Optical control of submicron objects for nanoscale imaging

Abstract

A microfluidic device 100 for accommodating a sample during nanoscale imaging comprises a microfluidic channel 101 and one or more sets of opposing optical waveguides 103a,103b, wherein each set of op

Description

Field of the invention The present invention relates to systems and methods for controlling the position and orientation of submicron objects during nanoscale imaging, in particular, by controlling the position and orientation of the submicron objects using optical forces. Background Mechanical goniometers are widely used for specimen orientation in electron microscopy (including transmission electron microscopy (TEM) and scanning electron microscopy (SEM)), x-ray microscopes / diffractometers, and scattering experiments. These systems have a number of limitations, including: • low speed: mechanical systems are often slow, hindering rapid data acquisition; • low precision: precision in the positioning of samples is limited, affecting the reproducibility of experiments; • a large sphere of confusion: the mechanical design of multi-axis goniometers introduces a significant sphere of confusion (the more axes that a goniometer has, the larger the sphere of confusion, leading to a decrease in precise control); • mechanical constraints: mechanical stages are constrained by their physical design, limiting the range and fluidity of specimen orientation, and often resulting in a "missing wedge" of data in tomographic analysis; and • sample damage: mounting nanoscale materials on grids can deform the specimen, affecting the accuracy of imaging and analysis. Given these limitations, there is a pressing need for an advanced control system for specimen orientation that is faster, more precise, and capable of manipulating samples without physical contact or mechanical constraints. Such a system should also be adaptable to various microscopy and diffraction techniques, while minimising sample handling and damage. Summary According to a first aspect of the disclosure, there is provided a microfluidic device for accommodating a sample during nanoscale imaging, wherein the microfluidic device comprises: a microfluidic channel provided between opposing window layers; and one or more sets of opposing optical waveguides, wherein each set of opposing optical waveguides is configured to guide one or more optical fields into the microfluidic channel, to control, by optical forces, the position and / or orientation of one or more objects (e.g. submicron objects) present in a sample in the microfluidic channel. Nanoscale imaging includes imaging by methods capable of achieving a resolution of 100 nm or smaller. For the purposes of the present disclosure, nanoscale imaging is also not limited to the generation of images in real space (e.g. by electron or x-ray microscopy), but also includes, for example, methods involving the generation of diffraction patterns in reciprocal space (e.g. electron or x-ray diffraction). Optical waveguides are structures capable of directing and / or confining the propagation of optical light. The optical fields may be of any optical wavelength suitable for the trapping of particles (e.g. of visible or (near) infrared wavelengths, for example in the range of 400-7,000 nm). In embodiments, the objects present in the sample have a maximum diameter of 20 pm or less, or 10 pm or less, or the objects are submicron objects. In embodiments, the opposing optical waveguides in each set are configured to guide respective optical fields into the microfluidic channel, wherein said optical fields are counter-propagating. In embodiments, one or more of the opposing optical waveguides in each set are configured to collect optical light scattered by the one or more objects, and to guide the collected scattered light to one or more detectors. In embodiments, the nanoscale imaging is by electron or x-ray microscopy and / or electron or x-ray diffraction, and the window layers are substantially transparent to electron and / or x-ray radiation. In embodiments, the window layers each have a thickness of 100 nm or less, 50 nm or less, or 30 nm or less, optionally wherein the window layers each have a thickness of between 10 nm and 30 nm. Thinner window layers generally are advantageous for imaging, e.g. in order to reduce background noise. However, the thickness of the window layers can be adapted to particular applications. For example, window layers having a thickness of 100 nm or less may be suitable for x-ray-based imaging techniques, whereas a thickness of 50 nm or less may be particularly suited for use in electron-based methods. In embodiments, the window layers are each formed substantially of silicon nitride, amorphous silicon, graphene, or 2D materials. In embodiments, at least a portion of each of the optical waveguides, adjacent to the microfluidic channel, is provided between the opposing window layers. In embodiments, a refractive index of the optical waveguides is equal to or higher than a refractive index of the window layers. In embodiments, the optical waveguides each have a thickness of 500 nm or less. In embodiments, the optical waveguides are each formed substantially of silicon nitride, silicon, or polysilicon. In embodiments, the optical waveguides are selected from slot waveguides, wire waveguides, and rib waveguides. In embodiments, the optical waveguides are selected from single mode waveguides and multimode waveguides. In embodiments, the device comprises a plurality of the sets of opposing optical waveguides. In embodiments, the microfluidic channel further comprises one or more fluid ports to allow the flow of liquid into and out of the microfluidic channel. In embodiments, the microfluidic channel further comprises a frame structure configured to support the window layers and / or the optical waveguides. In embodiments, the frame structure is substantially transparent to optical light, and is configured to allow optical light to pass through the material of the frame structure from an optical light source into the optical waveguides. In embodiments, the frame structure is substantially formed from silicon, silicon nitride, aluminium nitrite, gallium nitride, or diamond. In embodiments, the optical waveguides each comprise, at an end opposite to the microfluidic channel, a tapered portion configured to receive the optical light passing through the material of the frame structure. In embodiments, the frame structure comprises: an opening adjacent to each of the window layers; and / or one or more openings adjacent to respective fluid ports of the microfluidic channel. According to a second aspect, there is provided a sample holder for use in nanoscale imaging, wherein the sample holder comprises: a mount for a microfluidic device as described above, and wherein the sample holder is configured to insert a microfluidic device present in the mount into an imaging region of a nanoscale imaging device. In embodiments, the nanoscale imaging device is selected from an electron or x-ray microscope or an electron or x-ray diffractometer. In embodiments, the sample holder is configured to accommodate one or more optical fibres for coupling an optical light source to the optical waveguides of the microfluidic device. In embodiments, the sample holder further comprises: one or more optical elements for coupling the one or more optical fibres to the optical waveguides, optionally wherein the one or more optical elements comprise one or more lenses. In embodiments, the sample holder is configured to accommodate one or more fluid conduits coupled to one or more fluid ports of the microfluidic channel of the microfluidic device. According to a third aspect, there is provided a system for controlling the position and / or orientation of one or more objects (e.g. submicron objects) in a sample during nanoscale imaging, the system comprising: a microfluidic device according to any one preceding claim; one or more optical sources optically coupled to the optical waveguides of the microfluidic device; and one or more processors configured to control the one or more optical light sources to control, by optical forces, the position and / or orientation of one or more objects present in a sample in the microfluidic channel of the microfluidic device. In embodiments, the objects present in the sample have a maximum diameter of 20 pm or less, or 10 pm or less, or the objects are submicron objects. In embodiments, the system further comprises: one or more detectors configured to detect scattering of the optical fields by the one or more objects. In embodiments, the one or more processors are configured to, based on the detected scattering: determine a position and / or or orientation of the one or more objects based on the scattered light detected by the one or more detectors; and / or perform an optical analysis of the sample based on the scattered light detected by the one or more detectors, wherein the optical analysis is selected from: Raman spectroscopy; photoluminescence analysis; and cathodoluminescence analysis, optionally wherein the optical analysis is performed simultaneously with the nanoscale imaging. In embodiments, the one or more processors are configured to: control the one or more one optical light sources to manipulate an orientation of the one or more objects by manipulating a polarization of the optical fields (e.g. of counter-propagating optical fields); and / or control the one or more optical light sources to manipulate a position of the one or more objects by manipulating a phase shift and / or an intensity of the optical fields (e.g. a relative phase shift and / or a relative intensity of counter-propagating optical fields). In embodiments, the system further comprises: a user interface configured to allow a user to control the position and / or orientation of the one or more objects via the one or more processors. According to a fourth aspect, there is provided a method of nanoscale imaging, the method comprising: providing a sample comprising one or more objects (e.g. submicron objects) in an imaging region of a nanoscale imaging device; controlling a position and / or orientation of the one or more objects within the imaging region using optical forces produced by optical fields; and imaging the one or more objects, wherein the imaging comprises: illuminating the one or more objects using electron or x-ray radiation; and detecting electron or x-ray radiation scattered by the one or more objects in the sample to generate image data. In embodiments, the objects have a maximum diameter of 20 pm or less, or 10 pm or less, or the objects are submicron objects. In embodiments, the controlling of the position and / or orientation of the one or more objects within the imaging region is by using optical forces produced by counterpropagating optical fields. In embodiments, controlling the position and / or orientation of the one or more objects comprises controlling the one or more objects to be substantially stationary during imaging. In embodiments, the method comprises: imaging the one or more objects in a first position and / or orientation; manipulating the one or more objects to one or more different positions and / or orientations, by modulating the optical fields; and imaging the one or more objects in each of the different positions and / or orientations. In embodiments, the method further comprises combining image data generated by imaging the one or more objects in the plurality of different positions and / or orientations to reconstruct an image of the one or more objects, optionally by tomographic reconstruction. In embodiments, modulating the optical fields comprises: modulating a polarization of the optical fields (e.g. of counter-propagating optical fields) to manipulate an orientation of the one or more objects; and / or modulating a phase shift and / or an intensity of the optical fields (e.g. a relative phase shift and / or a relative intensity of counter-propagating optical fields) to manipulate a position of the one or more objects. In embodiments, the method further comprises: detecting scattering of the optical fields by the one or more objects. In embodiments, the method further comprises: based on the detected scattering, determining a position and / or or orientation of the one or more objects. In embodiments, the method further comprises: based on the determined position and / or orientation of the one or more objects, modulating the optical fields. In embodiments, modulating the optical fields comprises: actively stabilising the position and / or orientation of the one or more objects, by modulating the optical fields to compensate for Brownian motion of the one or more objects within the sample. In embodiments, modulating the optical fields is based on any one or more of: velocity damping; phased-locked loop control; kalman filtering; and directional light scattering feedback. In embodiments, the method further comprises: controlling the position and / or orientation of the one or more objects within the imaging region by electrical forces produced by electrical fields within the microfluidic channel. In embodiments, the method further comprises, based on the detected scattering: performing an optical analysis of the sample, wherein the optical analysis is selected from: Raman spectroscopy; photoluminescence analysis; and cathodoluminescence analysis, optionally wherein the optical analysis is performed simultaneously with the imaging of the one or more objects. Brief description of the drawings Fig. 1 shows schematically a cross-sectional view of a microfluidic device according to the present disclosure. Fig. 2 shows schematically a plan view of a microfluidic device according to the present disclosure. Fig. 3 shows schematically cross-sectional views of example optical waveguide structures of a microfluidic device according to the present disclosure. Fig. 4 shows schematically additional views of a microfluidic device according to the present disclosure. Figs. 5A and 5B show schematically arrangements for coupling light into optical waveguides of a microfluidic device according to the present disclosure. Fig. 6 shows a close-up image of a microfluidic device according to the present disclosure. Figs. 7A-7F show plots of relative intensities from finite difference time domain calculations, at different regions of a microfluidic device according to the present disclosure. Fig. 8 shows schematically a sample holder for mounting a microfluidic device according to the present disclosure. Fig. 9 shows schematically a system according to the present disclosure, for controlling the position and / or orientation of one or more submicron objects in a sample during nanoscale imaging. Fig. 10 is a flow diagram illustrating a method of nanoscale imaging according to the present disclosure. Detailed description Microfluidic device Fig. 1 shows schematically a cross-sectional view of a microfluidic device (or “sample chip”) 100 according to the present disclosure. The microfluidic device 100 is configured to accommodate a sample during nanoscale imaging (e.g. by electron microscopy or electron / x-ray diffraction). As described herein, the microfluidic device 100 facilitates the control of the position and orientation of submicron objects present in the sample using optical forces. The microfluidic device 100 comprises a microfluidic channel 101 for accommodating the sample. The microfluidic channel 101 may accommodate the sample within a liquid or vacuum environment, depending upon the nature of the sample or the desired imaging application. The microfluidic channel 101 is provided between a pair of opposing window layers 102a, 102b. The window layers 102a, 102b are configured to seal the microfluidic channel 101 from the external environment of the microfluidic device 100 (e.g. from the internal vacuum of an electron microscope). The window layers 102a, 102b are thin (e.g. 10-30 nm thick, for example 20 nm thick) and are formed of a material that produces low electron / x-ray scattering, such as silicon nitride, amorphous silicon, graphene, or suitable 2D-materials, in order to allow the passage of electron or x-ray radiation (indicated by intersecting arrows) into and out of the microfluidic channel 101 for imaging of the sample. The microfluidic device 100 further comprises a pair of opposing optical waveguides 103a, 103b provided between the opposing window layers 102a, 102b. For the purposes of the present disclosure, the term waveguide may be considered to mean a structure capable of directing and / or confining the propagation of waves (in particular, optical waves, for example visible or near-infrared waves). The optical waveguides 103a, 103b are configured to guide optical fields (e.g. in a wavelength range of 400-7,000 nm) (shown by dashed lines) into the microfluidic channel 101, in order to control the position and / or orientation of one or more submicron objects 104 (shown by ellipse) present in the sample in the microfluidic channel 101. A first waveguide 103a of the pair of opposing optical waveguides has a first end located on one side of a region of the microfluidic channel 101 (wherein said region is accessible to illumination radiation through the window layers 102a, 102b), and a second waveguide 103b of the pair of opposing optical waveguides has a first end located on an opposite side of the region of the microfluidic channel 101. In one example, the optical waveguides 103a, 103b are configured to guide respective counter-propagating fields into the microfluidic channel 101. Such counterpropagating fields interfere to create an interference pattern in which the submicron objects may be trapped and manipulated. Alternatively, during use, one of the optical waveguides 103a may be used to direct an optical field(s) into the microfluidic channel for trapping and positional / orientational control, and the other, opposing, waveguide 103b may be used only to collect the optical light scattered by the sample (e.g. to enable a feedback process as described in detail below). In one example, the pair of opposing optical waveguides 103a, 103b are configured to function as optical tweezers. The optical waveguides 103a, 103b are formed from a material that is suitable for guiding light at visible and near-infrared wavelengths, such as silicon nitride, silicon, or polysilicon. The optical waveguides 103a, 103b also have a higher refractive index than both the window layers 102a, 102b and the surrounding environment (e.g. air or vacuum). Waveguides (e.g. wire waveguides as described below) have a minimum width and height, that is dependent on both the refractive index of the waveguide material and the wavelength of light carried. However, the thickness (or vertical extent) of the optical waveguides 103a, 103b also determines the minimum spacing between the window layers 102a, 102b. Therefore, for electron microscopy applications in particular, by forming the optical waveguides 103a, 103b from a higher refractive index material, and / or by using the waveguides to carry light of a shorter wavelength, the dimensions of the waveguides can be minimised in order to reduce scattering of the radiation used for imaging. For electron microscopy or diffraction applications, optical waveguides 103a, 103b having a thickness of 500 nm or less may allow for an improved image due to low scattering and attenuation of the electron beam. Thicker waveguides may be more suitable for x-ray diffraction applications, for example. In one example, as shown in Fig. 2, a microfluidic device 200 comprises a plurality of pairs (201-1.,.201-n) of opposing optical waveguides (e.g. 10 pairs), each intersecting a microfluidic channel 202. This allows for the control of multiple submicron objects within the microfluidic channel simultaneously. As shown in Fig. 2, the microfluidic channel 202 also comprises fluid ports 203a, 203b, to allow the flow of fluids into and out of the microfluidic channel 202. Fig. 3 shows schematically a cross-sectional view of two examples of suitable structures for the optical waveguides of the microfluidic device. Panel A of Fig. 3 shows a wire waveguide 301a with a square cross-section. The wire waveguide 301a allows for the propagation of any light polarisation in a single longitudinal mode, and allows for the propagation of light with transverse-electric (TE) or transverse-magnetic (TM) polarisation. Alternatively, panel B of Fig. 3 shows a slot waveguide 301b, comprising two regions of material having a high refractive index, separated by a slot region. The use of a slot waveguide can further reduce the vertical dimensions of the microfluidic device, and thus further reduce scattering losses. Slot waveguides can also result in larger optical forces for the same light intensity when compared to wire waveguides, as the gradients produced are more localised. Other suitable waveguide structures, such as rib waveguides, are also contemplated. In some examples, the optical waveguides are multimode waveguides configured to generate optical fields having multiple intensity maxima within the microfluidic channel. This allows for the control and manipulation of multiple submicron objects simultaneously, even when the objects are in close proximity. Fig. 4 shows schematically additional views of a microfluidic device 400 as shown in Fig. 1. As shown in Fig. 4, the assembly of the window layers 401a, 401b and the optical waveguides 402a, 402b is supported and surrounded by a frame structure 403, formed of materials such as silicon, silicon nitride, aluminium, aluminium nitride, gallium nitride, or diamond. The frame structure 403 comprises openings 404a, 404b adjacent to each of the window portions 401 a, 401 b, to allow illumination radiation to enter the microfluidic chip 400 through a first window portion 401a, interact with the sample in the microfluidic channel, and exit the microfluidic chip 400 through the second window portion 401b for detection. Additional openings 405a, 405b are also provided in the frame 403 adjacent to each of the fluid ports of the microfluidic channel, to allow the flow of fluid into and out of the microfluidic channel. The microfluidic device also comprises means for coupling optical light from a source into the optical waveguides. The coupling means are polarisation insensitive (unlike, for example, grating couplers), to allow for orientational control of the sample objects by modulating the polarisation of the optical fields. The same coupling means also allow light to be collected after being scattered by the sample in the microfluidic channel. As described in more detail below, this scattered light can then be directed (e.g. by optical fibres) to photodetectors located external to the microfluidic device, to be used for position / orientation feedback, or for in-situ optical analysis methods such as Raman spectroscopy, photoluminescence, and cathodoluminescence. Figs. 5A / 5B show schematically two suitable arrangements for coupling light into the optical waveguides of the microfluidic device. In the example shown in Fig. 5A, light is directed into the optical waveguides 501a, 501b through the material of the frame structure 502 via a high numerical aperture lens 503. In the figure, only the optical coupling for the right-hand waveguide 501b is shown in each case, but it will be appreciated the same or similar coupling may be applied to the other, opposing waveguide 501a. In this example, the frame 502 is formed from a material that is substantially transparent to optical light of a suitable wavelength (e.g. silicon, which is substantially transparent to light having a wavelength of approximately 1550 nm, for example). A waveguide taper (not shown) may also be provided within the frame structure 502, to enhance the coupling of the light into the optical waveguides 501a, 501b. In the alternative arrangement shown in Fig. 5B, an optical fibre 504 passes through the frame 502 and abuts or is directly coupled onto (e.g. using a waveguide taper 505) an end of the optical waveguides 501a, 501b opposite to the microfluidic channel 506. Fabrication This section outlines suitable fabrication processes for constructing a microfluidic device (or sample chip) according to the present disclosure. In one example, the process commences by providing two silicon wafers, each with a thickness of 200 pm. In an initial stage, both wafers are coated with a 20 nm silicon nitride layer. Subsequently, one of these wafers is further processed to add a 200 nm thick silicon layer. This silicon layer undergoes a photolithographic and etching process to delineate microfluidic channels and optical waveguide structures. Following the patterning process, the two prepared wafers undergo a direct bonding procedure, which fuses them into a single, integrated structure. Windows are then etched through the top and bottom 200 pm silicon layers down to the silicon nitride layer, creating transparent areas for imaging beam interrogation and access to the underlying channels. Another example method of fabrication may include steps of: 1. Substrate Preparation: • A suitable (e.g. smooth) substrate is selected to ensure good adhesion and to prevent lift-off issues. 2. Mask Alignment: • Utilizing a mask aligner, a design is transferred onto the substrate. 3. Use of Adhesion Promoters: • Adhesion promoters may be applied to enhance the attachment of the photoresist to the substrate, thereby further mitigating the risk of pattern lift-off during development. 4. Pattern Transfer: • The sample chip design is imprinted onto the substrate using photolithography, which allows for the optical waveguides and liquid guides to be defined with high dimensional accuracy. 5. Etching and Development: • An etching process removes material to create the channels and features that constitute the optical pathways and fluidic channels. Features (e.g. as small as 1 micron in width) can be formed with high precision. 6. Deposition of Functional Layers: • Layers of materials with different refractive indices are deposited. The choice of materials, such as silicon nitride or polysilicon, may be determined based on etching characteristics as well as compatibility with the overall design. 7. Assembly: • The completed components are assembled. This may include the integration of the sample chip within a holder, and the alignment of the optical and liquid guides with respect to the sample holder. A close-up view of a section of a microfluidic device 600 prepared according to a method as described herein is shown in Fig. 6. By using precision manufacturing processes such as photolithography, it is possible to achieve high uniformity of the microchannels 601. As shown by the scale bar, the width of the channels 601 and the separation between features can be of order of micrometres, and walls of material provided between the liquid channel 601 and the waveguides 602a, 602b, 603a, 603b can be of the order of hundreds of nanometers. Alternative processes, that are similarly capable of producing a refractive index differentiation between the waveguide structures and the window layers, are also contemplated. For example, electron beam lithography may be used in order to achieve higher resolution patterning (e.g. for features with dimensions of the order of 500 nm). Backside etching techniques may also be advantageous for specific design requirements, such as for removing silicon from the frame's backside and filling with silicon nitride, to ensure structural integrity while maintaining the requisite refractive index contrast. Position and orientation control During use, optical or near-infrared light is coupled from a light source into the optical waveguides of the microfluidic device. The opposing optical waveguides direct the light into the microfluidic channel, creating fields capable of exerting forces upon objects present in a sample in the microfluidic channel. Such forces can be used to trap the objects in a particular position and orientation for imaging. In particular, an optical tweezer based set-up allows for the precise manipulation of microscopic particles, by leveraging light momentum transfer and the dielectric properties of the particles. When a particle is introduced into a high-intensity focal point (e.g. of a laser beam), gradient forces of the light field act to trap the particle at the beam's centre, where the light intensity is maximum. This trapping results from the particle's refractive index being higher than the surrounding medium, leading to a net force that pulls the particle towards the region of highest light intensity. Figs. 7A-7F show plots of relative intensities from finite difference time domain calculations, at different regions of a microfluidic device. Longer / narrower rectangles 701 outline the locations of silicon nitride windows, and the larger rectangles 702 indicate a 400x400 nm silicon waveguide. Panel A shows the relative intensity inside the waveguide, for light polarised along the y-axis. Panel B shows the relative intensity inside the microfluidic channel after exiting the waveguide, for light polarised along the y-axis. Panel C shows relative intensity from one of two possible counter-propagating beams in the microfluidic channel after exiting the waveguide, for light polarised along the y-axis (and propagating from right to left along the negative x-axis). Panel D shows the relative intensity inside the waveguide for light polarised along the z-axis. Panel E shows relative intensity inside the microfluidic channel after exiting the waveguide for light polarised along the y-axis. Panel F shows relative intensity from one of two possible counter-propagating beams in the microfluidic channel after exiting the waveguide, for light polarised along the z-axis (and propagating from right to left along the negative x-axis direction). By using optical forces to hold the objects in position, physical contact with the sample can be avoided, thereby reducing sample deformation. The position and orientation of the objects can also be changed, by modulating the fields present in the microfluidic channel (e.g. by a computer-processor located external to the microfluidic device, configured to control the light produced by the optical light source). For linearly polarised light, the longest length of the object aligns with the polarisation direction. Therefore, the orientation of the submicron objects may be changed by modulating the polarisation of the fields. The positions of the objects can be controlled by modulating the relative phase shifts between two counter-propagating beams, which create a moving interference pattern in which the sample is trapped, as well as by modulating the relative intensity between the beams. Therefore, the use of optical forces allows for the control of the submicron objects with six degrees of freedom (three translational and three rotational), and overcomes the “missing wedge” problem caused by mechanical goniometers in tomographic reconstruction. Precise oscillation of the submicron objects is possible without any backlash relative to the size of the objects. Feedback mechanism The optical light used to control the position and orientation of the submicron objects in the sample will also be scattered by the sample. This scattering can be detected by detectors (e.g. photodiodes), and used to determine the current position and orientation of the submicron objects. This allows for the implementation of feedback control loops, whereby a position and orientation of the submicron objects is determined based on the optical light scattered by the objects, and the optical fields are modulated based on the determined position and orientation. In this way, it is possible to regulate the positional and angular motion of a charged, optically trapped specimen with nanometer precision. It is also possible to achieve both cooling and stabilisation of the specimen during high-resolution imaging processes. This enables the system to counteract thermal fluctuations and other perturbative forces, providing robust control over the specimen's spatial and rotational degrees of freedom. The feedback mechanism operates by monitoring the position of the trapped particle, typically through the light it scatters when illuminated by the trapping laser. This scattered light is collected and analysed, often in real-time, to determine any deviation of the particle from the trap centre. If a displacement is detected, the feedback system promptly adjusts the laser's properties — such as its position, intensity, or phase — to re-centre the particle. This responsive adjustment allows the system to counteract the randomising effects of Brownian motion, which can displace the particle due to thermal collisions with surrounding molecules. The feedback control in an optical tweezer set-up relies on the precise modulation of the optical forces that are inherently part of the light field's interaction with the particle. By continually correcting the particle's position, the feedback mechanism ensures that the optical trap maintains a firm hold on the particle, allowing for its stable manipulation and the ability to perform tasks with remarkable precision at the microscopic scale. Additionally, polarisation feedback may be employed to control the angular alignment of particles, based on the orientation-dependent interaction between the polarised light and the anisotropic features of the particle. By analysing the polarisation of the scattered light, the feedback system can discern the particle's angular orientation. Subsequent adjustments to the trapping light's polarisation then exert torques on the particle, aligning it to a desired orientation. This method allows for the precise rotational control of particles, enabling imaging requiring specific angular positioning within the optical trap on the sample chip. Some particular implementations of the feedback control mechanism include: 1. Velocity Damping Feedback Velocity damping is a technique where the feedback loop applies a force proportional to and opposite to the velocity of the specimen. This is realised by measuring the Doppler shift of the scattered light, which indicates the specimen's velocity. This technique effectively reduces the kinetic energy of the specimen, leading to a reduction in thermal motion and a consequent decrease in image blur. 2. Phase-Locked Loop (PLL) PLL is a control system that generates an output signal whose phase is related to the phase of an input signal. In the present system, PLL may be employed to maintain a constant phase relationship between the motion of the specimen and the feedback signal. By locking the phase, PLL ensures consistent specimen orientation, which is particularly advantageous for techniques such as electron tomography, where angular precision is especially important. 3. Kalman Filtering Kalman filtering may be used to estimate the state of the system by minimising the mean of the squared error. It can predict the specimen's future position and orientation based on a series of measurements over time, which are affected by statistical noise and other inaccuracies. This predictive capability allows for anticipatory adjustments in the feedback loop, enhancing the system’s responsiveness and stability. 4. Electro-Optical Feedback When the specimen is charged, electro-optical feedback can also be applied. This technique combines optical monitoring with an electric field application to manipulate the specimen's position and orientation. Optical sensors detect positional and angular deviations, and in response, electric field vectors are dynamically altered to exert precise forces on the charged specimen, complementing the optical trapping forces. 5. Directional Light Scattering Feedback Directional light scattering feedback utilises the angular distribution of scattered light to determine the specimen's orientation. The intensity and direction of the scattered light provide real-time data on the orientation and allow for corrective adjustments. A photodetector array positioned around the specimen captures the scattered light profile. The feedback system then analyses this profile to maintain the specimen in the desired orientation through controlled light modulation. 6. Combined Feedback Systems The described feedback techniques can be employed individually or in any combination to achieve both positional and orientational stabilisation. For example, velocity damping can be used in conjunction with PLL to maintain both the position and angular frequency of the specimen. The feedback loop parameters for each technique can be adjusted based on the specimen's properties, such as charge, mass, and optical response, enabling a tailored control strategy for different imaging scenarios. This advanced feedback control system, with its multifaceted techniques, allows for the maintenance of the specimen in a state conducive to high-resolution imaging. The incorporation of velocity damping, PLL, Kalman filtering, and electro-optical feedback, along with directional light scattering feedback, provides a robust and adaptable approach. These techniques ensure that regardless of environmental perturbations, the specimen remains stable and precisely controlled, both positionally and rotationally, throughout the imaging process. This level of control allows for ground-breaking resolution and clarity in nanoscale imaging applications. In one example, the feedback mechanism allows for the submicron objects to actively stabilised. This can be used to prevent imaging blurring by compensating in real-time for Brownian motion of the sample within the microfluidic channel. In this way, it is possible to reduce the sphere of confusion (that is, the range of uncertainty in the specimen’s position and orientation) to sub-nanometre or even picometer precision, which is orders of magnitude beyond the capability of conventional mechanical or piezo sample stages. By continuously monitoring and adjusting the specimen's position, it is possible to significantly reduce errors caused by environmental factors such as thermal fluctuations. This leads to enhanced stability and precision in specimen handling, and yields sharper imaging. The feedback mechanism also allows for the control of the input light intensity in order to compensate for variation in the transmitted power with different light polarisations. Sample holder During use, the microfluidic device is inserted into a nanoscale imaging device, such as an electron microscope or an x-ray diffractometer. In one example, the microfluidic device may first be mounted onto a sample holder, which is configured to insert the microfluidic device into an imaging region of the nanoscale imaging device (e.g. such that the imaging region is sealed from the external environment, to allow for the creation of a vacuum in the imaging region). Fig. 8 shows an example of a sample holder 800. The sample holder 800 comprises a elongate body (or “rod”) 801, formed of materials such as metal (e.g. brass, non-magnetic steel, or aluminium) or metallised plastic. At a proximal end of the body 801, there is provided a handle 802 to allow a user to insert the sample holder 800 into the nanoscale imaging device. At a distal end of the body 801 is provided a mount 803 comprising an accommodating portion 804 for securing the microfluidic device 805. An opening extends through the body 801, configured to accommodate: one or more additional optical waveguides (e.g. optical fibres 806) for coupling an external optical light source to the optical waveguides of the microfluidic device 805; and / or one or more fluid conduits 807 for providing a flow of liquid through a microfluidic channel of the microfluidic device 805 via one or more fluid ports provided in the microfluidic device 805. Also shown in the figure are lenses 808a, 708b (e.g. gradient-index lenses) for directing light from the optical fibres 806 into the respective optical waveguides of the microfluidic device 805, as well as for collecting the light after scattering by the sample in the microfluidic channel (although alternative coupling means, such as fibre-butt couplings, are also contemplated). System for position and orientation control The microfluidic device described herein may be implemented as a part of a system for controlling the position and / or orientation of one or more submicron objects in a sample during nanoscale imaging. An exemplary system 900 is shown schematically in Fig. 9. As shown in the figure, a microfluidic device 901 (e.g. as shown in Fig. 1) is mounted on a sample holder 902 (e.g. as shown in Fig. 8), and is inserted into an imaging region 903 of a nanoscale imaging device 904 (e.g. an electron microscope or x-ray diffractometer). External to the nanoscale imaging device 904, the system 900 includes one or more optical light sources (e.g. laser source 905), that are optically coupled to the optical waveguides of the microfluidic device 901, for example via one or more optical fibres 906 passing through an opening in the body of the sample holder 902. One or more detectors 907 are configured to detect scattering of the optical fields by the one or more submicron objects. In one example, the detectors 907 are provided in the form of photodiodes that are located external to the microfluidic device, and coupled via the optical fibres 906 to the microfluidic device 901. A liquid pump 908 is also provided, to allow for liquids to be flowed through the microfluidic channel of the microfluidic device 901 during imaging, via one or more fluid conduits 909 passing through the opening in the body of the sample holder 902. One or more computer processors 910 are configured to control the one or more optical light sources 905 in order to modulate the fields present in the microfluidic channel of the microfluidic device 901, thereby controlling the position and / or orientation of one or more submicron objects provided in a sample within the microfluidic channel. The photodetectors 907 and the liquid pump 908 may also be under the control of the one or more processors 910. The processors 910 may be configured to control the various components of the system 900 via a field-programmable gate array (FGPA) or electric circuit 911. The system may also comprise a user interface (e.g. implemented via a graphic display), configured to allow a user to control the system via the one or more processors 910 (e.g. to control the position and / or orientation of the one or more submicron objects during imaging). Nanoscale imaging According to one aspect of the present disclosure, there is provided a method of nanoscale imaging a sample, wherein the position and orientation of one or more submicron objects present in the sample are controlled by using optical forces (as opposed to by bulk movement / rotation of a sample stage). Fig. 10 is a flow chart illustrating a method 1000 of nanoscale imaging according to the present disclosure. In step 1001, a sample is provided within an imaging region of a nanoscale imaging device (e.g. an electron microscope or an x-ray diffractometer). In step 1002, counterpropagating optical or near-infrared fields are used to trap one or more submicron objects present in the sample in a particular position and orientation. The submicron objects are then imaged by illuminating the objects with illumination radiation (e.g. electron orx-ray radiation), and detecting scattering of the illumination radiation by the sample. After imaging, the submicron objects may be repositioned and re-orientated, by modulating the optical fields (step 1004). As described above, orientation of the submicron objects may be changed by modulating the polarisation of the optical fields, and the position of the submicron objects may be changed by modulating a relative phase shift or intensity of the fields. Steps 1003 and 1004 may then be repeated, to produce image data for a plurality of different positions and orientations of the submicron objects. The image data for the plurality of different positions and orientations may then be combined, for example to produce a 3D reconstruction of the submicron objects by tomographic reconstruction. During the performance of steps 1002 to 1005, scattering of the optical or near-infrared fields by the submicron objects may be detected. The detected scattered light can then be analysed and used to enable a process of feedback control in real-time, as described in detail above. For example, the detected scattered light can be analysed to determine a position and / or orientation of the one or more submicron objects. Then, based on the determined position and / or orientation, the counter-propagating fields may be modulated accordingly. In one example, the fields can be modulated to actively stabilise the position and / or orientation of the one or more submicron objects, by modulating the counterpropagating optical or near-infrared fields to compensate for Brownian motion. The detected scattered optical light can also allow for other optical analysis techniques to performed simultaneously with the nanoscale imaging of the sample. Suitable optical analysis techniques include Raman spectroscopy, photoluminescence analysis, and cathodoluminescence analysis. Unlike conventional methods where the beam is precessed, the present methods enable the precession of the specimen itself. This approach effectively reduces the dynamical diffraction content in electron diffraction data. The high precision control provided by optical trapping allows for the detailed orientation of subregions within a specimen, which is key in assembling complete three-dimensional data sets for diffraction analysis, whether for crystalline structures or amorphous regions within a larger sample. The ability to orient specimens to any desired point enables the enhancement or reduction of dynamical diffraction effects. This feature is a powerful tool for phasing methods, overcoming the phase problem in crystallography by allowing measurements under kinematic, pseudo-kinematic, and dynamical conditions. The need for repositioning the specimen within the field of view after each rotation is also eliminated, resulting in a significant increase in the speed of tomographic data collection. This efficiency is a substantial improvement over current goniometers used in electron microscopes. Further benefits of the present methods over conventional mechanical goniometerbased imaging methods include: • Precise orientation control: The system allows for extremely fine-tuned orientation of the specimen in three-dimensional space. This precision is crucial for tomographic imaging, where the quality of the reconstructed image heavily depends on the accuracy of specimen alignment; • Six degrees of freedom: With control over all six degrees of freedom (three translational and three rotational), the system facilitates comprehensive specimen orientation. This capability allows for the capture of a complete projection data set from all required angles; • Continuous data acquisition: Unlike traditional systems where repositioning the specimen may require intermittent halts in data collection, our system enables continuous acquisition. This results in faster and more efficient tomography; • Reduced Missing Wedge Issue: Traditional mechanical stages have a limited range of motion, often leading to a 'missing wedge' of data in tomographic reconstructions. The system overcomes these limitations, allowing for complete data acquisition without mechanical constraints; • Motion Blur Reduction: Through the feedback mechanism, any thermal or mechanical-induced specimen motion is rapidly corrected, significantly reducing motion blur. This leads to clearer and more accurate tomographic images; and • Improved Image Resolution: The stability provided by the system, combined with its precision control, allows for higher-resolution imaging. This is particularly beneficial in reconstructing detailed structures at the nanoscale. The present systems and method are suitable for use with various nanoimaging techniques, such as electron microscopy (including transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and scanning electron microscopy (SEM), x-ray or neutron diffraction and scattering experiments (both using synchrotrons and x-ray tube sources), and even optical microscopy. The application of these techniques covers a broad spectrum of industry and research fields, including metrology, metallurgy, catalysis, inks, pharmaceutical sciences, structural chemistry, materials science, materials and condensed matter physics. The systems and methods are suitable for use with various different sample types, comprising various different types of submicron objects, such as: particles comprising various nanomaterials, pharmaceutical products, catalysts, or inks; nanocrystals of any suitable material type; polymers (including polymer vesicles, brushes, micelles, spherulites, fibres and rods); and biological materials such as proteins (and protein complexes), viruses, cells, and DNA self-assembled objects. It will be appreciated that a number of modifications may be made to the present systems and methods. For example, whilst a microfluidic device has been described above having two opposed window layers, alternative arrangements are also contemplated, in which the microfluidic device comprises only a single window layer provided adjacent to the microfluidic channel (e.g. for use in SEM applications). Whilst the technology has been described above in relation to nanoscale imaging (e.g. at resolutions of 100 nm or less (i.e. in the direction of atomic resolution)), the technology may also be suitable for other imaging applications having higher resolution limits (e.g. at the microscale). Similarly, the technology has been described above in relation to submicron particles (e.g. having a maximum diameter of 1 pm or less). However, the technology could be applied to larger sample objects, provided that a maximum diameter of the objects does not exceed the capability of optical trapping techniques (e.g. having a maximum diameter of 20 pm or less, or 10 pm or less). In some implementations, the optical or near-infrared fields used to control the position and orientation of the submicron objects may also be used to perform additional functions, such as for heating, cooling, or light excitation of the sample. In one example, optical fields may be directed into the microfluidic channel to induce the crystallisation of molecules in a sample solution, including small molecules or macromolecules proteins / protein complexes, for example while the sample solution is in situ in an imaging device (e.g. an electron microscope, x-ray microscope, or x-ray diffraction set-up). The position and orientation of the crystals formed can then be controlled as described herein, resulting in an improved method for studying a variety of crystal forms and structures in solution. The submicron objects can also be held in place while the surrounding fluid is flowed through the microfluidic channel, facilitating dynamic environmental studies, reactions, interactions and analysis.

Claims

1. A microfluidic device for accommodating a sample during nanoscale imaging, wherein the microfluidic device comprises:a microfluidic channel provided between opposing window layers; and one or more sets of opposing optical waveguides,wherein each set of opposing optical waveguides is configured to guide one or more optical fields into the microfluidic channel, to control, by optical forces, the position and / or orientation of one or more objects present in a sample in the microfluidic channel.

2. The microfluidic device of claim 1, wherein the objects present in the sample have a maximum diameter of 20 pm or less, or 10 pm or less, or wherein the objects are submicron objects.

3. The microfluidic device of claim 1 or claim 2, wherein the opposing optical waveguides in each set are configured to guide respective optical fields into the microfluidic channel, wherein said optical fields are counter-propagating.

4. The microfluidic device of any one preceding claim, wherein one or more of the opposing optical waveguides in each set are configured to collect optical light scattered by the one or more objects, and to guide the collected scattered light to one or more detectors.

5. The microfluidic device of any one preceding claim, wherein the nanoscale imaging is by electron or x-ray microscopy and / or electron or x-ray diffraction, and wherein the window layers are substantially transparent to electron and / or x-ray radiation.

6. The microfluidic device of any one preceding claim, wherein the window layers each have a thickness of 100 nm or less, 50 nm or less, or 30 nm or less, optionally wherein the window layers each have a thickness of between 10 nm and 30 nm.

7. The microfluidic device of any one preceding claim, wherein the window layers are each formed substantially of silicon nitride, amorphous silicon, graphene, or 2D materials.

8. The microfluidic device of any one preceding claim, wherein at least a portion of each of the optical waveguides, adjacent to the microfluidic channel, is provided between the opposing window layers.

9. The microfluidic device of any one preceding claim, wherein a refractive index of the optical waveguides is equal to or higher than a refractive index of the window layers.

10. The microfluidic device of any one preceding claim, wherein the opticalwaveguides each have a thickness of 500 nm or less.

11. The microfluidic device of any one preceding claim, wherein the opticalwaveguides are each formed substantially of silicon nitride, silicon, or polysilicon.

12. The microfluidic device of any one preceding claim, wherein the opticalwaveguides are selected from slot waveguides, wire waveguides, and rib waveguides.

13. The microfluidic device of any one preceding claim, wherein the opticalwaveguides are selected from single mode waveguides and multimode waveguides.

14. The microfluidic device of any one preceding claim, wherein the device comprisesa plurality of the sets of opposing optical waveguides.

15. The microfluidic device of any one preceding claim, wherein the microfluidic channel further comprises one or more fluid ports to allow the flow of liquid into and out of the microfluidic channel.

16. The microfluidic device of any one preceding claim, further comprising a frame structure configured to support the window layers and / or the optical waveguides.

17. The microfluidic device of claim 16, wherein the frame structure is substantially transparent to optical light, and is configured to allow optical light to pass through the material of the frame structure from an optical light source into the optical waveguides.

18. The microfluidic device of claim 16 or claim 17, wherein the frame structure is substantially formed from silicon, silicon nitride, gallium nitride, aluminium nitrite, or diamond.

19. The microfluidic device of claim 17 or claim 18, wherein the optical waveguides each comprise, at an end opposite to the microfluidic channel, a tapered portion configured to receive the optical light passing through the material of the frame structure.

20. The microfluidic device of any one of claims 16 to 19, wherein the frame structure comprises:an opening adjacent to each of the window layers; and / orone or more openings adjacent to respective fluid ports of the microfluidic channel.

21. A sample holder for use in nanoscale imaging, wherein the sample holder comprises:a mount for a microfluidic device according to any one preceding claim, and wherein the sample holder is configured to insert a microfluidic device present in the mount into an imaging region of a nanoscale imaging device.

22. The sample holder of claim 21, wherein the nanoscale imaging device is selected from an electron or x-ray microscope or an electron or x-ray diffractometer.

23. The sample holder of claim 21 or claim 22, wherein the sample holder is configured to accommodate one or more optical fibres for coupling an optical light source to the optical waveguides of the microfluidic device.

24. The sample holder of any one of claims 21 to 23, further comprising one or more optical elements for coupling the one or more optical fibres to the optical waveguides, optionally wherein the one or more optical elements comprise one or more lenses.

25. The sample holder of any one of claims 21 to 24, wherein the sample holder is configured to accommodate one or more fluid conduits coupled to one or more fluid ports of the microfluidic channel of the microfluidic device.

26. A system for controlling the position and / or orientation of one or more objects in a sample during nanoscale imaging, the system comprising:a microfluidic device according to any one preceding claim;one or more optical sources optically coupled to the optical waveguides of the microfluidic device; andone or more processors configured to control the one or more optical light sources to control, by optical forces, the position and / or orientation of one or more objects present in a sample in the microfluidic channel of the microfluidic device.

27. The system of claim 26, wherein the objects present in the sample have a maximum diameter of 20 pm or less, or 10 pm or less, or wherein the objects are submicron objects.

28. The system of claim 26 or claim 27, further comprising:one or more detectors configured to detect scattering of the optical fields by the one or more objects.

29. The system of any one of claims 26 to 28, wherein the one or more processors are configured to, based on the detected scattering:determine a position and / or or orientation of the one or more objects based on the scattered light detected by the one or more detectors; and / orperform an optical analysis of the sample based on the scattered light detected by the one or more detectors, wherein the optical analysis is selected from: Raman spectroscopy; photoluminescence analysis; and cathodoluminescence analysis, optionally wherein the optical analysis is performed simultaneously with the nanoscale imaging.

30. The system of any one of claims 26 to 29, wherein the one or more processors are configured to:control the one or more one optical light sources to manipulate an orientation of the one or more objects by manipulating a polarization of the optical fields; and / orcontrol the one or more optical light sources to manipulate a position of the one or more objects by manipulating a phase shift and / or a intensity of the optical fields.

31. The system of any one of claims 26 to 30, further comprising a user interface configured to allow a user to control the position and / or orientation of the one or more objects via the one or more processors.

32. A method of nanoscale imaging, the method comprising:providing a sample comprising one or more objects in an imaging region of a nanoscale imaging device;controlling a position and / or orientation of the one or more objects within the imaging region using optical forces produced by optical fields; andimaging the one or more objects,wherein the imaging comprises:illuminating the one or more objects using electron or x-ray radiation; and detecting electron or x-ray radiation scattered by the one or more objects in the sample to generate image data.

33. The method of claim 32, wherein the objects have a maximum diameter of 20 pm or less, or 10 pm or less, or wherein the objects are submicron objects.

34. The method of claim 32 or claim 33, wherein the controlling of the position and / or orientation of the one or more objects within the imaging region is by using optical forces produced by counter-propagating optical fields.

35. The method of any one of claims 32 to 34, wherein controlling the position and / or orientation of the one or more objects comprises controlling the one or more objects to be substantially stationary during imaging.

36. The method of any one of claims 32 to 35, wherein the method comprises: imaging the one or more objects in a first position and / or orientation;manipulating the one or more objects to one or more different positions and / or orientations, by modulating the optical fields; andimaging the one or more objects in each of the different positions and / or orientations.

37. The method of claim 36, further comprising combining image data generated by imaging the one or more objects in the plurality of different positions and / or orientationsto reconstruct an image of the one or more objects, optionally by tomographic reconstruction.

38. The method of claim 36 or claim 37, wherein modulating the optical fields comprises:modulating a polarization of the optical fields to manipulate an orientation of the one or more objects; and / ormodulating a phase shift and / or an intensity of the optical fields to manipulate a position of the one or more objects.

39. The method of any one of claims 32 to 38, further comprising: detecting scattering of the optical fields by the one or more objects.

40. The method of claim 39, further comprising:based on the detected scattering, determining a position and / or or orientation of the one or more objects.

41. The method of claim 40, further comprising:based on the determined position and / or orientation of the one or more objects, modulating the optical fields.

42. The method of claim 41, wherein modulating the optical fields comprises: actively stabilising the position and / or orientation of the one or more objects, by modulating the optical fields to compensate for Brownian motion of the one or more objects within the sample.

43. The method of claim 41 or claim 42, wherein modulating the optical fields is based on any one or more of: velocity damping; phased-locked loop control; kalman filtering; and directional light scattering feedback.

44. The method of any one of claims 32 to 43, further comprising:controlling the position and / or orientation of the one or more objects within the imaging region by electrical forces produced by electrical fields within the microfluidic channel.

45. The method of any one of claims 39 to 44, further comprising, based on the detected scattering:performing an optical analysis of the sample, wherein the optical analysis is selected from: Raman spectroscopy; photoluminescence analysis; and 5 cathodoluminescence analysis, optionally wherein the optical analysis is performed simultaneously with the imaging of the one or more objects.

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