GB2618378A

GB2618378A - Optical measurement apparatuses for use with a cutting apparatus

Info

Publication number: GB2618378A
Application number: GB2206626.0A
Authority: GB
Inventors: Margaret Collinson Lucy; Leonard Jones Martin; Guan Wei
Original assignee: Francis Crick Institute Ltd
Current assignee: Francis Crick Institute Ltd
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2023-11-08
Also published as: WO2023214147A1

Abstract

Apparatus for characterising thickness of slices obtained from a sample 210 using a microtome, comprises; detector 250 configured to receive optical signals reflected and/or scattered from at least first and second portions of the surface 211 of a sample or sample holder; controller configured to: determine first information indicative of a position of the first surface portion prior to obtaining slices using a blade 230, the first information is determined based on a first detector output, determine second information indicative of a position of the second surface portion subsequent to obtaining slices, the second information is determined based on a second detector output; and determine slice thickness based on the first and second information. Apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome is also disclosed. A first detector is configured to receive optical signals reflected and/or scattered from each of a plurality of surface portions of the sample or sample holder. A controller is configured to determine first information indicative of a respective position of each of the plurality of surface portions and determine an orientation of the surface based on the first information.

Description

Optical Measurement Apparatuses For Use With A Cutting Apparatus

Field

The present disclosure relates to measurement and control apparatuses for use with a cutting apparatus and particularly for use with a microtome or ultramicrotome apparatus.

Background

Cutting apparatuses are known to the skilled person, being configured to hold a sample and obtain slices from a surface of the sample via the use of a blade assembly. Such cutting apparatuses typically comprise a sample holder coupled to a multi-axis manipulator, allowing the sample to be positioned and oriented in space; and a blade coupled to a further multi-axis manipulator allowing the blade to be positioned and oriented in space. The obtaining of slices from the surface of a sample mounted on the sample holder is typically effected by providing control inputs to the sample holder and / or blade manipulators to cause a cutting edge of the blade to be aligned to an outer surface of the sample, and the sample and blade to be actuated towards one another to obtain a slice. In cutting apparatuses of this type, and particularly where such a cutting apparatus comprises a microtome or ultramicrotome configured to obtain very thin slices of potentially significantly less than 1000 nm in thickness, it is desirable to provide a user with a high degree of control over slice thickness, in order to allow the obtaining of high quality slices, to provide accurate tracking of the progress of slicing as multiple slices are obtained from a sample, and to prevent damage to the blade caused by suboptimal alignment of the blade to the sample surface.

Typically, the slice thickness of one or more slices obtained from a sample by a cutting apparatus, and the relative position of the cutting edge of the blade and the sample surface, are inferred by a controller of the cutting apparatus based on user observation. However, not only does this require exercise of skill by an experiences operator, but positioning errors in manipulators of cutting apparatuses can lead to positioning errors between the expected and actual positions of the blade and the sample surface in three-dimensional space. These positioning errors can lead to inaccuracy and / or inconsistency in the thickness of slices obtained by the cutting apparatus, and to potential damage to the sample and / or blade by misalignment of the sample to the blade during slicing / cutting. Various approaches are described herein which seek to help address or mitigate at least some of the issues discussed above.

Summary

According to a first aspect of the disclosure, there is provided an apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, the apparatus comprising: a detector configured to receive optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with the microtome apparatus; and provide an output indicative of at least one characteristic of the received optical signals; and a controller configured to: determine first information indicative of a position of a first surface portion of a sample or a sample holder assembly of the microtome apparatus prior to the obtaining of one or more slices from the outer surface of a sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determine second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

According to a second aspect of the disclosure, there is provided a method of characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, comprising: receiving, at a detector, optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with a microtome apparatus; providing, from the detector to a controller, an output indicative of at least one characteristic of the received optical signals; and determining, at the controller, first information indicative of a position of a first surface portion of the sample or sample holder assembly of the microtome apparatus prior to the obtaining of the one or more slices from the outer surface of the sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determining, at the controller, second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of the one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determining, at the controller, a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

According to a third aspect of the disclosure, there is provided a computer program comprising instructions which, when executed on a controller of an apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, cause the controller to: receive from a detector of the apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample an output indicative of at least one characteristic of optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with the microtome apparatus; determine first information indicative of a position of a first surface portion of the sample or a sample holder assembly of the microtome apparatus prior to the obtaining of one or more slices from the outer surface of the sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determine second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

According to a fourth aspect of the disclosure, there is provided an apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, the apparatus comprising: a first detector configured to receive optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly and provide an output indicative of at least one characteristic of each of the received optical signals; and a controller, wherein the controller is configured to: determine first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, and determine an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.

According to a fifth aspect of the disclosure, there is provided a method of characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, comprising: receiving, at a first detector, optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly; providing, from the detector to a controller, an output indicative of at least one characteristic of each of the received optical signals; determining, at the controller, first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly; and determining, at the controller, an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.

According to a sixth aspect of the disclosure, there is provided a computer program comprising instructions which, when executed on a controller of an apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, cause the controller to: receive, from a detector, an output indicative of at least one characteristic of optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly; determine first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, and determine an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.

Respective aspects, features and advantages of the present disclosure will be apparent from the following description of examples, which is to be read in conjunction with the accompanying drawings.

Brief Description of the Drawings

Embodiments of the disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 is a schematic diagram of a cutting apparatus with which apparatuses according to

the present disclosure may be configured for use.

Figures 2 to 4 are schematic diagrams of a slice thickness determination apparatus in use with a cutting apparatus.

Figures 5 to 7 are schematic diagrams of a sample surface orientation characterisation apparatus in use with a cutting apparatus.

Figure 8 is a schematic diagram of a sample surface orientation characterisation apparatus in use to characterise an orientation of a cutting edge of a blade Figure 9 is a schematic diagram of an approach for determining an orientation of a cutting edge of a blade of a cutting apparatus.

Figure 10 is a flowchart schematically detailing aspects of operation of an apparatus according

to an aspect of the present disclosure.

Figure 11 is a flowchart schematically detailing aspects of operation of an apparatus according to an aspect of the present disclosure.

Detailed Description

Aspects and features of certain examples and embodiments are discussed! described herein.

Some aspects and features of certain examples and embodiments may be implemented conventionally and these are not discussed / described in detail in the interests of brevity. It will thus be appreciated that aspects and features of apparatus and methods discussed herein which are not described in detail may be implemented in accordance with any conventional techniques for implementing such aspects and features. In particular, the present disclosure relates to apparatuses which may be used with or integrated into a cutting apparatus configured to obtain slices from a sample of material, such as a microtome or ultramicrotome, and aspects of these devices not described in detail herein in the interest of brevity may be implemented in accordance with the common general knowledge of the skilled person.

The present disclosure relates to apparatuses configured to perform measurement and / or characterisation functions, and optionally control functions, in conjunction with apparatus designed for the cutting of samples, particularly the cutting of samples at levels of precision around the micron level, or at the sub-micron level. In particular, the present disclosure is directed to apparatuses configured to perform measurement and / or characterisation functions, and optionally control functions, in conjunction with cutting apparatuses (e.g. microtome or ultramicrotome apparatuses), configured for the obtaining of thin slices of samples, and particularly for the obtaining of slices with a slice thickness of less than and / or substantially less than 1000 nm. In embodiments of the present disclosure, the apparatuses are configured to perform measurement and / or characterisation functions, and optionally control functions, in the context of an ultramicrotome apparatus configured to obtain slices with a thickness in the range of 50 to 200 nm.

More specifically, the present disclosure is directed to apparatuses, methods, and computer program products for characterising a thickness of one or more slices obtained from an outer surface of a sample using a cutting apparatus such as a microtome apparatus or ultramicrotome apparatus. The present disclosure is further directed to apparatuses, methods, and computer program products for characterising an orientation of an external surface of a sample or sample holder associated with a cutting apparatus such as a microtome apparatus or ultramicrotome apparatus. It will be appreciated that a single apparatus may be configured for characterising a thickness of one or more slices obtained from an outer surface of a sample and for characterising an orientation of an external surface of a sample or sample holder, according to approaches set out herein, or that separate apparatuses may be provided to carry out respective ones of these functions. It will also be appreciated herein that whilst the apparatuses described, or sub-components of said apparatuses, may be integrated into a cutting apparatus such as a microtome or ultramicrotome apparatus at manufacture, and may be considered to comprise integrated features of such a cutting apparatus as provided to users, apparatuses as described herein may be provided separately, for example for retrofitting to a cutting apparatus such as a microtome or ultramicrotome in order to augment / enhance the existing functionality of the cutting apparatus.

Figure 1 shows schematically a cutting apparatus 1 configured for the obtaining of slices from a sample, to which a measurement apparatus according to aspects of the disclosure has been attached for use. In Figure 1, the cutting apparatus 1 comprises an ultramicrotome apparatus configured for the obtaining of slices with a thickness of 1000 nm or less, preferably with a thickness of between 20 to 500 nm, and more preferably with a thickness of between 20 to 200 nm. However it will be appreciated the cutting apparatus with which measurement apparatuses described herein may be configured for use may comprise a microtome, cryomicrotome, a cryoultramicrotome, vibratome, or other cutting apparatus configured to obtain slices from samples, and that herein, references to a microtome or ultramicrotome may be generalised to other cutting apparatus comprising the same generic components (e.g. sample holder, blade assembly, etc). Such a cutting apparatus may generally operate according to the general principles describes in relation to the ultramicrotome 1 of Figure 1, with variations in respect of the positioning accuracy of various manipulators comprised in the system, a sample holder configuration, and a configuration of a cutting / blade assembly (e.g. in terms of knife! blade material and configuration). A sample 110 from which slices are to be obtained by an ultramicrotome 1 may comprise any sample type known to the skilled person, such as, for example, prokaryotic or eukaryotic cells or tissues embedded in a suitable embedding media known to the skilled person. For example, a sample 110 comprising such cells and! or tissues may be perfusion fixed or fixed in vitro using a suitable fixing agent known to the skilled person (e.g. a combination of 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer solution), may be stained using a suitable staining media (e.g. Walton's Lead Aspartate), may be dehydrated using a suitable dehydrating agent (e.g. ethanol or acetone), and then embedded in a suitable embedding medium (e.g. an epoxy or methacrylate resin) using an embedding and curing protocol known to the skilled person. It will be appreciated many different sample preparation approaches are known to the skilled person, and the most suitable sample preparation approach will be dependent on, for example, the type of sample to be embedded, and the thickness of the slices to be obtained. In the context of a microtome apparatus for obtaining of samples with a thickness of greater than 1 micron, paraffin wax may be used as an embedding medium. In other embodiments, measurement apparatuses as described herein may be configured for use with or integrated into a cryoultramicrotome, where the fixing of the sample for slicing is effected by freezing or vitrification of the sample using liquid nitrogen, liquid ethane or a similar cryogen in a high pressure freezer, plunge freezer or other apparatus known to the skilled person. However, whilst the sample type and embedding medium (where used) may influence the minimum obtainable slice thickness, it will be appreciated that the approaches and apparatuses described herein for slice thickness characterisation and sample or sample holder orientation characterisation may be applied to samples prepared in accordance with any sample preparation approach known to the skilled person, and as such, though they may be particularly useful in microtome and ultramicrotome contexts, they are not limited to use with samples prepared in any particular manner. For the purpose of providing a concrete example of a use case for the slice thickness determination and sample or sample holder orientation characterisation approaches described herein, the context of an ultramicrotome apparatus 1 is used. However it will be appreciated the approaches and apparatuses described herein may be applied to a wide range of cutting apparatuses configured to obtain slices from samples of material, and are not limited to histological contexts.

An ultramicrotome 1 as shown schematically in Figure 1 comprises a sample holder assembly 11, comprising a sample holder 101 configured to hold a sample 110 from which one or more slices are to be obtained, such as, for example, a cell and / or tissue sample embedded in a block of epoxy resin, or an unembedded and / or frozen cell and / or tissue sample mounted to a suitable base-layer known to the skilled person. The sample holder 101 may be configured to retain the sample using mechanical or adhesive means, such as, for example a clamp arrangement configured to retain a portion of a sample block or base layer between opposable jaws. Typically the sample holder 101 is mounted to a multi-axis manual or automated manipulator or sample stage 120, configured to allow positioning of the sample 110 in three-dimensional space. The manipulator 120 may be configured in any manner known to the skilled person, and known for use in cutting apparatuses such as microtomes and ultramicrotomes. Different manipulator types are known to the skilled person, and the specific implementation is not of particular significance, and as discussed below, in some cutting apparatuses, the sample holder is fixed, not being provided with a manipulator 120. The required positioning accuracy of a sample holder assembly manipulator 120 in each of its degrees of freedom is typically a function of the required slice thickness range, such that in the context of an ultramicrotome configured for obtaining samples of between 50 to 200 nm thickness, the manipulator will generally be configured with a positioning accuracy of better than 1000 nm, and preferably better than 200 nm, or better than 50 nm, in each translational axis. Accuracy in rotational axes may range from less than 2 degrees, to less than 0.01 degrees, for example using stepper motor actuators, down to less than or equal to 2x10-6 degrees. The sample holder 101 and manipulator 120 are together configured to hold a sample 110, and orient a surface of the sample 110 towards a blade assembly 12 such that a blade 130 of the blade assembly 12 may obtain a slice from the sample surface without colliding with any portion of the sample holder assembly 11.

The blade assembly 12 is configured for obtaining slices from a sample 110 retained in the sample holder 101. The blade assembly 12 comprises a blade 130 with a cutting edge configured to cut through a sample 110 to obtain one or more slices from the sample surface.

In an ultramicrotome context, where the target slice thickness is typically less than 1000 nm, and more preferably 50 to 200 nm, the blade 130 will typically comprise a glass or diamond material, with a straight cutting edge of high sharpness and hardness. In a microtome context, where the target slice thickness may be more than 1000 nm, the blade 130 may comprise an alternative material such as a metal. The blade 130 is typically mounted to a multi-axis manipulator 140 to allow positioning of the blade in three-dimensional space. This manipulator 140 will typically be configured in a similar manner to that described in relation to the sample holder manipulator 120. The blade assembly 12 may comprise a slice collecting assembly, such as an open liquid! fluid reservoir or 'boat' (visible in in Figure 1 behind the blade 130), configured relative to the blade 130 such that slices obtained as the cutting edge of the blade moves through the sample 110 are received intact onto the surface of a suitable liquid! fluid (e.g. water or aqueous buffer solution) retained in the reservoir, for collection and further processing.

Though the manipulator(s) 120 and / or 140 of an ultramicrotome 1, or any relevant other manipulators, may be manually actuated, they will typically comprise electronically controlled actuators, connected to a controller (not shown) configured to provide control outputs to the actuators to drive the sample holder assembly manipulator 120 and / or blade assembly manipulator 140 to move a sample 110 mounted to the sample holder 101 relative to the cutting edge of the blade assembly 12. The controller may be considered to logically comprise various sub-units / circuitry elements associated with different aspects of the operation of the ultramicrotome 1 and / or of a slice thickness determination apparatus and / or of a sample or sample holder orientation characterisation apparatus (where these apparatuses are integrated into the ultramicrotome). The controller is typically configured to provide a supply of power to any motorised / automated elements of the ultramicrotome 1, such as the sample holder and blade assembly manipulators, and / or to provide control signals to these and other components of the ultramicrotome 1. In particular, the controller will typically be configured to provide inputs to actuators comprised in said manipulators to allow the absolute positions of the sample 110 and the cutting edge of the blade assembly 12, and! or their relative positions, to be controlled! manipulated in three-dimensional space. The controller may also comprise display driving circuitry and user input detection circuitry, to provide functionality associated with one or more user displays, and / or one or more peripheral user input devices such as a keyboard, mouse, joystick, track-pad, and / or touch-screen display (not shown). It will be appreciated the functionality of the controller can be provided in various different ways, for example using one or more suitably programmed programmable computer(s) and / or one or more suitably configured application-specific integrated circuit(s)! circuitry! chi p(s) / chi pset(s) configured to provide the desired functionality. The controller may comprise a wireless transceiver and associated control circuitry enabling transfer of data between the ultramicrotome and external computing devices such as a personal computer (not shown) and / or cloud storage server, via a wireless transfer protocol such as WiFi or Bluetooth, or a wired transfer protocol such as Ethernet (e.g. IEEE 802.3). The controller also comprises one or more data storage elements (e.g. a memory element such as a ROM or RAM element) which can be used to store data associated with operation, control, and usage of the ultramicrotome according to established techniques for data storage and transfer. It will be appreciated that where a slice thickness determination apparatus and / or sample or sample holder orientation characterisation apparatus according to aspects of the present disclosure are integrated into an ultramicrotome, aspects of control of components of these apparatuses (e.g. of optical detectors, illumination sources, and controllers) may be provided by one or more controllers associated with the ultramicrotome. In other embodiments, aspects of control of subcomponents of these apparatuses as described herein may be provided by one or more separate controllers provided with the apparatus.

In use, the ultramicrotome of Figure 1 will typically acquire slices of a sample in the following manner. The sample 110 will typically comprise a block which has been prepared by embedding a cell and! or tissue sample in a suitable embedding medium as described herein, though the sample may also comprise an unembedded sample of cell and / or tissue (e.g. a frozen sample, or a sample mounted to a back-plate such as a glass slide), or a block of a material such as a plastics or metal material, or a sample of any other material of interest which is known to the skilled person. The term 'block' is not intended to imply any particular external shape, though commonly the block will comprise a trapezoid or truncated pyramidal shape with a flat 'working surface' cut broadly parallel to an intended cutting plane. The shape of the sample block 110 prior to sectioning in the ultramicrotome 1 may be prepared by a different cutting apparatus, such as a microtome, typically requiring lower positioning and cutting accuracy than the cutting apparatus to be used to obtain slices from the sample 110.

Commonly the working surface comprises a substantially flat outer surface of the sample 110. However, the working surface may in other instances comprise a non-flat outer surface, such as may be the case for an unembedded sample mounted to a base layer such as a slide. The working surface comprises an external surface which is to be presented towards a blade assembly 12 for slicing, and typically comprises an outer surface which is to be completely or substantially removed by the passing of the cutting edge of the blade assembly 12 through the sample 112 along a plane substantially parallel to the working surface (e.g. along a cutting axis parallel to the axis y in Figure 1).

In a first step, the prepared sample or sample block 110 is mounted to the sample holder 101, and the sample holder manipulator 120 is controlled via manual inputs from a user, and / or inputs from the controller, to orient the working surface towards the cutting edge of the blade 130. The sample holder 101 may be removable from the manipulator to aid mounting of the sample.

In a second step, the blade 130 is mounted to the blade assembly manipulator 140, typically in conjunction with a reservoir (e.g. a 'boat') of a liquid! fluid such as water, positioned on an opposite side of the cutting edge to the position of the sample holder. In an ultramicrotome context, the blade assembly 12 typically comprises a diamond or glass blade! knife 130 with a straight cutting edge, though in microtome contexts a metal blade may be used. It will be appreciated in other cutting apparatuses, the blade may be substituted for a water jet, laser, spark erosion wire, or other cutting means. Where a liquid / fluid reservoir is used, this is typically filled with a working liquid! fluid such as water to a level sufficient to bring a meniscus up to wet the cutting edge of the blade 130. It will be appreciated the order of the first and second steps is not significant.

In a third step, the cutting edge of the blade 130 and the working surface of the sample 110 are advanced towards each other along a sample advance axis x via actuation of one or both of sample holder manipulator 120 and blade assembly manipulator 140. One or both of the sample holder and blade assembly manipulators are controlled, for example via control signals provided by the controller, to bring the cutting edge of the blade assembly 130 into an orientation parallel to the working surface of the sample 110, to ensure that the slice thickness is consistent across the width of the slice.

In a fourth step, one or more slices are obtained from the exterior surface of the sample 110. The obtaining of a slice is typically achieved by moving the sample 110 and! or blade 130 towards one another along a cutting axis y oriented substantially perpendicular to the cutting edge of the blade assembly 12 and parallel to the working surface of the sample 110, causing the cutting edge to pass through a portion of the sample 110. Prior to slicing, the sample 110 is advanced towards the cutting edge (i.e. along a sample advance axis x oriented substantially perpendicular to both the cutting edge of the blade assembly and the working surface of the sample) to a position where the cutting edge of the blade assembly 12 overlaps the sample 110 as viewed along the cutting axis y, with the degree of overlap typically corresponding to the target thickness of a slice to be obtained from the sample 110. The sample advance may be effected by actuation of one or both of the sample holder manipulator 120 and/or blade assembly manipulator 140. Following the obtaining of a slice n by actuation of the sample holder towards the blade assembly along the cutting axis y, the sample 110 and / or blade 130 are advanced along the sample advance axis x by a distance corresponding to the target slice thickness, and the next slice n+1 obtained by driving sample 110 and! or blade along cutting axis y. This process can be repeated multiple times to obtain further slices. In some applications of a microtome / ultramicrotome, the working surface may be imaged using an imaging apparatus (e.g. scanning electron microscope (SEM) !transmission electron microscope (TEM) / confocal imaging apparatus / light microscope / atomic force microscope (AFM)) after each slice has been obtained, to implement a serial block face microscopy protocol, known to the skilled person.

In a typical ultramicrotome 1, slices are obtained by moving the sample 110 relative to a fixed blade 130, via a manipulator 120 attached to the sample holder 101, which drives the working surface of the sample 101 along a cutting axis y towards a cutting edge of the blade assembly 12. However, it will be appreciated that in other embodiments, the sample holder 101 may be stationary during slice acquisition, with the blade 130 being moved along a cutting plane y to intersect with the sample 110, via control input to the blade assembly manipulator 140. In other embodiments, both of the sample holder 101 and blade 130 may be configured to be moved along cutting axis y by respective manipulators during slice acquisition. In other embodiments, a first one of the blade 130 and sample holder 101 may be moved along sample advance axis x to bring the sample 110 into proximity to the blade 130, and the second one of the blade 130 and sample holder 101 is moved along cutting axis y to obtain a slice from the sample 110.

The inventors have recognised that there are aspects of operation of a cutting apparatus such as the ultramicrotome of Figure 1 which may be improved. A first issue is in determining the thickness of slices obtained from a sample 110. The thickness is typically inferred by determining the degree of sample advance 0.e. the relative movement of the sample holder towards the blade assembly) between slices. Following the obtaining a slice n it is typically assumed that the cutting edge of the blade 130 is at a position along the sample advance axis x which is coincident with the working surface of the sample. Thus when the sample 110 is advanced towards the blade 130 (by moving the sample holder 101 towards the blade 130, or the blade 130 towards the sample holder 101, or both) the degree of sample advance is typically assumed to correspond to the control input to the respective manipulator(s) along the sample advance axis x, whether this is manually input (e.g. via a micrometer gauge) or automatically actuated via a signal from the controller. In known slice obtaining protocols used in cutting apparatuses such as microtomes and ultramicrotomes, the slice thickness of obtained slices is typically inferred based on a skilled operator observing the interference colour of each obtained slice as it floats on the surface of a region of sample-collecting liquid held in a 'boat' positioned behind the cutting edge! blade, with inputs to one or more of the manipulators of the cutting apparatus being used to manually adjust the slicing depth to target thicker or thinner slices, based on visual feedback from one or more obtained slices. However, positioning errors may lead to a divergence between the target sample / and / or blade advance distance and the actual advance distance of the sample and / or blade. Without wishing to be bound by any particular theory, it is thought that factors such as mechanical backlash of gearing in manipulator apparatus may be one source of such positioning error. Whilst the positioning error for a single slice may be small relative to the slice thickness, in scenarios where a large number of slices (for example, at least 10, at least 50, at least 100, at least 200, or at least 400, slices) are to be obtained by serial sectioning of the sample 110, the cumulative effect of positioning errors of manipulators 120 and 140 may lead to a significant deviation between the expected position of the current working surface, and the actual position of the current working surface (e.g. exposed cut surface from the most recently obtained slice). This may be a particular problem, for example, in imaging-guided slicing approaches, where a complementary imaging methodology (e.g. confocal microscopy, X-ray computed tomography, or ultrasound) is used to identify a particular region of interest within the internal volume of the sample 110, and this information is used to guide slicing down towards the region of interest (for example aiming to align the working surface / cutting edge axis to a notional plane of interest identified within the sample). Typically, characterisation of the sample using the complementary imaging methodology will be used to determine a depth of cut from an initial working surface of the sample which will be required to bring the working surface coincident with a particular point or plane identified within the sample. This distance is then converted into a target number of slices of predefined depth which must be obtained to reach the particular point or plane. It is of interest to be able to track the actual thickness of one or more of the slices obtained during this process, as opposed to relying on the target sample advance value input to the manipulator(s) 120 and / or 140, to provide an actual measurement of the progress of slicing through the sample towards the point or plane of interest and improve the accuracy with which a region of interest can be reached when using image-guided slicing approaches.

A second issue identified by the inventors is a desire to automate the alignment of a working surface of a sample 110 to the axis or plane of a cutting edge of a blade 130 in a cutting apparatus 1 such as a microtome or ultramicrotome. The cutting edges of blades in these apparatuses are susceptible to damage if slicing is attempted without the cutting edge being aligned parallel to the plane of the working surface of the sample to a high degree of precision.. For example, in an ultramicrotome context, it is generally considered that the partial or complete cut depth should be kept to less than 1000 nm, to avoid damage to the blade and / or sample. Furthermore, if the cutting axis y along which the cutting edge travels relative to the sample is oriented non-parallel to the working surface of the sample 110, the slice thickness will vary as the cutting edge passes through the sample 110. Not only is this typically disadvantageous as it leads to slices of non-uniform thickness, but blade damage commonly results if the depth of cut exceeds a certain limit, for example 1000 nm in an ultramicrotome context. Thus it is of interest to improve the alignment of samples to cutting edges of blade assemblies in cutting apparatuses such as microtomes and ultramicrotomes. In addition, in use cases where slicing is guided by complementary imaging of the internal volume of the sample as set out above, providing more precise orientation of the sample to the cutting edge, and / or to a plane of interest within the sample, in order to maximise the number of slices obtainable of a region of interest is desirable. VVhere a plane of interest in the sample is nonparallel to the plane of the working surface, the cutting edge must typically be first aligned to the plane of the current working surface, then backed off, the cutting edge and / or sample rotated to align the cutting edge parallel to the plane of interest, and the proximity of the cutting edge to the sample for obtaining slices adjusted so that the cutting depth is not more than the slice thickness limit (e.g. 1000 nm as above), based on the part of the sample projecting closest to the blade axis.

In embodiments of the present disclosure, apparatuses are described which are configured to carry out characterisation / determination of a thickness of one or more slices obtained from an outer surface of a sample using a cutting apparatus such as a microtome or ultramicrotome apparatus, and characterising an orientation of an external surface of a sample or sample holder associated with using a cutting apparatus, preferably comprising a microtome or ultramicrotome. It will be appreciated that these two functions may be carried out by a single apparatus, or that an apparatus may be configured to carry out one or other of these functions.

Thus according to a first aspect of the present disclosure, there is provided an apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, the apparatus comprising: a detector configured to receive optical signals reflected and! or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with the microtome apparatus, and provide an output indicative of at least one characteristic of the received optical signals; and a controller configured to: determine first information indicative of a position of a first surface portion of a sample or a sample holder assembly of the microtome apparatus prior to the obtaining of one or more slices from the outer surface of a sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determine second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

Figure 2 shows schematically an apparatus 23 in accordance with an embodiment of the first aspect of the present disclosure, configured for use with a cutting apparatus 2 such as a microtome or ultramicrotome as described herein. Elements of the cutting apparatus 2 are shown schematically, but may generally be configured as described in accordance with the cutting apparatus of Figure 1, and comprise a sample holder assembly 21, comprising a sample holder 201, upon which is mounted a sample 210, with a working surface 211 facing substantially away from the sample holder 201. A sample holder manipulator 220, separate to or integrated into the sample holder 201, may be provided, configured with at least one or more linear / translational degrees of freedom, corresponding to a block advance axis x, and / or a cutting / slicing axis y. It will be appreciated Figure 2 is highly schematic, and that in practice a sample holder manipulator 220 may comprise further degrees of freedom provided by actuators, enabling drive of the sample holder 201 to translate and! or rotate the sample 210 relative to a plurality of axes. In other embodiments, where a blade assembly manipulator 240 comprises degrees of freedom corresponding to at least the sample advance axis x and cutting axis y, the sample holder 201 may not be provided with a manipulator 220. A blade assembly 22 comprises a blade 230, mounted to a blade assembly manipulator 240, wherein the blade 230 comprises a cutting edge. The blade 230 may be configured as described in accordance with blade 130 of Figure 1, comprising, for example, a glass, diamond, or metal, cutting edge. A liquid / fluid boat (not shown) may be provided in conjunction with the blade 230, as described further herein to collect slices. Typically the blade assembly 22 is provided with a blade assembly manipulator 240 configured to provide at least two linear/translational degrees of freedom, corresponding to the block advance axis x, and the slicing axis y. As with the sample holder manipulator 220, further translational and / or rotational degrees of freedom may be provided. In other embodiments, where the sample holder manipulator 220 provides degrees of freedom to translate the sample along at least sample advance axis x and cutting axis y, a blade assembly manipulator 240 may not be provided.

An apparatus 23 for characterising a thickness of one or more slices obtained from an outer surface of a sample 210 comprises a detector 250, configured to receive optical signals reflected and / or scattered from at least first and second portions of the surface of the sample 210 or sample holder assembly (where references to a sample holder assembly herein may be considered to refer to sample holder 201 and / or manipulator 220, or indeed any portion of the cutting apparatus (e.g. a frame or housing part) which is configured to be in fixed spatial relationship to the sample 210 in use), and provide an output indicative of at least one characteristic of the received optical signals to a controller 260.

In embodiments of the apparatus 23, the detector 250 is configured to provide first and second outputs to the controller 260 which are indicative of degrees of spatial separation between a reference position associated with the detector 250 and, respectively, first and second surface portions of the sample 210 or sample holder assembly. The apparatus 23 may optionally comprise an illumination source, for example associated with detector 250, or provided as a separate component, which is configured to direct optical signals at surface portions of the sample 210 or sample holder assembly 21, generating reflected or scattered optical signals which may be received by the detector 250. In some embodiments the detector 250 and illumination source (not shown) are comprised in an integrated imaging device configured for optical proximity measurement. For example, in some embodiments, a detector 250 and illumination source are integrated into a confocal chromatic measuring device, as known to the skilled person, wherein white light from an illumination source is passed through a lens array configured to induce colour aberration, causing different wavelength components of light in the optical signal directed at the sample or sample holder assembly to comprise focal points distributed at varying distances from the detector 250 along an optical signal receiving axis 251 (which will typically comprise an optical signal transmitting axis where an illumination source is included in apparatus 23). A detector 250 comprising a spectrometer receives reflected or scattered optical signals from a surface portion of a sample or sample holder assembly at which the incident polychromatic signal is transmitted, and the wavelength spectrum of received optical signals is analysed (e.g. by controller 260, or a separate controller integrated into the detector apparatus) to determine a distance between a reference point comprised in the apparatus 23 (e.g. in fixed relation to the detector and / or illumination source) and the position of a reflecting and / or scattering surface portion from which the optical signals are received. The relationship between the output of the detector and the distance between the reference position and a surface portion from which optical signals are received is typically configured according to approaches known to the skilled person via a calibration procedure, whereby the detector output is determined for a range of distances, and used to configure a look-up table or other reference (e.g. one or more functions mapping detector output(s) to distance) which can be used by a controller 260 to determine a distance between a surface portion and a reference position comprised in an illumination source based on detector output for optical signals received from the surface portion. Typically such a calibration procedure is carried out in manufacture. In other embodiments, a detector 250 and illumination source are comprised in an interferometer apparatus, wherein an output of the detector 250 indicative of at least one characteristic of optical signals received from each portion of the surface of a sample 210 or sample holder assembly 21 comprises a measure of a phase shift between the received optical signals and a reference signal (e.g. via an interference pattern created by constructive and destructive interference between a reference optical signal generated by the illumination source, having travelled a fixed path-length within the interferometer apparatus, and an optical signal received along optical signal reception axis 251 from a surface portion of a sample of sample holder assembly 21, comprising reflected / scattered optical signals generated by an incident optical signal directed from the illumination source towards the surface portion along an optical signal transmission axis 251). The interferometer apparatus may comprise, for example, a diffraction grating interferometer, a vertical scanning or coherence probe interferometer, or a scatter-plate interferometer, known to the skilled person, utilising a white light source to provide optical signals which may be directed at first and second surface portions of the sample or sample holder assembly, where the optical signals are directed at the sample 210 or sample holder assembly 21 along optical signal transmission axis 251. An interferometer apparatus may alternatively comprise a Michelson-type (e.g. beam-splitting) interferometer using white light or laser light to provide optical signals which may be directed at first and second surface portions of the sample 210 or sample holder assembly 21, along an optical signal transmission axis 251. Where the detector 250 is comprised in an interferometer apparatus, the distance between a surface portion from which optical signals are received and a reference position associated with the interferometer apparatus (e.g. in fixed spatial relation to the detector 250 and / or illumination source) may be determined using a calibration procedure known to the skilled person and similar to that described in relation to embodiments where the detector is comprised in a confocal chromatic measuring device. Reference is made to an optical signal transmission axis and an optical signal receiving axis, and whilst these may typically comprise the same axis, with a beam splitter being used in an apparatus comprising the illumination source and the detector 250, in some embodiments, a light/illumination source may be angled relative to a detector, such that the axes along which optical signals are transmitted towards the sample, and received from the sample, may be angled to one another, whilst intersecting the sample surface at the same point / region. Typically in such instances, the light / illumination source and the detector 250 are mounted to separately controllable manipulators, allowing the relative positions and / or orientations of the light / illumination source and the detector 250 to be varied under control of the controller 260 such that optical signals transmitted at the surface of the sample 210 or sample holder assembly 21 are received at the detector 250 down the optical signal receiving axis (e.g. by varying the distance of the detector 250 to the surface of the sample 210 or sample holder assembly 21, and adjusting the angle so that the optical signal receiving axis falls within the acceptance angle range of the detector 250). Furthermore, though for the purposes of explanation, axes of optical signal transmission / reception are described, it will be appreciated that optical signals may be transmitted towards and received from the sample 210 or sample holder assembly 21 surface over beam paths which are not constrained to the axes, as a consequence of the divergence properties of optical signals and / or focusing of the signals. However, typically the optical signals are transmitted / received along an orientation parallel to said axes (e.g. in the case of a laser illumination source), and / or comprise one or more focal points distributed along said axes (e.g. in the case of embodiments using a confocal chromatic measuring device).

In some embodiments, the detector 250 is fixed such that the optical signal transmission / receiving axis 251 along which optical signals are substantially transmitted and / or received is oriented parallel to the sample advance axis, x. However, typically, the detector 250 assembly will be provided with a manipulator apparatus 270 for positioning of the detector assembly according to a plurality of translational and / or rotational degrees of freedom, and which may be configured in the same or a similar manner to that described for sample holder manipulator 220 and blade assembly manipulator 240 of a cutting apparatus. In embodiments herein, and particularly in embodiments used for characterising the orientation of sample and / or sample holder and / or blade surfaces, the translational positioning accuracy of the detector manipulator apparatus 270 will typically be less than 10 microns. The manipulator 270, where provided, is connected to the controller 260, such that the controller 260 provides control signals to the manipulator 270 to orient the illumination source and detector 250 towards target surface portions of the sample 210 and / or sample holder assembly 21, to allow optical signals to be collected from different target surface portions (e.g. first and second surface portions as described herein), and optionally to allow optical signals generated by an illumination source associated with detector 250 to be directed at the target surface portions. It will be appreciated that whilst an illumination source, where provided, may generally direct optical signals along substantially the same axis 251 used to receive optical signals at the detector, these axes may be different, with the manipulator 270 (or separate manipulators) being configured to orient the illumination source and detector 250 to cause the optical signal transmission axis and optical signal reception axis to coincide at the same target surface portion of the sample holder assembly 21 or sample 210. It will further be appreciated that in some embodiments, an illumination source is not provided in the apparatus 23, and ambient illumination provided by one or more sources not comprised in the apparatus is used to provide illumination of the sample or sample holder assembly, generating reflected and / or scattered optical signals which can be received by the detector 250.

The controller 260 of the apparatus 23 is configured to receive output of the detector 250 associated with reception of optical signals at the detector, and to determine first information indicative of a position of a first surface portion of a sample or a sample holder assembly, wherein the first information is determined based on a first output of the detector 250 associated with optical signals received from the first surface portion of the sample or sample holder assembly, and to determine second information indicative of a position of a second surface portion of a sample or a sample holder assembly, wherein the second information is determined based on a first output of the detector 250 associated with optical signals received from the second surface portion of the sample or sample holder assembly. The controller may comprise a controller of a cutting apparatus such as a microtome or ultramicrotome, as described in association with Figure 1, or may be a standalone controller configured generally as described in the same manner as the cutting apparatus controller. The output of the detector 250 to the controller 260 may comprise raw information comprising one or more wavelength spectra, or information representing phase shift and / or interference patterns between a reference optical signal and a received optical signal, or may comprise data processed by a separate controller comprised in the detector 250 apparatus, such as data directly indicative of an absolute or relative distance between a reference position associated with the detector 250 and / or an illumination source (e.g. output based on calibration data as described above), depending on the mechanism of operation of the detector 250. Where the controller 260 receives raw output from the detector 250 which is not directly indicative of a position of the first or second surface portion, the controller 260 may be configured to determine first and second information indicative of positions of, respectively, the first and second surface portions of the sample or sample holder assembly with respect to a sample-advance axis, x, oriented substantially perpendicular to a cutting plane, y, along which the blade assembly of the microtome apparatus is configured to move relative to the sample in order to obtain one or more slices from the surface of the sample, by using predefined calibration data as described above, which relate specific characteristics of output of the detector 250 to a distance between the detector and / or illumination source and the surface portion from which optical signals are received. In other embodiments, where a controller / processor (not shown) integrated in the detector 250 apparatus analyses the raw detector output and determines this positional information from the output based on calibration data, the determining of first and second information by the controller 260 may comprise receiving this positional information from the detector 250 apparatus. The latter may be the case when the detector 250 is comprised in an off-the-shelf interferometer or confocal chromatic measuring device known to the skilled person, such as, for example, a Keyence (TM) CL-3000 confocal displacement sensor.

Thus the controller 260 is configured to determine first information prior to the obtaining of one or more slices from the outer surface of a sample 210 using a blade assembly associated with the cutting apparatus (e.g. ultramicrotome) 2. This first information may be expressed, for example, as an absolute or relative position of a first surface portion of a sample 210 or sample holder assembly 21, relative to a reference point associated with the detector 250, with respect to the sample advance axis x. Typically the axis 251 along which optical signals are directed at the sample 210 or sample holder assembly 21 by an illumination source, and / or along which optical signals are received from the sample 210 or sample holder assembly 21 is oriented parallel to the axis of sample advance, x, such that changes in distance between surface portions of a sample 210 or sample holder assembly 21 and the detector 250 position with respect to the optical signal receiving axis 251 are directly indicative of changes in distance with respect to the sample advance axis, x. However, it will be appreciated that where the optical signal receiving axis 251 is oriented non-parallel to the sample advance axis, x, changes in distance / position with respect to the optical signal axis 251 may be converted to changes in distance / position with respect to the sample advance axis, x, via trigonometric methods known to the skilled person, taking into account the angle between the optical signal receiving axis 251 and the sample advance axis x.

Thus, in embodiments of the present disclosure, prior to obtaining of a slice n, and optionally a plurality of further slices n+1 to m, from the surface of a sample 210, the controller 260 determines first information indicative of the position of a first surface portion of the sample 210 or sample holder assembly 21, with respect to sample advance axis x, expressed for example as a scalar distance from the detector 250 assembly to the first surface portion (e.g. a one-dimensional coordinate system), or using at least a three-dimensional coordinate system to describe a position of a point comprised on the first surface portion in three-dimensional space, relative to suitable reference axes, for example, a plurality of axes parallel to the translational axes of one or more of the manipulators of the cutting apparatus. The obtaining of slice n, and optionally further slices n+1 to m, may follow obtaining of a previous slice n-1 to produce an exposed working surface from which a subsequent slice n is to be obtained, and such that slice n will incorporate some or all of the working surface exposed by the obtaining of slice n-1. Following the obtaining of slice n, and optionally of further slices n+1 to m (where m>n), by advancing the sample 210 and / or blade 230 along sample advance axis x and moving the blade 230 and! or sample holder 201 along cutting axis y, a number of times corresponding to the number of slices obtained, the controller 260 determines second information indicative of the position of a second surface portion of the sample 210 or sample holder assembly following obtaining of the slice(s), with respect to sample advance axis, x, expressed for example as a scalar distance from the detector 250 assembly, or using a coordinate system to describe a position of a point comprised on the second surface portion in three-dimensional space. The approach for determining the second information may be the same as that used to determine the first information, in terms of operation of the detector 250 and controller 260.

The controller 260 is configured to determine the thickness of at least one slice obtained by the blade assembly from the sample 210, by determining a difference in the positions of the first and second surface portions of the sample or sample holder indicated by the first and second information. Where a sample advance distance corresponding to a single slice was input between the receiving at the detector 250 of optical signals associated with determining of the first information, and receiving of optical signals associated with determining of the second information, the determination of slice thickness may be based on determining the difference with respect to the sample advance axis x of the positions of respective first and second surface portions of the sample 210 or sample holder assembly. Where a plurality of slices (e.g. h slices) are obtained from the surface of the sample 210 between the receiving at the detector 250 of optical signals associated with determining of the first information, and receiving of optical signals associated with determining of the second information, the thickness of each of the plurality of slices may be estimated by dividing the difference in the positions of the first and second surface portions of the sample or sample holder with respect to the sample advance axis, x, by the number of slices, h, in the plurality of slices obtained from the surface of the sample. The difference in positions of the first and second surface positions may be determined, for example, by determining the absolute value of the difference in scalar values representing respective distances with respect to sample advance axis x between the first and second surface portions and a reference position associated with the detector 250 assembly, or by calculating the magnitude of a vector linking points in three-dimensional space representing the positions of the first and second surface portions.

It will be appreciated that in different embodiments of an apparatus 23 according to the first aspect of the disclosure, the optical signals received by the detector 250 for determination of the first and second information by the controller 260 may be received from first and second surface regions associated with either of the sample 210 and the sample holder assembly 21 (e.g. a regions of a surface of the sample holder 201 and / or sample holder manipulator 220 oriented relatively perpendicular to the sample advance axis).

In the embodiment shown in Figure 2, the detector 250 is configured to receive optical signals from a first surface portion comprising a region of an outer surface 211 of a sample to be removed from the sample by the obtaining of the one or more slices using the blade assembly of the cutting apparatus 2 (e.g. microtome / ultramicrotome). The outer surface is typically referred to as a working surface 211, and as described herein may have been exposed by the obtaining of previous slices by the cutting apparatus, or prepared by machining or other preparation steps using a different apparatus. Thus in this embodiment, the manipulator 270 is controlled by controller 260 to orient the optical signal axis 251 towards the working surface, preferably in a direction parallel to the sample advance axis x, such that the optical signal axis 251 intersects with the working surface at a target first surface portion. Optical signals are received by the detector 250 from the first surface portion, and output provided to the controller 260 to enable the determination of the first information by the controller 260. Similarly, the optical signal axis 251 may be oriented towards the working surface, preferably parallel to the sample advance axis, x, such that the optical signal axis 251 intersects with the working surface at a target second surface portion. Optical signals are received by the detector 250 from the second surface portion, and output is provided to the controller 260 to enable the determination of the second information by the controller 260. The first and second surface portions may comprise the same portion of the working surface, or different portions of the working surface, and / or portions respectively of a first working surface prior to the obtaining of one or more slices from the sample, and a second working surface exposed by the obtaining of the one or more slices from the sample, as described further herein.

Figure 3 will be recognised from Figure 2, and shows an embodiment in which the detector 250 is configured to provide output to the controller 260 used to determine the first and second information, based on receiving optical signals from first and second surface portions comprising one or more regions of an outer surface of a sample holder assembly 21, where the sample holder assembly will be understood to comprise any element of the cutting apparatus which is in fixed spatial relationship to the sample 210 with respect to at least the sample advance axis, x. VVhat may be considered significant is that one or more regions of an outer surface of a sample holder assembly 21 comprised in the first and second surface portions remain at a fixed offset distance to the back face of the sample 210 with respect to the sample advance axis, x, as the sample holder translates along the sample advance axis. In the embodiment shown schematically in Figure 3, the first and second surface portions from which optical signals are received by the detector 250 are comprised in respective first and second surface regions of a reference object 280 mounted to the sample holder 201 of the cutting apparatus. As in the embodiment of Figure 2, the detector 250 will typically be provided with a manipulator 270 to allow adjustment of the orientation of an optical signal axis 251 along which optical signals are received by the detector 250 (and illumination signals may optionally also be transmitted), in order to receive optical signals from different target surface regions of the sample holder assembly, and the detector will typically be oriented such that the optical signal axis 251 is parallel to the sample advance axis x. In some embodiments, and typically when the optical signal axis 251 is parallel to the sample advance axis, x, and the first and second surface regions of the reference object 280 (or surface portion of the sample holder where a reference object is not used) are aligned parallel to a plane normal to the sample advance axis, the first and second surface regions, comprising the first and second surface portions, will be coincident, since translation of the sample holder assembly along the sample advance axis, x, will not alter the intersection of the optical signal axis 251 with the reference object / sample holder assembly surface.

Where a reference object 280 is used to provide the first and second surface regions / portions, this may comprise a reflective element, such as a mirror. The mirror may, for example, comprise a retroreflector configured to reflect incident optical signals from an illumination source (e.g. along optical signal axis 251, or a parallel incident optical signal axis not shown) back towards detector 250 along optical signal axis 251. In some embodiments the first and second information respectively are determined based on first and second outputs of the detector 250 respectively associated with receiving optical signals which have travelled a fixed distance from the respective surface portion to the detector 250. However, in other embodiments, the first and second information respectively are determined based on first and second outputs of the detector 250, respectively associated with optical signals received from a plurality of portions of the sample holder assembly, where the spatial separation of the portions and the detector 250 varies across the plurality of portions, but whilst the position of the sample 210 relative to the sample advance axis x remains constant. Thus, in these embodiments, the first information indicative of a position of a first surface portion of the reference object 280 is determined by receiving, at the controller 260, a first output of the detector associated with a plurality of optical signals received from a respective plurality of portions distributed across the first surface region of the reference object, determining information indicative of the respective position of each of the plurality of portions based on the first output, and estimating the position of the first portion of the reference object based on the positions of the plurality of portions of the first region.

Thus, in embodiments of the present disclosure, the first and second surface regions of the reference object 280 are co-planar (e.g. comprising the same surface region of a planar mirror element such as a reference object 280), and are mounted non-parallel to a cutting axis y along which the sample holder 201 and / or blade 230 are translated to obtain slices from sample 210. Thus with respect to the arrangement shown schematically in Figure 3, the surface of the reference object 280 oriented towards the detector 250 may be oriented at a non-zero angle to the cutting axis y, this angle being set to be less than or equal to half of the acceptance angle of the detector 250, such that where optical signals are transmitted at the reference object 280 along an axis parallel to the cutting axis y, as shown in Figure 3, the angle of the optical signal receiving axis does not exceed the acceptance angle range of the detector 250, assuming the detector 250 is oriented such that an axis parallel to the cutting axis y bisects the acceptance angle range. The detector position along the direction of cutting axis y may be set by the manipulator 270 to a position such that the detector 250 intercepts the optical signal receiving axis. In embodiments where slices are obtained from the sample 210 by translating the sample holder assembly along cutting axis, y, towards the blade assembly 22, the detector 250 may be kept in a fixed position relative to the blade, with optical signals being received along optical signal axis 251 from different portions of the surface of the reference object 280 as it passed across optical signal axis 251 along cutting axis y.

Alternatively, the detector 250 may be translated parallel to cutting axis, y, to receive the plurality of optical signals from the respective plurality of portions distributed across the first surface region of the reference object, with the sample holder assembly 21 (and thus reference object 280) remaining stationary, as in cases where slices are obtained from the sample 210 by translating the blade 230 along cutting axis y relative to a fixed sample position. In other embodiments, the first surface region of the reference object may be oriented parallel to the cutting axis, y, and the plurality of optical signals may be received from the respective plurality of portions distributed across the first and second surface regions of the reference object by rotating the detector 250 to cause the optical signal axis 251 to sequentially intersect each of the plurality of portions, with varying detector to reference object distances along the optical signal axis 251. What may be considered significant in each of these examples is that the distance between each of the plurality of portions and the detector 250 is different, such that the first information determined by the controller 260 on the basis of output of the detector 250 takes into account information about a plurality of different degrees of spatial separation between the detector 250 and the surface of the sample holder assembly / reference object 280.

In a similar manner, the second information indicative of a position of a second surface portion of the sample holder assembly 21 (e.g. reference object 280) may be determined by receiving, at the controller 260, a second output of the detector 250 associated with a plurality of optical signals received from a respective plurality of portions distributed across the second surface region of the reference object, determining information indicative of the respective position of each of the plurality of portions based on the second output, and estimating the position of the second portion of the reference object based on the positions of the plurality of portions of the second region. This can be achieved as described for the first information, with the difference that the second output from the detector 250 used as the basis for determining the second information is associated with optical signals received by the detector 250 after the sample holder 201 has been translated along the sample advance axis x following the receiving by the detector 250 of optical signals associated with the provision to the controller 260 of first output used as the basis for determining the first information. Typically, the first and second portions / regions will be coincident.

In embodiments where the first and second outputs of the detector 250, used as a basis to determine first and second information by the controller 260, respectively comprise information about the positions of a plurality of portions of a first surface region of reference object 280, and a plurality of portions of a second surface region of reference object 280, wherein the positions vary across each plurality of portions, the position of each of the first surface portion and second surface portion is determined by interpolation (e.g. linear regression, or another fitting scheme known to the skilled person) among respectively, the positions of the plurality of portions distributed across the first surface region, and the positions of the plurality of portions distributed across the second surface region. The first and second information can be used to determine the thickness of at least one of one or more slices obtained from a sample using a blade assembly of a cutting apparatus (e.g. microtome or ultramicrotome), according to approaches set out further herein.

There have been described approaches in which the thickness of one or more slices obtained from a sample is determined based on first and second information, indicative respectively of positions of first and second surface portions of a sample (e.g. one or more working surfaces of a sample 210) or sample holder assembly 21 (e.g. outer surface of a sample holder 201, sample holder manipulator 220, or reference object 280 comprised in or affixed to the sample holder 201 or sample holder manipulator 220). The determination of the thickness of the one or more slices based on the first and second information is typically based on determining a difference in the indicated positions of the first and second surface portions with respect to the sample advance axis x and dividing this by the corresponding number of slices obtained from the sample, as described further herein.

The indicated positions of the first and second portions may be different for different reasons, depending on the manner in which the cutting apparatus (e.g. microtome or ultramicrotome) is configured to obtain slices from the sample, as described below. In a first configuration of the cutting apparatus, the slices are obtained by advancing the sample holder 201 along sample advance axis x relative to a blade! knife 230 position which is fixed with respect to the sample advance axis x. Slices are obtained by advancing the sample holder 201 along axis x to overlap the working surface of the sample 210 with the blade cutting edge position in the x axis, then passing the sample holder 201 and! or blade 230 along cutting axis y (perpendicular to sample advance axis x) to obtain a slice. In these embodiments, the detector 250 (and illumination source where used) will typically be fixed in position relative to the blade assembly with respect to the sample advance axis x. This may be via mounting of the detector 250 on the blade assembly 22, or mounting the detector separately to the blade assembly, but maintaining constant x axis separation between the blade assembly and the detector 250.

In embodiments according to Figure 2, where the first and second surface portions from which optical signals are received are defined on the sample 210 (e.g. on the working surface 211), the following approach may be used for slice thickness determination. In a first step, the manipulator 220 advances the sample holder 201 towards the blade assembly along axis x to a slicing / cutting position. In a second step, the detector 250 receives optical signals from a first surface portion of the sample 210, and transmits output to the controller 260 used to determine the first information. In a third step, a slice is obtained by translation of sample holder 201 and / or blade 230 along cutting axis y In a fourth step, the detector 250 receives optical signals from a second surface portion of the sample 210, comprising a portion of a freshly exposed working surface 211, and transmits an output to the controller 260 used to determine the second information. It will be appreciated subsequent to the second step, a plurality of sample advances may be effected, and a plurality of slices obtained, prior to the fourth step, such that the first and second surface portions of the sample represent working surfaces prior to and subsequent to a plurality of slices being removed from the sample. Embodiments according to Figure 2 may be considered advantageous in that the thickness of material removed from the sample 210 (e.g. the slice thickness) is directly measured, instead of being inferred by assuming the sample advance of the sample 210 between slices corresponds to the thickness of material removed. This may be advantageous when, for example, thermal expansion effects or deflection of the blade assembly during slicing may cause the actual thickness of material removed from the sample 210 to differ from the sample advance distance between subsequent slices.

In embodiments according to Figure 3, where the first and second surface portions from which optical signals are received comprise portions of the sample holder assembly 21, the following approach may be used for slice thickness determination. In a first step, an initial slice is obtained from the sample 210, such that following slicing, the cutting edge of the blade assembly can be considered to be coincident with the position of the working surface of the sample with respect to the sample advance axis x. In a second step, the detector 250 receives optical signals from a first surface portion of the sample holder assembly 21, and transmits an output to the controller 260 which is used to determine the first information. In a third step, the sample holder 201 is translated towards the blade assembly 22 along the sample advance axis x by an amount corresponding to a target slice thickness. In a fourth step, a slice is obtained by translation of sample holder 201 and / or blade 230 along cutting axis y. In a fifth step, the detector 250 receives optical signals from a second surface portion of the sample holder assembly, and transmits output to the controller 260 which is used to determine the second information. It will be appreciated subsequent to the second step, a plurality of sample advances may be effected, and a plurality of slices obtained, prior to the fifth step, such that the positions of the first and second surface portions of the sample holder assembly represent positions before and after a plurality of slices are obtained from the sample. It will further be appreciated that the fifth step may be carried out prior to the fourth step (e.g. following advance of the sample, but prior to obtaining of the slice). In embodiments according to Figure 3, where the sample holder moves relative to a fixed detector position, the advance of the sample holder, as determined based on the difference between positions of first and second surface portions indicated by first and second information, is assumed to equate to the thickness of one or more slices obtained from the sample between the determining of the first and second surface portion positions.

In a second configuration of the cutting apparatus 2, one or more slices are obtained from sample 210 by advancing the blade 230 along the sample advance axis x relative to a sample holder 201 position which remains fixed with respect to sample advance axis x. In a first subset of such embodiments, the detector 250 (and illumination source where used) may be fixed in position relative to the blade 230 with respect to the sample advance axis x, for example by mounting of the detector 250 with the blade 230 on the blade assembly manipulator 240. In these scenarios, the procedures described above in relation to a fixed blade assembly and an advancing sample holder assembly may be used, where advance of the blade assembly along the sample advance axis x is substituted for advance of the sample holder assembly along the sample advance axis x.

In other embodiments, such as when it is impractical to mount the detector 250 to a moving blade assembly, the detector 250 (and illumination source where included) may be separately mounted in fixed spatial relationship to the sample holder assembly, in at least the sample advance axis x, such that the blade 230 moves relative to the detector position as it translates towards the sample 210. In this configuration, the embodiments described herein wherein optical signals are received from surface portions of the sample holder assembly (such as described in association with Figure 3) are not usable, as the sample holder assembly to detector distance will not vary as slices are obtained from the sample by advancing the blade towards the sample. Accordingly, where the detector and sample holder are maintained at a fixed distance during slicing, a procedure must be used in which the optical signals are received by the detector 250 from the working surface of the sample.

Thus, with reference to the configuration shown in Figure 2, In a first step, the blade assembly manipulator 240 advances the blade 230 towards the sample holder 201 along sample advance axis x to a slicing position. In a second step, the detector 250 receives optical signals from a first surface portion of the sample 210 (e.g. working surface 211), and transmits output to the controller 260 which is used to determine the first information. In a third step, a slice is obtained by translation of sample holder and! or blade assembly along the cutting axis y. In a fourth step, the detector 250 receives optical signals from a second surface portion of the sample 210, comprising a portion of a freshly exposed working surface 211, and transmits output to the controller 260 which is used to determine the second information. It will be appreciated subsequent to the second step, a plurality of blade advances may be effected, and a plurality of slices obtained, prior to the fourth step, such that the first and second surface portions of the sample represent working surface positions prior to and subsequent to a plurality of slices being removed from the sample. It will further be appreciated that the second step may be effected prior to the first step.

In embodiments of the present disclosure where the controller is configured to determine the first and second information based on output of the detector associated with optical signals received from first and second surface portions of a sample holder assembly (for example, where the first and second surface portions are comprised in respective first and second surface regions of a reference object mounted to a sample or sample holder assembly), the first and second surface portions of the sample holder assembly may comprise first and second surface portions of a reference object which comprises a plurality of reference marks with a predefined spatial separation. Such a reference object is typically mounted to a sample holder assembly of a cutting apparatus such that the positions of the first and second surface portions of the reference object move relative to the location of the detector 250 as the sample holder 201 is actuated along the sample advance axis x.

Figure 4 will be recognised from Figures 2 and 3, and schematically shows an embodiment wherein a reference object 380 comprising a plurality of reference marks with a predefined spatial separation is mounted to a portion of sample holder manipulator 320 which moves along the sample advance axis x with a fixed spatial separation relative to the sample 310 (e.g. mounted on a portion of a linear actuator oriented in the x axis direction, to which the sample holder 301 is fixed, or directly mounted to the sample 310 or sample holder 301). The detector 350 is positioned such that the reference object 380 moves relative to the detector 350 as the sample holder 301 is actuated along the sample advance axis x. The detector 350 comprises an optical signal receiving axis 351, along which optical signals are received, and is oriented such that the optical signal receiving axis 351 intersects the reference object 380. The reference marks of the reference object 380 are distributed along the x axis, such that respective reference marks pass across the optical signal receiving axis 351 as the sample holder 301 is actuated along the sample advance axis x. The detector 350 is comprised in an optical encoder read-head, known to the skilled person, optionally comprising an illumination source to illuminate the reference object 380 via transmission of optical signals along the axis 351. The output of the detector 350 to the controller 360 may comprise information related to the location of at least one reference mark defined on the reference object 380, which is within the field of view of the detector 350 at the point in time at which optical signals are received from the reference object 380 by the detector 350.

The thickness of at least one of one or more slices obtained from a sample 310 using a blade assembly of a cutting apparatus, using a slice thickness determining apparatus 33 comprising detector 350 and controller 360 as shown schematically in Figure 4, can be determined according to the following procedure. In a first step, an initial slice is obtained, according to approaches set out further herein, such that a cutting edge of the blade 330 of the blade assembly 32 can be considered to be coincident with the position of the working surface 311 of the sample 310 with respect to the sample advance axis x. In a second step, the detector 350 receives optical signals from a first surface portion of the reference object, comprising at least one reference mark, and transmits output to the controller 360 used to determine the first information. Based on the output of the detector 350, the controller 360 establishes an indication of a position of the first surface portion of the reference object 380 with respect to the sample advance axis x. In a third step, the sample holder 301 is translated towards the blade assembly 32 along the sample advance axis x by a displacement corresponding to a target slice thickness. In a fourth step, a slice is obtained by translation of sample holder 301 and / or blade 330 along cutting axis y. In a fifth step, the detector 350 receives optical signals from a second surface portion of the reference object 380, comprising at least one reference mark, and transmits output to the controller 360 which is used to determine the second information. Based on the output of the detector 350, the controller 360 establishes an indication of a position of the second surface portion of the reference object 380 with respect to the sample advance axis x. Typically, the position of the first surface portion corresponds to an offset along the x axis of a first reference mark relative to the position of the optical signal receiving axis 351, and the position of the second surface portion corresponds to an offset along the x axis of a second reference mark relative to the position of the optical signal receiving axis 351. Because the reference marks are configured with a predefined spatial separation along the x axis, known at the controller 360, the translated distance of the reference object 380 between the second step and the fifth step can be determined by the controller 360 using simple arithmetic functions. It will be appreciated subsequent to the second step, a plurality of sample advances may be effected, and a plurality of slices obtained, prior to the fifth step, such that the positions of the first and second surface portions of the reference object 380 represent positions before and after a plurality of slices are obtained from the sample. It will further be appreciated that the fifth step may be carried out prior to the fourth step (e.g. following advance of the sample, but prior to obtaining of the slice). The sample advance of the sample holder assembly along sample advance axis x, as determined based on the difference between positions of first and second surface portions indicated by the first and second information, is assumed to equate to the thickness of one or more slices obtained from the sample between the determining of the first and second surface portion positions.

In the foregoing, it will be appreciated that reference objects 280 / 380 may be provided as separate elements to a sample holder assembly 21 / 31, being mounted to the sample holder assembly using suitable adhesive or mechanical fixing approaches known to the skilled person. However, in other embodiments, a reference object such as a reflective reference object (e.g. a mirror) or a reference object comprising a plurality of reference marks with predefined spatial separation, may be integrated into the sample holder assembly by, for example, polishing a portion of the sample holder assembly to provide a reflective surface, or machining reference marks of predefined separation onto a portion of the sample holder assembly configured to translate with the sample down the sample advance axis x.

In the embodiments described above and illustrated in Figures 1 to 4, the detector is positioned to receive optical signals scattered or reflected from a sample surface or surface of a sample holder or manipulator assembly. However, in embodiments where signals are received by the detector from a reference object, and the received signals are used to determine first and second positions of respective first and second portions of a reference object prior to and subsequent to obtaining one or more slices from a sample, it will be appreciated this may equally be achieved if the locations of detector and reference object are in effect switched, by mounting the detector to the sample holder or sample holder manipulator, in fixed spatial relationship to the sample with respect to the sample advance axis x, and mounting the reference object the opposite side of the sample to the sample holder, at a position separated from the detector along the sample advance axis x. Thus an apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, may comprise a detector mounted to a portion of a sample holder assembly which is in fixed spatial relationship to a sample with respect to at least a sample advance axis, the detector being configured to receive optical signals reflected and / or scattered from at least first and second portions of the surface of a fixed reference object, and provide an output indicative of at least one characteristic of the received optical signals; and a controller configured to: determine first information indicative of a position of a first surface portion of the reference object prior to the obtaining of one or more slices from the outer surface of a sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the reference object; determine second information indicative of a position of a second surface portion of the reference object subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the reference object; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

According to a further aspect of the present disclosure, there is provided there is provided an apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, the apparatus comprising: a first detector configured to receive optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly and provide an output indicative of at least one characteristic of each of the received optical signals; and a controller, wherein the controller is configured to: determine first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, and determine an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.

Figure 5 will be recognised from Figures 2 and 3, and shows schematically an apparatus 43 for characterising an orientation of an external surface of a sample or sample holder associated with a cutting apparatus 4 (e.g. a microtome or ultramicrotome), the apparatus 43 comprising a detector 450, controller 460, and optional detector manipulator 470. The cutting apparatus elements can be considered to be the same as those described in relation to exemplary cutting apparatuses of the first aspect of the present disclosure, and comprise a sample 410 mounted to a sample holder assembly comprising a sample holder 401 and manipulator 420, and a blade assembly comprising a blade 430 and a blade manipulator 440. Except where explicitly set out, it can be assumed the functionality of elements of the cutting apparatus 4 operate as described in accordance with the prior disclosure (e.g. accompanying Figures 1 to 4).

The detector 450 comprises an optical proximity sensor, configured to receive optical signals from a plurality of surface portions of the sample 410 or sample holder assembly along an optical signal receiving axis 451, and may be configured in the same manner as the detector described in association with the apparatus of the first aspect of the present disclosure. Thus, the detector 450 may be comprised in a detector apparatus comprising a confocal chromatic measuring device, or interferometer apparatus comprising, for example, a diffraction grating interferometer, a vertical scanning or coherence probe interferometer, a scatter-plate interferometer, using a white light illumination source, or a Michelson-type (e.g. beam-splitting) interferometer using a white light or laser light illumination source. An illumination source is typically included, configured to direct optical signals at target surface portions of the sample 410 or sample holder assembly along optical signal receiving axis 451, such that scattered and / or reflected signals are received by the detector 450 along optical signal receiving axis 451.

The detector 450 is configured to receive, substantially along an optical signal receiving axis 451, optical signals reflected and / or scattered from each of a plurality of surface portions of the sample 410 or sample holder assembly 41, and provide an output to the controller 460 indicative of at least one characteristic of each of the received optical signals. In order to receive optical signals from a plurality of surface portions of the sample 410 or sample holder assembly, a manipulator 470 may be provided to which the detector 450 is mounted, the manipulator 470 being configured according to principles already described for a sample holder manipulator and blade assembly manipulator of the cutting apparatus, and optional detector manipulator comprised in embodiments of the slice thickness determination apparatus according to the first aspect of the present disclosure. Where provided, the manipulator 470 is configured to position / orient the detector 450 in different spatial orientations relative to the sample holder assembly 41, such that the optical signal receiving axis 451 can be directed to intersect the surface of the sample 410 or sample holder assembly 41 at each of a plurality of different target positions. In other embodiments, the detector may not be provided with a manipulator 470, but is fixed in position with the optical signal receiving axis 451 typically oriented parallel to the sample advance axis x, and the sample holder manipulator 420 being used to drive the sample 410 to different positions in a plane oriented normal to the sample advance axis x, in order to cause the fixed optical signal receiving axis 451 to intersect with each of a plurality of different target positions on the surface of the sample 41001 sample holder 401.

The controller 460 of the apparatus 43 may be configured in the broadly the same manner as described for the controller of the slice thickness determination apparatus of the first aspect of the disclosure. In particular, the controller 460 of the second aspect is configured to determine first information indicative of a respective position of each of the plurality of portions of the surface of a sample or sample holder assembly of a cutting apparatus, and determine an orientation of the surface of the sample or sample holder assembly based on the first information, wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller 460 on the basis of the output received from the first detector 450. Determination of the position of a given portion of the surface of the sample or sample holder assembly on the basis of output of the detector 450 associated with optical signals received from said portion may be carried out in the manner described in accordance with embodiments of the apparatus of the first aspect. For example, where the output of a detector 450 comprised in an interferometer apparatus or confocal chromatic measuring device is indicative of a separation distance along an optical signal receiving axis 451 between a reflecting and / or scattering surface portion of a sample 410 or sample holder assembly 41, and a reference position comprised in the interferometer apparatus or confocal chromatic measuring device, a position of the surface portion in three-dimensional space may be determined by the controller 460 on the basis of the separation distance, the position of the reference point in three-dimensional space (according to a suitable frame of reference), and the orientation of the detector 450 (and thus of the optical signal receiving axis 451). The latter two parameters may be inferred by receiving, at the controller 460, positioning information relating to drive inputs applied to the detector manipulator 470, and / or the use of optical encoders (not shown) associated with degrees of freedom of the detector manipulator 470 to provide the controller with feedback as to the position and orientation of the detector 450 in three-dimensional space.

Figure 6 will be recognised from Figure 5, and shows an enlarged view of the sample 410 mounted on sample holder 401 (sample holder manipulator 420 and blade assembly are not shown for simplicity). The working surface 411 of the sample 410 is shown, and in this example is oriented non-parallel to the cutting axis y, as may typically be the case when a sample is first mounted to a sample holder of a cutting apparatus. The controller 460 is configured to drive the detector manipulator 470 to raster the optical signal receiving axis 451 of the detector 450 across the working surface 411 of the sample 410, for example, by driving the detector 450 along a cutting axis y so that the detector may receive optical signals from a plurality of portions of the working surface 411 with y axis separation (represented by the x symbols of Figure 4). It will be appreciated that Figure 6 is a highly schematic two-dimensional representation, and in practice, the controller 460 is configured to position / orient the detector 450 to receive optical signals from surface portions of the working surface 411 of the sample 410 distributed in three-dimensional space. More particularly, the plurality of surface portions from which optical signals are received will typically comprise at least three surface portions, comprising three surface points distributed across the working surface 411 of the sample 410, wherein a third one of the points does not lie on a straight line passing through the first and second points. However, as set out further herein, any number of surface portions of the working surface 411 or sample holder 401 may be scanned by the detector 450 via the receiving of optical signals from said surface portions.

The position of each of a plurality of points / positions comprised respectively in a plurality of portions of a surface of the sample 410 or sample holder assembly may be determined by the controller 460 based on output of the detector 450 associated with receiving optical signals from the portions, in the same manner as described further herein for the determination of the positions of first and second surface portions of a sample or sample holder assembly in embodiments of an apparatus according to the first aspect of the disclosure. Thus, the position in three-dimensional space of each surface portion from which optical signals are received may be determined by the controller 460 based on the orientation of the detector 450 (and thus of the optical signal receiving axis 451), as inferred for example by control inputs used by the controller 460 to drive detector manipulator 470, and! or output from one or more encoders (not shown) associated with the different degrees of freedom of the manipulator 470, providing explicit feedback to the controller 460 about the position and / or orientation of the detector 450 in three-dimensional space. The distance from a surface portion of the sample 410 (e.g. of working surface 411) to the detector 450 along the optical signal receiving axis 451 may be inferred by the controller 460 on the basis of output of the detector 450 associated with receiving optical signals from the surface portion, based on interferometry or confocal chromatic measurement approaches described further herein in association with the first aspect of the present disclosure. On the basis of the information about the orientation of the detector 450 (and thus of the optical signal receiving axis 451) in three-dimensional space, and the distance from a reference point associated with the detector to a surface portion of the sample or sample holder along the optical signal receiving axis 451, the controller 460 is configured to determine coordinates of a point comprised in the surface portion (e.g. the centre of the surface portion and / or the intersection of the surface portion with the optical signal receiving axis 451).

The coordinates of a plurality of points comprised in a plurality of surface portions of the working surface 411 of a sample, or of the surface of a sample holder 401, may be determined by sequentially positioning the detector 450 in different orientations and / or translating the sample 410 into different positions in a plane perpendicular to the sample advance axis x, as optical signals are received by the detector 450. For example, the detector 450 and! or sample 410 may be translated! rotated such that the optical signal receiving axis 451, which will often also comprise an incident optical signal transmission axis for signals generated by an illumination source comprised in the apparatus 43, scans a plurality of lines, for example comprising a grid, across the working surface 411 of the sample 410, or dwells at each of a plurality of points distributed across the working surface 411, for optical signals to be received from a plurality of surface portions. In the schematic illustration of Figure 6, a series of points are shown coincident with the working surface 411 of the sample 410 (via cross symbols), whose positions have been determined by the controller 460 based on output received from the detector 450 associated with optical signals received from a portion of the working surface 411 coincident with each point, whilst the detector 450 is oriented such that the optical signal receiving axis 451 intersects the working surface 411 at each point.

In embodiments of an apparatus 43 according to the second aspect of the disclosure, the controller 460 is configured to estimate a spatial position and orientation of a surface region of the surface of a sample 410 and! or sample holder 401 based on the first information by fitting a surface profile through at least three positions (e.g. represented as three-dimensional points) comprised in the plurality of positions of the plurality of surface portions from which optical signals have been received by the detector 450 by, for example, fitting a plane or non-uniform (i.e. non-flat) topology through the plurality of positions / points, using geometric approaches known to the skilled person (for example, using fitting routines known in software such as Matlab (TM), and Origin (TM)). This information may be used by the controller 460 in a variety of ways. For example, where the plurality of portions whose positions are determined by the controller 460 comprise portions of a working surface 411 of a sample 410 exposed by the obtaining of one or more slices using a blade assembly of the cutting apparatus 4, the controller 460 may be configured to analyse the fitted surface to determine a measure of surface uniformity using approaches known to the skilled person for quantifying the deviation of points from a plane of best fit. According to some embodiments of an apparatus 43 according to the second aspect of the disclosure, the controller 460 is configured to analyse the fitted surface to determine a parameter representative of the degree of surface uniformity, and provide an indication to a user if the parameter is determined to be below a predefined threshold. The threshold may be based on calibration information provided by a user indicating a degree of deviation from a flat surface which is associated with sample slices of unusable quality. In some embodiments, the surface of the sample 410 comprises a region which is to be removed by the obtaining of the one or more slices using a blade assembly of the microtome apparatus, and an indication given to a user may be used to alert the user that, for example, a blade 430 of the blade assembly 42 of the cutting apparatus 4 may need checking to determine if the sub-standard working surface uniformity is the result of damage and / or bluntness of the blade. In some embodiments, the controller 460 is configured to characterise a first orientation of a surface of the sample 410 or sample holder 401 prior to obtaining one or more slices from the sample, and to characterise a second orientation of a surface of the sample 410 or sample holder 401 subsequent to obtaining the one or more slices from the sample, and to estimate a uniformity of at least one of the one or more slices based on comparing the first and second orientations. This comparison may be carried out, for example, by analysing the extent to which first and second planar surface profiles fitted to respectively to the two surfaces are parallel to one another, using geometric approaches known to the skilled person.

In embodiments of an apparatus according to the second aspect of the present disclosure, the fitting of a surface profile through positions of a plurality of portions of a surface of a sample 410 or sample holder 401 by the controller 460 may be used to provide control outputs to a manipulator 440 of a blade assembly 42 and / or a manipulator 420 of a sample holder assembly 41, as shown in Figure 5, to align the sample 410 and! or blade 430 for obtaining slices from the sample 410 using the blade 430. Thus the controller 460 may be configured to provide control outputs to a cutting apparatus 4 (e.g. a microtome or ultramicrotome), the control outputs indicating or being usable to determine target translations and / or rotations of a sample holder 401 and! or a blade 430 of the microtome to be used in obtaining slices from a sample 410 mounted on a sample holder 401 of the cutting apparatus 4 using a blade assembly 42. Suitable control outputs are determined based on the relative orientations of the surface of the sample or sample holder, determined from the fitted surface profile (e.g. a plane fitted to a plurality of points characterised across the working surface 411 of the sample) and information describing the orientation in three-dimensional space of a cutting edge of the blade 430 of the blade assembly 42. Typically, the control outputs indicate target translations and! or rotations of the sample holder 401 or blade 430 required to cause the cutting edge of the blade 430 to be aligned substantially parallel to an external surface (e.g. working surface 411) of the sample 410, and to cause the external surface of the sample 410 to be aligned substantially parallel to an axis of travel (e.g. cutting axis y) along which the sample holder 401 is configured to travel relative to the blade 430 to obtain one or more slices from the sample 410. Figure 7 will be recognised from Figure 6 and shows an example in which the sample holder 401 and sample 410 of Figure 6 have been reoriented following a control input to the sample holder assembly manipulator (not shown) by the controller 460, wherein the control inputs to the manipulator are selected to cause a plane fitted through a plurality of points characterised on the working surface 411 of the sample 410 to be brought into parallel alignment with a cutting axis y along which the sample holder 410 and / or blade 430 of the cutting apparatus are translated to obtain slices from the sample 410.

In some embodiments, the controller 460 may be further configured to determine a position and / or orientation of a cutting edge of a blade assembly of a cutting apparatus (e.g. microtome or ultramicrotome), providing additional information which can be used by the controller 460 in providing control inputs to manipulators of the cutting apparatus to cause a sample 410 to be aligned to a cutting edge of a blade 430 of the cutting apparatus. In some embodiments, the controller 460 is configured to determine the orientation of a cutting edge of the blade assembly based on calibration information input by a user, for example, based on characterisation of the blade 430 by user measurement. The orientation of the cutting edge may also be determined based on information provided to the controller 460 from one or more encoders or other positional sensors associated with the blade assembly manipulator 440. Thus, the controller 460 may be configured to determine the position and / or orientation of the cutting edge of a blade 430 of a blade assembly 42 of a cutting apparatus 4 (e.g. a microtome or ultramicrotome) based on output received by the controller 460 from one or more sensors, wherein the output is indicative of the orientation of the blade. The one or more sensors may comprise a second detector 450 configured to receive optical signals reflected and / or scattered from each of a plurality of portions on at least one surface of a blade assembly 42, and to provide an output indicative of at least one characteristic of each of the received signals to the controller 460, wherein the controller 460 is configured to determine second information indicative of a respective position of each of the plurality of portions of the surface of the blade assembly 42, and determine an orientation of a cutting edge of a blade 430 of the blade assembly 42 based on the second information, wherein the respective position of each of the plurality of portions of the surface of the blade assembly 42 is determined by the controller 460 on the basis of the output received from the second detector 450. Typically, the second detector will comprise a first detector as described for use in characterising the orientation of a surface of a sample 410 or sample holder assembly 41 as described above in accordance with the first aspect of the present disclosure.

Typically, the approach for using a second detector comprising an optical detector 450 to characterise an orientation of a cutting edge of a blade 430 may follow the same approaches described herein for characterising the orientation of a surface of a sample 410 or sample holder assembly 41. Thus said approaches may be used to determine the positions of one or more points on a surface of a blade assembly 42 (e.g. of a blade 430, blade holder, or blade assembly manipulator 440), such as points comprised in one or more reference portions or marks on the surface of the blade assembly 42. The controller 460 may be configured to determine the orientation of the cutting edge of the blade 430 based on a predefined spatial relationship between the positions of each of the plurality of portions of the at least one surface of the blade assembly and the position of the cutting edge of the blade 430, with this information provided, for example, via manual input by a user. The second detector 450 may be a separate detector to that used for determining the orientation of the sample 410 or sample holder assembly 41, or may be the same detector 450, with the controller 460 being configured to control manipulator 470 to orient the detector 450 towards the blade assembly 42 to receive optical signals from surface portions of the blade assembly 42.

In one embodiment, the second detector 450 is configured to receive optical signals reflected and / or scattered from each of a plurality of portions of a first surface region of the blade assembly 42 and a plurality of portions of a second surface region of the blade assembly 42, wherein the first and second surface regions of the blade assembly 42 converge at a cutting edge, wherein the second information is indicative of a respective position of each of the plurality of portions of the first and second surface regions of the blade assembly. Figure Swill be recognised from Figure 7, and shows the same apparatus 43 comprising detector 450, manipulator 470, and controller 460, in this case configured to receive optical signals from a plurality of portions of a first surface region 431 of a blade 430 of a cutting apparatus 4, and to receive optical signals from a plurality of portions of a second surface region 432 of the blade 430, where the first and second surface regions of the blade converge at a cutting edge (e.g. the first and second surface regions may be front and back faces of a generally wedge-shaped microtome or ultramicrotome blade). The controller 460 will typically be configured to provide control outputs to manipulator 470 to position the detector 450 (and thus optical signal receiving axis 451) to sequentially scan the first and second surface regions 431, 432, of the blade 430 in a similar manner as described in respect of scanning the sample working surface 411 and / or sample holder assembly 41 in other embodiments described herein. The controller 460 is configured to determine the orientation of the cutting edge of the blade assembly by determining a plurality of points in three-dimensional space, each point corresponding to the position of one of the plurality of portions of the first surface region 431 and second surface region 432 of the blade 430, fitting a first surface profile through a first subset of the plurality of points, the first subset of points being coincident with the first surface region 431 of the blade assembly, fitting a second surface profile through a second subset of the plurality of points, the second subset of points being coincident with the second surface region 432 of the blade assembly, and determining the orientation of the cutting edge of the blade by determining the intersection of the first and second surface profiles (e.g. of first and second planes).

The manipulator 470 may be configured to orient the detector 450 under control of the controller 460 to first scan the first surface region 431 from a first side of the blade 430, then orient the detector 450 to secondly scan the second surface region 432 of the blade 430 from a second side of the blade 430. Alternatively, separate dedicated detectors 450 positioned on different sides of the blade may be used to respectively scan each of the first and second surface regions. In other embodiments, a single detector 450 positioned on one side of the blade 430 may be used to characterise surface profiles of first / front surface region 431 and second / back surface region 432 of the blade 430, by scanning the second / back surface region 432 through the first! front surface region 431. In these embodiments, the blade 430 comprises an optically transparent material such as glass or diamond, and the detector 450 is configured to receive optical signals from a first surface region 431 comprising an external surface of the blade 430 (e.g. such that incident optical signals are scattered! reflected from the surface into air), and to receive optical signals from a second surface region 432 comprising an internal surface of the blade assembly (e.g. such that incident optical signals are scattered / reflected from the blade / air interface through the blade thickness via internal scattering! reflection), and wherein the detector 450 is configured to receive the optical signals reflected and / or scattered from the second surface region 432 of the blade following their transmission through at least a portion of the blade 430. Figure 8 shows schematically how detector 450 receives optical signals along an optical signal receiving axis 451 from a first point on the second surface region 432, and a first point on the first surface region 431. The path of optical signals from the second surface region 432 is shown schematically as a dotted line through the blade 430 (noting that in physical embodiments optical signals will be refracted as they cross the boundary from blade to air, and these effects are not shown in Figure 8 for simplicity of representation), and the path of optical signals from second and first surface regions is shown as a dot-dash line along axis 451. Typically an illumination source comprised in apparatus 43 will direct optical signals down the axis 451 at the first and second surface regions of the blade to induce the scattering / reflection of optical signals which are received by the detector. Where optical signals received at the detector comprise light reflected from two surfaces offset in the direction of the optical signal receiving axis, the detector 450 (for example a chromatic confocal sensor) and controller 460 are configured to determine the contributions of the received optical signals which respectively comprise light reflected / scattered from the nearer surface, and light reflected! scattered from the further surface, with a longer path length and greater optical losses for the light received from the further surface. When the detector output is processed by the controller 460, the optical signals received from the nearer and further surfaces (i.e. 431 and 432 respectively) will be represented by different peaks in the detected spectrum, allowing the contributions to the detected optical signals of reflection! scattering from nearer and further surfaces respectively to be separately analysed, and surface to detector distances separately determined. As the skilled person is aware, optical sensors comprising confocal chromatic measuring devices (e.g. such as a Keyence CL-3000 (TM) sensor) may be configured in manufacture to provide the functionality to measure distances of two offset surfaces of a transparent or semi-transparent object simultaneously in this manner.

Thus the controller 460 determines the positions (e.g. coordinates) of a plurality of points on each of first and second convergent surface regions of the blade 430, wherein the convergence of the surface regions corresponds to the cutting edge of the blade 430. Figure 9 schematically shows an abstract representation of a plurality of points corresponding to positions of respective ones of the plurality of portions of first and second surface regions of an exemplary blade 430 as shown schematically in Figure 8, wherein the points 570 are points distributed across the first surface region, and the points 550 are points distributed across the second surface region. It will be appreciated that Figure 9 is a highly schematic two-dimensional representation, and that in practice the points 550 and 570 will be distributed in three-dimensional space. The controller 460 is configured to fit a first surface profile (e.g. a plane) through the plurality of points 570 coincident with the first surface region of the blade assembly, and to fit a second surface profile (e.g. a plane) through the plurality of points 550 coincident with the second surface region of the blade assembly, and to determine the position and orientation of the line / axis defining the cutting edge of the blade 430 by determining the intersection of the first and second surface profiles. In the schematic two-dimensional representation of Figure 9, the intersection of a first profile fitted through points 570 and a second profile fitted through points 550 is given by a point 490. However it will be appreciated in a three-dimensional context, the intersection will comprise a line / axis, and the approach can be generalised to three-dimensions by the skilled person.

Where the apparatus 43 is configured to characterise the orientation / position of a cutting edge of the blade 430, this information may be used by controller 460 in conjunction with information about the orientation! position of the working surface 411 of the sample 410, as determined using approaches described herein, to compute control outputs to be provided to a sample holder manipulator 420 and / or blade assembly manipulator 440 of a cutting apparatus 4, the control outputs being suitable for aligning the cutting edge of the blade 430 to the working surface 411 of the sample 410 in order to obtain slices from the working surface of the sample using the cutting apparatus 4 (e.g. microtome or ultramicrotome). The control outputs provided by the controller 460 to the cutting apparatus 4 indicate or are usable to determine target translations and! or rotations of a sample holder 401 of the cutting apparatus 4 and! or a blade 430 to be used in obtaining slices from a sample 410 mounted on a sample holder 401 of the cutting apparatus using a blade assembly 42, and are determined by the controller 460 based on the relative orientations of the surface of the sample 410 (e.g. a working surface 411) or sample holder 401, and the cutting edge of the blade 430, as determined using approaches described herein. The required rotations and / or translations may be calculated by the controller 460 based on geometric approaches known to the skilled person. Typically, the control outputs computed by the controller 460 indicate target translations and / or rotations of the sample holder 401 and / or a blade 430 of the cutting apparatus required to cause the cutting edge of the blade 430 to be moved into alignment substantially parallel to the working surface 411 of the sample 410, and to cause the working surface 411 of the sample 410 to be moved into alignment substantially parallel to an axis of travel along which the sample holder is configured to travel relative to the blade to obtain slices from the sample (e.g. cutting axis y). By providing automated alignment of the blade 430 and sample 410, approaches such as those described in accordance with embodiments of apparatuses according to the second aspect of the present disclosure may provide for the obtaining of higher quality slices with a greater degree of control on uniformity of thickness and absolute thickness, and may further help prevent damage to the blade due to collisions between the blade and sample, and / or preventing the obtaining of slices of a thickness greater than the safe slice thickness limit of the blade.

Thus there have been described a first apparatus for slice thickness determination in the context of cutting apparatuses for the obtaining of slices from samples, and a second apparatus for determination of an orientation of a surface of a sample and / or sample holder assembly, and optionally of a cutting edge of a blade, and optionally for aligning of a cutting edge of a blade to a sample working surface, in the context of cutting apparatuses for the obtaining of slices from samples. As set out further herein, it will be appreciated that these first and second apparatuses may in some embodiments comprise the same apparatus, with the controller configured to control the detector (and optionally an illumination source and manipulator where included) to carry out functions described in association with both apparatuses. It will further be appreciated that in some embodiments, a first or second apparatus as described herein may be integrated into a cutting apparatus such as a microtome or ultramicrotome, such that the controller of the apparatus comprises a controller associated with the cutting apparatus. However, apparatuses according to first and second aspects of the present disclosure as described herein may be provided as one or more standalone units which may be fitted (e.g. retrofitted) to a cutting apparatus to provide the cutting apparatus with the functions described herein. In these latter embodiments, it will be appreciated the controller of the apparatus will be provided with a suitable data connection interface to enable transfer of information (such as positional information associated with control of cutting apparatus and / or detector manipulators, and control outputs indicating translational and / or rotational shifts to be applied to sample holder and / or blade assemblies of the cutting apparatus) to a controller of the cutting apparatus, according to a suitable wired or wireless data connection protocol known to the skilled person. Such connectivity may be implemented in accordance with standard data transfer protocols commonplace to networking in computing systems.

Though the first and second apparatuses have been predominantly described in the context of use with a microtome or ultramicrotome apparatus, it will be appreciated the principles described herein may be applied to other types of cutting apparatuses, such as industrial cutting apparatuses used in engineering contexts, for the determination of slice thicknesses and / or for the characterisation of orientation of a surface of a sample or sample holder.

Similarly, any general references to cutting apparatuses herein may be taken to optionally refer specifically to microtome or ultramicrotome apparatuses.

Thus there has been described a method of characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, as shown schematically in Figure 10, in which: In a first step, Si, there are received, at a detector, optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with a microtome apparatus; In a second step, S2, there is provided, from the detector to a controller, an output indicative of at least one characteristic of the received optical signals; In a third step, 33, there is determined, at the controller, first information indicative of a position of a first surface portion of the sample or sample holder assembly of the microtome apparatus prior to the obtaining of the one or more slices from the outer surface of the sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly; In a fourth step, S4, there is determined, at the controller, second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of the one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and In a fifth step, S5, there is determined, at the controller, a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.

Thus there has also been described a method of characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, as shown schematically in Figure 11, in which: In a first step, Ti, there are received, at a first detector, optical signals reflected and / or scattered from each of a plurality of surface portions of a sample or sample holder assembly of a microtome apparatus; In a second step, T2, there is provided, from the detector to a controller, an output indicative of at least one characteristic of each of the received optical signals; In a third step, T3, there is determined, at the controller, first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector; In a fourth step, T4, there is determined, at the controller, an orientation of the surface of the sample or sample holder assembly based on the first information.

The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and/or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc, other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

Claims

Claims 1. An apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, the apparatus comprising: a detector configured to receive optical signals reflected and/or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with the microtome apparatus, and provide an output indicative of at least one characteristic of the received optical signals; and a controller configured to: determine first information indicative of a position of a first surface portion of a sample or a sample holder assembly of the microtome apparatus prior to the obtaining of one or more slices from the outer surface of a sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determine second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.
2. The apparatus of claim 15 wherein the detector is configured to provide first and second outputs to the controller which are indicative of degrees of spatial separation between a reference position associated with the detector and, respectively, first and second surface portions of the sample or sample holder assembly.
3. The apparatus of any preceding claim, wherein the first and second information are indicative of positions of, respectively, the first and second surface portions of the sample or sample holder assembly with respect to an axis oriented substantially perpendicular to a cutting plane along which the blade assembly of the microtome apparatus is configured to move relative to the sample in order to obtain one or more slices from the surface of the sample.
4. The apparatus of any preceding claim, wherein the controller is configured to determine the thickness of the at least one slice by determining a difference in the positions of the first and second surface portions of the sample or sample holder indicated by the first and second information.
5. The apparatus of any preceding claim, wherein the second information is determined based on second output of the detector received subsequent to obtaining a plurality of slices from the surface of the sample, and the thickness of each of the plurality of slices is estimated by dividing the difference in the positions of the first and second surface portions of the sample or sample holder by the number of slices in the plurality of slices obtained from the surface of the sample.
6. The apparatus of any preceding claim, wherein the first surface portion comprises a region of an outer surface of a sample to be removed from the sample by the obtaining of the one or more slices using the blade assembly of the microtome apparatus.
7. The apparatus of any preceding claim, wherein the second surface portion comprises a region of an outer surface of the sample exposed by the obtaining of the one or more slices using the blade assembly of the microtome apparatus.
8. The apparatus of any of claims 1 to 5, wherein the first and second surface portions are comprised in respective first and second surface regions of a reference object mounted to a sample or sample holder assembly of the microtome apparatus.
9. The apparatus of claim 8, wherein the reference object comprises a plurality of reference marks with a predefined spatial separation.
10. The apparatus of any of claims 8 and 9, wherein the detector is comprised in an optical encoder read-head, and the reference object comprises an encoder strip configured to be read by the optical encoder read-head.
11. The apparatus of claim 8, wherein the first and second regions of the reference object comprise reflective regions configured to reflect incident optical signals towards the detector.
12. The apparatus of claim 9, wherein the first and second surface regions of the reference object are coincident.
13. The apparatus of any of claims 11 to 12, wherein the first and second surface regions are co-planar, and are mounted non-parallel to a first axis of travel of the sample holder assembly, wherein the first axis of travel comprises an axis along which the sample holder assembly is translated in use by the microtome apparatus, relative to a sample holder assembly, to obtain one or more slices from the sample.
14. The apparatus of claim 13, wherein: the first information indicative of a position of a first surface portion of the reference object is determined by receiving, at the controller, a first output of the detector associated with a plurality of optical signals received from a respective plurality of portions distributed across the first surface region of the reference object, determining information indicative of the respective position of each of the plurality of portions based on the first output, and estimating the position of the first portion of the reference object based on the positions of the plurality of portions of the first region; and the second information indicative of a position of a second surface portion of the reference object is determined by receiving, at the controller, a second output of the detector associated with a plurality of optical signals received from a respective plurality of portions distributed across the second surface region of the reference object, determining information indicative of the respective position of each of the plurality of portions based on the second output, and estimating the position of the second portion of the reference object based on the positions of the plurality of portions of the second region.
15. The apparatus of claim 14, wherein the position of the first surface portion is estimated by interpolation among the positions of the plurality of portions of the first surface region, and the position of the second surface portion is estimated by interpolation among the positions of the plurality of portions of the second surface region
16. The apparatus of any preceding claim, further comprising an illumination source configured to direct optical signals at surface portions of the sample or sample holder assembly.
17. The apparatus of claim 18, wherein the detector and illumination source are comprised in an interferometer apparatus, wherein the output of the detector indicative of at least one characteristic of optical signals received from each portion of the surface of a sample or sample holder comprises a measure of a phase shift between the received optical signals and a reference signal.
18. The apparatus of any of claims 16 to 17, wherein the illumination source comprises a white light source.
19. The apparatus of any of claims 16 to 17, wherein the illumination source comprises a laser.
20. The apparatus of any preceding claim, wherein the apparatus comprises the microtome apparatus.
21. The apparatus of any preceding claim, wherein the microtome apparatus comprises an ultramicrotome apparatus.
22. A method of characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, comprising: receiving, at a detector, optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with a microtome apparatus; providing, from the detector to a controller, an output indicative of at least one characteristic of the received optical signals; and determining, at the controller, first information indicative of a position of a first surface portion of the sample or sample holder assembly of the microtome apparatus prior to the obtaining of the one or more slices from the outer surface of the sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determining, at the controller, second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of the one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determining, at the controller, a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.
23. A computer program comprising instructions which, when executed on a controller of an apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample using a microtome apparatus, cause the controller to: receive from a detector of the apparatus for characterising a thickness of one or more slices obtained from an outer surface of a sample an output indicative of at least one characteristic of optical signals reflected and / or scattered from at least first and second portions of the surface of a sample or sample holder assembly associated with the microtome apparatus; determine first information indicative of a position of a first surface portion of the sample or a sample holder assembly of the microtome apparatus prior to the obtaining of one or more slices from the outer surface of the sample using a blade assembly associated with the microtome apparatus, wherein the first information is determined based on a first output of the detector associated with optical signals received from the first surface portion of the sample or sample holder assembly, determine second information indicative of a position of a second surface portion of the sample or sample holder assembly of the microtome apparatus subsequent to the obtaining of one or more slices from the outer surface of the sample using the blade assembly associated with the microtome apparatus, wherein the second information is determined based on a second output of the detector associated with optical signals received from the second surface portion of the sample or sample holder assembly; and determine a thickness of at least one of the one or more slices obtained from the sample using the blade assembly of the microtome, based on the first and second information.
24. An apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, the apparatus comprising: a first detector configured to receive optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly and provide an output indicative of at least one characteristic of each of the received optical signals; and a controller, wherein the controller is configured to: determine first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, and determine an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.
25. The apparatus of claim 24, wherein the position of each of the plurality of portions of the surface of the sample or sample holder assembly comprises the position in three-dimensional space of a point coincident with the portion of the surface of the sample or sample holder assembly.
26. The apparatus of claim 25, wherein the controller is configured to estimate a spatial position and orientation of a surface region of the surface of the sample or sample holder based on the first information by fitting a surface profile through at least three positions comprised in the plurality of positions.
27. The apparatus of claim 26, wherein the fitted surface comprises a plane.
28. The apparatus of claim 26, wherein the fitted surface comprises a non-uniform topology.
29. The apparatus of claim 28, wherein the plurality of portions comprise portions of a surface of a sample exposed by the obtaining of one or more slices using a blade assembly of the microtome apparatus, and the controller is configured to analyse the fitted surface to determine a measure of surface uniformity.
30. The apparatus of any of claims 24 to 29, wherein the surface of the sample or sample holder assembly comprises a region of the sample which is to be removed by the obtaining of the one or more slices using a blade assembly of the microtome apparatus.
31. The apparatus of claim 30, wherein the controller is configured to analyse the fitted surface to determine a parameter representative of the surface uniformity, and provide an indication to a user if the parameter is determined to be below a predefined threshold.
32. The apparatus of any of claims 24 to 31, wherein the apparatus is configured to characterise a first orientation of a surface of the sample or sample holder prior to obtaining one or more slices from the sample, and characterise a second orientation of a surface of the sample or sample holder subsequent to obtaining the one or more slices from the sample, and to estimate a uniformity of at least one of the one or more slices based on comparison of the first and second orientations.
33. The apparatus of any of claims 24 to 32, wherein the controller is further configured to determine an orientation of a cutting edge of a blade assembly of the microtome.
34. The apparatus of claim 33, wherein the controller is configured to determine the orientation of the cutting edge of the blade assembly based on calibration information input by a user.
35. The apparatus of any of claims 33 and 34, wherein the controller is configured to determine the orientation of the cutting edge of the blade assembly based on output received by the controller from one or more sensors, wherein the output is indicative of the blade orientation.
36. The apparatus of claim 35, wherein the one or more sensors comprise a second detector configured to receive optical signals reflected and / or scattered from each of a plurality of portions on at least one surface of a blade assembly configured to obtain slices from the surface of a sample, and provide an output indicative of at least one characteristic of each of the received signals; wherein the controller is configured to: determine second information indicative of a respective position of each of the plurality of portions of the surface of the blade assembly, and determine an orientation of a cutting edge of the blade assembly based on the second information; wherein the respective position of each of the plurality of portions of the surface of the blade assembly is determined by the controller on the basis of the output received from the second detector.
37. The apparatus of claim 36, wherein the second detector comprises the first detector.
38. The apparatus of any of claims 36 to 37, wherein the controller is configured to determine the orientation of the cutting edge of the blade based on a predefined spatial relationship between the positions of each of the plurality of portions of the at least one surface of a blade assembly and the position of the cutting edge of the blade.
39. The apparatus of any of claims 36 to 38, wherein the second detector is configured to receive optical signals reflected and / or scattered from each of a plurality of portions of a first surface region of the blade assembly and a plurality of portions of a second surface region of the blade assembly, wherein the first and second surface regions of the blade assembly converge at a cutting edge of the blade assembly; wherein the second information is indicative of a respective position of each of the plurality of portions of the first and second surface regions of the blade assembly.
40. The apparatus of claim 39, wherein the controller is configured to determine the orientation of the cutting edge of the blade assembly by: determining a plurality of points in three-dimensional space, each point corresponding to the position of one of the plurality of portions of the first and second surface regions of the blade assembly; fitting a first surface profile through a first subset of the plurality of points, the first subset of points being coincident with the first surface region of the blade assembly; fitting a second surface profile through a second subset of the plurality of points, the second subset of points being coincident with the second surface region of the blade assembly; and determining the orientation of the cutting edge of the blade by determining the intersection of the first and second surface profiles.
41. The apparatus of any of claims 39 to 40, wherein the blade assembly comprises an optically transparent material, and wherein the second detector is configured to receive optical signals from a first surface region comprising an external surface of the blade assembly, and to receive optical signals from a second surface region comprising an internal surface of the blade assembly, wherein the detector is configured to receive optical signals reflected and / or scattered from the second surface region of the blade following transmission through a portion of the blade assembly.
42. The apparatus of any of claims 24 to 41, wherein the first detector is comprised in an interferometer apparatus comprising an illumination source configured to direct optical signals at portions of a surface of the sample, sample holder assembly, and / or blade assembly, wherein the output indicative of at least one characteristic of signals received from each portion comprises a measure of phase shift between the received signals and a reference signal.
43. The apparatus of claim 42, wherein the illumination source comprises a white light source.
44. The apparatus of claim 42, wherein the illumination source comprises a laser.
45. The apparatus of any of claims 33 to 44, wherein the controller is further configured to provide control outputs to the microtome apparatus, the control outputs indicating or being usable to determine target translations and / or rotations of a sample holder assembly of the microtome and / or a blade assembly of the microtome to be used in obtaining slices from a sample mounted on a sample holder of the microtome using a blade assembly; wherein the control outputs are determined based on the relative orientations of the surface of the sample or sample holder and the cutting edge of the blade assembly.
46. The apparatus of claim 45, wherein the control outputs indicate target translations and / or rotations of a sample holder assembly of the microtome and / or a blade assembly of the microtome required to cause the cutting edge of the blade to be aligned substantially parallel to an external surface of the sample, and to cause the external surface of the sample to be aligned substantially parallel to an axis of travel along which the sample holder is configured to travel relative to the blade.
47. The apparatus of any of claims 24 to 46, wherein the apparatus comprises the microtome apparatus.
48. The apparatus of any of claims 24 to 47, wherein the microtome apparatus comprises an ultramicrotome apparatus.
49. A method of characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, comprising: receiving, at a first detector, optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly; providing, from the detector to a controller, an output indicative of at least one characteristic of each of the received optical signals; determining, at the controller, first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly; and determining, at the controller, an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.
50. A computer program comprising instructions which, when executed on a controller of an apparatus for characterising an orientation of an external surface of a sample or sample holder associated with a microtome apparatus, cause the controller to: receive, from a detector, an output indicative of at least one characteristic of optical signals reflected and / or scattered from each of a plurality of surface portions of the sample or sample holder assembly; determine first information indicative of a respective position of each of the plurality of portions of the surface of the sample or sample holder assembly, and determine an orientation of the surface of the sample or sample holder assembly based on the first information; wherein the respective position of each of the plurality of portions of the surface of the sample or sample holder assembly is determined by the controller on the basis of the output received from the first detector.