Patent Description:
Light sheet fluorescence microscopy (LSFM) employs an illumination optical system to produce a thin sheet of laser illumination to excite fluorescence from a thin plane within the specimen. Fluorescence is then collected using a second detection optical system aligned to image the plane that is illuminated.

LSFM has the advantage that it causes minimal photobleaching (light induced destruction of fluorophores) and phototoxicity (light induced killing of the sample) away from the plane that is currently being imaged. It also has the advantage that - in its simplest form - it requires no moving parts or image processing to acquire an optically sectioned image; this makes it able to acquire optically sectioned images at high frame rates that are only limited by the frame rate of the camera employed.

Conventional LSFM employs two microscope objectives oriented at <NUM>° with respect to one another. The first is used to provide the light sheet illumination and the second is used to collect the resulting fluorescence. The requirement to use two microscope objectives that are placed close to the sample complicates the sample preparation and mounting and makes it harder to use conventional sample mounting methods such as microscope coverslips and multiwell plates.

As described by the present inventor in <CIT> and [<NUM>], and as illustrated in the present <FIG>, oblique plane microscopy (OPM) uses a high numerical aperture microscope objective to provide both illumination and detection for LSFM. The high numerical aperture objective (O1) is used to provide an illumination sheet that is tilted with respect to the optical axis, and correction optics (TL2, O2, O3, TL3) placed after the first microscope (O1 and TL1) are used to tilt the detection plane so that it co-aligns with the illuminated plane. The correction optics are achieved by placing a second microscope (TL2 and O2) at the output of the first microscope (O1 and TL1) in order to produce an intermediate image that is close to the actual size of the sample. Together, the first and second microscopes are designed to ensure that the lateral and axial magnifications are equal in the intermediate image and therefore that the intermediate image is not distorted (stretched) compared to the object. A tilted plane in the intermediate image - conjugate to that of the illumination plane - can then be imaged onto a camera by using a third microscope (O3 and TL3) placed at an angle to the optical axis of the first two microscopes.

As well as achieving LSFM with a single microscope objective for illumination and collection of fluorescence from the sample, OPM also enables rapid remote scanning of the plane being imaged, thus enabling high speed volumetric (3D) imaging. This remote scanning has been achieved by adjusting the axial position of the remote objective O2 [<NUM>]. It is also possible to introduce additional optics to an OPM system to provide lateral scanning of the light sheet in a method called swept confocally-aligned planar excitation (SCAPE) microscopy [<NUM>], as illustrated in the present <FIG> and described in <CIT>.

Both pre-existing OPM and SCAPE require quite a long physical optical path in order to accommodate the three microscopes placed one after the other. In addition, both OPM and SCAPE require the remote objectives (O2 and O3) to have high numerical apertures and be able to be placed at a significant angle (typically <NUM>-<NUM>°) to one another. This condition can be difficult to achieve as their focal planes of O2 and O3 must also intersect at the point of intersection of their respective optical axes.

There is a desire to improve the optical configuration in OPM, including making the arrangement of the beam paths more compact.

There is also a desire to be able to implement scanning in OPM, to enable 3D imaging, in an efficient and reliable manner that does not involve adjusting the axial position of the remote objective O2.

The present invention provides a new (so-called "folded") OPM configuration that enables a more compact arrangement of the beam paths to be achieved, whilst also enabling scanning of the illumination sheet and field of view.

More particularly, the present invention provides an optical arrangement for oblique plane microscopy as defined in Claim <NUM> of the appended claims. Details of certain embodiments are set out in the dependent claims. Also provided is a method of performing oblique plane microscopy as defined in Claim <NUM>.

Thus, according to a first aspect of the present invention there is provided an optical arrangement for oblique plane microscopy, the optical arrangement comprising: an illumination generator arranged to provide a beam of illumination through a first objective lens to illuminate or excite an oblique plane of a sample in use, wherein the first objective lens is also arranged to receive a beam of emitted light from the oblique plane of the sample in use; first and second relay lenses and a second objective lens sequentially arranged to receive the beam of emitted light from the first objective lens and to form, at the focal plane of the second objective lens, an intermediate image having a tilted plane conjugate to that of the oblique plane of the sample; a first mirror located at said focal plane of the second objective lens, arranged to receive and reflect the beam of emitted light; a third relay lens; and an image detector; wherein the second objective lens and the third relay lens are arranged to relay the intermediate image from the first mirror to the image detector; wherein the first mirror is also arranged to receive the beam of illumination from the illumination generator and to reflect the beam of illumination through the second objective lens; and wherein the optical arrangement further comprises a beam splitter disposed between the second objective lens and the second relay lens, the beam splitter being configured to: (i) direct the beam of illumination from the second objective lens to the second relay lens, and thence to the first relay lens, the first objective lens and the sample; (ii) direct the beam of emitted light from the second relay lens to the second objective lens, and thence to the first mirror; and (iii) direct the reflected beam of emitted light from the second objective lens to the third relay lens and thence to the image detector.

As a consequence of the first mirror being arranged to receive the beam of illumination from the illumination generator and to reflect the beam of illumination through the second objective lens, as well as the first mirror reflecting the emitted light from the sample, and by virtue of the operation of the abovementioned beam splitter, this provides an improved OPM configuration in which the arrangement of the beam paths is more compact. Moreover, by essentially reusing the second objective lens (which has a high numerical aperture) for both the emission and illumination beams, the optical arrangement is not constrained by needing to situate two separate objective lenses (O2 and O3 in <FIG> and <FIG>) close to one another.

Preferably the first mirror is a translatable mirror, operable to cause the plane of imaging to be scanned through the sample in use. This advantageously enables 3D imaging to be carried out in an efficient and reliable manner that does not involve adjusting the axial position of the remote objective. Rather, using the present technique, scanning can be achieved by moving the first mirror in any direction that has a component parallel to the mirror normal. Such translation of the first mirror can be performed rapidly, not least since a single mirror is very light in weight in comparison to an objective lens. Further benefits of scanning by translating the first mirror - including that the illumination beam can be scanned across the sample in synchrony with the scanned detection plane - are set out below.

The beam splitter may comprise a non-polarising beam splitter. Preferably, though, the beam splitter may comprise a polarising beam splitter together with a quarter-wave plate, to increase the optical throughput compared to a non-polarising beam splitter.

A half-wave plate and/or quarter-wave plate (or alternative retarder or retarder combination) may also be provided between the illumination generator and the first mirror, to adjust the polarisation state of the illumination light to maximise its reflection from the beam splitter used, and thereby maximise throughput to the sample.

Optionally a second mirror may be provided in the illumination beam path, closely before the first mirror, so as to avoid clipping of the illumination beam, e.g. by the front face of the second objective lens, so as to increase the numerical aperture of the illumination beam and to decrease the achievable illumination sheet waist size in use.

In certain embodiments the beam splitter may be configured such that the beam of emitted light is reflected through the beam splitter on its path to the first mirror.

In other embodiments the beam splitter may be configured such that the beam of emitted light is transmitted through the beam splitter on its path to the first mirror.

Advantageously the first mirror may be rotatable about the optical axis of the second objective lens, thereby enabling the angle of the oblique plane of imaging to be rotated.

For example, the optical arrangement may further comprise a rotatable optical subassembly which includes the first mirror. The components of the rotatable optical subassembly may be mounted on a common rotatable stage, to facilitate rotation.

Alternatively, or in addition, the illumination may be provided by the illumination generator via an optical fibre. An optical fibre rotating coupler may be disposed between the optical fibre and the first mirror, to enable the mirror to be rotated without undesirable twisting of the optical fibre.

In certain embodiments the first mirror may be rotatable to enable the selection of one of two illumination beam paths. Alternatively, the first mirror may be one of two mirrors orientated in different directions, the said two mirrors being translatable with at least a component of their motion being perpendicular to the optical axis of the second objective lens. The said two mirrors may or may not be commonly mounted.

Optionally the optical arrangement may further comprise a bulk optical circulator, to increase the optical collection efficiency. The optical circulator may include a half-wave plate placed so as to only act on the illumination beam.

To minimise vibrations during rapid translation of the first mirror, the first mirror may be coupled to a compensating mass, the compensating mass having the same mass as the first mirror, and arranged to oscillate in antiphase with the first mirror and with the same amplitude as the first mirror. For example, the first mirror and compensating mass may form part of a flexure stage, the flexure stage also having an actuator operable to drive the first mirror and the compensating mass.

Optionally the optical arrangement may further comprise means for generating an activation beam to cause photoactivation and/or photoconversion in a portion of the sample. The activation beam may be arranged to pass through the second objective lens and be reflected by the first mirror back through the second objective lens and thence along the path taken by the emitted light from the sample to the second objective lens in reverse.

According to a second aspect of the present invention there is proved a method of performing oblique plane microscopy, the method comprising: providing a beam of illumination through a first objective lens to illuminate or excite an oblique plane of a sample, wherein the first objective lens is also arranged to receive a beam of emitted light from the oblique plane of the sample; sequentially using first and second relay lenses and a second objective lens to receive the beam of emitted light from the first objective lens and to form, at the focal plane of the second objective lens, an intermediate image having a tilted plane conjugate to that of the oblique plane of the sample; using a first mirror located at said focal plane of the second objective lens to receive and reflect the beam of emitted light; and relaying the intermediate image from the first mirror to an image detector using the second objective lens and a third relay lens; wherein the first mirror also receives the beam of illumination from the illumination generator and reflects the beam of illumination through the second objective lens; and wherein the method further comprises using a beam splitter disposed between the second objective lens and the second relay lens to: (i) direct the beam of illumination from the second objective lens to the second relay lens, and thence to the first relay lens, the first objective lens and the sample; (ii) direct the beam of emitted light from the second relay lens to the second objective lens, and thence to the first mirror; and (iii) direct the reflected beam of emitted light from the second objective lens to the third relay lens and thence to the image detector.

The present disclosure also provides an optical arrangement for oblique plane microscopy, comprising a bulk optical circulator.

The present disclosure also provides a flexure stage for use in microscopy or spectrometry, the flexure stage comprising: a translatable mirror coupled to a compensating mass, the compensating mass having the same mass as the mirror, and arranged to oscillate in antiphase with the mirror and with the same amplitude as the mirror; and an actuator operable to drive the mirror and the compensating mass.

The present disclosure also provides an optical arrangement for oblique plane microscopy, comprising means for generating an activation beam to cause photoactivation and/or photoconversion in a portion of the sample, wherein the activation beam is arranged to pass in a reverse direction along at least part of the path taken by the emitted light from the sample.

Embodiments of the invention will now be described, by way of example only, and with reference to the drawings in which:.

In the figures, like elements are indicated by like reference signs throughout.

The present embodiments represent the best ways known to the Applicant of putting the invention into practice. However, they are not the only ways in which this can be achieved.

The present work provides an improved implementation of oblique plane microscopy (OPM) from that of <CIT>, where the beam path of the emitted light (fluorescence from the sample) is now "folded" about a mirror placed at the focal plane of the second OPM microscope objective, O2. If desired, scanning of the imaged plane can be achieved by translating the fold mirror. Advantageously, the illumination light is coupled in via this mirror so that the illumination beam can be scanned across the sample in synchrony with the scanned detection plane. Amongst other things, this approach has the following benefits:.

A first embodiment of a "folded" OPM configuration, that provides a new beam scanning mechanism and enables a more compact beam path, is illustrated in <FIG>.

In brief, in the new "folded" configuration, the light sheet illumination pattern (generated by illumination generator <NUM>) is produced at mirror M1 (via lens L1) and is then relayed to the sample S via so-called "microscope <NUM>" (objective lens O2 and relay lens TL2) and "microscope <NUM>" (relay lens TL1 and objective lens O1). The illumination generator <NUM> may provide any number of light sheet illumination beams, e.g. Gaussian, scanned Bessel, Airy, or lattice light sheet.

Emitted light (fluorescence) from the sample S is then relayed back to mirror M1 via "microscope <NUM>" (objective lens O1 and relay lens TL1) and "microscope <NUM>" (relay lens TL2 and objective lens O2). The emitted light reflects off mirror M1 and is then collected back into objective lens O2 and relayed to form an image of a tilted plane in the sample on the camera <NUM> by a third microscope ("microscope <NUM>") formed by objective lens O2 and relay lens TL3. A beam splitter is used to separate the beam paths to the microscope and the camera and may comprise a non-polarising or polarising beam splitter. The beam splitter PBS1 shown in <FIG> is a polarising beam splitter that, when combined with quarter-wave plate QWP1, increases the optical throughput compared to a non-polarising beam splitter.

In more detail, still with reference to <FIG>, the presently-disclosed arrangement <NUM> for oblique plane microscopy comprises an illumination generator <NUM> arranged to provide a beam of illumination (e.g. laser light, illustrated using broken lines) through the first objective lens O1, to illuminate or excite an oblique plane (shaded in the figure) of the sample S in use. The first objective lens O1 is also arranged to receive a beam of emitted light (e.g. fluorescence, illustrated using solid lines) from the oblique plane of the sample S in use. As described in <CIT>, only a part of the available numerical aperture of the first objective lens O1 is used to image the sample S at an oblique angle, while another part of the first objective lens O1 is used to illuminate the sample S at a different angle.

First and second relay lenses TL1, TL2 and the second objective lens O2 are sequentially arranged to receive the beam of emitted light from the first objective lens O1.

As illustrated, the first objective lens O1 and the first relay lens TL1 form the first microscope (so-called "microscope <NUM>") arranged to produce a magnified image of the oblique plane of the sample, and the second relay lens TL2 and the second objective lens O2 form the second microscope ("microscope <NUM>") arranged to demagnify the magnified image and thereby form, at the focal plane of the second objective lens, an intermediate image having a tilted plane conjugate to that of the oblique plane of the sample. As those skilled in the art will appreciate, to achieve the optical relay effect, the second relay lens TL2 may be separated from the first relay lens TL1 by the sum of their focal distances.

The optical arrangement <NUM> further comprises the mirror M1 (also referred to as the "first" mirror herein) located at said focal plane of the second objective lens O2, arranged to receive and reflect the beam of emitted light; the third relay lens TL3; and the image detector <NUM>. The intermediate image produced by microscope <NUM>, having a tilted plane conjugate to that of the oblique plane of the sample, is formed in the plane of the first mirror M1.

The second objective lens O2 and the third relay lens TL3 form the third microscope ("microscope <NUM>") arranged to relay the intermediate image from the first mirror M1 to the image detector <NUM>. In the illustrated embodiment the image detector <NUM> is a charge-coupled device (CCD), although in other embodiments alternative image detectors or cameras may be used.

Significantly, the first mirror M1 is also arranged to receive the beam of illumination from the illumination generator <NUM> and to reflect the beam of illumination through the second objective lens O2.

The optical arrangement <NUM> further comprises a beam splitter (in this case, polarising beam splitter PBS1 together with quarter-wave plate QWP1) disposed between the second objective lens O2 and the second relay lens TL2, the beam splitter being configured to:.

For the sake of clarity, the enlarged inset <NUM> in <FIG> (and in some of the subsequent figures) shows that the beams passing through the second objective lens O2 are, in sequence, as follows:.

Ideally, the second objective lens O2 has a high numerical aperture, i.e. a numerical aperture that is sufficiently high such that it does not restrict or reduce the numerical aperture of the first and second microscopes, and accommodates the arrangement of rays <NUM>, <NUM> and <NUM> as shown in the enlarged inset <NUM> in <FIG>. More particularly, as illustrated, the extent of the numerical aperture of O2 needs to encompass ray <NUM> to one side and ray <NUM> to the other side (with ray <NUM> being between the two).

As a consequence of the first mirror M1 being arranged to receive the beam of illumination from the illumination generator <NUM> and to reflect the beam of illumination through the second objective lens O2, as well as the first mirror M1 reflecting the emitted light from the sample S, and by virtue of the operation of the abovementioned beam splitter, this improves the optical performance of the OPM (as quantitatively evaluated below) and also makes the arrangement of the beam paths more compact.

Optical arrangements according to the present work may be used to image static samples, or may be employed to image particles or cells flowing through the oblique image plane, e.g. in a microfluidic device. Such particles or cells may be intentionally flowed through the oblique image plane, as part of the imaging procedure.

Image contrast may arise from fluorescent light excited at one or more wavelengths and detected in corresponding detection bands at longer (for single photon excitation) or shorter (for multiphoton excitation) wavelengths than each excitation wavelength. Alternatively, the image contrast may arise from differences in the fluorescence lifetime of the sample.

In practice, any one of the optical arrangements described herein may be integrated in a microscope, or provided as a "bolt-on" attachment for an existing microscope.

To evaluate the improvements afforded by the present work, the pre-existing OPM setup (from <CIT>) was analysed using the following microscope objectives: O1, 60x/<NUM> NA; O2, 50x/<NUM> NA; O3, 40x/<NUM> NA. The overall effective NA of this system is <NUM> and the collection efficiency (fraction of light collected over the range of angles subtended by a hemisphere) is <NUM>% if the OPM angle (angle between the optical axis of microscopes <NUM>&<NUM> and microscope <NUM> - see <FIG> of <CIT>) is <NUM>°.

On the other hand, in the new "folded" OPM configuration of the present work, the specification of O2 and O3 become the same - as it is the same physical objective - and so therefore O3 is effectively formed by a 50x/<NUM> NA microscope objective, i.e. the new configuration allows the numerical aperture of O3 to be greatly increased. There is a <NUM>% loss in signal imposed by the use of the polarising beam splitter cube PBS1 but, despite this, the new folded configuration has an effective NA of <NUM> and a collection efficiency of <NUM>%, so both parameters exceed those of the original OPM system. Therefore, the new folded OPM system is more compact and has improved spatial resolution compared to the pre-existing setup, and also provides an improved optical fluorescence collection efficiency.

Advantageously and conveniently, the new "folded" OPM configuration also enables the plane of imaging to be scanned rapidly through the sample by translating mirror M1, thereby enabling 3D imaging to be performed. Such scanning may be implemented using a suitable actuator coupled to the mirror M1. Such an actuator may be computer controlled, as those skilled in the art will appreciate. For the purpose of supporting the translated mirror M1 whilst minimising vibrations, the present work also provides a flexure stage design with a compensating mass, as described below with reference to <FIG>.

The direction of motion of the remote scanning is defined by the angle of mirror M1 with respect to the optical axis of the second objective lens O2, so remote scanning can be achieved if mirror M1 is moved in any direction that has a component parallel to the mirror normal. Some possible directions of motion for mirror M1 are indicated by arrows A1, A2 and A3 in <FIG> and the subsequent figures. As illustrated, arrow A1 indicates movement of mirror M1 forwards or backwards along the optical axis of the second objective lens O2, arrow A2 indicates movement of mirror M1 forwards or backwards in a direction perpendicular to the optical axis of the second objective lens O2, and arrow A3 indicates movement of mirror M1 forwards and backwards in a direction parallel (or substantially parallel) to the normal of mirror M1.

In order to maximise the transmission of the illumination light through the system, the polarisation state of the illumination light should be adjusted to maximise its reflection from the beam splitter used. In the case of a polarising beam splitter as shown in <FIG>, quarter-wave plate QWP2 can be inserted to provide the polarisation state with maximum throughput to the sample.

As shown in <FIG>, in the case that the working distance of the second objective lens O2 limits the achievable numerical aperture for the illumination beam, hence limiting the minimum sheet waist size, then a small stationary second mirror M2 can be incorporated close to the first mirror M1 and the second objective lens O2 to overcome this and avoid clipping of the illumination beam.

The beam splitter can be configured so that fluorescence emitted from the sample either reflects (as in <FIG>) or transmits (as in <FIG>) through the beam splitter on its path to mirror M1.

More particularly, in the configuration of <FIG>, the fluorescence emitted from the sample is reflected by the polarising beam splitter PBS1 and then transmitted through the quarter-wave plate QWP1 before being focused by the second objective lens O2 onto the mirror <NUM>. Then, the reflected fluorescence from the mirror M1 passes back through the second objective lens O2 and the quarter-wave plate QWP1 and is transmitted through the polarising beam splitter PBS1 towards the detector <NUM>.

On the other hand, in the configuration of <FIG>, the fluorescence emitted from the sample is transmitted through the polarising beam splitter PBS1 and the quarter-wave plate QWP1 before being focused by the second objective lens O2 onto the mirror <NUM>. Then, the reflected fluorescence from the mirror M1 passes back through the second objective lens O2 and the quarter-wave plate QWP1 and is then reflected by the polarising beam splitter PBS1 towards the detector <NUM>.

Alternatively, PBS1 in <FIG> may be rotated by <NUM>° about the optical axis of O1, TL1, TL2 and O2 such that the beam reflected by PBS1 is emitted vertically into (or out of) the plane of the figure.

In the case of isotropically distributed fluorophore dipoles, the configuration shown in <FIG>, or a configuration as shown in <FIG> where the PBS is rotated by <NUM>° about the optical axis of O1, TL1, TL2 and O2, will lead to a higher fluorescence collection efficiency due to fluorescence anisotropy effects [<NUM>].

The angle of the oblique plane of imaging with respect to the plane of the page can be rotated by rotating mirror M1 about the optical axis of the second objective lens O2. This can be achieved for example by means of the configuration shown in <FIG>, which includes a rotatable optical subassembly <NUM> (the dashed box). The rotatable optical subassembly <NUM> includes mirror M1, and also further mirrors M2, M3 and M4, and lens L1. The components of the rotatable optical subassembly <NUM> may be mounted on a common rotatable stage. Such an arrangement enables a set of images or image volumes to be acquired from the sample with different light sheet angles that can then be fused together in software, e.g. to produce a more isotropic system point spread function.

<FIG> illustrates a variant of the setup of <FIG>, and shows that, if the illumination light is delivered via an optical fibre F, then an optical fibre rotating coupler R can be used to allow the mirror M1 and illumination optics (including mirrors M2, M3 and M4, and cylindrical lens C1) to be rotated without undesirable twisting of the optical fibre F. This advantageously enables the angle of illumination and detection to be changed with respect to the optical axis. The rotating fibre optic coupler R could for instance consist of two plane polished fibre ends placed in close proximity with the intermediate gap filled by a refractive index matching fluid. The collimation and illumination sheet generation optics (within the brace <NUM> in <FIG>), including cylindrical lens C1, are then all rotated together.

Other well-known types of illumination optics to produce Gaussian, Bessel, Airy or other beam profiles can be used.

Alternatively, as illustrated in <FIG>, two separate illumination beam paths - formed either by M4, M3, L1 and M2, or by M5, L2 and M6 - can be used. The mirror M1 is rotatable to select one of the two illumination beam paths, i.e. via either M2 or M6. The axial position of the image plane can be controlled by the component of the translation of mirror M1 in the direction normal to the mirror M1, and one of the two illumination beam paths can be selected by rotating mirror M1 through <NUM>° about the optical axis of the first and second microscopes.

By being able to change between the two illumination beam paths, images of the sample may be acquired from two different directions that can then be fused together in software to produce a near-isotropic spatial resolution. This is particularly important when performing quantitative analyses of cell morphology as non-isotropic resolution leads to many unwanted sources of bias.

Another alternative is that shown in <FIG>, where two small mirrors M1 and M2, orientated in different directions, can be translated perpendicular to the optical axis of the second objective lens O2 (i.e. forwards and backward in the direction of arrow A2), or with at least a component of their motion being perpendicular to the optical axis of the second objective lens O2. Fine translation of the mirrors M1 and M2 allows the height of the tilted illumination and image plane to be chosen, whereas coarse translation, to select the use of either M1 or M2 at the focus of the second objective lens O2, allows one of two different tilt angles to be chosen and to utilise illumination light from either L1 or L2 respectively.

The mirrors M1 and M2 could also be translated in multiple directions, with one actuation to switch between M1 and M2, and another direction to perform scanning while acquiring an image volume with one of the mirrors in use.

For ease of translation the mirrors M1 and M2 may be commonly mounted, e.g. on a translatable stage or other mount, thereby enabling them to be translated together, although in other embodiments they may not be commonly mounted.

Additional mirrors at different angles may be added to further increase the number of illumination/detection angles and volumes that can be imaged.

In an alternative embodiment shown in <FIG>, the mirrors M1 and M2 may be illuminated by a common lens L1 and the beam switched between the different beam paths by the use of a galvo mirror GM1.

In order to increase the optical collection efficiency of configurations where the emitted fluorescence double-passes the second objective lens O2, a bulk optical circulator configuration as illustrated in <FIG> can be employed, the bulk optical circulator configuration being formed by PBS1, PBS2, M2, M3, FR1, FR2, HWP1 and HWP2. FR1 and FR2 are Faraday rotators, which rotate the light differently depending on which direction the light is passing through. HWP1 and HWP2 are half-wave plates. In the configuration shown in <FIG>, which does not represent an embodiment of the present invention, a separate illumination beam path is provided via dichroic filter D1. The illumination generation optics are configured to provide axial scanning of a tilted light sheet and to match the scanning of the tilted plane in the emission path provided by translation of M1.

Preferably, the illumination optics provide the ability to tilt the angle of the illumination light sheet to match the image plane tilt angle determined by the angle of mirror M1, to translate the illumination light sheet perpendicular to the plane of the light sheet, and also for the axial position of the illumination beam waist to be translated so as to follow the centre of the field of view of the region imaged by the detection beam path as mirror M1 is translated.

To achieve the above, a synchronisation controller <NUM> may be provided, controllably coupled to an adjustable telescope and scanner <NUM> (within the illumination path from the illumination generator <NUM>) and to an actuator provided
for translating mirror M1, to control the scanning of the illumination light sheet and to synchronise it with translation of the mirror M1. The synchronisation controller <NUM> may be provided by a suitably programmed computer.

As illustrated in <FIG>, the principle of using a bulk optical circulator can be applied to a system in which the excitation light is coupled in via mirror M1, in the manner of the embodiments of <FIG>. In this case, the optical circulator needs to be modified to allow additional polarization optics (half-wave plate HWP3 in the configuration shown in <FIG>) to be placed so as to only act on the illumination beam, in order to allow the excitation light to be efficiently coupled to the sample.

The optical system shown in <FIG> places HWP3 in a position where it preferably only interacts with the excitation light and not the detected fluorescence. Positioning HWP3 to achieve this may be easier if HWP3 is positioned so that it is conjugate to the pupil of O2. As shown in <FIG>, this can be achieved by the addition of an extra pair of relay lenses, TL3 and TL4.

In order to translate the mirror M1 at the focus of the second objective lens O2, ideally a method for rapidly and accurately translating the mirror is required. However, left unchecked, rapid translation of the mirror may produce mechanical vibrations that may affect other parts of the optical setup. Such mechanical vibrations can be avoided or at least mitigated by coupling the translated mirror to a compensating mass having the same mass as the translated mirror, that oscillates in exactly the opposite direction (i.e. in antiphase) and with the same amplitude as the mirror.

One possible setup for achieving this is shown in <FIG>, which illustrates (in plan view) a flexure stage design having a compensating mass C so that the unit produces no vibration when the mirror M is translated. As mentioned above, the compensating mass C has the same mass as the translated mirror M. Both the mirror M and the compensating mass C are coupled to an actuator A that is disposed equidistantly between the mirror M and the compensating mass C, and which drives the mirror M and the compensating mass C so that they translate in antiphase with the same amplitude. Ideally, the line between the centre of mass of the mirror M and the centre of mass of the compensating mass C is parallel with their direction of motion. Translational motion of the mirror M and the compensating mass C is enabled by virtue of flexible hinges H. As illustrated, the flexible hinges H are located either side of the mirror M and either side of the compensating mass C, coupling the mirror M and the compensating mass C to opposing frame members F.

It should be noted that the principles illustrated in <FIG> may also be applied to other techniques in which a scanning mirror is used, such as other areas of microscopy, or spectrometry, as those skilled in the art will appreciate.

In some biological experiments it is desirable to photoactivate and/or photoconvert a specific portion of the sample, e.g. only specific cells. <FIG> shows the incorporation of an additional activation beam (illustrated using a thick line) provided by a scanner or spatial light modulator (SLM) <NUM> via dichroic beam splitter DM. It can be seen that the activation beam is delivered to the sample S along much of the beam path taken by the emitted light from the sample, but in the reverse direction. More particularly, the activation beam passes through the second objective lens O2 and is reflected by the first mirror M1 back through the second objective lens O2 and thence along the path taken by the emitted light from the sample S to the second objective lens O2 in reverse.

This technique may be used for single or two-photon photoactivation or photoconversion of the sample. Because the activation beam passes through the second objective lens O2 and is reflected by the tilted mirror M1, changing the angle of the activation beam as it leaves the scanner <NUM> causes the activation beam to scan over a tilted plane in the sample. Activation from the activation beam scanner <NUM> could be combined with activation light from the light sheet illumination path in order to achieve two-photon (instantaneous or step-wise) photoactivation or photoconversion from orthogonal directions.

As illustrated in <FIG>, a similar approach can be incorporated in the non-folded original OPM configuration (as per <CIT>). Again, changing the angle of the activation beam scanner causes the activation spot to scan in a tilted plane in the sample that matches the tilted imaging plane. Again, the activation beam could be combined with light from the illumination beam to achieve two-photon (instantaneous or step-wise) photoactivation or photoconversion.

As described above, the present work provides a device where an illumination light pattern is reflected off a mirror M1 and imaged to a sample S via an optical relay. Light from the sample S is then collected back through the same optical relay onto the mirror M1. Light from the sample S then reflects off the mirror and is collected by a subset of the same optical relay before being separated by a beam splitter onto an imaging detector.

Detailed embodiments and some possible alternatives have been described above. As those skilled in the art will appreciate, a number of modifications and further alternatives can be made to the above embodiments whilst still benefiting from the claimed invention.

It will therefore be understood that the invention is not limited to the described embodiments and encompasses modifications apparent to those skilled in the art lying within the scope of the claims appended hereto.

For example, in the embodiments described above, the emitted light is primarily described as being fluorescence. However, in alternative embodiments, reflected or scattered light instead of fluorescence may be collected.

Moreover, in most of the embodiments described above, the optical relay between the first objective lens O1 and the mirror M1 comprises only the first and second relay lenses, TL1 and TL2. However, in alternative embodiments, additional relay lenses may be provided, for example between the first relay lens TL1 and the second relay lens TL2, if desired. In other examples not according to the present invention, it may also be possible that lenses O1 and O2 are designed so that they can be placed with just the beam splitter PBS1 in-between, without the need for any relay lenses.

It should be noted that, in <CIT>, an embodiment was described with respect to <FIG> of that document, whereby a mirror is placed along the plane indicated by the line <NUM> in that figure, at the intermediate focal plane (coincident with image <NUM>), so as to cause the detected image <NUM> to be produced via mirror <NUM> and lens <NUM>. However, this in no way suggests that the illumination light may also be introduced via the same mirror (i.e. at the plane indicated by the line <NUM> in <FIG>), thereby improving the optical performance of the OPM and making the arrangement of the beam paths more compact. Furthermore, <CIT> also does not suggest that said mirror (at the plane indicated by the line <NUM> in <FIG>) may be translated to implement scanning in OPM, to enable 3D imaging in an efficient and reliable manner that does not involve adjusting the axial position of any of the objective lenses.

Claim 1:
An optical arrangement (<NUM>) for oblique plane microscopy, the optical arrangement comprising:
an illumination generator (<NUM>) arranged to provide a beam of illumination through a first objective lens (O1) to illuminate or excite an oblique plane of a sample (S) in use, wherein the first objective lens (O1) is also arranged to receive a beam of emitted light from the oblique plane of the sample (S) in use;
first and second relay lenses (TL1, TL2) and a second objective lens (O2) sequentially arranged to receive the beam of emitted light from the first objective lens (O1) and to form, at the focal plane of the second objective lens (O2), an intermediate image having a tilted plane conjugate to that of the oblique plane of the sample (S);
a first mirror (M1) located at said focal plane of the second objective lens (O2), arranged to receive and reflect the beam of emitted light;
a third relay lens (TL3); and
an image detector (<NUM>);
wherein the second objective lens (O2) and the third relay lens (TL3) are arranged to relay the intermediate image from the first mirror (M1) to the image detector (<NUM>); and
wherein the optical arrangement further comprises a beam splitter disposed between the second objective lens (O2) and the second relay lens (TL2), the beam splitter being configured to:
direct the beam of emitted light from the second relay lens (TL2) to the second objective lens (O2), and thence to the first mirror (M1); and
direct the reflected beam of emitted light from the second objective lens (O2) to the third relay lens (TL3) and thence to the image detector (<NUM>);
the optical arrangement characterised in that the first mirror (M1) is also arranged to receive the beam of illumination from the illumination generator (<NUM>) and to reflect the beam of illumination through the second objective lens (O2);
and in that the beam splitter is further configured to direct the beam of illumination from the second objective lens (O2) to the second relay lens (TL2), and thence to the first relay lens (TL1), the first objective lens (O1) and the sample (S).