Background and Field

The invention relates to an optical microscope assembly, and a focussing method for such an assembly, in particular a modular optical microscope assembly.

Optical microscopes are widely used for many applications, such as fast characterization for disease analyses, real-time food quality monitoring, and examining semiconductor structures. For many important fields, such as biological research, food industry, medicine, and semiconductor manufacturing, conventional optical microscopes that work in ambient air are useful as the operation depends directly on fast detection of chemicals, microbes or defects.

However, such fields may employ characterizations involving a variety of techniques and/or magnifications. Complex characterizations may be cumbersome and time-consuming using conventional optical microscopes which generally have inflexible optical set-ups and therefore may not be able to meet the needs of different optical imaging applications.

It is desirable to provide a sample stage for an optical microscope assembly and a method of focussing an optical microscope assembly which address at least one of the drawbacks of the prior art and/or to provide the public with a useful choice.

Summary

In a first aspect, there is provided a focussing method for an optical microscope assembly including at least one objective lens having an optical axis and a sample support. The method comprises receiving, by one or more processors, an image captured via the at least one objective lens by the optical microscope assembly of a sample carried by the sample support; calculating, by one or more processors, a sharpness value of the received image; and in response to the sharpness value, causing, by one or more processors, the sample support to move to alter a distance along the optical axis between the at least one objective lens and the sample support until a predetermined sharpness value is obtained. In particular, the sharpness value may comprise a Canny value.

By moving the sample support to alter a distance along the optical axis between the at least one objective lens and the sample support until a predetermined sharpness value is obtained, an optimal focussing position may be automatically determined regardless of the sample or the focal length of the objective lens. This may facilitate the implementation of a modular microscope assembly, in which optical columns are interchangeable without time consuming manual repositioning of the sample support.

In a specific embodiment, the predetermined sharpness value may be a maximum sharpness value of a plurality of sharpness values, each calculated at a different distance along the optical axis between the at least one objective lens and the sample support.

It is envisaged that the focussing method may comprise causing, by one or more processors, the sample support to move in at least one direction perpendicular to the optical axis of the at least one objective lens to move the sample support onto the optical axis. In particular, the optical microscope assembly may comprise a plurality of objective lenses, each of the plurality of objective lenses having an associated optical axis, and the focussing method may comprise causing, by one or more processors, the sample support to move between at least one first axis position on a first associated optical axis of a first objective lens of the plurality of objective lenses and at least one second axis position on a second associated optical axis of a second objective lens of the plurality of objective lenses.

Specifically, one or more processors may cause the sample support to move between a plurality of first axis positions along the first associated optical axis, each of the plurality of first positions corresponding to a different first axis distance between the sample support and the first objective lens, and between a plurality of second axis positions along the second associated optical axis, each of the plurality of second positions corresponding to a different second axis distance between the sample support and the second objective lens. Thus, the microscope assembly may have different optical columns and the sample support may move between them and the optimal focussing position for the sample support may be determined for each respective optical column. Specifically, it is envisaged that the first objective lens and the second objective lens may have different focal lengths.

In a specific embodiment, the focussing method may further comprise replacing the at least one objective lens with a further at least one objective lens having a further optical axis. Correspondingly, the focussing method may further include receiving, by one or more processors, a further image captured via the further objective lens by the optical microscope assembly of the sample carried by the sample support; calculating, by one or more processors, a further sharpness value of the received further image; and in response to the further sharpness value, causing, by one or more processors, the sample support to move to alter a further distance along the further optical axis between the further at least one objective lens and the sample support until a further predetermined sharpness value is obtained. In particular, the at least one objective lens and the further at least one objective lens may have different focal lengths. Thus, the microscope assembly may be altered according to the requirements of the particular user and/or optical characterization being performed while enabling the optimal focussing position for the sample support to be determined at every stage.

In a second aspect, there is provided an optical microscope assembly. The optical microscope assembly comprises at least one objective lens having an optical axis; a sample stage having a sample support for carrying a sample; the sample support movable between a plurality of positions along the optical axis, each of the plurality of positions corresponding to a different distance between the sample support and the at least one objective lens; at least one sensor for capturing an optical image of the sample via the at least one objective lens; and an autofocus controller operable to calculate a sharpness value of the captured optical image and, in response to the sharpness value, move the sample support to another position in the plurality of positions until a predetermined sharpness value is obtained. In a specific embodiment, the predetermined sharpness value may be a maximum sharpness value of a plurality of sharpness values, each calculated at a different position in the plurality of positions.

It is envisaged that the sample support may be further movable in at least one direction perpendicular to the at least one objective lens. In particular, the optical microscope assembly may comprise a plurality of objective lenses, each of the plurality of objective lenses having an associated optical axis, and the sample support may be further movable between at least one first axis position on a first associated first optical axis of a first objective lens of the plurality of objective lenses and at least one second axis position on a second associated optical axis of a second objective lens of the plurality of objective lenses.

It is also envisaged that the sample support may be movable between a plurality of first axis positions along the first associated optical axis, each of the plurality of first positions corresponding to a different first axis distance between the sample support and the first objective lens, and between a plurality of second axis positions along the second associated optical axis, each of the plurality of second positions corresponding to a different second axis distance between the sample support and the second objective lens. In particular, the first objective lens and the second objective lens may have different focal lengths.

Advantageously, the optical microscope may further comprise a further at least one objective lens having a further optical axis, and a further at least one sensor for capturing a further optical image of the sample via the further at least one objective lens. The further at least one objective lens may be operable to replace the at least one objective lens, and the further at least one sensor may be operable to replace the at least one sensor. In addition, the sample stage may be further movable, upon replacement of the at least one objective lens with the further at least one objective lens, between a further plurality of positions along the further optical axis, each of the further plurality of positions corresponding to a further different distance between the sample support and the further at least one objective lens. Correspondingly, the autofocus controller may be further operable to calculate a further sharpness value of the further captured optical image and, in response to the further sharpness value, cause the sample support to move to a further position in the further plurality of positions until a further predetermined sharpness value is obtained. In particular, the at least one objective lens and the further at least one objective lens may have different focal lengths.

In a specific embodiment, the sample stage may comprise three mechanical stages, each independently operable to actuate movement of the sample support in a respective direction, each respective direction of the three mechanical stages being mutually orthogonal to enable movement of the sample support in three- dimensional space.

In a third aspect, there is provided an autofocus controller for an optical microscope assembly including at least one objective lens having an optical axis and a sample support, the autofocus controller comprising: an input configured to receive an image captured by the optical microscope assembly of a sample carried by the sample support; an output configured to control a position of the sample support; a processor configured to: calculate a sharpness value of the received image; and in response to the sharpness value, cause the output to move the sample support to alter a distance along the optical axis between the at least one objective lens and the sample support until a predetermined sharpness value is obtained.

In a fourth aspect, there is provided a tangible or non-tangible computer readable medium comprising computer executable instructions that, when executed by a processor, cause the processor to perform a focussing method for an optical microscope assembly including at least one objective lens having an optical axis and a sample support, the focussing method comprising: receiving, by one or more processors, an image captured via the at least one objective lens by the optical microscope assembly of a sample carried by the sample support; calculating, by one or more processors, a sharpness value of the received image; and in response to the sharpness value, causing, by one or more processors, the sample support to move to alter a distance along the optical axis between the at least one objective lens and the sample support until a predetermined sharpness value is obtained. It is envisaged that features relating to one aspect may be applicable to the other aspects.

Brief Description of the Drawings

An exemplary embodiment will now be described with reference to the accompanying drawings in which:

Fig. 1 illustrates a simplified block diagram of an optical microscope assembly according to a preferred embodiment;

Fig. 2 is a schematic diagram illustrating a portion of the optical microscope assembly of Fig. 1 in more detail;

Fig. 3 illustrates a schematic front view of a sample stage of the optical microscope assembly of Fig. 1 ;

Fig. 4 further illustrates a plan view of the sample stage of Fig. 3;

Fig. 5 illustrates cross-section views of two optical columns of the optical microscope assembly of Fig. 1 to show internal components of the optical columns;

Fig. 6 is a schematic diagram of a mounting arrangement for the two optical columns of Fig. 5;

Fig. 7 is a schematic block diagram of an autofocus controller of the optical microscope assembly of Fig. 1 ;

Fig. 8 illustrates a method of capturing an image of a sample using the optical microscope assembly of Fig. 1 ;

Fig. 9 illustrates experimental results obtained using the optical microscope assembly of Fig. 1 ; and

Fig. 10 illustrates potential configurations for one or more optical columns of the optical microscope assembly of Fig. 1 .

Detailed Description of Preferred Embodiment Fig. 1 illustrates a simplified block diagram of an optical microscope assembly 100 according to a preferred embodiment. The optical microscope assembly 100 includes three objective lenses 5, 6 (not visible in Fig. 1 ) each comprised within a respective one of three optical columns 203, 205, 207, each of the objective lenses 5, 6 having an optical axis 213, 215, 217. As will be described in more detail further below, each of the three optical columns 203, 205, 207 also includes a sensor for capturing an optical image of a sample via the respective objective lens 5, 6.

It should be appreciated that the meaning of the term “objective lens”, as used herein is not intended to be limited to that of a single lens, but is also intended to encompass several optical elements cooperating to gather light from an object and produce a real image (sometimes known simply as an “objective”).

The optical microscope assembly 100 further includes a sample stage 201 , of which only a movable sample support 101 for carrying the sample is shown in Fig. 1.

In the described embodiment, each optical column 203, 205, 207 is mounted approximately vertically in elevation above the sample stage 201 from a mechanical support 109 in the form of a flat arch structure, the roof of the flat arch structure being defined by an approximately horizontal support beam 9. As such, the optical axes 213, 215, 217 of the three objective lenses 5, 6 are approximately vertical (and as such parallel) and a focal point of each of the objective lenses 5, 6 is positioned below each of the optical columns 203, 205, 207, towards the sample stage 201 . This can be appreciated from Fig. 2 in which the focal points 209, 211 of two of the optical columns 203, 205 are illustrated. Note that although the optical axes 213, 215, 217 appear in Fig. 1 to be positioned in the same plane, it should be appreciated that this may not be the case, for example one of the optical columns 203, 205, 207 may be set back from the other two. In Fig. 1 , the sample support 101 is illustrated as being positioned on the optical axis 215 of optical column 205. As further illustrated in Fig. 1 , in the described embodiment, the sample support 101 is movable horizontally, i.e. in a plane approximately perpendicular to the direction of the optical axes 213, 215, 217 of the objective lenses 5, 6 of each of the optical columns 203, 205, 207, thereby enabling it to move between each of the individual optical axes 213, 215, 217. Although not shown in Fig. 1 , the sample support 101 is also movable vertically, i.e. the sample support 101 is also movable between a plurality of positions on each individual one of the optical axes 213, 215, 217, each position corresponding to a different distance between the sample support 101 and the corresponding objective lens 5, 6. In other words, the sample support 101 may be moved between a plurality of positions along the optical axis 213, a plurality of positions along the optical axis 215 and a plurality of positions along the optical axis 217.

The optical microscope assembly 100 also includes an autofocus controller 10 (see Fig. 2) for controlling movement of the sample support 101 which, in the described embodiment, forms part of a wiring and connection unit 1011 which is mounted on the mechanical support 109 above the optical columns 203, 205, 207, specifically on top of the support beam 9. The autofocus controller 10 is communicatively coupled to the sensor of each of the optical columns 203, 205, 207 and also to the sample stage 201 . The operation of the autofocus controller 10 will be described in more detail below.

It will be appreciated that additional components in the wiring and connection unit may also provide other control and/or power functionality employed in the operation of the microscope assembly 100, for example, it may provide power to the sensors of the optical columns 203, 205, 207.

The microscope assembly 100 also includes a base 107 in the form of an approximately horizontal planar mechanical frame on which the mechanical support 109 and sample stage 201 are themselves mounted and which forms a base for the overall microscope assembly 100. Three lights 105 are also mounted on the base 107 below the sample stage 201 , each light 105 being positioned on the optical axis 213, 215, 217 of a respective one of each of the three optical columns 203, 205, 207. In other words, each optical column 203, 205, 207 is associated with a respective light 105, thereby defining an optical column/light arrangement sandwiching the sample stage. These optical column/light arrangements may enable each optical column 203, 205, 207 to capture an image of a sample supported on the sample support 101 when the sample support 101 is positioned on the optical axis 213, 215, 217 of the corresponding objective lens 5, 6, illumination of the sample by the respective light 105 being achieved, for example, via a window in the sample stage 201 and sample support 101 (not shown) in a transmission mode of the optical microscope assembly 100 or respective optical column 203, 205, 207.

Fig. 2 shows a more detailed schematic diagram of a portion of the optical microscope assembly 100 illustrating only two of the optical columns 203,205, support beam 9, the sample stage 201 (including sample support 101 ), and the autofocus controller 10. The communicative connections 219, 221 between the optical columns 203, 205 and the autofocus controller 10, and the communicative connection 223 between the sample stage 201 and the autofocus controller 10 are also illustrated. It should be appreciated that although these communicative connections 219, 221 , 223 are illustrated as being wired connections, they may instead be wireless connections.

As discussed above, sample stage 201 is mounted on mechanical frame 107 (not shown in Fig. 2) and includes three independent mechanical transition stages: an x-axis stage 1 , a y-axis stage 2 mounted on top of the x-axis stage 1 , and a z-axis stage 3 which is mounted on top of the y-axis stage 2. The sample support 101 is defined by an upper surface of the z-axis stage 3. Optionally, an extension 4 may be added to the x-axis stage 1 . Likewise, an extension may be optionally added to the y-axis stage 2 (not shown). Fig. 3 illustrates a schematic front view of the sample stage 201 . Each of the mechanical transition stages comprises a moving stage and a stage base. Specifically, the x-axis stage 1 includes an elongate stage base 301 and a moving stage 311 mounted on top the stage base 301 and operable to move along the stage base 301 in the direction of elongation of stage base 301 .

Likewise, the y-axis stage 2 includes an elongate stage base 302 and a moving stage 322 mounted on top of the stage base 302 and operable to move along the stage base 302 in the direction of elongation of stage base 302.

The z-axis stage 3 includes a substantially cuboid stage base 303 and a moving stage 333 mounted approximately centrally within the stage base 303 so as to project upwards out of the stage base 303. The moving stage 333 is operable to move up and down with respect to the stage base 303, i.e. in such a way that more or less of the moving stage 333 projects from the stage base 303.

Stage base 302 of the y-axis stage 2 is fixedly mounted to the moving stage 311 of x-axis stage 1 , for example by nailing the stage base 302 to the moving stage 311 , and, likewise, the stage base 303 of the z-axis stage 3 is fixedly mounted to the moving stage 322 of the y-axis stage 2. As will be appreciated from Fig. 4, which illustrates the sample stage 201 in plan view, stage base 301 of x-axis stage 1 and stage base 302 of y-axis stage 2 are both elongate and arranged so as to extend approximately perpendicular to each other, stage base 302 of y-axis stage 2 extending beyond an outer edge of the moving stage 311 of x-axis stage 1. In contrast, stage base 303 of z-axis stage 3 is positioned approximately centrally on an upper surface of the moving stage 322 and does not extend beyond an outer edge of moving stage 322, having a smaller footprint than moving stage 322.

Each of the mechanical transition stages further includes an independent motor (not shown), for example a linear motor arranged to drive linear motion of the respective moving stage with respect to the corresponding stage base in a single direction. Stage bases 301 , 302, 303 thereby each define a linear track for the respective moving stage 311 , 322, 333, each linear track being arranged perpendicularly to the linear tracks defined by the other two moving stages, as can be appreciated from Fig. 4. In other words, moving stage 311 of x-axis stage 1 is arranged to move along a notional x-axis 304 of the sample stage 201 via the stage base 301 , moving stage 322 is arranged to move along a notional y-axis 306 of the sample stage 201 via the stage base 302, and moving stage 333 is arranged to move along a notional z-axis 308 of the sample stage 201 via the stage base 302, the notional x- and y- axes 304, 306 being approximately parallel to the plane of the base 107 and the notional z-axis 308 being approximately parallel to the optical axes 213, 215, 217 of the objective lenses 5, 6, i.e. the notional z-axis 308 defines an axis of elevation of the sample surface 101 .

All of the independent motors (one included in each of the x-axis stage 1 , the y- axis stage 2 and the z-axis stage 3) are controlled by the autofocus controller 10 thereby enabling the autofocus controller 10 to control the position of the sample support 101 in three dimensional directions, as will be discussed in more detail below.

It should be appreciated that the sample stage 201 may be manufactured in a variety of ways. For example, the sample stage 201 may be fabricated by combining three standalone mechanical sample stages and fixedly attaching each of the standalone mechanical sample stages to the moving platform of another, as appropriate. Alternatively, the sample stage 201 may be manufactured as a unitary structure with respective movable parts for the x, y and z-axis movements.

Returning now to Fig. 2, each optical column 203, 205 comprises three principal components, namely: the objective lens 5, 6 (which may comprise an integrated system functioning as an objective); an imaging unit 8; and a sensor in the form of a charge-coupled device (CCD) array 7. As discussed above, in relation to Fig. 1 , the optical columns 203, 205, 207 are mounted approximately vertically from the mechanical support 109, specifically from support beam 9. The objective lens 5, 6 of each of the optical columns 203, 205 is positioned at the base of the respective optical column 203, 205, adjacent to the path of the sample support 101. In the described embodiment, the objective lenses 5, 6 have different focal lengths, as can be appreciated from their respective focal points 209, 211 , as illustrated in Fig. 2, the focal point 209 of objective lens 6 being higher than the focal point 211 of objective lens 5.

Fig. 5 shows the components of optical columns 203, 205 in more detail according to the described embodiment. The sample support 101 and associated lights 105 are also illustrated in Fig. 5 for reference.

In addition to objective lenses 5, 6 optical columns 203, 205 both further include imaging units 8 mounted vertically above the respective objective lens 5, 6, the imaging units 8 themselves each including a tube lens 507 and a mechanical frame 509 for maintaining the tube lens 507 in a correct position. Vertically above each of the imaging units 8 is mounted an optional beam splitter 503 and LED 501 assembly operable to produce sample illumination in a reflective mode of the optical columns 203, 205.

The objective lens of optical column 203 further includes an additional optional lens 511 integrated at the lower end of the objective lens 6, i.e. adjacent to the path of the sample support 101. An example of such an optional component is a microsphere lens, which may be capable of nano-scale imaging.

As noted above, the optical sensors of the optical columns 203, 205 are in the form of charge-coupled device (CCD) arrays 7 and are located at the top of each of optical column 203, 205. It will be appreciated from Fig. 5 that each CCD array 7 is positioned to receive light from the sample via the respective additional lens 511 (if applicable), objective lens 5, 6, tube lens 507 and beam splitter 503 and thereby capture an optical image of the sample carried by the sample support (when positioned on the respective optical axis 213, 215). As discussed above, the optical sensors - CCD arrays 7 in the case of optical columns 203, 205 - are communicatively coupled to the autofocus controller 10, thereby enabling image data from the optical columns 203, 205, 207 to be received by the autofocus controller 10.

As illustrated in Fig. 1 , the optical columns 203, 205, 207 are mounted to the mechanical support 109 which holds them in the correct position for imaging of the sample, namely substantially vertical in the described embodiment. Fig. 6 illustrates the mounting arrangement in more detail according to the described embodiment. The support beam 9 of the mechanical support 109 is fixedly attached perpendicular to the mechanical frame 509 of the image unit 8, for example via screws 601 passing through the support beam 9 and into the mechanical frame 509. If necessary, an additional adaptor 603 may be positioned around the image unit 8 and the mechanical support 9 fixedly attached to the adaptor 603 in place of the mechanical frame 509.

The attachment of the optical columns 203, 205, 207 to support beam 9 may be reversable, for example by unscrewing screws 601 , thereby enabling removal and replacement of one or more of the optical columns 203, 205, 207. As such the optical microscope assembly 100 may be described as a modular optical assembly.

Returning again to Fig. 2, autofocus controller 10, which is communicatively coupled to the optical columns 203, 205 and the sample stage 201 is in the form of a computer system. Fig. 7 illustrates the autofocus controller 10 according to the described embodiment in more detail. In this embodiment, the autofocus controller 10 includes a processor 382 (which may be referred to as a central processor unit or CPU) that is in communication with memory devices including secondary storage 384, read only memory (ROM) 386, random access memory (RAM) 388, input/output (I/O) devices 390, and network connectivity devices 392. The processor 382 may be implemented as one or more CPU chips. It is understood that by programming and/or loading executable instructions onto the autofocus controller 10, at least one of the CPU 382, the RAM 388, and the ROM 386 are changed, transforming the autofocus controller 10 in part into a particular machine or apparatus having the novel functionality taught by the present disclosure. It is fundamental to the electrical engineering and software engineering arts that functionality that can be implemented by loading executable software into a computer can be converted to a hardware implementation by well-known design rules. Decisions between implementing a concept in software versus hardware typically hinge on considerations of stability of the design and numbers of units to be produced rather than any issues involved in translating from the software domain to the hardware domain. Generally, a design that is still subject to frequent change may be preferred to be implemented in software, because re-spinning a hardware implementation is more expensive than re-spinning a software design. Generally, a design that is stable that will be produced in large volume may be preferred to be implemented in hardware, for example in an application specific integrated circuit (ASIC), because for large production runs the hardware implementation may be less expensive than the software implementation. Often a design may be developed and tested in a software form and later transformed, by well- known design rules, to an equivalent hardware implementation in an application specific integrated circuit that hardwires the instructions of the software. In the same manner as a machine controlled by a new ASIC is a particular machine or apparatus, likewise a computer that has been programmed and/or loaded with executable instructions may be viewed as a particular machine or apparatus.

Additionally, after the autofocus controller 10 is turned on or booted, the CPU 382 may execute a computer program or application. For example, the CPU 382 may execute software or firmware stored in the ROM 386 or stored in the RAM 388. In some cases, on boot and/or when the application is initiated, the CPU 382 may copy the application or portions of the application from the secondary storage 384 to the RAM 388 or to memory space within the CPU 382 itself, and the CPU 382 may then execute instructions that the application is comprised of. In some cases, the CPU 382 may copy the application or portions of the application from memory accessed via the network connectivity devices 392 or via the I/O devices 390 to the RAM 388 or to memory space within the CPU 382, and the CPU 382 may then execute instructions that the application is comprised of. During execution, an application may load instructions into the CPU 382, for example load some of the instructions of the application into a cache of the CPU 382. In some contexts, an application that is executed may be said to configure the CPU 382 to do something, e.g., to configure the CPU 382 to perform the function or functions promoted by the subject application. When the CPU 382 is configured in this way by the application, the CPU 382 becomes a specific purpose computer or a specific purpose machine.

The secondary storage 384 is typically comprised of one or more disk drives or tape drives and is used for non-volatile storage of data and as an over-flow data storage device if RAM 388 is not large enough to hold all working data. Secondary storage 384 may be used to store programs which are loaded into RAM 388 when such programs are selected for execution. The ROM 386 is used to store instructions and perhaps data which are read during program execution. ROM 386 is a non-volatile memory device which typically has a small memory capacity relative to the larger memory capacity of secondary storage 384. The RAM 388 is used to store volatile data and perhaps to store instructions. Access to both ROM 386 and RAM 388 is typically faster than to secondary storage 384. The secondary storage 384, the RAM 388, and/or the ROM 386 may be referred to in some contexts as computer readable storage media and/or non-transitory computer readable media.

I/O devices 390 include communicative connections 219, 221 to the optical columns 203, 205, 207 and the communicative connection 223 to the sample stage 201 , which may be wired or wireless, as discussed above. I/O devices 390 may further include printers, video monitors, liquid crystal displays (LCDs), plasma displays, touch screen displays, keyboards, keypads, switches, dials, mice, track balls, voice recognizers, card readers, paper tape readers, or other well-known input devices.

The network connectivity devices 392 may take the form of modems, modem banks, Ethernet cards, universal serial bus (USB) interface cards, serial interfaces, token ring cards, fiber distributed data interface (FDD I) cards, wireless local area network (WLAN) cards, radio transceiver cards that promote radio communications using protocols such as code division multiple access (CDMA), global system for mobile communications (GSM), long-term evolution (LTE), worldwide interoperability for microwave access (WiMAX), near field communications (NFC), radio frequency identity (RFID), and/or other air interface protocol radio transceiver cards, and other well-known network devices. These network connectivity devices 392 may enable the processor 382 to communicate with the Internet or one or more intranets. With such a network connection, it is contemplated that the processor 382 might receive information from the network, or might output information to the network in the course of performing the herein-described method steps, for example control instructions for controlling the sample stage 201 . Such information, which is often represented as a sequence of instructions to be executed using processor 382, may be received from and outputted to the network, for example, in the form of a computer data signal embodied in a carrier wave.

The baseband signal or signal embedded in the carrier wave, or other types of signals currently used or hereafter developed, may be generated according to several methods well-known to one skilled in the art. The baseband signal and/or signal embedded in the carrier wave may be referred to in some contexts as a transitory signal.

The processor 382 executes instructions, codes, computer programs, scripts which it accesses from hard disk, floppy disk, optical disk (these various disk based systems may all be considered secondary storage 384), flash drive, ROM 386, RAM 388, or the network connectivity devices 392. While only one processor 382 is shown, multiple processors may be present. Thus, while instructions may be discussed as executed by a processor, the instructions may be executed simultaneously, serially, or otherwise executed by one or multiple processors. Instructions, codes, computer programs, scripts, and/or data that may be accessed from the secondary storage 384, for example, hard drives, floppy disks, optical disks, and/or other device, the ROM 386, and/or the RAM 388 may be referred to in some contexts as non-transitory instructions and/or non-transitory information.

In an embodiment, the autofocus controller 10 may comprise two or more computers in communication with each other that collaborate to perform a task. For example, but not by way of limitation, an application may be partitioned in such a way as to permit concurrent and/or parallel processing of the instructions of the application. Alternatively, the data processed by the application may be partitioned in such a way as to permit concurrent and/or parallel processing of different portions of a data set by the two or more computers. In an embodiment, virtualization software may be employed by the autofocus controller 10 to provide the functionality of a number of servers that is not directly bound to the number of computers in the autofocus controller 10. For example, virtualization software may provide twenty virtual servers on four physical computers. In an embodiment, the functionality described herein may be provided by executing the application and/or applications in a cloud computing environment. Cloud computing may comprise providing computing services via a network connection using dynamically scalable computing resources. Cloud computing may be supported, at least in part, by virtualization software. A cloud computing environment may be established by an enterprise and/or may be hired on an as-needed basis from a third-party provider. Some cloud computing environments may comprise cloud computing resources owned and operated by the enterprise as well as cloud computing resources hired and/or leased from a third-party provider. In an embodiment, some or all of the functionality disclosed herein may be provided as a computer program product or implemented by computer. The computer program product may comprise one or more computer readable storage medium having computer usable program code embodied therein to implement the functionality disclosed. The computer program product may comprise data structures, executable instructions, and other computer usable program code. The computer program product may be embodied in removable computer storage media and/or non-removable computer storage media. The removable computer readable storage medium may comprise, without limitation, a paper tape, a magnetic tape, magnetic disk, an optical disk, a solid state memory chip, for example analogue magnetic tape, compact disk read only memory (CD-ROM) disks, floppy disks, jump drives, digital cards, multimedia cards, and others. The computer program product may be suitable for loading, by the autofocus controller 10, at least portions of the contents of the computer program product to the secondary storage 384, to the ROM 386, to the RAM 388, and/or to other non-volatile memory and volatile memory of the autofocus controller 10. The processor 382 may process the executable instructions and/or data structures in part by directly accessing the computer program product, for example by reading from a CD-ROM disk inserted into a disk drive peripheral of the autofocus controller 10. Alternatively, the processor 382 may process the executable instructions and/or data structures by remotely accessing the computer program product, for example by downloading the executable instructions and/or data structures from a remote server through the network connectivity devices 392. The computer program product may comprise instructions that promote the loading and/or copying of data, data structures, files, and/or executable instructions to the secondary storage 384, to the ROM 386, to the RAM 388, and/or to other non-volatile memory and volatile memory of the autofocus controller 10.

In some contexts, the secondary storage 384, the ROM 386, and the RAM 388 may be referred to as a non-transitory computer readable medium or a computer readable storage media. A dynamic RAM embodiment of the RAM 388, likewise, may be referred to as a non-transitory computer readable medium in that while the dynamic RAM receives electrical power and is operated in accordance with its design, for example during a period of time during which the autofocus controller 10 is turned on and operational, the dynamic RAM stores information that is written to it. Similarly, the processor 382 may comprise an internal RAM, an internal ROM, a cache memory, and/or other internal non-transitory storage blocks, sections, or components that may be referred to in some contexts as non-transitory computer readable media or computer readable storage media.

The autofocus controller 10 may be a dedicated control unit for the sample stage 201 or it may additionally perform control functions of other components within the optical microscope assembly 100 and, as such, may perform the role of a central control unit for the whole assembly.

Fig. 8 illustrates a method of capturing an image of the sample carried by the sample support 101 using the microscope assembly 100.

In step 801 , a software module for carrying out imaging by a particular optical column of the microscope assembly, for example column 203, is executed on the CPU 382 of the autofocus controller 10. For example, the software module may be executed in response to a manual command via an input from a user via an I/O device 390 of the autofocus controller 10, or via a network connectivity device 392, or in response to a command issued as a result of the execution of software on the CPU 382 of the autofocus controller 10 or the CPU of another computing device.

In step 803, execution of the software module causes a pre-set XY position for the sample support 101 to be read by the CPU 382 from the ROM 386 or Secondary Storage 384 of the autofocus controller 10, the positions having being input (manually, or otherwise) in, for example, a database stored on the respective memory device of autofocus controller 10 upon installation of the optical column 203 in the microscope assembly 100.

In step 805, the software module causes actuation of movement of the sample support 101 in the notional X-Y direction by causing control instructions to be sent to the respective motor of the x-axis stage 1 and the y-axis stage 2, via the communicative connection 223 to the sample stage, for moving the sample support 101 to the pre-set XY position, i.e. to move the sample support 101 perpendicular to the optical axis 213. It will be appreciated that the pre-set XY position is located on the optical axis 213 of the objective lens 6, thereby enabling an image of the sample to be captured at the corresponding CCD array 7 of the optical column 203 via the objective lens 6.

In step 807, image data from the CCD array 7 of the optical column 203 is received at the autofocus controller 10, for example via the communicative connection 219 between the optical column 203 and the autofocus controller 10. In other words, the communicative connection 219 functions as an input to the autofocus controller for receiving an image captured by the optical column 203. The software module causes processing of the image data by the CPU 382 in order to calculate a sharpness value for the image received by the CCD array 7. For example, the raw image data may be received at the CPU 382 and converted into black and white. From the black and white image, the Canny value may be calculated. Open source tools for calculating the Canny value of an image are available, for example, the emgu.cv OpenCV library.

In step 809, the software module causes actuation of movement of the sample support 101 in the Z direction by causing control instructions to be sent to the respective motor of the z-axis stage 3, for example via communicative connection 223 between the sample stage 201 and the autofocus controller 10, i.e. the communicative connection 223 functions as an output configured to control a position of the sample support 101. The sample support 101 is moved to a new position along the notional z-axis corresponding to an altered distance along the optical axis between the objective lens 6 and the support surface 101 , relative to the position from which an image was captured previously.

The method then returns to step 807 and the sharpness value for the image captured at the new z-axis position of the sample support 101 is calculated as described above. Steps 807 and 809 are repeated for a predetermined number of positions of the sample support 101 along the z-axis position. Once the sharpness value (for example, the Canny value) has been calculated for each of the predetermined positions, the position having a sharpness value corresponding to the sharpest image (e.g. the highest Canny value) is selected and movement of the sample support 101 into the selected position is actuated.

For example, the sample support 101 may first be moved into a position along the notional z-axis corresponding to the lowest possible position for the moving stage 333 with respect to the stage base 303, i.e. corresponding to the greatest possible distance along the optical axis 213 between the sample support 101 and the objective lens 6, and the Canny value calculated at this position. The moving stage 333 may then be moved incrementally upwards, for example in steps of approximately 100nm or less, until the moving stage 333 reaches its most extreme position of projection from the stage base 303 (i.e. corresponding to the smallest distance along the optical axis 213 between the sample support 101 and the objective lens 6 that the sample stage is capable of achieving). Once the sharpness value (e.g. the Canny value) has been calculated at each incremental position, the sample support 101 is then moved into the position corresponding to the highest Canny value, or sharpest image. Thus, the optimal stage position may be determined down to a resolution of 100nm.

Thus, the sample support 101 is moved, in response to the sharpness value of the image received at the CCD array 7, to alter a distance along the optical axis 213 between the objective lens 6 and the sample support 101 until a predetermined sharpness value is obtained, in the described embodiment a maximum sharpness value of a plurality of sharpness values, each calculated at a different distance along the optical axis 213 between the objective lens 213 and the sample support 101 .

Hence, step 807 and step 809 together defined a method of focussing the optical microscope assembly for imaging via objective lens 6.

In step 811 , imaging of the sample is performed as desired. Image data may be received by the autofocus controller 10 and output for example via one or more of the I/O devices 390 or the network devices 392. Alternatively, image data may be output to a user or network directly from the optical column 203, for example from another processor forming part of the wiring and connection unit 1011.

The method then optionally returns to 801 and a software module (which may be the same or a different software module for carrying out imaging of the optical column 203) for carrying out imaging by another of the optical columns 203, 205, 207, for example optical column 205 of the microscope assembly, may then be executed by the CPU 382, and steps 803 to 811 are repeated for optical column 205. In other words, after moving the sample support 101 (and sample) onto the first optical axis 213 and moving the sample support 101 along the first optical axis until the predetermined sharpness value is obtained and an image is captured, the CPU then proceeds to cause the sample support 101 (and sample) to move onto a second optical axis 215 and move the sample support 101 along the second optical axis until the corresponding predetermined sharpness value is obtained for the second optical axis, i.e. the position for obtaining the maximum sharpness along the second optical axis 215, according to the described embodiment.

Alternatively, a user may manually give a command to initiate another cycle of measurement, or the method may end if all desired or selected characterization tasks have been performed. Further alternatively, the optical column 203 may be removed from the optical microscope assembly replaced with a new, or further optical column, and the method of Fig. 8 repeated for imaging with the new optical column. In other words, after moving the sample support 101 (and sample) onto the first optical axis 213 and moving the sample support 101 along the first optical axis until the predetermined sharpness value is obtained and an image is captured, the first optical column 203 may be replaced and the sample support 101 is moved along the further optical axis corresponding to the objective of the further optical column until the corresponding predetermined sharpness value is obtained for the further optical axis, i.e. the position for obtaining the maximum sharpness along the further optical axis, according to the described embodiment.

In summary, therefore, for performing a set of characterization tasks on a sample, the sample may be loaded on to the sample support 101 and sent, by the autofocus controller 10, to the position of the first optical column 203 of choice for the first characterization task. The auto-focus mechanism of the autofocus controller 10 is enabled, which ensures that the first optical column 203 can focus on the point of interest by adjusting the height (i.e. z-axis position) of the sample support 101 to the correct level so that the image is clear for data collection. Data collection is done by the CCD array 7. The collected data are sent to the autofocus controller 10 for analysis, which decides whether fine tuning of the sample position (via repositioning of the sample support 101 ) is required. When sufficient information is collected by this first optical column 203, the first characterization task may be completed. Then, the sample stage may move to the second optical column 205 for the next characterization task. Auto-focus, height adjustment, data collection, and software analysis may follow as discussed above. This cycle may repeat until all of the pre-set characterization tasks are completed.

Thus, the microscope assembly 100 may provide a fully automated, modular optical system, which may enable smooth, accurate, and fast transition between different optical modules, regardless of the make up of each optical column 203, 205, 207. For example, different optical columns 203, 205, 207, having different optical components, may require specific sample positions and heights. For example, the objective lenses 5, 6 have different focal lengths. The autofocus method performed by the autofocus controller 10 as described above may enable automatic adjustment of the position of the sample, based on image sharpness which may enable an optimal sample position and height to be obtained for each individual optical column 203, 205, 207. As such, the microscope assembly 100 and above-described autofocus method may provide a “one-button” imaging solution; the single sample stage 201 may be employed with any combination of optical columns 203, 205, 207 to perform a wide variety of sample characterization tasks.

The control mechanism of the microscope assembly 100 via the autofocus controller 10 and independent motors of the three independent mechanical transition stages may enable optimal positioning of the sample for every optical column 203, 205, 207, no matter how diverse the optical functionality between optical columns 203, 205, 207. A user may need only to press one control button, for example on an I/O device 390 of the autofocus controller 10 and the microscope assembly 100 may do the rest, including all adjustment and focussing. The microscope assembly 100 may therefore be user friendly.

In particular, as the z-axis position may be highly sample-dependent, adaptably determining the optimal z-axis position using the algorithm of Fig. 8 may enable improved accuracy and imaging compared with pre-storing a z-axis value for a particular optical column. Further, as the optimal z-axis position of the sample support 101 is determined for each characterization cycle, the optical columns 203, 205, 207 may be altered or replaced by completely new optical columns without any reduction in image sharpness; the method described above in association with Fig. 8 enables automatic adjustment of the position of sample support 101. The independent column design may enhance flexibility in designing optical systems with different magnifications, which may ensure a balance between high performance and cost-efficiency. In addition, the module-based design may enable each optical column 203, 205, 207 to be independently upgraded, undergo maintenance, or simply replaced without any reduction in imaging accuracy. Further, the microscope assembly 100 may support micro/nano- scale imaging (for example using a microsphere lens 511 , which can provide observation powers beyond conventional optical microscopes, potentially down to ~20 nm), and/or may enable the combination of different characterization techniques, such as 3-D confocal imaging, fluorescence, phase contrast and Raman within one apparatus, thereby potentially enabling the combination of characterization techniques to generate novel characterization. The apparatus may also not be limited by the properties of the light source.

The microscope assembly 100 may be employed in a wide variety of industrial applications not limited to conventional microscopy applications. For example, the microscope assembly 100 may enable nano-imaging capability without requiring vacuum conditions. Further, the microscope assembly 100 may enable ambient air operation and dynamic real-time detection, and sample treatment and preparation may not be required.

The microscope assembly 100 may enable the provision of sufficient observation power to explore the key mechanisms of biological and chemical samples and may be compatible with most optical imaging processing techniques. As such, the microscope assembly 100 may provide a flexible, universal platform enabling the examination of the nano-world using fast, noncontact and real-time methods.

In view of the above described advantages, potential fields of application for the microscope assembly include the fast detection of defects and flaws in the semiconductor industry; nano-imaging capability for biological applications in hospitals and biological research labs; flexible and customized functionalities for schools and educational institutions.

Experimental characterizations were performed in order to demonstrate the applicability of the microscope assemblies according to embodiments. Fig. 9 illustrates a sample characterization employing a prototype microscope assembly according to an embodiment using three example optical columns 203, 205, 207 each having a different magnification: 10x, 20x and 100x, the sample being a S$10 note.

Image “a” is a reference image of the note taken without any magnification (i.e. not using the microscope assembly 100). The square 901 indicates the area of the note captured by each of the three optical columns 203, 205, 207 of the microscope assembly 100.

Image “b” is an image captured by the optical column having a 10x magnification. At this magnification, the shape of the letter “in” may be characterized from the image.

Image “c” is an image captured by the optical column having a 20x magnification. At this magnification, more details are revealed, such as the surface roughness and other morphology features.

Image “d” is an image captured by the optical column having 100x magnification. This magnification may be sufficient to even test the quality of the materials to fabricate the sample, which may have application in detecting counterfeit currency.

The described embodiment should not be construed as limitative.

Although the sharpness value employed in the described embodiment is the Canny value, it should be appreciated that other methods of evaluating the 1 sharpness of an image are known and could be employed in place of the Canny value according to embodiments.

Although specific arrangements of the optical components of optical columns 203, 205 are described above, it is envisaged that the optical columns 203, 205, 207 may be adaptable according to the requirements of the sample characterization to be performed. Fig. 10 shows a 3x3 matrix of squares 1101 , each representing a potential configuration for one or more of the optical columns 203, 205, 207 for use in the microscope assembly 100 according to embodiments. It is envisaged that any combination of the optical components represented by each of the squares 1101 could be employed as part of optical columns 203, 205, 207 in the microscope assembly 100. It should be appreciated that further optical columns not represented within the matrix of Fig. 10 could also be employed according to embodiments.

Further, it is envisaged that one or more of the optical columns 203, 205, 207 may be replaceable with a differently configured optical column, which may have a different focal length.

It should be appreciated that the optical columns 203, 205, 207 and/or their replacements may be pre-fabricated, i.e. out of the box optical columns, or they may be custom built for use in the optical assembly 100 according to requirements.

Although the mechanical support 109 is shown schematically in Fig. 1 as a flat arch structure, it should be appreciated that a wide variety of supports for the optical columns 203, 205, 207 may be employed, for example, one or more of the optical columns 203, 205, 207 may be mounted in separate, independent support structures. Likewise, a wide variety of mounting methods other than that illustrated in Fig. 6 may be employed in order to support the optical columns 203, 205, 207 in position for imaging, for example, a clamp may be employed according to a variation of the described embodiment. Indeed, it is envisaged that any method or structure of supporting an optical column in a fixed position with the optical axis unobstructed may be employed according to embodiments.

Further, the wiring and connection unit 1011 may not be mounted on the mechanical support 109 or other supports in which the optical columns 203, 205, 207 are mounted, or may not be provided at all, for example, each of the components of the wiring and connection unit 1011 may be provided as individual and separate components.

Although the microscope assembly 100 is described as having three optical columns 203, 205, 207, it should be appreciated that the number of optical columns may be fewer or greater than three and may be adaptable according to the requirements of a user, for example the microscope assembly 100 may include only a single optical column. Increases in the number of columns may or may not require extension 4 of the mechanical transition stage 1 , for example by extending stage base 301.

Although the z-axis stage 3 is described as being mounted on the y-axis stage 2, which is itself mounted on the x-axis stage 1 , any possible stacking order of the stages is envisaged. For example, the vertical transition stage, or z-axis stage 3 may not be the uppermost transition stage in the assembly.

Although the optical columns 203, 205, 207 are described as being mounted in a substantially vertical configuration above the sample stage, it is envisaged that one or more of the optical columns could be mounted at an angle to the vertical and, as such, the optical axes 213, 215, 217 may not be parallel.

Although it is described above that the final position of the sample support 101 is selected at the position at which a maximum value of the sharpness value is obtained, it is envisaged that the position of the sample support 101 may instead be varied until a threshold value for the sharpness value is obtained or exceeded and that the position at which the threshold value for the sharpness value is obtained is selected as the final position for imaging. For example, the sample support 101 may be initially set at its lowest z-position and the sample incrementally moved upwards towards the objective until the threshold value of the sharpness value is obtained. This method of determining a z-axis position for the sample support 101 may be faster than selecting the position at which the sharpness value is a maximum.

Although light sources 105 are described as being included in the microscope assembly 100 thereby enabling a transmission mode of the microscope, it is envisaged that no light sources 105, or light sources 105 only for certain of the optical columns 203, 205, 207 may be included in the microscope assembly 100 according to embodiments. For example, one or more of the optical columns 203, 205, 207 may be configured to operate only in a reflective mode using beam splitter 503 and LED 501 assembly.

Although the sample support 101 for carrying the sample is shown in Fig. 4 as being defined by an upper surface of the z-axis stage 3 and thereby not extending beyond the z-axis stage, it is envisaged that in a variation of the described embodiment, the sample support 101 may extend beyond the z-axis stage, for example but not limited to, via an additional elongate surface mounted on the moving stage 333. This variation may enable a sample carried by the elongate sample support 101 to be moved into an extended position beyond the respective footprints of the x-axis stage 1 , the y-axis stage 2 and the z-axis stage 3, i.e. such that at least a portion of the sample support 101 is not positioned directly above any other component of the sample stage 201. In this variation, one or more of the lights 105 may be correspondingly positioned so as to be offset from the sample stage 201 so that, in the transmission mode, the sample support 101 may be positioned directly above it, i.e. without obstruction from any other component of the sample stage 201 . As such, only the sample support 101 may be capable of transmitting light (e.g. via a window) from the lights 105 to a sample received thereon, transmission of light may not be possible through any other component of the sample stage 201 . Likewise, although optical columns 203, 205 are described as including a beam splitter 503 and LED 501 assembly, one or more of the optical columns 203, 205, 207 may not include a beam splitter 503 and LED 501 assembly and, as such, may not be capable of operating in a reflective mode.

Although only a CCD array 7 is described as being included in optical columns 203, 205, it is envisaged that other data collection devices may be installed in addition to, or instead of the CCD array 7 one or more of the optical columns 203, 205, 207.

Although autofocus controller 10 and the method of Fig. 8 are described as being employed with sample stage 201 , it is envisaged that autofocus controller 10 and the method of Fig. 8 could be employed with any sample stage capable of moving a sample in three-dimensions. It is further envisaged that autofocus controller 10 and step 807 and step 809 of the method of Fig. 8 could be employed with a sample stage capable of moving a sample automatically in only a single direction, for example, it is envisaged that the autofocus controller 10 and step 807 and step 809 of the method of Fig. 8 could be employed with a sample stage having only z-axis stage 3. Alternatively, the sample stage 201 could be manually movable in the x-y plane, as opposed to under the control of the autofocus controller 10, as described.

Although manual replacement of one objective lens with another is described above, it is envisaged that objective lenses could be swapped automatically, for example, two or more objective lenses may be mounted to a revolving turret and the rotation of the turret actuated to enable imaging via each of the two or more objective lenses in turn.

Having now fully described the invention, it should be apparent to one of ordinary skill in the art that many modifications can be made hereto without departing from the scope as claimed.