WO2021064434A1 - Transmission microscopy - Google Patents

Abstract

Transmission microscopy apparatus (2) comprises an illumination apparatus (10) arranged to illuminate a sample region (6) with first and second monochromatic coherent light beams (12, 14), and an objective lens (4), having an imaging axis (A), for collecting light emanating from a sample (8) within the sample region (6). In a first configuration the first light beam (12) enters the sample region (6) along a first linear path from a first reflecting element (328; 406a; 506a) and the second light beam (14) enters along a second linear path from a second reflecting element (330; 406b; 506b) such that the first and second light beams interfere within the sample region to illuminate the sample with a first interference pattern (16). In a second configuration the first light beam (12) enters the sample region (6) along a different, third linear path such that the first and second light beams (12, 14) illuminate the sample (8) with a different, second interference pattern (18) which is not the same as any translation, rotation, or translation and rotation of the first pattern (16). The first, second and third paths are at respective angles α1, α2, α3 to the imaging axis and at least one is oblique to the imaging axis.

Description

Transmission Microscopy

BACKGROUND OF THE INVENTION

This invention relates to transmission microscopy and particularly, but not exclusively, to transmission microscopy apparatus for imaging a sample using Structured Illumination Microscopy (SIM).

Optical microscopes are used within histology, cell biology and related fields to view biological samples such as cells. However, the resolving power of optical microscopes is limited due to the diffraction limit of light. This limitation restricts the resolution of visible light microscopy to around 200 to 300 nm. In order to overcome this limit, several techniques have been developed in the art, termed “nanoscopy”, “super- resolution imaging”, or “super-resolution microscopy”.

These super-resolution imaging techniques allow imaging of a biological sample with a resolution finer than 200 nm, and possibly down to around 20 to 50 nm. They typically process light emitted from markers, such as photo-switchable fluorophores or quantum dots, that have been attached to, or embedded within, the biological sample. Known examples of such super-resolution techniques include ensemble techniques such as Structured Illumination Microscopy (SIM) and Stimulated Emission Depletion Microscopy (STED), single-molecule localisation techniques such as Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), and fluctuation-based super-resolution techniques such as Multiple Signal Classification ALgorithm (MUSICAL).

Single-molecule localisation techniques typically provide better resolution (e.g.

20-50 nm) but they are generally slower than the ensemble techniques and therefore not usually suitable for imaging live cell samples.

In general, STED has a practical optical resolution of around 30 nm. However, because it is a scanning based technique (e.g. a raster scanning based technique), it also suffers from slow imaging speeds (around 0.1 Hz for a wide field of view) that are not well suited to live cell imaging. This problem is exacerbated when a wide field-of- view is needed (i.e. when a large sample area needs to be scanned). Moreover, STED techniques typically require high-power illumination to excite the light emitting markers (e.g. fluorophores) and this is problematic because it can lead to photo-bleaching effects and photo-toxicity.

Fluctuation-based super-resolution techniques have so far used the photo-kinetic nature of the fluorescence phenomenon for super-resolution. Fluorescent molecules’ photon emissions are independent of each other and fluctuate over time. This results in fluctuation of intensity of fluorescence images of the sample. Statistical methods unique to each approach under such techniques analyse the fluctuations to provide super-resolved images. These techniques are significantly faster than single molecule localization techniques and less photo-toxic than other techniques. Nonetheless, they are not as fast as SIM.

In SIM based techniques, the sample is illuminated with different periodic light patterns. Typically, the periodic light pattern is a sinusoidal striped light pattern that is generated using diffraction gratings. During the imaging processing, the fringes of the periodic light pattern are usually shifted relative to the sample and also rotated relative to the sample, over a sequence of frames, so as to generate and illuminate the sample with a plurality of different periodic light patterns. A super-resolution image of the sample can then be constructed based on Fourier analysis of the different emission patterns from the light-emitting markers that result from the different illumination patterns. These emission patterns reveal information about the structure of the sample beyond the resolution limit of the imaging system, because of the Moire effect arising from frequency mixing with the periodic illumination patterns. In particular, high- frequency sample information that lies outside the support region of the system’s optical transfer function (OTF) can be down-shifted into this support region by the structured illumination.

In general, the optical resolution of SIM-type set-ups is limited by the fringe spacing in the periodic light pattern (e.g. the spacing between the fringes in a stripped illumination pattern). However, other factors may further limit the resolution. In known SIM set-ups based on total internal reflectance (TIR) illumination, the illumination pattern is formed by the imaging objective lens. The diffraction limit of an objective lens is given as λ₀ /

(2 * N.A.) where λ₀ is the wavelength, and N.A. is the numerical aperture of the objective lens. The resolution enhancement factor of SIM is double for a given objective lens, i.e. λ₀ / (4 * N.A.), which is typically 100-130 nm for visible wavelengths (400-650 nm).

A drawback with using the imaging objective lens to generate the interference pattern for SIM is that it will illuminate the area determined by the field of view of the given objective lens. In conventional TIR-SIM-type set ups, as the field of view increases, the fringe period increases and thus the resolution supported by the SIM technique worsens.

Conventional SIM set-ups typically have an optical resolution of around 100-130 nm and acquire images faster than STED and single-molecule localisation set-ups (e.g. SIM imaging speeds of around 0.1-1 Hz are typical for a wide field of view). Furthermore, unlike scanning-based set-ups, conventional SIM based set-ups can capture a relatively wide field image (e.g. 100 μm² or more) of the sample in a single shot.

To increase the imaging speed of SIM set-ups further it is possible to reduce the field of view over which an image is taken. Using this approach, SIM based set-ups have been reported that take images at speeds of 11 Hz over an 8 μm x 8 μm field of view. However, reducing the field of view can be undesirable because, if a wide-field view is required, the sample and imaging apparatus then need to be repositioned and realigned relative to each other in order to obtain a composite image of the whole sample. This is particularly problematic when imaging live cell samples.

The present invention aims to provide an approach to transmission microscopy that can support high resolution imaging across a wide field of view.

SUMMARY OF THE INVENTION

From a first aspect, the invention provides an apparatus for transmission microscopy comprising: an illumination apparatus arranged to illuminate a sample region with a first monochromatic coherent light beam and with a second monochromatic coherent light beam; and an objective lens, having an imaging axis, for collecting light emanating from a sample within the sample region, wherein: the illumination apparatus has a first configuration in which the first light beam enters the sample region along a first linear path from a first reflecting element and the second light beam enters the sample region along a second linear path from a second reflecting element, such that the first and second light beams interfere within the sample region to illuminate the sample with a first interference pattern; the illumination apparatus has a second configuration, in which the first light beam enters the sample region along a third linear path, different to the first path, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern; and the first, second and third paths are at respective first, second and third angles to the imaging axis, and at least one of the first, second and third angles is oblique to the imaging axis.

From a second aspect, the invention provides a method of imaging a sample using transmission microscopy, wherein the method comprises: in a first time period, i) illuminating a sample with a first monochromatic coherent light beam entering the sample along a first linear path from a first reflecting element and with a second monochromatic coherent light beam entering the sample along a second linear path from a second reflecting element, such that the first and second light beams interfere within the sample to illuminate the sample with a first interference pattern, and ii) collecting light emanating from the sample, illuminated with the first interference pattern, with an objective lens; and in a second time period, i) illuminating the sample with the first light beam entering the sample region along a third linear path, different to the first path, and with the second light beam, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern, and ii) collecting light emanating from the sample, illuminated with the second interference pattern, with the objective lens, wherein the first, second and third paths are at respective first, second and third angles to an imaging axis of the objective lens, and at least one of the first, second and third angles is oblique to the imaging axis. ln accordance with the present invention, it will be seen that the sample need not be illuminated using the imaging objective lens, but is instead illuminated with two different interference patterns created by interfering light beams entering the sample, at different respective angles to the imaging axis, from reflecting elements. The first light beam may enter the sample region directly from the first reflecting element, which may be a first mirror, and the second light beam may enter the sample region directly from the second reflecting element, which may be a second mirror. Illuminating the sample region directly from mirrors, or other reflecting elements, without any focusing optics between the mirrors and the sample region, facilitates versatile wide-field illumination of a sample. Since the first and second patterns are different (by more than mere rotation and/or translation) they can emulate illumination optics with two different numerical apertures. Thus, in the first configuration, the illumination apparatus has a first effective numerical aperture and in the second configuration, the illumination apparatus has a second, different effective numerical aperture.

This has been found to facilitate the generating of wide-field illumination patterns that have higher spatial frequencies than can be generated when using a low magnification imaging objective lens to provide both illumination and imaging, while still enabling an objective lens of relatively low magnification to be used to provide for wide-field imaging of the sample. In general, the greater the spatial frequency of the interference fringes (i.e. the smaller the interference fringe period), the higher the possible imaging resolution. Therefore, to image large areas with high resolution, it is desirable to generate interference fringes with smaller fringe spacing over large areas or field of views. For example, with some embodiments of the invention it has been found to be possible to generate high-visibility and stable interference patterns over cm²-scale areas.

By illuminating the sample region with light reflected from reflecting elements, as disclosed herein, it can also be possible to achieve wider illumination, at high spatial- frequencies, than is possible using conventional transmission-geometry systems, in which fringe patterns are created using an illumination objective lens that is separate from the imaging objective lens. In such conventional approaches, generating fringe patterns with high-spatial frequency requires an illumination objective lens with high numerical aperture (N.A.) and high magnification; however, higher magnifications limit the area over which the fringes can be formed (and hence limit the field of view). There is a physical limit on making objective lenses of very high N.A. but low magnification. The back-aperture of the illumination objective lens also limits the size of the input beam and the effective N.A. (and fringe spacing) in such conventional transmission- based approaches. Because embodiments of the present invention introduce the first and second light beam to the sample region along direct paths from respective reflecting elements, instead of from an illumination objective lens, they are not dependent on the properties of an illumination objective lens and back-aperture. This makes it possible for the light beams to illuminate the sample region with very large plane waves (e.g. of a few centimetres), with the effective N.A. of the illumination apparatus being determined by the configuration of the reflecting elements with respect to the sample region, almost independently of the size of the light beams.

It will be appreciated that the present invention requires light beams to enter the sample region along respective linear paths, and thus differs from approaches that illuminate a sample with an evanescent field, such as those disclosed in US 6,255,642 (Massachusetts Institute of Technology).

Having at least one illumination angle that is oblique (i.e. , neither parallel nor perpendicular) to the imaging axis means that one or both of the interference patterns can be non-uniform (e.g. periodically varying) along at least one path that is parallel to the illumination axis. Moreover, spatial frequencies in one or both of the interference patterns may be determined by the illumination angles, rather than depending solely on the wavelength of the monochromatic light beams. Setting at least one angle to be oblique thus enables greater control of the first and/or second interference patterns.

A property, such as fringe spacing (spatial wavelength) along one or more axes, intensity, or spatial extent, of the first and/or second interference pattern may at least partially depend on one or more of the first, second and third angles. It is thus possible to control one or more such properties of the first and second interference patterns by controlling the first, second and third angles of the first, second and third paths. For example, the first and second angles may be selected to produce a first interference pattern having a first fringe spacing (e.g. along an axis perpendicular to the imaging axis), and the third angle may be selected to produce a second interference pattern having a second, different, fringe spacing.

The first and/or second interference patterns with which the sample is illuminated may comprise respective periodic light patterns. They may comprise lines or bands having an intensity that varies sinusoidally with distance. The first interference pattern may have a first spatial frequency over some or all of an extent of the first pattern within the sample or sample region. The second interference pattern may have a second spatial frequency over some or all of an extent of the second pattern within the sample or sample region.

The interference patterns may be suitable for use with super-resolution microscopy techniques (e.g. SIM imaging or MUSICAL imaging).

The light collected by the objective lens may comprise light resulting from the interference patterns being scattered by the sample. However, the microscope is particularly suitable for fluorescence microscopy and so, in some preferred embodiments, the light collected by the objective lens may be produced in the sample via fluorescence, i.e. wherein the first and second interference patterns excite fluorescence in the sample, creating fluorescence patterns which are emitted from the sample and captured by the objective lens. For example, the light collected by the objective lens may be emitted by markers attached to, or embedded within, the sample such as photo-switchable fluorophores or quantum dots.

In some embodiments, the light collected by the objective lens (e.g. a fluorescence pattern) may be imaged. It may be analysed using known SIM processing methods. The microscopy apparatus may comprise imaging apparatus. The imaging apparatus may comprise an imaging sensor such as a CCD or CMOS array. The imaging sensor may be arranged to receive light passing through the objective lens. The objective lens may focus an image of the sample region on the imaging sensor. The imaging apparatus may comprise a controller or processor for performing one or more image processing operations — e.g. comprising software executing on a computer.

Methods embodying the invention may thus further comprise receiving light from the objective lens at an imaging sensor. Electronic signals from the imaging sensor may be processed to determine information about the sample — e.g., imaging information for the sample, or other information about the structure of the sample. Image data may be processed using a super-resolution technique such as SIM.

Because the sample is illuminated by interfering the first and second light beams within the sample region, the objective lens is not used for sample illumination and its optical properties need not therefore restrict the spatial frequency (i.e. resolution) or spatial extent of the illumination patterns. The objective lens may therefore have a lower numerical aperture (NA) (and thus a wider field-of-view) than is traditionally possible with microscopes where the objective lens is also used for illumination. In some embodiments, the objective lens may have a numerical aperture that is less than the first and/or second effective numerical aperture of the illumination apparatus.

In some preferred embodiments the objective lens has a NA of less than 1 (e.g. 0.8 or 0.6) and potentially of less than 0.5, such as 0.25 or lower.

Decoupling the illumination and collection optics in this manner may enable illumination patterns with high spatial frequencies (e.g. higher than that supported by the objective imaging lens) to be introduced which are stable (e.g. having a consistent fringe spacing or a uniform fringe pattern) over a wide field of view, increasing the inherent achievable imaging resolution of the microscope without sacrificing imaging scale and/or speed. The first and second patterns may be stable across a large area of the sample region (e.g. in a sample plane extending perpendicular to the imaging axis) — for example being periodic in one or two dimensions within a planar illumination area of at least 1 mm² or 5 mm², e.g., of 8 mm², 12 mm², 16 mm² or more. Each pattern may have a constant period along at least one path over a plurality of cycles — e.g., over tens, thousands, millions or more spatial wavelengths.

The first and/or second interference patterns may also vary in a direction parallel to the imaging axis. One or both patterns may vary periodically — e.g., with sinusoidal intensity — along one or more paths that are parallel to the imaging axis. The periodic variation may be consistent along the path over a plurality of cycles — e.g., two, ten, a thousand, a million or more spatial wavelengths of the pattern in the path direction.

One or both patterns may have a volumetric component (e.g. extending parallel to the imaging axis) which is stable across a path within the sample region that is at least 0.1mm, 1mm or more long. For example, the first and/or second interference pattern may be stable across a common region where the first and second beams overlap. In some such examples the depth across which the first and/or second interference pattern(s) is stable is related to the diameters of the first and second light beams and the first, second and third angles. In one particular example in the first configuration where the first and second beams have a diameter of 8 mm and the first and second angles are both equal to 30°, the stable depth is approximately 16 mm. An illumination pattern (i.e. the first and/or second interference pattern) may have a constant period along a path parallel to the imaging axis (i.e. a depth) over a distance that is longer than a depth of field of the objective lens.

Embodiments of the present invention may be particularly useful for imaging applications requiring a relatively large field of view (FOV), such as pathological tissue samples. The FOV of the objective lens preferably extends over an area (i.e. in the sample plane) approximately equal to or less than the area over which the first and second interference patterns are stable (i.e. have a constant respective spatial frequency or wavelength). In other words, the first and second interference patterns are preferably stable over the entire intersection of the FOV of the objective lens with the sample or the sample region.

In some embodiments, the first and/or second interference patterns may comprise a spatial frequency that is greater than can be imaged using the objective lens. For example, a spatial wavelength (e.g. a fringe spacing) of the first and/or second interference pattern may be smaller than a diffraction limit of the objective lens at a wavelength of the illumination light. Preferably, the first and/or second interference pattern comprises a fringe spacing (spatial wavelength) that is less than 1.5 times the wavelength of the first and/or second beam, e.g. 1l, 0.75A or even 0.6A or less.

Using two different interference patterns (e.g. with different spatial frequencies) means the sample is illuminated in two different ways (i.e. representing illumination by optics with two different numerical apertures or cut-off frequencies), allowing more data about the sample to be recovered than if a single illumination pattern were used. However, the applicant has recognised that illuminating the sample with more than two illumination patterns may further improve imaging quality and/or resolution. In some embodiments, therefore, the illumination apparatus may have one or more additional configurations — e.g. a third configuration, a fourth configuration, etc. — in which the first and second light beams interfere within the sample region to illuminate the sample with one or more further respective unique interference patterns that are not the same as the first or second patterns, and are not the same as any translation, rotation, or translation and rotation, of the first or second patterns (i.e. corresponding to unique effective numerical apertures). In particular, the illumination apparatus may have a third configuration, in which the first or second light beam enters the sample region along a third-configuration linear path, such that the first and second light beams interfere within the sample region to illuminate the sample with a third interference pattern, wherein the third pattern is not the same as the first or second patterns, and is not the same as any translation, rotation, or translation and rotation, of the first or second patterns. The third- configuration path may also be at an oblique angle to the imaging axis, although this is not essential.

The first and second configurations may result from respective first and second physical arrangements of the illumination apparatus.

The first reflecting element may be a first mirror and the second reflecting element may be a second mirror.

When the illumination apparatus is in the second configuration, the first light beam may enter the sample region along a third linear path from a reflecting element, which may be the first reflecting element, or which may be a further reflecting element — e.g. a third mirror or other reflective element.

In some sets of embodiments the illumination apparatus may comprise a first adjustable reflecting apparatus. The first adjustable reflecting apparatus may be arranged to support a first setting (e.g. a first position and/or inclination and/or orientation) associated with the first configuration of the illumination apparatus and a second setting (e.g. a second position and/or inclination and/or orientation) associated with the second configuration of the illumination apparatus. The first reflecting apparatus may be arranged, when in the first setting, to reflect the first light beam such that it enters the sample region along the first path and, when in the second setting, to reflect the first light beam such that it enters the sample region along the third path.

The use of a first reflecting apparatus in this manner may enable a user to exert fine control over the first and second configurations, without requiring a highly complex illumination apparatus.

The first adjustable reflecting apparatus may comprise a reflective member (e.g. mirror), which may be the first reflecting element, whose position and/or inclination and/or orientation can be adjusted between the first and second settings (e.g. a moveable planar mirror).

In one set of embodiments the first adjustable reflecting apparatus may comprise a plurality of reflective members (e.g. each with a fixed orientation), wherein the first beam is directed at a first of the reflective members (e.g. the first reflecting element) in the first setting, and at a second of the reflective members (e.g. said third reflecting element) in the second setting.

The first adjustable reflecting apparatus may comprise a plurality of mirrors (e.g. with different fixed orientations or angles associated with different respective settings of the reflecting apparatus) mounted on a common structure. The common structure may be movable relative to the sample region and/or an illumination source. The common structure may be configured to be moved (e.g. rotated) to bring a mirror associated with a particular setting into a position in which the first light beam is directed at, and reflected by, said mirror, when travelling to the sample region.

The adjustable reflecting apparatus may comprise the first reflecting element. The adjustable reflecting apparatus may direct light directly to the sample region — i.e. along a respective path at which the beam enters the sample. Alternatively, the adjustable reflecting apparatus may direct light to one or more further path-diverting components, which may include the first reflecting element, before the light enters the sample.

In embodiments in which the illumination apparatus has additional configurations (i.e. three or more configurations), the additional configuration(s) may result from additional unique physical arrangements of the illumination apparatus.

In some sets of embodiments, the second light beam may enter the sample region along the second path when the illumination apparatus is in the first and second configurations (i.e. the path of the second beam may be unchanged between the first and second configurations). However, the inventors have recognised that changing the paths of both the first and second beams between the first and second configurations may allow greater and/or finer control over the first and second interference patterns.

In some sets of embodiments, therefore, in the second configuration of the illumination apparatus, the second light beam enters the sample region along a fourth path, different to the second path, which is at a fourth angle to the imaging axis.

In some embodiments, the first and second angles may be equal. The first and second paths may be coplanar. When both of these hold true, the first and second beams enter the sample region along symmetrical paths extending either side of the imaging axis at a common angle to the imaging axis. In such embodiments, adjusting the common angle may adjust the period of the resulting interference pattern. Increasing the common angle may decrease the period (i.e. increase the spatial frequency) of the resulting interference pattern. The third and fourth angles may be equal. The third and fourth paths may be coplanar.

The illumination apparatus may comprise a second adjustable reflecting apparatus. As with the first adjustable reflecting apparatus, the second adjustable reflecting apparatus may be arranged to selectively have a first setting associated with the first configuration of the illumination apparatus and a second setting associated with the second configuration of the illumination apparatus. The second adjustable reflecting apparatus may comprise the second reflecting element. The second adjustable reflecting apparatus may be arranged, when in a first setting, to reflect the second light beam such that it enters the sample region along the second path and, when in a second setting, to reflect the second light beam such that it enters the sample region along the fourth path. As with the first reflecting apparatus, the second reflecting apparatus may be provided as a single configurable reflective member (e.g. comprising the second reflecting element), or a plurality of fixed reflective members (e.g. comprising the second reflecting element, and a further — e.g. fourth — reflecting element, which may be a further mirror). Such a plurality of fixed reflective members may be attached to a common structure.

The first adjustable reflecting apparatus and second adjustable reflecting apparatus may comprise a common structure — e.g. comprising respective mirrors on a common mount. In one embodiment, the first and second adjustable reflecting apparatus are provided as multiple pairs of opposing mirrors mounted (e.g. symmetrically) on a toroidal common structure. The first and third reflecting elements may be a first opposing pair of mirrors on the structure. The second and fourth reflecting elements may be a second opposing pair of mirrors on the structure. In some such embodiments, the illumination apparatus may be configured between different configurations by rotating the toroidal structure and/or by changing which reflecting elements are illuminated by the first and second light beams.

In some embodiments, the illumination apparatus is configured to be put into the first and/or second configurations through a manual user interaction — e.g. by a user manually moving or adjusting the position or orientation of one or more components of the illumination apparatus, e.g. by adjusting a position or orientation of the first reflecting element. The illumination apparatus may comprise a mechanism for resiliently holding the illumination apparatus in each configuration (e.g. a clamp or detent) and/or for indicating to a user when the apparatus is in one of the configurations. Alternatively, the apparatus may be continuously variable between the first and second configurations.

In some sets of embodiments the transmission microscopy apparatus comprises an electronic controller which is configured to control the configuration of the illumination apparatus (e.g. to control the illumination apparatus to adopt the first and second configurations). The electronic controller may be configured to control the actuation of one or more electronic actuators or motors (e.g. to control one or more adjustable reflecting apparatuses). This may enable more precise and/or faster and/or more replicable control of the first and second configurations than purely manual configuration. The electronic controller may comprise digital logic, such as a processor, or it may comprise analogue circuitry.

The first and second light beams preferably have a common wavelength, which may be within the visible spectrum (e.g., λ ≈ 400 - 800 nm), near-IR (e.g., λ ≈ 800 - 1500 nm) or even mid-IR or higher (e.g. λ ≥ 1500 nm). The first and second light beams may be produced by a common light source, such as a laser or other spatially- coherent light source, although in some embodiments separate first and second light sources (e.g. first and second lasers) are used to produce the first and second light beams. A suitable light source includes a laser light source such as a solid state laser, fibre laser or diode laser. It will be appreciated that the monochromaticity and coherence of the first and second beams may be limited in practice due to physical limitations in the illumination apparatus. The first and second light beams may comprise plane waves. The first and second light beams may be collimated beams. In embodiments featuring a common light source, the first and second light beams may be produced from an initial light beam produced by the common light source using a beam splitter device such as a half-silvered mirror. However, in some preferred embodiments the first and second light beams may comprise diffraction fringes (e.g. first order fringes) produced by an initial light beam interacting with a diffraction grating or spatial light modulator (SLM). Thus, the illumination apparatus may comprise a diffracting component such as a diffraction grating or an SLM. The SLM may be controller by an electronic controller.

The apparatus may comprise a beam expander. The beam expander may be arranged to expand an initial light beam from which the first and second light beams are produced. The apparatus may comprise a diaphragm for selecting a region of the initial beam, e.g. a region having an approximately flat beam profile.

As well as being dependent on the first, second and third angles, one or more properties of the first and second interference patterns (e.g. a lateral or phase offset) may also be dependent on one or more properties of the first and second light beams (e.g. wavelength, intensity, phase, shape). In some embodiments, therefore, the illumination apparatus may comprise means for adjusting one or more properties of the first and/or second light beams (e.g. before they are reflected towards the sample region by a reflecting element). For example, the illumination apparatus may comprise a controllable optical component (e.g. a rotatable diffraction grating or an electronically controlled SLM) arranged to alter one or more properties of the first and/or second light beams and thus of the resulting interference patterns. The controllable optical component may be arranged to adjust the phase of the first and/or second light beams. The controllable optical component may comprise more than one element and there may be more than one controllable optical component provided.

In some embodiments a controllable optical component (e.g., an SLM, grating, beam splitter or mirror holder) may be arranged to control the orientation of the first and/or second interference patterns relative to the sample region. For example, the apparatus may be configured for rotating a pattern on an SLM so as to alter the orientation of a resulting interference pattern relative to the sample region. In relevant embodiments, this may require movable or additional reflecting elements (e.g. additional to the first and second reflecting elements, such as third and/or fourth reflecting element) to reflect the first and/or second light beams such that they continue to enter the sample region along the first, second or third path (i.e. at the first, second or third angles) even when the orientation is altered. In another set of embodiments, the sample region itself may be rotatable — e.g., by manually or electronically rotating a sample holder.

The adjustment provided by the controllable optical component may be associated with the first and second configurations of the illumination apparatus. For example, in the first configuration, the controllable optical component may be arranged to add a first phase shift to the first and/or second light beams and, in the second configuration, the controllable optical component may be arranged to add a second phase shift to the first and/or second light beams. This may enable finer control over the first and second interference patterns.

However, in some sets of embodiments a controllable optical component (e.g. SLM, grating, beam splitter or mirror holder) may be used to change one or more properties of the first and/or second light beams whilst the physical configuration of the rest of the illumination apparatus remains the same. In such embodiments the controllable optical component may be arranged to produce multiple variants of the first and second interference patterns (e.g. with different combinations of pattern orientation and phase/lateral offset). These variants may be used to illuminate the sample across the Fourier space (i.e. by filling in Fourier space piecewise using different orientations and phase offsets for each interference pattern). Illumination across the Fourier space (e.g. gapless and isotropic illumination) may enable higher quality imaging.

The invention allows the creation of temporally varying illumination patterns. These may be used to introduce fluctuation in fluorescence images (e.g. for use with the MUSICAL algorithm). The achievable resolution depends on the amount of fluctuations in the sample’s fluorescence images introduced by illuminations patterns with respect to the average intensity over all the illumination patterns and also the average signal to background ratio in fluorescence microscopy images. Even with small signal to background ratio and/or fluctuations in fluorescence images, MUSICAL still provides contrast enhancement over the average image.

In embodiments where the first and second light beams are produced from a common initial beam (e.g. from a common light source), the controllable optical component may be arranged to interact with the initial beam (i.e. before it has been split into the first and second beams). Where the first and second light beams comprise diffraction fringes produced from an initial beam, these may be produced by the initial light beam interacting with the controllable optical component. An electronically controllable SLM, for instance, can adopt precisely defined complex periodic patterns, enabling the relative energy of diffraction fringes (such as those forming the first and second beams) to be precisely engineered, allowing the intensity of the first and second light beams to be controlled.

As explained above, the use and control of first and second monochromatic coherent light beams enables the first and second interference patterns to be produced in the sample region, with control over the fringe spacing of the interference patterns. However, the Applicant has recognised that using one or more additional light beams (e.g. third, fourth, etc. light beams) may enable more complex interference patterns to be produced and controlled (e.g. interference patterns similar to conventional speckle patterns but with greater predictability and control). More complex interference patterns may allow super-resolution imaging techniques such as Multiple Signal Classification Algorithm (MUSICAL) or Entropy-Based Super-Resolution Imaging (ESI) to be used.

One such technique, MUSICAL, can exploit fluctuating (or temporally varying) illumination patterns instead of photokinetics of fluorescent molecules to provide super-resolution. With this possibility, the super-resolution supported depends upon the spatial frequency, contrast, and variability of illumination patterns, allowing a superior determinism in the expected resolution.

In some embodiments, therefore, the illumination apparatus is further arranged to illuminate the sample region using one or more additional monochromatic coherent light beams. In one particular embodiment, the illumination apparatus is arranged to illuminate the sample region using four monochromatic coherent light beams, simultaneously or at different times. The one or more additional light beams may contribute to the first and/or second interference patterns (i.e. the first and second light beams may also interfere with the one or more additional light beams in the sample region to produce the first and/or second interference patterns) and/or they may be used to produce one or more additional unique interference patterns (e.g. corresponding to one or more additional configurations of the illumination apparatus). For example, a multi-periodic illumination pattern along one direction can be generated by the mutual interference of four coplanar beams. A path along which each light beam enters the sample region may be controlled (e.g. using an adjustable reflecting apparatus for each beam) to provide control over the resulting interference pattern(s). The angle to the imaging axis at which each path extends may, for instance, be changed between configurations of the illumination apparatus. In some embodiments the path and/or path angle of every light beam may be changed between configurations, but in other embodiments only a sub-set of light beam paths is changed between configurations. One or more controllable optical components may be used to control one or more properties of each light beam.

The apparatus may be in the first configuration for a first time period, and in the second configuration for a second time period. The first and second time periods may be non-overlapping. However, the first and second time periods (and any further time periods in which the illumination apparatus is in further respective configurations) may, in total, span a maximum duration that is less than 0.1, 1, 2 or 10 seconds, thereby enabling the imaging (e.g. super-resolution imaging) of samples that may be in motion or in flux.

The sample region may be defined by a sample holder or receptacle — e.g. being defined by one or more faces of a glass container. A sample may occupy some or all of the sample region.

As mentioned above, the present invention is particularly suited to super-resolution microscopy imaging techniques. In some sets of embodiments, therefore, the microscope further comprises imaging apparatus (e.g., an imaging unit) arranged to perform super-resolution imaging using the light captured by the objective lens. The imaging apparatus may comprise one or more of: image sensors, processors, memory, ASICs, FPGAs, DSPs, inputs and outputs. It may comprise memory storing software instructions for instructing the imaging apparatus to perform one or more steps of SIM imaging. The imaging apparatus may be arranged to perform frequency analysis, such as Fourier analysis, with the collected light. The imaging apparatus may use knowledge of the configuration of the illumination apparatus when performing super-resolution imaging. In embodiments featuring an electronic controller, the electronic controller may be in communication with or in control of the imaging apparatus. More generally, from another aspect, the invention provides an apparatus for transmission microscopy comprising: an illumination apparatus arranged to illuminate a sample region with a first monochromatic coherent light beam and with a second monochromatic coherent light beam; and an objective lens, having an imaging axis, for collecting light emanating from a sample within the sample region, wherein: the illumination apparatus has a first configuration in which the first light beam enters the sample region along a first linear path and the second light beam enters the sample region along a second linear path, such that the first and second light beams interfere within the sample region to illuminate the sample with a first interference pattern; the illumination apparatus has a second configuration, in which the first light beam enters the sample region along a third linear path, different to the first path, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern; and the first, second and third paths are at respective first, second and third angles to the imaging axis, and at least one of the first, second and third angles is oblique to the imaging axis.

From a further aspect, the invention provides a method of imaging a sample using transmission microscopy, wherein the method comprises: in a first time period, i) illuminating a sample with a first monochromatic coherent light beam entering the sample along a first linear path and with a second monochromatic coherent light beam entering the sample along a second linear path, such that the first and second light beams interfere within the sample to illuminate the sample with a first interference pattern, and ii) collecting light emanating from the sample, illuminated with the first interference pattern, with an objective lens; and in a second time period, i) illuminating the sample with the first light beam entering the sample region along a third linear path, different to the first path, and with the second light beam, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern, and ii) collecting light emanating from the sample, illuminated with the second interference pattern, with the objective lens, wherein the first, second and third paths are at respective first, second and third angles to an imaging axis of the objective lens, and at least one of the first, second and third angles is oblique to the imaging axis.

Features of any other aspects and embodiments disclosed herein may be features of embodiments of these aspects also.

In particular, in some embodiments, the apparatus of this aspect may comprise an adjustable reflecting apparatus. The adjustable reflecting apparatus may be arranged to direct light directly to the sample region (i.e. along a respective path at which each beam enters the sample). It may have any of the features of the first and/or second adjustable reflecting apparatus disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain preferred embodiments of the invention will now be described, byway of example only, with reference to the accompanying drawings, in which:

Figures 1 and 2 are schematic cross-sectional side views of a transmission microscope according to a first embodiment;

Figure 3 is a schematic cross-sectional side view of a transmission microscope according to a second embodiment;

Figure 4 is schematic cross-sectional side view of parts of a transmission microscope according to a third embodiment;

Figure 5 is a perspective view of a reflecting apparatus for a transmission microscope according to some embodiments;

Figure 6 is schematic cross-sectional side view of a transmission microscope according to a fourth embodiment;

Figure 7 shows frequency-domain plots illustrating the collection of high frequency information through a low NA objective lens using embodiments of the invention; Figure 8 shows four scanning electron microscopy images of an interference pattern created using illumination apparatus embodying the invention;

Figure 9 shows plots of the Fourier spectrum of the interference pattern of Figure 7; Figure 10 contains images of an interference pattern at different phase shifts along with a graph of line profiles; Figures 11, 12, 13 & 14 show simulations of images captured by a microscope embodying the invention and associated Fourier spectra;

Figures 15a, 15b & 15c are examples of interference patterns that can be produced with embodiments of the present invention;

Figure 16 is a schematic cross-sectional side view of one configuration of a transmission microscope according to a fifth embodiment;

Figure 17 is a graph showing different possible orientations of an interference pattern that can be generated by the microscope of the fifth embodiment;

Figure 18 is a frequency spectrum of an interference pattern that can be generated by the microscope of the fifth embodiment; and

Figures 19a, 19b & 19c show an example interference pattern that can be generated by the microscope of the fifth embodiment, in different orientations.

DETAILED DESCRIPTION

Figures 1 and 2 show schematic views of a transmission microscope 2 according to an embodiment. The microscope 2 comprises an objective lens 4 with an imaging axis A, a sample holder 6 in which a sample 8 is contained, and an illumination apparatus 10, located on the opposite side of the sample 8 to the objective lens 4. The objective lens 4 is arranged to collect light emanating from the sample 8.

The illumination apparatus 10 is arranged to illuminate the sample 8 with a first monochromatic coherent light beam 12 and a second monochromatic coherent light beam 14. The beams 12, 14 may have the same wavelength.

In Figure 1, the illumination apparatus 10 is shown arranged in a first configuration, in which the first light beam 12 enters the sample holder 6 along a first path that extends at a first angle ai to the imaging axis A and the second light beam 14 enters the sample holder 6 along a second path that extends at a second angle c^to the imaging axis A. The first and second light beams 12, 14 thus interfere within the sample holder 6, producing a first interference pattern 16 with which the sample 8 is illuminated.

In Figure 2, the illumination apparatus 10 is shown arranged in a second configuration, in which the first light beam 12 enters the sample holder 6 along a third path that extends at a third angle α₃ to the imaging axis A. In the second configuration the second light beam 14 still enters the sample holder 6 along the second path that extends at a second angle α₂ to the imaging axis A. The first and second light beams 12, 14 again interfere within the sample holder 6, but because the first light beam 12 now enters the sample holder 6 along a different path, a different second interference pattern 18 is produced in the sample holder 6, thus illuminating the sample 8 in a different way to the first configuration.

Due to the change in angle of the first light beam 12, it can be seen that the spatial frequency of the second interference pattern 18 is higher than that of the first interference pattern 16. For clarity of illustration, the first and second interference patterns 16, 18 are shown in Figures 1 and 2 in cross-section as simple one dimensional series of fringes. However, in reality the interference patterns 16, 18 will have much more complex three-dimensional structures, including periodic variation in an image plane as well as parallel to the imaging axis A — i.e. having volumetric structure. Exemplary illumination patterns are shown in more detail in Figures 14a-14c.

The illumination apparatus 10 may enable the angle of the light beams 12 to be configured by using any one or more of the mechanisms disclosed herein.

The sample 8 may be sufficiently sparse that direct transmission microscopy is possible. However, in general, it is expected that the sample 8 will contain fluorescence markers, for performing fluorescence microscopy. The markers fluoresce when exposed to a sufficient intensity of light from the interference patterns 16, 18. This fluorescence is captured by the objective lens 4.

The light collected by the objective lens 4 can be used by an imaging apparatus (not shown) to perform super resolution imaging (e.g. SIM imaging) of the sample 8. This imaging apparatus may comprise a digital imaging sensor connected to a computer. The computer runs software for processing a series of images of the sample 8 under different illumination patterns and for combining frequency information from across the images so as to reconstruct information about the structure of the sample 8, potentially at a finer resolution than the objective lens 4 can resolve directly. Details of these processing steps, where unconventional, are explained in greater detail below.

A schematic diagram of a transmission microscope 302 in accordance with another embodiment is shown in Figure 3. The microscope 302 comprises an objective lens 304 arranged to collect light emanating from a sample (not shown) in a sample region 306 (e.g. comprising a sample holder such as a slide, well or plate on/in which the sample is located).

The microscope 302 comprises an illumination apparatus 308 arranged to illuminate the sample from an opposite side to the objective lens (i.e. transmission-mode illumination). The illumination apparatus 308 comprises featuring a laser light source 310 (e.g. a diode pumped solid-state laser source, such as the “BlueMode laser” manufactured by TOPTICA Photonics AG, Germany, which emits at a vacuum wavelength of 405 nm) that produces an initial light beam 312. The initial beam 312 is collimated and widened using a telescope 314 (i.e. a beam expander).

A region of the initial beam 312 with an approximately flat beam profile is selected with a diaphragm 316 and subsequently projected onto a spatial light modulator 318 (SLM, e.g. a reflective phase only spatial light modulator such as the Holoeye LETO).

A grating pattern displayed on the SLM 318 divides the input beam 312 into a 0^th order beam 320 and two 1^st order beams: a first beam 322 and a second beam 324. A mirror 326 reflects the beams 320, 322, 324 towards the sample region 306. The 0^th order beam 320 is blocked, and the first beam 322 and the second beam 324 are directed to respective first and second mirrors 328, 330.

The first and second mirrors 328, 330 reflect the first and second beams 322, 324 towards the sample region 306. The angle of the first and second mirrors 328, 330 is controllable, such that the first and second beams 322, 324 may be directed towards the sample region 306 along multiple different paths that extend at different angles to the imaging axis. The mirrors 328, 330 may be used to generate a sinusoidal interference pattern for super-resolution imaging (e.g. SIM). The spatial frequency of this interference pattern depends on the angle between the two interfering beams 322, 324, where a larger interference angle realizes a higher numerical aperture (NA) and thus a higher pattern frequency. Numerical apertures larger than the NA of the objective lens 304 are of particular interest as they provide a resolution improvement.

In Figure 3, the first and second mirrors 328, 330 are shown in a first configuration in which the first and second beams 322, 324 are directed along respective first and second paths 332, 334. The first path 332 extends at a first angle ai to the imaging axis I and the second path 334 extends at a second angle α₂ to the imaging axis I. In this first configuration, the first and second beams 322, 324 enter the sample region 306 and interfere to illuminate the sample with a first interference pattern (e.g. a sinusoidal interference pattern).

As can be seen from the overlays in Figure 3, the first and second mirrors 328, 330 can adopt second and third configurations, in which the first and second mirrors 328, 330 are oriented at different angles to the first configuration, such that the first and second beams 322, 324 enter the sample region 306 along different paths to the first configuration, producing respective second and third interference patterns which, for example, comprise different spatial frequencies to the first interference pattern (and to each other).

The illumination apparatus 308 is also arranged to generate one or more variants on each of the interference pattern using the SLM 318. A pattern on the SLM 318 may be rotated to adjust the orientation of the resulting interference pattern. A pattern on the SLM 318 may be changed to adjust the phase/lateral offset of the resulting interference pattern.

An imaging unit (not shown) may use the light collected by the objective lens 304 to perform super-resolution microscopy, using light collected from the sample illuminated with different interference patterns and variants of these.

Similar to Fourier ptychography, the Fourier space can be filled piecewise by using different pattern orientations and different interference angles mimicking different NAs. Since the illumination pattern does not depend on the imaging objective’s NA anymore, a low NA objective with a large FOV can be used.

Figure 4 shows a partial view of another embodiment, in which an illumination apparatus 401 comprises an SLM 402 and six mirrors 406a-406f, i.e. forming an adjustable reflecting apparatus. Six light beams 404a-404f are shown propagating from the SLM 402 towards the six mirrors 406a-406f. The mirrors 406a-406f direct each of the light beams 404a-404f towards a sample region 408 containing a sample (not shown).

As can be seen in Figure 4, a first pair 406a, 406b of the mirrors is configured for illuminating the sample at a first angle to an imaging axis; a second pair of the mirrors 406c, 406d is configured for illuminating the sample at a second angle to the imaging axis, greater than the first angle; and a third pair of the mirrors 406e, 406f is configured for illuminating the sample at a third angle to the imaging axis, greater than the second angle.

Any combination of beams 404a-404f may illuminate the sample region 408 at a time — e.g. the pair 404a and 404b, or all six beams 404a-404f together.

Some or all of the six light beams 440a-404f interfere within the sample region 408 to produce an interference pattern with a high spatial frequency. The interference pattern illuminates the sample, which can then be imaged with an objective lens (not shown).

The position(s) and angle(s) of at least one of the six mirrors 460a-406f can be adjusted to adjust the interference pattern generated within the sample region 408.

The six mirrors 406a-406f are adjustable between a first configuration (a first set of angles/positions), which results in a first interference pattern, and a second configuration which results in a second, different, interference pattern (e.g. with a different spatial frequency).

Figure 5 shows an exemplary toroidal mount structure 450 on which six mirrors 451 a-f (i.e. six reflecting elements) are mounted in three opposing pairs (451a & 451b; 451c & 451d; 451e & 451f), uniformly spaced around the mount structure 450. The angles of the mirrors 451 a-f can be adjusted. Such a structure 450 may form part or all of an adjustable reflecting apparatus for a transmission microscope. It may be used to reflect light along linear paths to a sample region in embodiments of a transmission microscope as disclosed herein. It could, for example, be used in an embodiment similar to that described above with reference to Figure 4.

Figure 6 shows another illumination apparatus 501 comprising an SLM 502, four beam splitters 504a-504d and two mirrors 506a, 506b, arranged symmetrically about an imaging axis A. Two initial light beams 508a, 508b propagate from the SLM 502 towards a first pair of beam splitters 504a, 504b, wherein part of the two initial beams’ energy is reflected as first and second beams 512a, 512b and the rest of the beams’ energy is allowed to pass through towards a second pair of beam splitters 504c, 504d. The first and second beams 512a, 512b propagate towards a sample region 510 at a first angle ai to the imaging axis A. The second pair of beam splitters 504c, 504d partially reflects the beams 508a, 508b, producing third and fourth beams 512, 512d which propagate towards the sample region 510 at a second angle α₂ to the imaging axis A. The second pair of beam splitters 504c, 504d allows the rest of the beams’ energy to pass through towards the two mirrors 506a, 506b. These reflect the beams 508a, 508b towards the sample 510 as fifth and sixth beams 512e, 512f which propagate at a third angle α₃ to the imaging axis A. The position(s) and angle(s) of at least one of the four beam splitters 504a-504d and the two mirrors 506a, 506b can be adjusted to adjust the interference pattern generated within the sample region 510. The four beam splitters 504a-504d and the two mirrors 506a, 506b are adjustable between a first configuration (a first set of angles/positions), which results in a first interference pattern, and a second configuration which results in a second, different, interference pattern (e.g. with a different spatial frequency).

The basic concept of high-frequency information collection over a large FOV through a low NA objective using SIM is described in Figure 6. As depicted in panel (a), given an imaging objective with NA = 0.25 and an excitation and emission wavelength of l (λ_ex = Aem), the OTF support is limited to

= 0.5λ^-1 (area 602 in panel (a)). Illumination patterns are generated by interfering the excitation light under an angle (of first and second beams) of l = 23°, 36°, and 54°, (mimicking NAs of 0.39, 0.59, and 0.81 respectively, with pattern frequencies of f = 0.78 λ^-1, 1.18 λ^-1 1.62 λ^-1 respectively), indicated by NA1 604, NA2606, NA3608. These provide additional information at higher frequencies in the Fourier space in the form of the shifted copies of the conventional OTF.

Images acquired using the mentioned illumination patterns contain additional information at higher frequencies in the Fourier space in form of the shifted copies of the conventional OTF. Here only the information gain in one direction, along the y-axis and only the positive direction are shown. Panel (b) of Figure 7 shows the acquisition of additional spectral components along three different directions d₁, d₂, d₃. These orientations are distributed in even 60° steps with an offset to present a more general case. The dash-dotted circle 610, the dashed circle 612 and the dotted circle 614 indicate the pattern frequencies of NA1, NA2, and NA3 respectively. For each of the represented pattern orientations only one set of spectral components acquired using one pattern frequency is illustrated in panel (b). However, to fill the Fourier space isotropically, the imaging acquisition is repeated for all three pattern fringe spacings at up to six orientations.

Further details of the theoretical underpinnings of embodiments of the invention will now be provided, as well as the results of simulations that demonstrate the principles in action.

Space-bandwidth product of transmission SIM

The space-bandwidth product (SBP) is a measure to characterize an imaging system.

It is the size of the field of view (FOV) divided by its resolution

(1)

where d is the achievable resolution (smallest resolvable distance) and the factor 0.5 stems from the Nyquist-Shannon sampling theorem. Since the FOV only depends on the imaging objective and camera size, and the SIM resolution depends mainly on the illumination pattern, the SBP can be increased to four times as compared to a conventional SIM approach if illumination patterns generated by three times the NA of the imaging objective are generated. This is sixteen times the SBP of conventional widefield imaging. Given an imaging objective with a numerical aperture of NA_obj = 0.25 and a FOV of 16 mm², the resolution can be calculated to be 190 nm.

(2)

This gives a SBP of

(3)

= 1.75 X 10⁹.

It is possible to generate many different interference patterns using the set up described above. To study one such illumination pattern, an interference pattern was generated and recorded photolithographically so that it could be characterized in a scanning electron microscope (SEM). A glass substrate was spin-coated with a layer of about 1 μm photoresist (AZ 1505, Microchemicals) and placed at the sample plane. A laser diode (BlueMode, Toptica) emitting at a vacuum wavelength of 405 nm replaces the source in the setup described above.

The interference pattern generated by the system is inscribed in the photoresist on the glass substrate in the sample plane. A set of two opposing mirrors at a half angle of 54° corresponding to an NA of 0.8 was used to generate a sinusoidal pattern 701 seen in panels (a), (b), (c), (d) of Figure 8. The interference patterns are recorded on the photoresist which is then developed and the pattern period is determined using SEM.

The size of the FOV where the interference fringes are formed can be increased by adjusting the beam diameter. The FOV over which the sinusoidal pattern was generated seen in Figure 8 panel (a) is roughly 4 mm². Figure 8 panels (b), (c), (d) show cropped parts from the upper left corner, the central region and the lower right corner respectively, suggesting a homogeneous pattern.

For a NA of 0.8 the pattern period was around 250 nm. For a NA of 0.6 (corresponding to two mirrors at 36°), a pattern period of 330 nm was found. For NA of 0.4 (23°), the pattern period was 510 nm. These values were obtained by peak localization in the Fourier spectra of the SEM images of the developed photoresist on the glass substrate.

In Figure 9 panel (a) the Fourier spectrum 801 of an exemplary SEM image is shown. The delta peaks closest to the DC peak in the centre stem from the sinusoidal pattern, their distance to the DC peak being the grating period. Figure 9 panel (b) depicts the results for different NAs and orientations at different regions of the samples. The results show homogeneity of the pattern with minor variations which may be attributed to variations at the photoresist. Since the exposure time in the photolithographic application is fairly large, the good visibility of the patterns in the photoresist also demonstrates high phase stability. As mentioned above, phase shifting of the structured illumination may be used in a SIM implementation to enable more complete information of the sample area to be gathered (i.e. to recover information about the sample beyond the diffraction limit). The ability to maintain a stable phase is demonstrated by the fact that the pattern can actually be recorded in photoresist. Since repeated imaging using a shifted pattern is required, the control over the phase shift using the SLM is characterized.

For this purpose, an interference pattern was generated a low NA and directly recorded on a CMOS camera. By introducing phase shift on the SLM, an equivalent phase shift of the pattern on the camera is achieved as presented in Figure 10. Along the top of the figure, four phase steps and the intensity profiles are shown, demonstrating the capability of phase control over the extent of the modulation frequency. The lower portion of the figure is a graph showing line profiles of the four phase steps.

The imaging performance of a SIM setup of the proposed type may be assessed by reconstructing simulated raw data. First, the theoretical background of SIM as it is used in the presented work is explained. In the second part, the expected results of SIM using different parameters are shown.

Theoretical formulation of the imaging process

The image formation in two-dimensional widefield fluorescence microscopy using a sinusoidal illumination pattern can be formulated as

The acquired image D is the dye distribution of the fluorescence labelled sample S multiplied with the illumination intensity / and convolved

with the point spread function or PSF (h) of the microscope; r refers to the spatial coordinate. In Fourier space this expression becomes:

Tilde (~) indicates the Fourier transform, and k is the Fourier space coordinate or spatial frequency. The extent of the optical transfer function (OTF) is limited to a cutoff frequency This is a basic property of the imaging objective and

results in a limited resolution in the image. If the fluorescence in the sample is excited using a sinusoidal illumination pattern:

with a modulation depth a_m, a wave vector p and a phase Φ. The Fourier transform of an acquired image can be written as

The Fourier transforms of the acquired images

contain bands that are shifted by mp with respect to their original position in

Fourier space before they are multiplied with the OTF. This way frequency components of the sample that actually lie outside the OTF support are now transmitted into image space. Taking N images of the sample with different phases Φ_h with n = 1, ··· , N, the resulting Fourier transforms of the images can be written

as

with the acquired images in the vector the matrix

M_nm = exp (imΦ_n) and a vector . If the inverse of

M (M^-1) exists, the different components can be separated by

The final image is the inverse Fourier transform of

the final estimate in the Fourier domain. It is obtained by shifting each band to its original position and recombining them using a generalized Wiener filter

The Wiener filter reduces the degrading influence of the OTF and weights the bands in regions where they overlap according to their expected SNR. The Wener parameter w is determined empirically, A(k) is an apodization function decreasing linearly from unity at the centre to zero near the end of the extended OTF support, shaping the overall spectrum in order to prevent ringing artefacts in the final image, and the asterisk (*) indicates the complex conjugate. Since the resolution improvement only takes place in the direction of p, the process of image acquisition and band separation is repeated for different orientations d to obtain isotropic resolution enhancement.

Simulations

For the presented simulations in Figures 11, 12, 13 & 14 an imaging objective with a numerical aperture of NA = 0.25 is assumed. The excitation and emission wavelength is set to be equal l = 10 px in the sample space.

Panel (a) in Figure 11 shows the sample which is a Siemens star. Panel (b) shows a simulation of the widefield deconvolution. Panel (c) shows the corresponding Fourier spectrum.

The simulated result for plain illumination is generated in the same way as results for structured illumination, just with an interference angle of 0° generating plane illumination. For the simulation of the raw data for SIM, three interference angles of 23°, 36° and 54° (referred to as NA1, NA2, and NA3) for the generation of the illumination pattern can be used. These interference angles determine the fringe spacing of the sinusoidal illumination pattern. For the orientations, three or six evenly distributed angles with a random overall offset are used. For each direction, a set of three evenly distributed phases, also with a random overall offset, is generated. The raw data images are then simulated according to Eqs. (4) and (6) using a modulation depth of am = 1. The PSF is simulated using a 2D distribution based on the Bessel function of first kind and first order. For best possible contrast the colour map “Morgenstemning” is used to represent the intensity here and in the following figures. The reconstruction results as presented in Figures 12, 13 & 14 show the resolution improvement that is to be expected in comparison to Figure 11.

Different NAs and orientations for the illumination pattern result in different pattern frequencies and wavevectors p in the description above. Since the data is acquired consecutively, the different frequency components can be separated one at a time and joined as described in Eq. (10) with additional terms d in the sum.

If the Fourier space is filled successively but only three pattern orientations are used the Fourier space is not filled isotropically anymore. In Figure 12 panel (a) and the corresponding Fourier spectrum (d), the reconstruction is done with simulated data using illumination patterns generated with NA1. This way three pattern orientations with three phase steps each equals nine raw data images are required. In panels (b) and (e) the raw data set is simulated with NA1 and NA2. An additional set of nine raw data images, in total eighteen, are acquired. In panels (c) and (f) all three suggested NAs: NA1 & NA2 & NA3 are used. A third set of raw data images, in total twenty-seven images are acquired. The reconstructions in Figure 12 use only three pattern orientations instead of six in order to create the raw data, which leads to an anisotropic filling of the Fourier space of the reconstruction.

Figure 13 illustrates what happens if the Fourier space is not filled successively but with six pattern orientations (and three phase steps for each). Panels (a) and (d) show the image reconstruction based on raw data simulated with patterns of NA2 only.

Since images of six pattern orientations each with three phase steps are used, a total of eighteen raw data images is acquired for the proposed reconstruction. In panels (b) and (e) the raw data is simulated using NA3 only, acquiring eighteen raw data images as well. Panels (c) and (f) show a reconstruction based on raw data using NA2 and NA3. This way twice as many raw data images (thirty-six) are acquired but there is a gap between the low and the high frequency components. This gap results in artefacts in the reconstruction and is clearly visible in the figure.

Figure 14 shows the expected reconstruction results for six orientations but using only NA1 in panels (a) & (b); NA1 and NA2 in panels (c) & (d); and NA1, NA2, and NA3 in panels (e) & (f). The Fourier space of the images is expanded successively and the resolution improves accordingly. There are no gaps in the Fourier space, neither is the Fourier space filled anisotropically. Figures 15a, 15b & 15c show examples of illumination patterns that can be achieved by interfering more than two beams and introducing phase difference between the beams. The illumination patterns shown in Figures 15a-c are produced by first and second beams entering a sample region along linear paths at 40 degrees to the imaging axis. The scales of the x- and y-axes are microns (1 x 10^-6 m).

These patterns are simulated using interference of four beams and introducing phase difference of different values between the beams. Such interference patterns create a set of diverse illuminations, illuminating different regions of the sample. Through this, several unique images of the same sample are generated, each carrying a different information. Then, methods like MUSICAL, SOFI and ESI, that use diversity or stochasticity in such images can be applied. These methods compute an indicator function, which indicates the presence of indicator. The indicator function uses mathematical concepts such as entropy in ESI, and projections into signal and null spaces in MUSICAL to be able to support super-resolution.

Figure 14 shows a schematic view of a transmission microscope set-up 1502 comprising an illumination apparatus 1504 and collection/imaging optics 1506, which are decoupled from each other. A sample 1508 is illuminated with non-sinusoidal illumination patterns generated using a spatial light modulator (SLM) 1510 and a multimirror arrangement 1512 (in this case, four mirrors M_-2, M_-1, M₊₁, M₊₂ are used). A common light source 1514 is diffracted from the SLM 1510 to produce four light beams tuned with different phases. The four light beams are reflected towards the sample 1508 by the multi-mirror arrangement 1512 where they interfere to produce complex structured illumination (interference patterns) in the sample plane of the microscope.

The collection/imaging optics 1506 comprises an objective lens that collects light emanating from the sample (e.g. via fluorescence in the sample), and an imaging sensor that images the collected light. The objective lens has a numerical aperture that is less than the effective numerical aperture of the illumination apparatus.

The illumination pattern is homogeneous over a large area (mm × mm) in the sample plane. In the collection/imaging optics 1506, a low magnification/NA objective lens is used, which enables imaging with large FOV. The multi-mirror arrangement 1512 is placed beneath the sample stage such that two plane waves interfere at the sample plane generating closely spaced interference fringes. The illumination apparatus 1504 is shown in a first configuration that generates a first complex illumination pattern with a first fringe period. However, the illumination apparatus 1504 is capable of generating illumination patterns with different fringe periods (different spatial frequency) through adjustment of the multi mirror arrangement 1512. Providing different illumination patterns supports isotropic resolution enhancement by covering the Fourier space.

The orientation of the illumination pattern may also be varied (e.g. by rotating the SLM 1510). For example, three different orientations R₁, R2 and R3 may be used with angular offsets of α₁ = 0^°, α₂= 60^° and α₃= 120^° respectively, as shown in Figure 17.

Figure 18 is a frequency spectrum of the illumination pattern generated by the illumination apparatus 1504 described above in the sample plane. The pattern comprises a mixture of four frequencies:

0.98 λ^-1 and f₂ = 1.28 λ^-1 in increasing order.

For first orientation of sinusoidal grating let the grating vector is along one direction x direction only and kG = kGx. Normal structured illumination imaging is done by considering first order beams only. Here our interest takes consideration of both the first and second orders simultaneously. Towards the enhancement of image resolution, it leads to an interesting phenomenon called double-more effect which is an extension of moire effect in conventional SIM imaging. The intensity expression I₁ signifies the double-moire illumination by interference of four beams when the plane of incidence R₁ is oriented at an angle ai with respect to x axis. This illumination incorporates higher frequency response in SIM imaging in one direction which eventually lead to high resolution image along that particular direction. The grating pattern displayed onto the SLM need to be rotated to achieve isotropic resolution enhancement in SIM imaging. The four-beam interference intensity profile corresponding to R, orientation is given by the equation:

Intensity distributions of four beam interference for the three different orientations R₁,

R₂ and R₃ are represented in Figs. 19a, 19b and 19c respectively. Each figure shows a stack of illumination patterns generated using four beam interference with phase step 0.04p between elements of each stack. There are 50 images in each orientation. The intensity values are normalized shown by the greyscale bar.Certain embodiments of the invention are able to enhance resolution by two to five or more times over the diffraction limit in the event of fluctuating fluorophores. The enhancement in resolution is of the same order as the fringe pattern periodicity, according to a preliminary analysis. Moreover, since the indicator functions in such methods are non-linearly related to the fluorescence intensity, they also provide considerable contrast enhancement. MUSICAL and ESI are not believed to have been applied in transmission-mode setup. With this innovation, transmission-MUSICAL and transmission-ESI can also provide large FOV with super-resolution imaging and contrast enhancement.

Embodiments of the invention not only decouple the pattern generation from the imaging path, but are also able to generate uniform interference patterns over a large FOV. This may find utility wherever it is necessary to image large areas of samples, such as pathological tissue samples. For examination of tissue samples, it may be useful to acquire image over large areas for accurate diagnoses. In addition to enhancement of resolution, structured illumination may also benefit the image contrast by providing better optical sectioning and removing out-of-focus light.

It will be appreciated by those skilled in the art that the invention has been illustrated by describing one or more specific embodiments thereof, but is not limited to these embodiments; many variations and modifications are possible, within the scope of the accompanying claims.

Claims

Claims

1. An apparatus for transmission microscopy comprising: an illumination apparatus arranged to illuminate a sample region with a first monochromatic coherent light beam and with a second monochromatic coherent light beam; and an objective lens, having an imaging axis, for collecting light emanating from a sample within the sample region, wherein: the illumination apparatus has a first configuration in which the first light beam enters the sample region along a first linear path from a first reflecting element and the second light beam enters the sample region along a second linear path from a second reflecting element, such that the first and second light beams interfere within the sample region to illuminate the sample with a first interference pattern; the illumination apparatus has a second configuration, in which the first light beam enters the sample region along a third linear path, different to the first path, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern; and the first, second and third paths are at respective first, second and third angles to the imaging axis, and at least one of the first, second and third angles is oblique to the imaging axis.

2. The apparatus as claimed in claim 1, wherein, in the first configuration, the illumination apparatus has a first effective numerical aperture and in the second configuration, the illumination apparatus has a second, different effective numerical aperture.

3. The apparatus as claimed in claim 1 or 2, comprising imaging apparatus arranged to image the light collected by the objective lens.

4. The apparatus as claimed in claim 3, wherein the imaging apparatus is arranged to perform super-resolution imaging using the light collected by the objective lens.

5. The apparatus as claimed in any preceding claim, wherein a spatial wavelength of the first and/or second interference pattern is less than a diffraction limit of the objective lens.

6. The apparatus as claimed in any preceding claim, wherein a spatial wavelength of the first and/or second interference pattern is less than 1.5 times a wavelength of the first and/or second beam,

7. The apparatus as claimed in any preceding claim, wherein the objective lens has a numerical aperture of less than one.

8. The apparatus as claimed in any preceding claim, wherein the first and second interference patterns are stable over the entire intersection of a field-of-view of the objective lens with the sample or the sample region.

9. The apparatus as claimed in any preceding claim, wherein the first and/or second interference pattern comprises a constant period along a path parallel to the imaging axis over a distance that is longer than a depth of field of the objective lens.

10. The apparatus as claimed in any preceding claim, wherein the illumination apparatus has a third configuration, in which the first or second light beam enters the sample region along a third-configuration linear path, such that the first and second light beams interfere within the sample region to illuminate the sample with a third interference pattern, wherein the third pattern is not the same as the first or second patterns, and is not the same as any translation, rotation, or translation and rotation, of the first or second patterns.

11. The apparatus as claimed in any preceding claim, wherein the first and second configurations result from respective first and second physical arrangements of the illumination apparatus.

12. The apparatus as claimed in any preceding claim, wherein the first reflecting element is a first mirror and wherein the second reflecting element is a second mirror.

13. The apparatus as claimed in any preceding claim, wherein the third linear path is a linear path to the sample region from the first reflecting element or from a third reflecting element.

14. The apparatus as claimed in any preceding claim, comprising an adjustable reflecting apparatus arranged to support a first setting associated with the first configuration of the illumination apparatus and a second setting associated with the second configuration of the illumination apparatus.

15. The apparatus as claimed in claim 14, wherein the adjustable reflecting apparatus comprises the first reflecting element, and wherein a position and/or inclination and/or orientation of the first reflecting element is adjustable between the first and second settings.

16. The apparatus as claimed in claim 14 or 15, wherein the adjustable reflecting apparatus comprises the first reflecting element and further comprises a third reflecting element, and wherein the illumination apparatus is arranged to direct the first beam at the first reflecting element when in the first setting, and to direct the first beam at the third reflecting element when in the second setting.

17. The apparatus as claimed in any preceding claim, wherein the first and second angles are equal and the first and second paths are coplanar.

18. The apparatus as claimed in any preceding claim, wherein, in the second configuration of the illumination apparatus, the second light beam enters the sample region along a fourth path, different to the second path, wherein the fourth path is at a fourth angle to the imaging axis.

19. The apparatus as claimed in claim 18, wherein the third and fourth angles are equal and the third and fourth paths are coplanar.

20. The apparatus as claimed in claim 18 or 19, comprising a second adjustable reflecting apparatus comprising said second reflecting element, wherein the second adjustable reflecting apparatus is arranged to support a first setting associated with the first configuration of the illumination apparatus and a second setting associated with the second configuration of the illumination apparatus, wherein, when in the first setting, the second adjustable reflecting apparatus is arranged to reflect the second light beam such that it enters the sample region along the second path from the second reflecting element and, when in the second setting, the second adjustable reflecting apparatus is arranged to reflect the second light beam such that it enters the sample region along the fourth path.

21. The apparatus as claimed in claim 20, wherein the first adjustable reflecting apparatus and the second adjustable reflecting apparatus comprise a common toroidal structure comprising a plurality of pairs of opposing mirrors mounted on the common toroidal structure.

22. The apparatus as claimed in any preceding claim, comprising an electronic controller configured to control the configuration of the illumination apparatus.

23. The apparatus as claimed in any preceding claim, wherein the first and second light beams are produced by a common light source, such as a laser.

24. The apparatus as claimed in claim 23, wherein the first and second light beams comprise diffraction fringes produced by an initial light beam produced by the common light source interacting with a diffraction grating or spatial light modulator.

25. The apparatus as claimed in any preceding claim, wherein the illumination apparatus comprises a controllable optical component arranged to alter one or more properties of the first and/or second light beams and thus of the resulting interference patterns.

26. The apparatus as claimed in claim 25, wherein the controllable optical component is a spatial light modulator.

27. The apparatus as claimed in claim 25 or 26, wherein the controllable optical component is arranged to adjust the phase of the first and/or second light beams.

28. The apparatus as claimed in any of claims 25 to 27, wherein the controllable optical component is arranged to control the orientation of the first and/or second interference patterns relative to the sample region.

29. The apparatus as claimed in any of claims 25 to 28, configured to adjust the controllable optical component when changing the illumination apparatus between the first and second configurations.

30. The apparatus as claimed in any of claims 25 to 29, wherein the controllable optical component is arranged to vary the pattern orientation and/or phase offset of the first and second interference patterns so as to illuminate the sample region across the Fourier space.

31. The apparatus as claimed in any preceding claim, wherein the illumination apparatus is configured for rotating a pattern on an spatial light modulator so as to alter the orientation of a resulting interference pattern relative to the sample region.

32. The apparatus as claimed in any preceding claim, wherein the illumination apparatus is further arranged to illuminate the sample region using one or more additional monochromatic coherent light beams.

33. A method of imaging a sample using transmission microscopy, wherein the method comprises: in a first time period, i) illuminating a sample with a first monochromatic coherent light beam entering the sample along a first linear path from a first reflecting element, and with a second monochromatic coherent light beam entering the sample along a second linear path from a second reflecting element, such that the first and second light beams interfere within the sample to illuminate the sample with a first interference pattern, and ii) collecting light emanating from the sample, illuminated with the first interference pattern, with an objective lens; and in a second time period, i) illuminating the sample with the first light beam entering the sample region along a third linear path, different to the first path, and with the second light beam, such that the first and second light beams interfere within the sample region to illuminate the sample with a second interference pattern, wherein the second pattern is not the same as the first pattern, and is not the same as any translation, rotation, or translation and rotation, of the first pattern, and ii) collecting light emanating from the sample, illuminated with the second interference pattern, with the objective lens, wherein the first, second and third paths are at respective first, second and third angles to an imaging axis of the objective lens, and at least one of the first, second and third angles is oblique to the imaging axis.

34. The method as claimed in claim 33, wherein the light collected by the objective lens is produced in the sample via fluorescence.

35. The method as claimed in claim 33 or 34, further comprising performing superresolution imaging of the sample using the light collected by the objective lens.