Microscope with focusing system

A microscope comprises a microscope objective, a camera and an imaging optical system for imaging an object through the objective to the camera along a first optical path. A projection optical system is provided for projecting a test image onto the object through the objective, and the imaging optical system is configured to image the projected test image from the object to the camera through the objective and along at least part of the first optical path. A focus adjustment system is provided for focusing the test image at the camera. Using the same objective and the same camera for both imaging and focusing allows reduction of the cost of the microscope in comparison with known microscopes that provide separate focusing systems.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to and claims priority to Great Britain Patent Application Serial No. 1905815.5, filed Apr. 25, 2019, entitled MICROSCOPE WITH FOCUSING SYSTEM, the entirety of which is incorporated herein by reference.

FIELD

This invention relates to focusing in microscopes. The invention relates particularly, but not exclusively, to compensating for focus drift in microscopes.

BACKGROUND

The term focus drift refers to a microscope's inability to maintain a selected focal plane over time, for example as a result of changes in temperature or mechanical shocks or vibrations. The imaging quality delivered by a microscope can be adversely affected by focus drift, particularly when operating at high axial resolutions.

It is known to compensate for focus drift in microscopes by providing a focus correction system that is separate from the imaging system. Typically, an infra-red (IR) reference beam, spatially separated from the microscope's imaging axis, is injected into the infinity space above or below the microscope's objective. The returning, also spatially separated, IR signal, is then detected and imaged using a dedicated imaging path that is separate from the microscope's primary imaging path. This arrangement enables continuous closed-loop feedback on a reference surface to provide focus correction, but at the expense of requiring a dedicated imaging path for focus correction.

It would be desirable to support focus drift compensation in a microscope without requiring a dedicated imaging path for this purpose. More generally, it would also be desirable to provide a relatively inexpensive focusing system for microscopes.

SUMMARY

This disclosure generally relates to a microscope and relate to a first aspect the invention provides a microscope comprising:

a microscope objective;

at least one camera;

an imaging optical system configured to image an object through said objective to said at least one camera along a first optical path;

a projection optical system configured to project a test image onto said object through said objective; and

a focus adjustment system,

wherein said imaging optical system is configured to image the projected test image from said object to said at least one camera through said objective and along at least part of said first optical path, said focus adjustment system being operable to focus said test image at said at least one camera.

Preferably, said microscope is operable in a focusing mode in which said projection optical system projects said test image onto said object, said imaging optical system images the projected test image from said object to said at least one camera, and said focus adjustment system focuses said test image at said at least one camera. The microscope is typically operable in an imaging mode in which said imaging optical system images said object to said at least one camera. The projection optical system is preferably disabled in said imaging mode.

In preferred embodiments, said projection optical system is configured to project said test image onto said object along part of said first optical path.

In preferred embodiments, a first beam splitter is included in said imaging optical system in said first optical path, and wherein said projection optical system is configured to project said test image onto said object by means of said first beam splitter. Said projection optical system may include an optical projector, said first beam splitter being configured to split light from said optical projector into first and second portions, and to direct said first portion to said objective to project said test image onto said object through said objective. Said beam splitter may be configured to direct said first portion to said objective to project said test image onto said object along part of said first optical path.

Typically, said optical projector includes a first light source configured to generate light in a first frequency band, said first beam splitter being configured to split light in said first frequency band. Said first beam splitter may be configured to reflect said first portion of light and to transmit said second portion of light. In preferred embodiments, said first light source comprises an infra red (IR) or near infra red (NIR) light source or a white light source.

In preferred embodiments, the microscope includes an irradiation optical system comprising a second light source for irradiating said object. The irradiation optical system may be configured to direct light from said second light source onto said object through said first beam splitter. The second light source may be configured to produce light in a second frequency band, and wherein said first beam splitter is configured to transmit light in said second frequency band. Preferably, in said focus correction mode, said second light source is disabled. Typically, said second light source comprises at least one laser device.

In preferred embodiments, said first beam splitter is configured to transmit light in a frequency band corresponding to fluorescent light emanating from said object in use.

In preferred embodiments, the microscope includes a stage for receiving said object, wherein said focus adjustment system is operable to focus said test image at said at least one camera by effecting relative movement between said stage and said objective along an optical axis.

Typically, said at least one camera is a digital camera having a digital image sensor, and wherein said focus adjustment system is operable to focus said test image on said image sensor.

The microscope may include at least one sensor for sensing one or more environmental parameter of the microscope, said microscope being configured to adopt said focusing mode in response to input from said at least one sensor. Said at least one sensor may comprise at least one temperature sensor, and wherein said microscope is configured to adopt said focusing mode in response to said at least one temperature sensor detecting a change in temperature, or temperature gradient, greater than a threshold amount. Said at least one sensor may comprise at least one shock sensor and/or at least one vibration sensor, and wherein said microscope is configured to adopt said focusing mode in response to said at least one shock or vibration sensor detecting shock or vibration greater than a threshold amount.

The microscope may include at least one movement sensor for detecting movement of said objective or of the stage, said microscope being configured to adopt said focusing mode in response to said at least one movement sensor detecting movement of said objective or stage greater than a threshold amount.

Said microscope may be configured to adopt said focusing mode periodically, or in response to user input. Said focus adjustment system may be operable manually.

Typically, said focus adjustment system comprises means for effecting relative movement between the objective and the object.

Typically, said focus adjustment system comprises a controller programmed to perform contrast detection autofocusing.

In preferred embodiments, said at least one camera comprises a first camera, said imaging optical system being configured to image said object through said objective to said first camera along said first optical path, and wherein said imaging optical system is configured to image the projected test image from said object to said first camera through said objective and along said first optical path, said focus adjustment system being operable to focus said test image at said first camera. Optionally, said at least one camera comprises only said first camera.

Alternatively, said at least one camera comprises a first camera and a second camera, said imaging optical system being configured to image said object through said objective to said first camera along said first optical path, and wherein said imaging optical system is configured to image the projected test image from said object to said second camera through said objective and along part of said first optical path, said focus adjustment system being operable to focus said test image at said second camera. A beam splitter may be provided in said first optical path for reflecting a test image beam towards said second camera.

From a second aspect the invention provides a method of focus adjustment in a microscope having an imaging optical system for imaging an object to at least one camera through an objective along a first optical path, the method comprising:

projecting a test image onto said object through an objective;

imaging the projected test image from said object to said at least one camera through said objective; and

focusing said test image at said at least one camera.

Advantageously, the same objective and preferably also the same camera are used both for imaging and focusing. This arrangement allows reduction of the cost of the microscope in comparison with known microscopes that provide separate focusing systems. In preferred embodiments, the optical imaging system and camera are used to implement focus drift correction. This arrangement dispenses with the need for an additional dedicated imaging system for focus drift correction, which reduces the expense and complexity of the microscope.

DETAILED DESCRIPTION

Referring now to the drawings there is shown, generally indicated as100, a microscope embodying one aspect of the invention. The microscope100is an optical microscope, and in the illustrated embodiment is a spinning disk confocal microscope, although microscopes embodying the invention may be of other conventional types as would be apparent to a skilled person.

The microscope100includes a stage20for receiving an object55to be imaged. The object55typically comprises a slide9on which a specimen, for example a biological specimen, is located. The specimen is typically immersed in a medium16, e.g. water. A cover slide8may be placed over the specimen, as required.

The microscope100includes an imaging optical system30for imaging the object55to a camera14along an optical path. In particular, it is desired that the imaging optical system30focuses an image of the object55at a focal plane of the camera14. The imaging optical system30comprises a train of optical devices, typically comprising at least one lens and optionally at least one mirror, arranged to image the object55to the camera14, i.e. form an image of the object55at the camera14via the optical train. The imaging optical system30comprises a microscope objective7, preferably an infinity-corrected microscope objective. The preferred imaging optical system30also comprises a tube lens10, configured to form an intermediate image of the object55at, for example, a confocal spinning pinhole disk11. Optionally, a mirror19is provided between the objective7and the tube lens10, and is configured to cause the excitation beam to be correctly aligned to the optical axis of the objective7. In the illustrated embodiment, the imaging optical system30includes first and second image plane relay lenses13,13′ between the tube lens10and the camera14. Optionally, a mirror21is provided between the relay lenses13,13′, the mirror21being configured to cause a fluorescence beam to be optimally aligned to the optical axis of the second relay lens13′. In alternative embodiments (not illustrated) the imaging optical system may include any other suitable arrangement of lenses and, if required, mirror(s).

In preferred embodiments, the camera14is a digital camera having a digital image sensor22, for example a CCD sensor. The imaging optical system30images the object55to the image sensor22. More particularly, it is desired that the imaging optical system30focuses an image of the object55on the sensor22(wherein the image sensing surface of the sensor22is located at the focal plane of the imaging optical system30).

The microscope100includes a focus adjustment system35for adjusting the imaging optical system30and/or the stage20in order to focus an image of the object55at the camera14. The focus adjustment system35comprises means for effecting relative movement between the stage20and the objective7in an axial direction that corresponds to the optical axis of the objective7. In typical embodiments, the objective7is movable with respect to the stage20, and therefore the object55, in the axial direction. To this end, the objective7is carried by a movable support structure15, typically an objective turret. In the illustrated embodiment, the turret15, and therefore the objective7, is movable in the direction indicated by arrows A-A′. The turret15may include, or be coupled to, a drive system (not shown), for example a motorised drive system or a piezo-electric drive system, for moving the turret15in the direction A-A′. Any suitable conventional motorised drive system may be used. Movement of the objective7towards and away from the object55in the axial direction adjusts the focus of the image at the camera14. As such the movable objective assembly7,15provides part of the focusing system35. Typically, the stage20is stationary during focusing and the objective7moves relative to it. Alternatively, the stage20may be moved axially with respect to the objective7, in which case the objective7may be held stationary during focusing. More generally, either one or both of the objective7and the stage20may be movable axially towards and away from one another to adjust the focus.

The focus adjustment system35also includes a controller50for controlling movement of the objective7(and/or of the stage20as applicable) in order to focus the image at the camera14. The controller50may take any conventional form, typically comprising a suitably programmed processor, e.g. a microprocessor or microcontroller. The focus adjustment system is preferably configured to perform autofocusing of the image at the camera14. To this end, the camera14and/or the microscope100may include any conventional autofocusing means. For example, the controller50may be programmed to perform contrast detection autofocusing using any conventional contrast detection autofocusing algorithm. To perform contrast detection autofocusing, the controller50may monitor the intensity of light detected by at least some of the pixels of the sensor22and move the objective7to control, e.g. maximize, the detected light intensity in accordance with the contrast detection autofocusing algorithm. Alternatively, the camera14and/or the microscope100may be configured to perform phase detection autofocusing.

The microscope100includes an irradiation optical system45for irradiating the object55, and in particular the specimen included in the object55. The irradiation optical system45comprises a light source25, which in preferred embodiments comprises one or more laser devices, but may alternatively comprise any other suitable conventional light source, for example one or more LEDs, or one or more incandescent bulb. The light source25may be configured to produce light in one or more frequency bands as suits the application and as would be apparent to a skilled person. For example, in cases where the object55comprises a specimen that is capable of fluorescence (either because it is inherently capable of fluorescence, i.e. auto-fluorescence, or because one or more fluorescent markers (e.g. proteins or dyes) have been added to the specimen), the light source25may be configured to provide light in one or more frequency bands that excites the specimen/markers and causes fluorescence. In preferred embodiments, the irradiation optical system45is configured to irradiate the object55by directing light (laser beam65in the present example, which may comprise light at any one of a plurality of wavelengths corresponding to the fluorescence characteristics of the specimen/markers) to the object along at least part of the optical path defined by the imaging optical system30. In particular, the irradiation optical system45is configured to irradiate the object55through the objective7. To facilitate this, a beam splitter12is included in the imaging optical system30. The beam splitter12is configured to be transmissive to light in one or more frequency bands corresponding to the light produced by the laser device25. The laser device25is arranged to direct the laser beam65through the beam splitter12and onto the optical path whereupon it is directed to the object55through the objective7. The beam splitter12is configured to be reflective (or at least partly reflective) to light in one or more frequency band corresponding to light that is reflected from, or emitted from, the object55. The beam splitter12may be said to have one or more reflection band corresponding to light that is reflected from, or emitted from, the object55, and a transmission band corresponding to the light produced by the laser device25. In the illustrated embodiment, the beam splitter12is located between the tube lens10and the first relay lens13, and arranged to reflect light that passes through the tube lens10to the first relay lens13. Typically, the beam splitter12comprises a dichroic mirror.

In the illustrated embodiment, the microscope100is configured to perform spinning disk confocal laser microscopy and the irradiation optical system45includes a spinning disk11onto which the laser beam65is directed. The spinning disk11includes pinholes (not shown) and may be part of a spinning disk assembly that includes a corresponding spinning collector disk (not shown) with microlenses. The spinning disk11, or spinning disk assembly, acts as a scanner and causes the object55to be irradiated with an array of laser beams produced from the laser beam65. The spinning disk11is located at an intermediate image plane in the optical path of the imaging optical system30. In the illustrated embodiment, the spinning disk11is located between the tube lens10and the beam splitter12.

In alternative embodiments in which the microscope100is not a confocal spinning disk microscope, the spinning disk11may be omitted. In embodiments in which the microscope uses laser scanning to irradiate the object55, any other conventional laser scanning system may be provided. In other embodiments, the irradiation optical system45may be arranged to irradiate the object55from behind, i.e. through the stage20.

In typical embodiments, the object55includes a specimen that fluoresces (either by auto-fluorescence or by means of fluorescent markers (or labels) included in the specimen) when excited by the light from the irradiation optical system45. Therefore, when the microscope100operates in an imaging mode, it is fluorescent light emitted from the specimen that is imaged by the imaging optical system30to the camera14.

During operation of the microscope100, the focusing of the image at the camera by the imaging optical system30can drift for various reasons, including shock, vibrations, changes in temperature or thermal gradients in the microscope, or as a result of slippage in the focusing mechanism. The microscope100includes a projection optical system40which, together with the focus adjustment system35, can be used to correct focus drift.

The projection optical system40is configured to project a test image onto the object55through the objective7. As such the objective7is shared by the projection optical system40and the imaging optical system30.

The projection optical system40includes an optical projector42for projecting the test image. The optical projector42comprises a light source1, which in preferred embodiments is a near-infrared (NIR) light source, for example comprising one or more LEDs. The light source1may alternatively take other forms, for example a lamp or any conventional non-coherent light source. Advantageously, the light source1is configured to produce light in a frequency band that is different than, and preferably non-overlapping with, the frequency band of the light source25. In preferred embodiments, the light produced by the light source1is also a frequency band that is different than, and optionally non-overlapping with, the frequency band of the light emitted from the object55during imaging, i.e. the fluorescence of the specimen. The preferred arrangement allows the beam splitter6to be fully transmitting in the fluorescence band, but a mirror only for NIR. In alternative embodiments, the light source1may be configured to produce light in any convenient frequency band(s), including the same frequency band(s) as the light source25and/or the fluorescence of the specimen. Optionally, the light source1may be configured to produce white light. The light source1may comprise any suitable conventional light source, for example one or more LEDs, or one or more laser.

To create the test image, the projector42includes an aperture4, which may be formed in a plate4A or other convenient structure, through which light from the light source1passes. In alternative embodiments, the test image may be created by any other convenient means.

The projector42may include a collimating lens2for collimating the light from the light source1, typically located between the light source1and the aperture4. The projector42may also include an optical diffuser3, which may be located between the collimating lens2and the aperture4. A collimating lens5may also be provided in front (with respect to the direction of travel of the light) of the aperture4for collimating the light passing through the aperture4.

The projector42produces a test image beam59which is directed to the objective7in order to project the test image onto the object55. Advantageously, the test image beam59is directed to the objective7along part of the optical path defined by the imaging optical system30. To this end, a beam splitter6is included in the optical path of the imaging optical system30. The projection optical system40is configured to project the test image onto the object8,9via the beam splitter6. In particular, the projector42is arranged to direct the test image beam59onto the beam splitter6. The beam splitter6is configured to split the test image beam59into a first portion62and a second portion63, to direct the first portion62to the objective7in order to project the test image onto the object8,9. In preferred embodiments, the beam splitter6reflects the first portion62from a reflecting face61towards the objective7. The second portion63of the beam59is transmitted through the beam splitter6and may be directed to a beam dump64. Accordingly, the beam splitter6introduces the first portion62of the test image beam59onto the optical path towards the objective7. As such, the part of the optical path between the splitter6and the object8,9, including the objective7, is shared by the projector optical system40and the imaging optical system30.

The beam splitter6is configured to split light in a frequency band corresponding to the light59projected by the projector42. This frequency band may be referred to as the reflection band. The beam splitter6splits the light59such that the first portion62is reflected and the second portion63is transmitted. Preferably, the beam splitter6is configured to cause a50/50split between the portions62,63. In the illustrated embodiment, the beam splitter6is located between the tube lens10and the objective7. Typically, the beam splitter6comprises a dichroic mirror. More generally however, the beam splitter6may be any optical element that reflects any portion of the light59to the objective7, while allowing at least some of the light reflected by or emitted from the object55to pass through it. For example, the beam splitter6may comprise a window with a suitable anti-reflective coating.

The beam splitter6is configured to be transmissive to light in a frequency band corresponding to the light produced by the laser device25. In the present context transmissive may be taken as meaning that approximately 90% or more of the relevant light is transmitted, although this definition is not limiting to the invention. This frequency band may be referred to as the first transmission band. In preferred embodiments, the beam splitter6is also transmissive to light that is emitted from the object55during imaging, i.e. the fluorescence of the specimen. This frequency band may be referred to as the second transmission band. The first and second transmission bands may be separate from each other, or may wholly or partly overlap, depending on the requirements of the application. For example, the first and second transmission bands may be within a single transmission band of the beam splitter6, or the beam splitter6may support separate transmission bands corresponding to the first and second transmission bands. The reflection band may or may not overlap with the transmission band(s), as suits the application.

The irradiation optical system45is configured to direct light from the laser device onto the object55through the beam splitter6. The beam splitter6is located in the optical path such that the laser beam65impinges upon the beam splitter6, and passes though the beam splitter6because the beam splitter6is transmissive to the laser light.

The microscope100is operable in an imaging mode in which the object55is imaged to the camera14by the imaging optical system30. In the imaging mode, the irradiation optical system45irradiates the object55with the laser beam65. In preferred embodiments, and as can best be seen fromFIGS. 2 and 3, the laser beam65is transmitted through the beam splitter12and the spinning disk11(when present), and is also transmitted through the beam splitter6, after which it reaches the objective7and is directed onto the object55. In applications where the object55comprises a fluorescent specimen, the laser beam65acts as an excitation beam that excites fluorescence of the specimen. The resulting fluorescent light651is gathered by the objective7, directed along the optical path and imaged at the camera14. In preferred embodiments, and as can best be seen fromFIGS. 4 and 5, the fluorescent beam651is transmitted through the beam splitter6and the spinning disk11(when present), and is reflected by the beam splitter12, directing it to the relay lenses13,13′ and subsequently to the camera14to create an image of the object55at the camera14. During imaging, the imaging optical system30may focus the image at the camera14by any conventional means, including the focus adjustment system35.

When the microscope100is in the imaging mode, the projection optical system42is optionally disabled such that it does not project the test image onto the object55. For example the projector42may be switched off or otherwise disabled (e.g. deflected or diverted) such that it does not direct the test image beam59onto the beam splitter6.

In order to allow any focus corrections that may be required, the preferred microscope100is operable in a focus correction mode. In the focus correction mode, the projection optical system40projects the test image onto the object55, and the imaging optical system30images the projected test image from the object55to the camera14. The focus adjustment system35is operated to focus the test image at the camera14, thereby correcting the focus as required.

In preferred embodiments, and as can best be seen fromFIG. 6, the test image beam59is split by the beam splitter6such that a portion62of it is directed to the objective7thereby projecting the test image onto the object55. The portion62of the test image beam59is reflected from the object55back to the objective7to allow it to be imaged by the imaging optical system30. Reflection may occur at one or more interfaces of the object55where a refractive index discontinuity exists.

FIGS. 7A, 7B and 7Cillustrate how the portion62of the test image beam59may be reflected from the object55to allow it to be imaged to the camera14. InFIG. 7A, it is assumed that the medium between the objective7and the object55is air. The test image beam portion62is reflected at the interface between the air and the obverse face of the cover slip8(illustrated by reflected light621), and also at the interface between the reverse face of the cover slip8and the specimen medium16(illustrated as reflected light622). InFIG. 7B, it is assumed that the medium17between the objective7and the object55is water. The test image beam portion62is reflected at the interface between the water17and the obverse face of the cover slip8(illustrated by reflected light623), and also at the interface between the reverse face of the cover slip8and the specimen medium16(illustrated as reflected light622). InFIG. 7C, it is assumed that the medium17between the objective7and the object55is oil. The test image beam portion62is reflected at the interface between the reverse face of the cover slip8and the specimen medium16(illustrated as reflected light622). More generally, depending on the type of objective7, and on the composition of the object55, the test image beam59may be reflected at one or more interfaces of the object55, in particular any interface where there is a change of refractive index. Commonly, for objects55having a cover slip8, the most detectable reflection occurs at the interface at the obverse surface of the cover slip8(as illustrated by reflections621and623).

In any case, the reflected light621,622,623(as applicable) is gathered by the objective7and imaged to the camera14along the optical path as a reflected test image beam. In preferred embodiments, and as can best be seen fromFIG. 8, the reflected test image beam621,622,623impinges upon the beam splitter6which splits the reflected test image beam621,622,623such that a portion625of it is transmitted though the beam splitter6and continues along the optical path. Another portion624of the reflected test image beam is reflected by the beam splitter6and is diverted from the optical path, typically towards the projector42. In preferred embodiments, and as can best be seen fromFIG. 5, the transmitted portion625of the reflected target image beam is transmitted through the spinning disk11(when present), and is reflected by the beam splitter12, directing it to the relay lenses13,13′ and subsequently to the camera14to create an image of the target image at the camera14. The focus adjustment system35may be operated to focus the test image at the camera14based on any one or other of the reflected light components621,622,623, or any other light reflected by the object55, as desired.

The microscope100may be caused to adopt the focus correction mode (typically by the controller50) periodically and/or in response to one or more detected events including any one or more of: a detected change in temperature or temperature gradient in the microscope100above a threshold amount; vibration above a threshold level; mechanical shock above a threshold level; mechanical drift in the focusing mechanism, e.g. movement of the turret15. To this end, one or more conventional sensors51(e.g. temperature sensor(s), vibration sensor(s), movement sensor(s) and/or shock sensor(s)) may be included in the microscope100. The sensor(s)51may provide signals to the controller50in response to which the controller50may determine whether or not to cause the microscope100to adopt the focus correction mode. If imaging is being performed, the microscope100may switch to the focus correction mode and then back to the imaging mode once focus correction has been performed. Alternatively, or in addition, the microscope100may be operable manually by a user to adopt the focus correction mode.

Optionally, the microscope100may be operable in a manual focusing mode in which a user can manually set the focus of the microscope100using the projection optical system40. To this end, the microscope100may be configured to allow the user to manually move the objective7(and/or stage20as applicable) in order to focus the test image at the camera. The microscope100may be operable in a user-initiated auto-focusing mode in which the microscope100, in response to activation by the user, operates the projection optical system40and focuses the test image at the camera. The microscope100may subsequently adopt the imaging mode after focusing is complete, or in response to user input.

When the microscope100is in any of its supported focusing modes (which in preferred embodiments include any one or more of the focus correction mode, the manual focusing mode or the user-initiated auto-focusing mode), the irradiation optical system45is optionally disabled such that it does not irradiate the object55. For example the laser device25may be switched off or otherwise disabled, e.g. deflected or diverted.

In preferred embodiments, an emission filter23A is provided in the optical path between the beam splitter12and the camera14for preventing any portion of the light65from light source25that is reflected by the beam splitter12towards the camera14from reaching the camera14. For example, the emission filter may be configured to pass fluorescent light emitted from the object55but to block light in the frequency band(s) emitted by the light source25. During focusing, unless the emission filter includes a pass band corresponding to the wavelength of the light from the projector system40, the emission filter is removed from the optical path. Conveniently, the emission filter23A is provided in a filter selector device23that is operable to move the emission filter into and out of the optical path as required. The filter selector device23may comprise a rotatable wheel. The filter selector device23may include an optically transparent window23B, or a suitable filter, or a void, that may be moved into alignment with the optical path during the relevant focusing mode.

In preferred embodiments, the microscope100operates in either the imaging mode, or any one of its supported focusing modes, but does not perform imaging and focusing simultaneously. When focusing, the microscope100uses the projection optical system40to focus the test image at the camera14, either automatically or with user input as applicable, as described above. The result of the focusing is to cause the microscope100to adopt a reference focus state. In the reference focus state there is a reference axial distance between the objective7and the object55, or more particularly between the objective7and whichever interface of the object55was used to perform the focusing. In some embodiments, during the imaging mode, the objective7is moved relative to the stage20in the axial direction, or vice versa, with respect to the reference focus state. For example in embodiments where the microscope100is a scanning microscope, the scanning that is performed during the imaging mode may involve relative axial movement of the objective7and the stage20with respect to the reference focus state.

FIG. 9shows an alternative embodiment of the microscope100′ in which like numerals are used to denote like parts and in which the same or similar description applies as is provided in respect ofFIGS. 1 to 8unless otherwise indicated. The microscope100′ differs from the microscope100in that it includes a second camera27. The second camera27is used to focus the test image in any of the supported focusing modes. To facilitate this, a beam splitter24is included in the imaging optical system30. The beam splitter24is configured to be transmissive to light in one or more frequency band corresponding to the light that emanates from the object55during the imaging mode (typically the fluorescence of the specimen), and to be reflective (or at least partly reflective) to light in one or more frequency band corresponding to light that is reflected from the object55during focusing, i.e. the light originating from the light source1. The beam splitter24is provided in the optical path of the imaging optical system30and arranged to reflect the relevant light to the second camera27, away from the optical path to the first camera. The beam splitter24may be a dichroic mirror.

The beam splitter24may be located between the first and second relay lenses13,13′. A third relay lens13″ may be provided between the beam splitter24and the second camera27, and may be arranged to be co-operable with the first relay lens13during focusing.

In the imaging mode, the first camera14is used to image the object55as described above. In any of the supported focusing modes, the test image reflected from the object55is imaged to and focused at the second camera25. This may be achieved in the same manner as described above in relation to the embodiment ofFIGS. 1 to 8.

In preferred embodiments, the same objective7and preferably also the same camera14are used both for imaging and focusing. This arrangement allows reduction of the cost and size of the microscope in comparison with known microscopes that provide separate focusing systems.

The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present invention. It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described herein above. In addition, unless mention was made above to the contrary, it should be noted that all of the accompanying drawings are not to scale. A variety of modifications and variations are possible in light of the above teachings without departing from the scope and spirit of the invention, which is limited only by the following claims.