one or more interfacing features configured to form an at least partially closed sample chamber for imaging, wherein the at least partially closed sample chamber is defined, at least in part, by the first and second optical elements and / or the interfacing features.

[0008] The sample may be located inside a vessel. At least part of the imaging apparatus may be provided inside the vessel. The at least partially close sample chamber may comprise a sample volume.

[0009] The first surface may be fixed relative to the vessel such that the at least partially closed sample chamber is formed by movement of the moveable spacer. The first and / or second optical elements may be at least partially transparent. The first and / or second optical elements may comprise a substantially flat surface. The first and / or second optical element may form part of the first and / or second interfacing surfaces. The first and / or second optical elements may comprise an index of refraction between 1 and 2, optionally between 1.4 and 1.6, optionally between 1.45 and 1.55. The index of refraction may be substantially close to the index of refraction of glass.

[0010] The at least partially closed sample chamber may comprises a 2D layer, optionally, wherein the 2D layer comprises a depth in the range 1 to 500 pm. The at least partially closed sample chamber may comprise a substantially two dimensional volume, optionally, wherein the two dimensional volume comprises a depth in the range 1 to 500 pm.

[0011] The resilient membrane may comprise one or more flexible resilient members. The moveable spacer may be moveable relative to the light source housing. The light source may moveable relative to the light source housing.

[0012] The one or more interfacing features may comprise one or more protrusions from the first and / or the second surface and / or one or more recesses formed in the first and / or second surface.

[0013] The one or more interfacing features may comprise one or more further spacer elements attached to the first and / or second surface.

[0014] The apparatus may be configured to collect a sample, for example, a liquid sample. The apparatus may be configured to collect a sample capable of being introduced into the sample chamber. The sample chamber may be at least partially closed to reduce a flow of liquid into a sample volume defined by the at least partially closed sample chamber.

[0015] The one or more interfacing features and / or first or second optical elements may form at least one structural part of the sample chamber. The at least one structural part may comprise at least one wall and / or at least part of a lower surface and / or at least part of an upper surface of the sample chamber. The apparatus of any preceding claim, wherein at least the height of the sample chamber may be defined by the one or more interfacing features.

[0016] The further spacers may have a height smaller than 1 mm, optionally smaller than 0.1 mm, optionally smaller than 100 nm. The further spacers may be detachable. The further one or more further spacer may be selected to modify the height of the imaging chamber.

[0017] The further spacers may be formed from a polymer or other material configured to precisely manufacture. The further spacers may formed by a spin-coating process. The further spacers may be sized and / or positioned to decrease a size of the sample volume. The further spacers may be secured to a first surface and may be sized to fit inside a corresponding recess of the second surface, such that the at least partially closed chamber is defined between the further spacer and the corresponding recess.

[0018] The at least partially closed sample chamber may defined a sample volume that comprise a fully closed or partially closed volume. In the protruding configuration, at least part of the first and / or second surface and / or the interfacing features, may restrict flow between first and second surface. The first and second surface may comprise interfacing and / or co-operating surfaces.

[0019] The apparatus may comprise or for part of an in-line imaging microbial cell imaging device. The one or more interfacing features may form at least one wall and / or a lower surface and / or an upper surface of the sample chamber.

[0020] The sample chamber may be formed by at least the first optical element of the first surface, the second optical element of the second surface and / or the surrounding walls are formed at least in part by at least part of the interfacing feature.

[0021] At least part of the first surface, for example, part of the optical element, and at least part of the distal surface are configured to contact to form the at least partially closed sample chamber.

[0022] The first surface may form part of a separate component, for example, an objective lens housing and / or a surface of a vessel for containing liquid. The separate component may be fixed relative to the vessel. The separate component may be independently moveable and / or fixed. At least part of the light source housing may be independently moveable relative to the separate component. The light source housing may be independently moveable relative to the separate component. The objective lens housing may be hermetically sealed. The first surface may be as part of a separate component to the light source housing.

[0023] The moveable spacer may be or may comprise a separable portion. At least part of the distal surface may be formed or comprise a separable portion. The moveable spacer may be removable from the light source. The moveable spacer comprises securing means for securing the resilient membrane to the moveable spacer.

[0024] The at least partially closed sample chamber may be defined at least partially between said optical elements. The surfaces comprise optical elements and / or recesses for receiving optical elements. The optical elements may be substantially transparent. The optical elements may permit light to pass through. The optical elements may comprise coverslip elements. The optical elements may comprise plastic or glass coverslips. The moveable spacer comprises an upper and lower part.

[0025] The apparatus may further comprise a motor or other drive arrangement configured to indirectly move the moveable spacer into the protruding position.

[0026] The motor or other drive arrangement may be configured to move the moveable spacer via a further component, for example, a pressing member.

[0027] The mechanism may comprises a pushing member moveable relative to light source housing and relative to the moveable spacer, wherein the pushing member is configured to push said moveable spacer into said protruding position thereby to form the sample chamber, optionally, wherein the pushing member is operable to push the moveable spacer from its natural position to a protruding position, wherein the moveable spacer is biased against movement to the protruding position by the resilient membrane.

[0028] The light source may be disposed on and / or fixedly coupled to the pushing member such that said light source moves together with said pushing member. The condenser lens may be disposed on and / or fixedly coupled to said pushing member. The pushing member may comprise an aperture. The light source may be disposed on and / or fixedly coupled to the pushing member such that that light is transmitted through said aperture.

[0029] The pushing member may form part of a pushing frame structure to which the light source and / or other optical elements are secured. The motor or other driving arrangement may be coupled to the pushing member. The motor or other driving arrangement may be coupled to the pushing member by a coupling. The coupling may comprise a rigid coupling. The coupling may comprise one or more springs and / or resilient members. The motor may be operable to move the pushing member to push the moveable spacer.

[0030] The pushing member and movable spacer may be arranged coaxially along a longitudinal axis of the light source housing, wherein the pushing member and movable spacer are movable along said axis. The objective lens housing may be arranged coaxially along said longitudinal axis.

[0031] The movable spacer may comprise an aperture for permitting light to pass through.

[0032] The first and second surfaces may form the at least partially closed chamber when at least part of the first surface and at least part of the second surface are contacted together.

[0033] At least one of the first and second surfaces may comprise a first recess for defining at least part of the sample chamber when contacting the corresponding interfacing surface of the movable spacer. At least one of the first and second surfaces may comprise a second recess for receiving a transparent optical element, wherein the second recess is provided inside the first recess.

[0034] The resilient membrane may substantially surround the moveable spacer. The resilient membrane may be secured at a groove provided in the moveable spacer. The moveable spacer may comprise a circular or ring shape. The resilient membrane may be circumferentially disposed about the spacer. The resilient membrane may comprise a silicone diaphragm. The flexible membrane and / or moveable spacer may form at least part of a barrier to fluid at the distal end to prevent entry of fluid into the light source housing.

[0035] In use, the resilient membrane and / or movable spacer may be in contact with said liquid. The movable spacer may be the only moveable part of the apparatus in contact with said liquid. The movable spacer and / or resilient membrane may be the only exposed movable parts.

[0036] The at least partially closed sample chamber may be defined by parts of the first and second surfaces and / or their respective optical elements and / or is not defined by part of the resilient membrane

[0037] The moveable spacer may be substantially contained inside the light source housing when in the retracted position.

[0038] The objective housing and light source housing may form two separate parts of the apparatus such that, when assembled, an open volume is defined between the moveable spacer and the objective lens housing when the moveable spacer is in the retracted position.

[0039] The open volume may comprise a gap of uniform thickness and / or wherein no part of the apparatus is present in said open volume. The moveable spacer may be configured to be moved into the open volume and / to collect a sample from said open volume when moved to the protruding position.

[0040] The first surface may be configured to be fixedly coupled to a bio-reactor and / or other vessel for containing liquid.

[0041] The moveable spacer may be moveable longitudinally along a longitudinal axis of the light source housing.

[0042] The motor may be coupled to the pushing member and the pushing member may be configured to push the movable spacer. The motor may comprise a stepper or piezo motor. The interfacing features may comprise a plurality of recesses sized to receive cover slips of different sizes and / or wherein the first surface and / or the second surface comprise a plurality of recesses having different depths for forming sample chambers of different depths.

[0043] The apparatus may be configured to perform fluorescence imaging and / or to image microbial objects.

[0044] The apparatus may comprise a controller configured to control the motor via control signals. The controller may be configured to operate the motor to move the movable spacer a predetermined distance. The apparatus may comprise a sensor, for example, as part of the motor, for generating a stall signal, wherein the controller is configured to cease operation of the motor and / or the motor is configured to cease movement in response to the detection of a stall signal.

[0045] The apparatus may further comprise or be configured for use with steering optics and / or an image sensor.

[0046] In accordance with a second aspect, there is provided a method of imaging a sample, comprising providing at least part of the apparatus of the first aspect in a liquid container such that liquid is present between the distal surface of the moveable spacer and the first surface, wherein the method further comprises causing the moveable spacer to move to the protruding position thereby to form the at least partially closed sample chamber and contain a sample inside the sample chamber.

[0047] The method may further comprise providing the container, fixing the first surface relative to the container and providing the light source housing in said container, and moving said moveable spacer in the container relative to the first surface.

[0048] The method may comprise performing an imaging process using the apparatus, the imaging process comprising operating the light source such that light is incident on the sample. The imaging process may further comprise capturing an image of the sample.

[0049] The imaging process may comprise a fluorescence imaging and / or imaging of microbial objects. In accordance with a third aspect, there is provided an in-line microbial cell imaging apparatus or system comprising the imaging apparatus of the first aspect.

[0050] Features of one aspect may be provided as features of another aspect. For example, apparatus features may be provided as method features and vice versa.

[0051] Brief Description

[0052] Various aspects of the invention will now be described by way of example only, and with reference to the accompanying drawings, of which:

[0053] Figure 1 (a) is a rendered view of the imaging apparatus, in accordance with an embodiment;

[0054] Figure 2(a) is a first cross section of the apparatus in the open configuration and Figure 2(b) is cross section of the apparatus in a closed configuration;

[0055] Figure 3(a) is a cut-away view of part of the apparatus in the open configuration and Figure 3(b) is a further cut-away view of the apparatus;

[0056] Figure 4 is a cross section of a pushing structure forming part of the apparatus;

[0057] Figure 5 is a rendered view of an objective lens housing forming part of the apparatus;

[0058] Figure 6 is a cross-sectional view depicting the interfacing surfaces of the apparatus;

[0059] Figure 7 is a further cross-section view depicting the interfacing surfaces of the apparatus forming an imaging chamber;

[0060] Figure 8(a) to (d) are technical drawings showing parts of the apparatus, in accordance with an embodiment;

[0061] Figure 9(a) to (d) are technical drawings showing parts of the apparatus, in accordance with an embodiment;

[0062] Figures 10(a) to (d) are photographic images of an imaging apparatus in situ in accordance with an embodiment;

[0063] Figures 11 (a) and (b) are views of an imaging apparatus including a further spacer element, in accordance with a further embodiment;

[0064] Figures 11 (a) and (b) are views of an imaging apparatus including a further spacer element, in accordance with a further embodiment;

[0065] Figure 12 depicts an embodiment including a stabilizing arrangement;

[0066] Figure 13 depicts a plot of experimental results;

[0067] Figure 14 depicts two images obtained using an embodiment. Detailed Description

[0068] The present invention relates to an imaging apparatus or system. The apparatus may comprise separate parts and be configured to be assembled in situ in a sample vessel, for example, a bio-reactor. The apparatus is configured to form an imaging chamber that is substantially planar. To form the imaging chamber the apparatus has two interfacing or mating surfaces. In particular, in embodiments, the apparatus has concentric rings of specified sizes and a moveable spacer to provide a temporary quasi 2D imaging chamber. The interfacing surfaces include surfaces that interface or otherwise cooperate to form the imaging chamber. The interfacing surfaces may be surfaces having features referred to as interfacing features that co-operate to form the sample chamber. The described embodiments may provide an imaging apparatus capable of achieving high resolution images of micron-sized, potentially fast swimming, microbes, in potentially turbid and viscous cultures.

[0069] To achieve good quality imaging for microbial samples, the following embodiments are described in which the sample chamber is formed by two parts: a part that is fixed relative to the sample vessel, and a moveable part that is moveable relative to the first part and therefore the vessel. As such, only one of the parts of the apparatus moves in the liquid. By having a fixed portion, stability and image quality may be improved.

[0070] The present invention is capable of collecting samples for imaging microbial object(s) of the size 0.5 to 20um (micron). The microbial object(s) may be have a size in the range 0.5um to 1um diameter and 1-3 urn in length. These objects can also move up to 100um / s. Part of the objects may elongate to be 1 um in width and 100s of microns in length. Due to the size of the objects, their movement and environment, a precise imaging setup is required.

[0071] Figure 1 is a rendered image of an imaging apparatus 10, in accordance with an embodiment. The apparatus has a first housing 100 and a second housing 200. The housing 100 has a moveable base element, in the present embodiment referred to as a moveable spacer element 300. Disposed inside the first housing is a mechanism for moving a moveable spacer from a first position where at least part of the interfacing surface of the moveable spacer is flush with a distal end surface of the light source housing to a second position in which the interfacing surface protrudes beyond the distal end surface of the light source housing. The mechanism includes a motor and a pushing member. The pushing structure is moved by a driving arrangement 500. The first housing 100 that may be referred to as light source housing and a second housing 200 may be referred to as objective lens housing 200. In the present embodiment, the first and second housings are cylinders, may be referred to as first and second cylinder. In use, the cylinders are aligned along a longitudinal axis of the first and second housing. It will be understood that an advantage of providing the moveable spacer flush with the distal end surface is to achieve uniform mixing of the sample, but not a strict requirement

[0072] Figure 2(a) is a first cross section of the apparatus in the open configuration and Figure 2(b) is cross section of the apparatus in a closed configuration. As can be seen in Figure 2(a), and as described in further detail in the following, the first and second housings provide two surfaces that co-operate to define an imaging chamber, specifically a quasi two-dimensional imaging chamber. In particular, the surfaces include interfacing features, in this embodiment the interfacing features include recesses that form the chamber. The interfacing features may include a recess on one surface that interfaces with a non-recessed surface and / or a protrusion on the other surface, to form a sample volume.

[0073] In the present embodiment, one of these surfaces is moveable to define the imaging chamber. The apparatus is configured for use inside a vessel chamber containing fluid and the interfacing surfaces are operable to trap fluid in the imaging chamber. In further detail, the apparatus is in an open configuration, as shown in Figure 2(a) then operable to be placed in the closed configuration, as shown in Figure 2(b), to define the imaging chamber. The vessel chamber and / or apparatus is sterilisable, for example, may be manufactured from a material that can be sterilized.

[0074] Alternative views of the apparatus 10 are described in the following. It will be understood that certain reference numerals are omitted from some Figures for the purpose of clarity. Figure 3(a) and (b) depict the first housing 100 and show the drive arrangement 500, pushing frame structure 400 and moveable spacer 300 provided inside the first housing. Figure 4 is a cross-sectional view of the pushing frame structure 400. Figure 5 is a rendered view of the objective lens housing 200. Figure 6 is a cross-sectional view of the movable spacer 300 and surface of the objective lens housing 200, in the open configuration. Figure 7 is a cross-section view showing a close-up region of the apparatus in the closed configuration, depicting co-operation of moveable spacer 300, pushing frame 400, flexible membrane and surface of the objective lens housing 200 to define the imaging chamber 600.

[0075] Figure 3(a) is a rendered cut-away view of the first housing 100 showing the inside of the housing. Figure 3(b) is an alternative rendered cut-away view of the first housing 100 from below.

[0076] At a distal end of the housing 100, the moveable spacer 300 is provided. A surface of the moveable spacer forms part of the first interfacing surface described above. The distal end of the housing 100 has an inwardly projecting protrusions, referred to as a lip 102. The lip 102 defines at least part of the distal surface of the housing 100. The lip 102 has a ring shape defining a void 104 at the distal end of the housing. As described in the following, the moveable spacer is provided in the void 104.

[0077] As described it the following, the apparatus has a moveable spacer 300 provided at the distal end of the housing 100. The moveable spacer 300 is provided in the void 104 of the housing 100. The moveable spacer 300 is attached or otherwise secured to the distal end of the housing, in particular to the lip 102, by a flexible membrane 302. The flexible membrane may also be referred to as a resilient membrane. By securing the resilient membrane to the moveable spacer, the moveable spacer is biased against movement from a retracted position to a protruding position by the resilient membrane. In the retracted position, the moveable spacer is at least partly contained in the light source housing.

[0078] A drive arrangement 500 is provided inside the housing 100. The drive arrangement 500 includes motor 502 and drive rod 504. In the present embodiment the motor is a piezoelectric motor 502 for generating linear movement. The motor 502 is provided inside the first housing 100. The piezoelectric motor 502 has a drive rod 504 and is operable to move the drive rod 504 in a linear direction. At least part of the rod 504 threads through the motor. Known piezeolectric motors may be used, such as PiezoMotor Piezo LEGS® LL06. In other embodiments, alternative types of motors may be provided, for example, stepping motors. The faces are brought into contact by pushing the base of the upper cylinder with the high-resolution motor located inside the cylinder. The motors are capable of high resolution movement. For example, the PiezoMotor Piezo LEGS® LL06 has sub-nanometer resolution, however, it will be understood that coarser resolutions may also be suitable.

[0079] The motor 502 is fixedly secured to the housing 100 by a securing means, in this embodiment, a securing plate 506 is attached to a securing beam 508 at an upper surface of the housing 100. The securing plate 506 and hence the motor 502 is positioned so that the drive rod 504 is centrally disposed in the housing and extends along a central, longitudinal axis of the cylinder. The drive rod 504 is aligned with the central, longitudinal axis of the cylinder.

[0080] In use, the motor 502 is configured to move the rod forward and backwards in a longitudinal direction along the central axis of the cylinder 100. It will be understood that alternative means of securing the motor to the housing can be used. The end of the rod 504 closest to the distal end of the housing 100 is referred to as the distal end.

[0081] At the distal end of the rod 504 the pushing frame structure 400, also referred to as a pushing frame 400 is provided. Figure 4 depicts a cross-sectional view of the pushing frame structure. The pushing frame 400 is formed by a circular member 402, a ring member 404 and a number of elongate members 406a, 406b, 406c. Due to their position in the pushing frame inside the housing 100, the first member 402 is referred to as an upper member 402 and the ring member 404 referred to as a lower member 404. The ring member 404 has a central void 405 or aperture provided at its centre. Each elongate member is secured at a first end to the upper member 402 and at a second end to the lower member 404. The elongate members extend between the upper and lower members and provide openings therebetween such that the frame 400 is partially open. The elongate members are secured to the upper member 402 and lower member 404 by any suitable securing means. The securing means comprise screws in the present embodiment. The disc shaped upper member 402 and the ring shaped lower member 404 are planar and fixed to lie in parallel planes. The elongate members 406a, 406b, 406c, are oriented substantially perpendicular or normal to the planes of the ceiling and ring members. The ring member 404 provides a distal end surface for the pushing frame with the void 405 in its centre. The pushing structure and moveable spacer are arranged coaxially along the longitudinal axis of the light source housing. The pushing structure and movable spacer are movable along said axis An open cavity is generally defined between the surfaces of the upper member 402 and lower member 404 and by the elongate members 406a, 406b, 406c. Inside the cavity a light source 408 and associated condenser lens 410 are fixedly attached. The light source and condenser lens are both is fixedly coupled to the pushing structure. The light source is attached to the upper member 402 by a light source support 412. The lens is attached to the upper member 402 by a lens support 414 that suspends the lens at centrally in the frame. The light source 408 and lens 410 are disposed centrally inside the cavity and supported in that position by their respective supports. The light source 408 and lens 410 are on the central axis of the pushing frame structure 400 such that a light path is defined from the light source 408 through a central void 405 of the lower, ring member 404 via the lens 410. The distal end surface of the pushing structure provided by lower surface 404 is annular or ring-shaped surface to permit light to pass through the void 405.

[0082] The pushing structure is configured to apply a pressure and / or pushing force to the movable spacer and may push the moveable spacer from a first position to a second position. The pushing structure may therefore be referred to or may comprise a pushing member. The drive arrangement is coupled to the pushing structure and is not coupled to the moveable spacer. The drive arrangement including the motor and drive rod is therefore configured to move the moveable spacer indirectly. The motor and drive rod therefore operate to move the moveable spacer without contacting the moveable spacer, as the pushing structure contacts the moveable spacer.

[0083] The pushing frame structure 400 is attached to the housing 100 as follows. The upper member 402 of the frame 400 is secured to the distal end of the rod 504 at the centre of the upper member 402. The frame 400 and rod 504 are attached such that the elongate members 406a, 406b, 406c are oriented parallel to the longitudinal axis of the rod 504 and the central axis of the cylinder 100. The frame 400 therefore extends in a longitudinal direction from the distal end of the rod 504.

[0084] The pushing structure 400 is mechanically coupled to the motor 502 by the rod 504 such that the motor is operable to move the pushing frame 400 in a linear direction along the central axis inside the cavity of the cylindrical housing 100. The pushing member 400 therefore moves up and down inside the housing 100. The motor is configured to cease movement when the pushing frame moves a predefined distance and / or when the moveable spacer contacts the object lens housing. This stopping distance can be implemented by moving the motor a pre-determined distance or by providing a sensor in the motor for sensing a physical stall caused by the objective housing and ceasing movement when the resistance is above a threshold level.

[0085] In further detail, the motor may be programmed to stop after a certain number of steps, or by stopping when it encounters a physical stall. By stopping on encountering a physical stall, the initial placement of the cylinder in the same position may be less important. In some embodiments, in which the chamber is not fully closed, stopping after movement of a fixed distance may be implemented.

[0086] Turning to the objective lens housing 200, Figure 5 is a rendered view of the objective lens housing 200 which is also cylindrical in shape. It will be understood that while the objective lens housing 200 is depicted as a cylinder protruding from a surface of a sample chamber, in some embodiments, the interfacing surface of the objective lens housing forms part of or is integrated into a wall of the sample chamber.

[0087] The objective lens housing 200 has an end surface 202 at its upper end. As depicted in Figure 5 and Figure 6, provided at a central void of the end surface is an optical element across a void forming a window. The optical element forming the window is a glass coverslip 216 but may be plastic or any other transparent and rigid material. The optical element is flat and planar. The end surface 202 has a recess structure. In the present embodiment, the recess structure is a nested ring structure. The nested ring structure is depicted in further detail with reference to Figure 5 and 6. The nested ring structure comprises two aligned circular recesses: a first circular recess 206 has a first radius at a first depth and a second circular recess 208 has a second radius at a second, deeper depth. The second, deeper, recess is contained inside and is axially aligned with the first shallower recess such that an intermediate rim 210 is formed about the second deeper, recess 208. The first recess has a cylindrical side wall around its edge, the side wall 218 (shown in Figure 7) having a size equal to the first depth and the second recess has a cylindrical side wall 220 having a size equal to the second depth.

[0088] The second recess is aligned to the void to provide a circular rimmed seating 212 for a glass coverslip 216. When positioned in the seating, the glass coverslip 216 spans the second deeper recess 208 forming a planar surface together with the intermediate rim 210 of the second, deeper recess 208. Inside the circular rimmed seating 212, a groove 214 is formed circumferentially in the second recess 208 for holding bonding material to bond the coverslip to the end surface.

[0089] The nested ring structure of the objective lens housing will be understood as an interfacing feature that forms part of the imaging chamber. The recess structure is such that a glass coverslip 216 is received and secured to the circular rimmed seating 212 inside the deeper, second recess 208, thereby defining a lower surface or floor for the first recess 206. When the glass coverslip is positioned in the seating, only the upper recess is accessible. The defined lower surface forms part of the substantially planar imaging chamber in use. The glass coverslips are examples of optical elements that allow light transmission.

[0090] Figure 6 also depicts the moveable spacer element 300 in cross-section. The moveable spacer element 300 is generally ring shaped and has a void 304 at its centre. The void 304 allows light, provided from the light source 408 in the pushing frame 400, to pass through the spacer element 300 into the imaging chamber formed by the interfacing surface.

[0091] As can be seen in Figure 6, the moveable spacer element has two surfaces: a first, upper surface 306 for contacting the pushing structure 400 and a second, lower surface 308. The second surface 308 is referred to or forms at least part of one of a pair of interfacing surfaces. The second surface 308 has a rim 310 at its circumference. The rim 310 defines a circular recess 312 at the second surface 308 for receiving a glass coverslip 318. At an inner radius of the rim 310 is a groove 314 disposed circumferentially for receiving bonding or glue for the glass coverslip 318. The rim 310 and glass coverslip 318 form a substantially planar outer surface. The rim and glass coverslip 318 together may be referred to as an interfacing surface. The circular recess 312 may be considered as an interfacing feature that forms part of the sample volume. In particular, the surrounding walls of the recess form the inner wall of the sample chamber.

[0092] As depicted in Figure 6, in the present embodiment, the moveable spacer 300 is formed by an upper part 300a and a lower part 300b. Both the upper part and lower part have respective voids that form void 304 when secured together. The moveable spacer 300 has a lateral surface having a depth between the first and second surface. At a mid-point of the lateral surface 316, the flexible resilient member 302 is secured to the moveable spacer. In the present embodiment, the moveable spacer is formed of two separable parts, a top part and a bottom part that are secured together using screws. The flexible membrane is thus pinched between these two parts and secured to the moveable spacer.. In alternative embodiments, the moveable spacer may have a groove formed around its mid-point for receiving the flexible membrane, the groove being sized to provide a good seal. The flexible resilient member may also be referred to as a diaphragm or a flexible resilient membrane. . The flexible resilient member is thus received in the channel and disposed circumferentially about the moveable spacer 300. As can be seen in the Figures, the flexible member 302 is provided in an annular gap between the movable spacer 300 and the light source housing 100. The moveable spacer is moveable relative to the distal end of the light source housing.

[0093] The resilient membrane 302, also referred to as a flexible member, is secured to the moveable spacer 300 at a central position by any suitable securing means. The resilient member 302 is secured at an outer position, at its outer radius to a separable ring element 106. The resilient member is secured to the separable ring element 106 and an inner surface of the distal end of the cylinder 100, namely the lip 102 by a further suitable securing means. In the present embodiment, the securing means is a screw.

[0094] The pushing member or structure 400 is configured to contact the moveable spacer 300. The lower, outer surface 404 of the pushing member contacts the upper surface 306 of the moveable spacer and thus moves the moveable surface from a first position to a second position. The pushing structure is configured to press the moveable spacer towards the recess structure of the corresponding surface of the objective lens. In the retracted configuration or position, the moveable spacer is retracted inside the first housing and the space between the housings is open for fluid to move into. In the extended or protruding configuration or position, the moveable spacer protrudes from the first housing until part of the moveable spacer contacts the corresponding surface of the objective housing thus forming an enclosed chamber. In situ, the enclosed chamber will contain fluid to be imaged. The apparatus is thus operable to form an imaging chamber in situ.

[0095] Figure 7 is a further cross-section view of the apparatus. In particular, Figure 7 is a closeup view of region 550 of Figure 2(b). Figure 7 depicts a number of elements described with reference to the above Figures. In particular, Figure 7 depicts first housing 100 and second housing 200. Figure 7 depicts moveable spacer 300 secured to flexible resilient membrane 302. The flexible resilient member 302 is secured to the first housing 100 between separable ring element 106 and lip 102 of first housing 100. Figure 7 also depicts a coverslip 318 seated and secured in recess 312 of moveable spacer 300 and a further coverslip 216 seated and secured in rimmed seating 212 formed by the recess structure of the objective lens housing 200. Figure 7 also depicts, in greater detail, a first groove 314 formed in each of the moveable spacer 300 and a second groove 214 formed in end surface of objective lens housing. Figure 7 depicts the first and second surfaces in a closed configuration defining the imaging chamber 600.

[0096] When in the closed configuration, an upper surface of the imaging chamber is formed by the coverslip 318 of the moveable spacer 300. The lower surface of the imaging chamber is formed by the coverslip 216 of the objective lens housing and part of the first recess, in particular the circular rimmed seating 212. The side walls of the imaging chamber are formed by the cylindrical side wall 218 of the first recess 206. The imaging chamber is therefore a thin disc shape.

[0097] In use, the apparatus is provided in a vessel containing a fluid, for example, a suspension culture. The objective lens housing 200 is provided in a protruding position at an internal surface of the vessel. The housing 100 is then provided in a position relative to the objective lens housing. The system is robust to small horizontal misalignments, and therefore a manual alignment of the two cylinders is possible. In situ, the moveable spacer of the upper cylinder and the membrane are the moving parts of the system in contact with the fluid, for example, a suspension culture. The flexible membrane which may be formed of silicone permits movement while providing a watertight barrier. The flexible membrane forms a barrier to water ingress to the light source housing together with, for example, the moveable spacer. The system is kept in the retracted position to allow for effective mixing of the culture and is then extended to the protracted position for imaging.

[0098] Once in situ, the apparatus is operable to form the imaging chamber. In the first open configuration, the liquid to be sampled is free to move between the lower surface of the moveable spacer and the surface of the objective lens. A stirring or mixing process may be performed on the liquid. In the open configuration, the moveable spacer is its natural position in which the lower surface is aligned with the lower surface of the housing 100. The moveable spacer is maintained in its natural position by the resilience of the resilient member and is therefore biased against movement from the natural position. The lower surface of the moveable spacer and the surface of the objective lens form first and second surfaces configured to form a sample chamber.

[0099] To form the sample chamber, the motor is operated through received control signals to move the pushing structure from its starting position to contact the moveable spacer. As both the pushing structure and moveable spacer are rigid, the pushing structure moves the moveable member from its natural position to a protruding position. In the protruding position, part of the moveable member contacts part of the correspond surface of the objective lens housing. In particular, the rim 310 and the coverslip 318 contact an outer region of the distal surface of the objective lens housing. The imaging chamber 600 is thus formed collecting any liquid present inside. The imaging chamber may also be referred to a sample chamber. The imaging chamber is thin and provides a quasi two- dimensional imaging chamber. The imaging chamber or the volume defined in the chamber may be considers as a 2D layer. The depth of the volume may vary in embodiments, but will be in the range 1 to 500 pm.

[0100] The imaging chamber defines a sample volume. In some embodiments, the recess for coverslip 318 is slightly shallower than the coverslip itself, so the coverslip is not exactly flush with rim 310, but slightly protruding outwards. In such embodiments, the contact is between coverslip 318 and the surface of the objective housing. In further embodiment, the rim may contact the surface of the objective housing.

[0101] An imaging process is then performed involving the light source 408. A light path is defined from light source 408 through the objective lens in the objective lens housing 200 via the condenser lens 410. The light path visits the condenser lens 410, void 304, the first coverslip 318, the imaging chamber 600, the second coverslip 216 and then enters the objective lens housing via the window in void 204. Light is emitted from light source 408 and travels to the objective lens via the light path. After the lens, the light is collimated to achieve Kohler illumination. Images are collected by image sensors in a further imaging apparatus (not shown). The image sensor may be any suitable charge- coupled device, image sensor or camera. Figure 8 depicts technical schematic drawings of the moveable spacer and lower member of the pushing structure. As described above, the moveable spacer 300 is formed of an upper part 300a and a lower part 300b. Figure 8(a) depicts a drawing of the lower part 300b of the moveable spacer 300, from above, in accordance with an embodiment. As can be seen from Figure 8(a), the lower part of the moveable spacer 300b is ring shaped. The outer diameter is 34 mm. The inner diameter is 15.00 mm. The surface has 6 apertures in a circular arrangement. Each aperture are threaded holes for receiving screws to secure the lower part to the upper part. As can be seen from Figure 8(a), the lower part of the moveable spacer has a void 304.

[0102] Figure 8(b) depicts a view of the lower part 300b of the moveable spacer 300 for receiving the glass coverslip, in particular, a view from below. The moveable spacer, also referred to as a diaphragm insert, has a central void 304. As described above, the lower part 300b of the moveable spacer has a rim 310 defining a circular recess 312 for receiving the glass coverslip. The groove 314 is provided for sealing the coverslip to the spacer 300.

[0103] Figure 8(c) is a perspective drawings of the lower part of the moveable spacer from above (left hand side) and below (right hand side), in accordance with an embodiment.

[0104] Figure 8(d) is a close up view of the circular recess structure of the moveable spacer 300. Figure 8(d) depicts the dimensions of the circular recess of the moveable spacer having depth d. The moveable spacer 300 itself has a depth of 4mm. The recess of dimension d in Detail 1 is used to mount a coverslip of appropriate thickness for the imaging. Dimension d can be in the range 0.08 - 0.25 mm

[0105] In situ, the apparatus is configured to be placed inside a bioreactor. The objective lens housing may be placed in a position that does not obstruct or interfere with any functions such as stirring. The optical apparatus 1002 provided outside the vessel include objective lens and camera. The objective lens is stationary and offers up to 100x magnification.

[0106] The above description of specific embodiments is made by way of example only. A skilled person will appreciate that variations of the described embodiments may be made without departing from the scope of the invention. For example, the first surface does not need to be part of an objective housing and may be, for example a window on the culture vessel.

[0107] In the above described embodiments, the surfaces are described as interfacing and having interfacing features in the form of recesses that form part of a sample volume. In further embodiment, further interfacing features are provided in addition to or as an alternative to the recessed. These include further spacer elements, for example, an insert, a washer or an O-ring formed out of a polymer, for example, a PDMS. The spacer elements are detachable and / or swappable. The size of the separable spacer may be precisely controlled by a spin coating manufacturing process therefore allowing additional control on the height of the imaging chamber. The spacer elements or inserts are secured to either of the end surfaces by, for example, a plasma bonding process.

[0108] Figure 11(a) and 11(b) depicts views of an apparatus in accordance with a further embodiment, in which a further spacer element 301 provided on the distal surface of the moveable spacer. Figure 11(a) will be understood to correspond to Figure 3(a) with a further spacer element 301. Likewise, Figure 11 (b) will be understood to correspond to Figure 6. The further spacer element 301 is bonded or otherwise secured to the distal surface of the moveable spacer element, in particular to the coverslip received in the moveable spacer element.

[0109] In the embodiment of Figure 11 , the spacer element 301 is a thin element formed out of Polydimethylsiloxane (PDMS). The spacer element 301 has a diameter sized to allow the element to protrude inside the corresponding recess 206 of the surface of the objective housing. It will be understood that without the additional spacer element 301 (as shown in Figure 3(a)) the formed sample chamber has a first depth and with the additional spacer element 301 the formed sample chamber has a second, smaller depth.

[0110] The surface area difference between these two may be small in that the additional spacer element 301 diameter can be quite large, as long as it fits into 206. However, it may also be smaller if required.

[0111] While Figure 11 (a) and (b) depicts a further spacer element bonded to a surface of the moveable element 300, the further spacer may be bonded to the corresponding surface of the objective housing. The height of such a further spacer offers very precise control over the depth of the formed chamber, above a tolerance level of the machined moveable spacer. The spacer may be formed using a spin-coating procedure through which the height may be precisely controlled using a spin-coater or other suitable apparatus. The additional spacer may be a washer or insert.

[0112] The spacers may have any suitable shape, for example, a rectangle, disc or ring shaped. The spacers may be secured to either interfacing surface to decrease the depth of the sample chamber. Such spacers may be made of Polydimethylsiloxane (PDMS), for example. In some embodiments, the depth of one or more of the interfacing surfaces may be varied, for example, by attaching additional spacers.

[0113] While a spin-coated PDMS spacer is described, other materials and other manufacturing processes may be suitable. For example, other polymers may be suitable. Additive processes or other 3-D printing processes that offer precise control over the size of the element may be used.

[0114] In addition or alternatively to recesses, it will be understood that protrusions may be provided on either surface as an example of an interfacing feature. In some embodiments, complimentary mating features may be provided on each surface for mating the two surfaces together.

[0115] In the above embodiments, glass coverslips are described, however it will be understood that other optical elements and materials may be used. For example, these may be plastic if the plastic has a suitable index of refraction. In alternative embodiments, other optical elements may be used in place of the coverslip. Optical elements that are at least transparent and present a substantially flat and planar surface may be suitable. A microscope slide may be used, for example, in place of the coverslip. Suitable optical elements include elements that have an index of refraction close to glass, for example, between 1 and 2, preferably between 1.45 and 1.55. The coverslips may therefore be referred to as first and / or second optical elements respectively.

[0116] A material such as rubber may be suitable. In the above-described embodiments, the flexible membrane is flexible enough to allow movement of the moveable spacer. Movement of the moveable spacer is controlled and limited, for example, by hitting the interfacing surfaces at the objective side. In terms of material for the flexible membrane, any material that is flexible and not water soluble and can provide a barrier to water is suitable.

[0117] In addition, while the housings are described as cylindrical, it will be understood that other shapes may be suitable. In particular, any shape that is generally aligned with the objective lens axis may be suitable.

[0118] In the above described embodiments, the light source is described as attached to the condenser lens and these two components are described as moving together. However, in alternative embodiments, these two components may remain static. In such circumstance, if the light is not ideally collimated then the distance between the condenser lens and the sample will affect the amount of light reaching the image plane.

[0119] In the above described embodiments, a piezo motor is used, however, alternative motor and drive arrangements can be provided. For example, a stepper motor configured to move predefined linear distances may be suitable. In the above described embodiments, the motor senses when the imaging chamber is formed and ceases moving.

[0120] The objective lens may also be moveable within the objective housing. In the above embodiments, the objective lens is movable using a dove tail type stage, but it could be motorised as well, piezo or stepper motor. The objective lens may be manually moveable or may be replaced by a different objective. However, it will be understood that the objective housing remains fixed in operation in the vessel.

[0121] In the above embodiments, a series of precisely designed recesses (rings) on the mating faces of the cylinders are used to accommodate appropriate glass coverslips for the imaging and thereby allow a thin layer of culture in the imaging plane to be confined between the two cylinders when the system is in the extended position. In some embodiments, the movable spacer may have concentric rings of different sizes to allow different imaging volume heights. This may be machined or could be achieved using a plasma bonded PDMS layer, as described with reference to Figure 11. In the above described embodiments, a moveable spacer is described that is a separate component to the motor / pushing member. This allows a decoupling of electronics (contained in the culture vessel) as far as possible for cleaning / decontamination purposes. If we want to autoclave the vessel, the inner elements that can't be autoclaved can be extracted from the light source housing without taking apart the spacer assembly thus maintaining sterility inside the vessel (i.e. all the surfaces in contact with the liquid will be autoclaved). The components to be sterilised include the light source housing, moveable spacer and the objective lens housing. In addition, the electronics may be accessed without impacting any alignment that has already been performed. The light source housing may therefore have an accessible interior, for example, accessible from an upper direction to allow access to the electronics without moving the aligned components and, therefore, without altering the fine alignment.

[0122] In the above embodiments, the motor or other driving arrangement may be coupled to the pushing member a rigid coupling. In other embodiments the coupling may be non- rigid, for example, including one or more springs and / or resilient members. These could compensate for misalignments.

[0123] In some embodiments, a mechanical stabilizing arrangement is provided for stabilizing one or more parts of the device. For example, a stabilizing arrangement may be provided for stabilizing the drive arrangement. Such a stabilizing arrangement may improve imaging.

[0124] Figure 12 depicts the embodiment depicted above with reference to Figures 1 to 7 together with a stabilizing arrangement in the form of a stabilizer plate 1200 secured at an upper part of the first housing 100 to the securing beam 508. The stabilizer plate 1200 may also be referred to as a stabilizer for brevity.

[0125] In this embodiment, the stabilizer 1200 is a disc-shaped plate with an aperture sized to fit first housing 100 therethrough. The stabilizer is depicted in Figure 12 in a cut-away view to show securing bolt 1202 or other fastening means. The securing bolt 1202 secures the beam 508 to the stabilizer 1200. The beam 508 is also secured to the securing plate 506. Therefore, the stabilizer 1200 acts to stabilize the drive arrangement 500 in operation. In particular, the stabilizer acts to prevent unwanted upward and / or downward movement of the drive arrangement. Alternative stabilizing arrangements may be provided in other embodiment. For example, stabilizing arrangements for stabilizing one or more parts of the device may be provided. Figure 13 depicts a plot 1300 of experimental results obtained using the device. The y- axis of the plot 1300 depicts a cell concentration (in terms of colony-forming unit - CFU - per mL). The x-axis of the plot depicts the time since the start of the measurement, in hours.

[0126] Figure 13 depicts a cell count 1302 obtained using the device, in accordance with an embodiment. In detail, the growth curve of MG1655 in Lysogeny broth media (LB) is depicted. Figure 13 depicts a control cell count 1304 obtained using known optical density (OD) measurements. OD measurements estimate the number of cells using light scattering. As can be seen in Figure 13, the device currently has capability to measure OD values up to the intersection point 1306 at which point the measured optical density value is 0.8. The limitation here is due to the image processing and cell counting algorithm used which is unable to distinguish cells at a higher density. This limit may be overcome using better suited software.

[0127] In accordance with embodiments, the cell count may be performed using any suitable image processing and / or cell counting algorithm. In an example, an artificial intelligence based algorithm may be used that identifies the cells (counters) and then the images of cells over different z (over the z-stack) may be clustered with a clustering algorithm.

[0128] Figure 14 depicts images obtained using the device, in accordance with an embodiment. Figure 14 depicts two images of E.coli K12 AD38 strain. Figure 14 (upper) depicts a first image of a sample corresponding to a low optical density (optical density of 0.4) and therefore low cell concentration. A single E.coli bacteria 1402 is indicated. Figure 14 (lower) depicts a second image in which the sample is focussed at a higher density (optical density of 0.92) and thus higher cell concentration. A single E.coli bacteria 1404 is indicated.

[0129] Although description of particular embodiments has been provided above, it should be understood that these embodiments are illustrative only and that the claims are not limited to those embodiments. Those skilled in the art will be able to make modifications and alternatives to the described embodiments which are contemplated as falling within the scope of the appended claims. Each feature disclosed or illustrated in the present specification may be incorporated in any embodiment, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein. In particular, one of ordinary skill in the art will understand that one or more of the features of the embodiments of the present disclosure described above with reference to the drawings may produce effects or provide advantages when used in isolation from one or more of the other features of the embodiments of the present disclosure and that different combinations of the features are possible other than the specific combinations of the features of the embodiments of the present disclosure described above.

Claims

CLAIMS1. An imaging apparatus for collecting and imaging a sample inside a vessel comprising: a first surface comprising or configured to receive a first optical element; a light source housing for a light source; a moveable spacer secured at a distal end of the light source housing by a resilient membrane and moveable relative to the distal end of the light source housing, wherein the moveable spacer comprises a second surface comprising or configured to receive a second optical element; a mechanism for controllably moving said moveable spacer from a retracted position to a protruding position relative to the distal end of the light source housing, wherein the movable spacer is biased against movement into the protruding position by the resilient membrane, wherein the first surface and / or the second surface of the moveable spacer comprise one or more interfacing features configured to form an at least partially closed sample chamber for imaging, wherein the at least partially closed sample chamber is defined, at least in part, by the first and second optical elements and / or at least part of the interfacing features.

2. The apparatus of claim 1 , wherein the first surface is fixed relative to the vessel such that the at least partially closed sample chamber is formed by movement of the moveable spacer.

3. The apparatus of any preceding claim, wherein the first and second surface comprise substantially planar portions, for example, comprising the first and / or second optical elements, such that the at least partially closed sample chamber and / or volume formed between said portions and / or a sample volume defined by said at least partially closed sample chamber is substantially planar.

4. The apparatus of any preceding claim, wherein the at least partially closed sample chamber comprises a 2D layer, optionally, wherein the 2D layer comprises a depth in the range 1 to 500 pm.

5. The apparatus as claimed in claim 1 , wherein the one or more interfacing features comprise one or more protrusions from the first and / or the second surface and / or one or more recesses formed in the first and / or second surface.

6. The apparatus as claimed in any preceding claim, wherein the one or more interfacing features comprise one or more further spacer elements attached to the first and / or second surface.

7. The apparatus as claimed in any preceding claim, wherein the one or more interfacing features and / or first or second optical elements form at least one structural part of the sample chamber, for example, at least one wall and / or at least part of a lower surface and / or at least part of an upper surface of the sample chamber.

8. The apparatus of claim 1 , wherein at least part of the first surface, for example, part of the optical element, and at least part of the distal surface are configured to contact to form the at least partially closed sample chamber.

9. The apparatus of any preceding claim further comprising a drive arrangement, optionally comprising a motor, configured to indirectly move the moveable spacer into the protruding position.

10. The apparatus of any preceding claim, wherein the mechanism comprises a pushing member moveable relative to light source housing and relative to the moveable spacer, wherein the pushing member is configured to push said moveable spacer into said protruding position thereby to form the sample chamber, optionally, wherein the pushing member is operable to push the moveable spacer from its natural position to a protruding position, wherein the moveable spacer is biased against movement to the protruding position by the resilient membrane.11 . The apparatus of claim 10, wherein the light source is disposed on and / or fixedly coupled to the pushing member such that said light source moves together with said pushing member, optionally wherein a condenser lens if disposed on and / or fixedly coupled to said pushing member.

12. The apparatus of claims 10 or 11 , wherein the pushing member comprises an aperture and wherein the light source is disposed on and / or fixedly coupled to the pushing member such that that light is transmitted through said aperture.

13. The apparatus of any preceding claim, wherein the pushing member and movable spacer are arranged coaxially along a longitudinal axis of the light source housing, wherein the pushing member and movable spacer are movable along said axis14. The apparatus of any preceding claim, wherein the movable spacer comprises an aperture for permitting light to pass through.

15. The apparatus of any preceding claim, wherein at least one of the first and second surfaces comprise a first recess for defining at least part of the sample chamber when contacting the correspond interfacing surface of the movable spacer, optionally a second recess for receiving a transparent optical element, wherein the second recess is provided inside the first recess.

16. The apparatus of any preceding claim, wherein the flexible membrane and / or moveable spacer forms at least part of a barrier to fluid at the distal end to prevent entry of fluid into the light source housing.

17. The apparatus of any preceding claim, wherein the at least partially closed sample chamber is defined by parts of the first and second surfaces and / or their respective optical elements and / or is not defined by part of the resilient membrane18. The apparatus of any preceding claim, wherein the moveable spacer is substantially contained inside the light source housing when in the retracted position.

19. The apparatus of any preceding claim, wherein the objective housing and light source housing form two separate parts of the apparatus such that, when assembled, an open volume is defined between the moveable spacer and the objective lens housing when the moveable spacer is in the retracted position.

20. The apparatus of claim 19, wherein the open volume comprises a gap of uniform thickness and / or wherein no part of the apparatus is present in said open volume.

21. The apparatus of any preceding claim, wherein the first surface is configured to be fixedly coupled to a bio-reactor and / or other vessel for containing liquid.

22. The apparatus of any preceding claim, wherein the interfacing features comprise a plurality of recesses sized to receive cover slips of different sizes and / or wherein the first surface and / or the second surface comprise a plurality of recesses having different depths for forming sample chambers of different depths.

23. A method of imaging a sample, wherein the method comprises: providing at least part of the apparatus of any of claims 1 to 22 in a liquid container such that liquid is present between the surface of the moveable spacer and the first surface; causing the moveable spacer to move to the protruding position thereby to form the at least partially closed sample chamber and contain a sample inside the sample chamber.

24. The method of claim 23 further comprising performing an imaging process using the apparatus, the imaging process comprising operating the light source such that light is incident on the sample.

25. The method of claim 24, wherein the imaging process comprises a fluorescence imaging and / or imaging of microbial objects.

Images