WO2021008819A1

WO2021008819A1 - Microscopy system

Info

Publication number: WO2021008819A1
Application number: PCT/EP2020/067374
Authority: WO
Inventors: Fergal O'reilly
Original assignee: University College Dublin, National University Of Ireland, Dublin
Priority date: 2019-07-18
Filing date: 2020-06-22
Publication date: 2021-01-21
Also published as: GB201910302D0

Abstract

A microscopy system comprises: an x-ray source (101), having a first dimension along a first axis; a sample holder (201) comprising a material opaque to wavelengths emitted by the x-ray source and defining a window transmissive to the wavelengths in which a sample is located, the sample holder being configured to be located with the window adjacent the x-ray source so that radiation from the x-ray source passes through the window and the sample to radiate around a solid angle intersecting at least a plane incorporating the first axis. A plurality of zoneplates (30102, 30101), each comprising a number of zones is disposed at respective locations around the intersection of the solid angle and the plane. A radiation detector intersects each path radiating through each zoneplate from said source so that a respective image is simultaneously produced on the detector by each zoneplate.

Description

Microscopy System

Field

The present invention relates to a microscopy system.

Background

“Pinhole Array Camera for Integrated Circuits”, by Phillip A. Newman, Applied Optics, Vol 5, No. 7, page 1225-1228, July 1966 discloses a pinhole camera for the production of multiple images for integrated circuits comprising an array of precisely positioned, uniform diameter pinholes and a vacuum back to hold the film or plate to replace a step-and-repeat camera method used for producing multiple images.

“Three-dimensional imaging of X-ray and gamma-ray objects in real time”, Yin et al, Applied Optics, Vol 19 No. 17, 1 September 1980 and US 4,521 ,688 disclose an instrument for obtaining three-dimensional and tomographic information relating to X- ray and gamma-ray emitting objects and for the orthoscopic viewing of such objects including a multiple-pinhole aperture plate held spaced from an x-ray or gamma ray to visible-light converter which is coupled to a visible-light image intensifier.

“Laboratory Cryo X-ray Microscopy for 3D Cell Imaging”, Emelie Fogelqvist et al, Scientific Reports, 18 October 2017 discloses a liquid-nitrogen-jet laser-plasma source producing 2.48 nm line emission. A normal incidence multilayer condenser mirror focuses the 500 eV x-rays onto a sample, which is imaged by a zone plate onto a CCD detector. A central stop creates a hollow-cone illumination and a 200 nm aluminium filter absorbs scattered visible light while transmitting the soft x-rays.

Summary

According to the present invention there is provided a microscopy system according to claim 1.

Systems according to the present application simultaneously illuminate multiple imaging optics with a plasma or electron beam x-ray source located near a sample object to make efficient use of available photons emitted into a wide solid angle by the source and transmitted through the sample object. These optics can also be engineered to be optimised for high resolution imaging over a wide spectral bandwidth, thus taking advantage of plasma source emission which can be highly efficient over this bandwidth.

Embodiments allow for fast 2D X-ray imaging simultaneously at multiple angles, from which a 3D tomogram of the sample object can be produced.

Embodiments can comprise an array of zoneplates, each with an associated detector to simultaneously image the sample object at different angles. If a sufficiently high flux laser plasma is used and coupled effectively into the system, then single shot tomographic imaging is possible with complete tomograph imaging times potentially lower than 1 nanosecond.

Systems can have a small footprint, a minimum of moving parts and therefore a relatively low cost of production. Systems can produce data at well defined, repeatable angles, thus reducing or removing the requirement for resource consuming 2D projection alignment which is currently required for tomograph production on sequential 2D imaging systems.

The spatial relationship between the individual images can be established using a calibrated sample with known spatial features and with known relative positions.

This spatial relationship of images will not substantially change during subsequent imaging, and so the tomographic reconstruction can use this data to simplify calculations with high accuracy.

Some embodiments allow for simultaneous bright-field images to be acquired, and also comprise optics disposed outside of the illumination solid angle, in such a way as to capture multiple dark-field images of the sample object, which can also contribute to the computation of a tomogram, particularly from higher spatial frequency features in the sample object.

Brief Description of the Drawings

Various embodiments of the invention will now be described, by way of example, with reference to the acompanying drawings, in which:

Figure 1 illustrates a microscopy system for simultaneously imaging a sample from multiple different directions according to an embodiment of the present invention; Figure 2 illustrates the system of Figure 1 with a sample in situ;

Figure 3 shows schematically a sample illuminated by a laser plasma in the system of Figure 1 ; and

Figure 4 illustrates schematically a microscopy system according to a second embodiment of the present invention.

Description of the Embodiment

Referring now to Figure 1 , there is shown schematically a microscopy system 100 according to an embodiment of the present invention. The system 100 includes a radiation source 101 (which produces photon wavelengths from approximately 13.5 nm to approximately 0.1 nm, but could also operate at lower or higher wavelengths) placed in close proximity to a sample holder 201. As will be discussed later, the source 101 is preferably an x-ray source emitting in the water window between 4.4nm and 2.3 nm, which is especially useful for imaging biological samples.

Nonetheless, alternative sources radiating at wavelengths of, for example, 1 nm to 0.1 nm could be employed for imaging silicon based electronic samples or geological samples primarily composed of material transparent at the source wavelength. The source 101 can be a laser induced plasma, a discharge plasma or an electron beam source, or any other source of photons emitting around a large angular range, for example, >20, Figure 2.

In the embodiment, the sample holder 201 is largely opaque to the incident radiation, apart from a window 202 comprising a membrane capable of supporting a sample and which has a sufficient transmission to allow for imaging with radiation from the source 101. In embodiments, the sample holder 201 can comprise a silicon body with a thin membrane of silicon nitride in the window 202. A window membrane with a thickness of less than 100 nm provides excellent transmisson below 3 nm for example, and is sufficiently robust over a diameter of 5pm to 10pm.

Because the sample holder 201 is opaque to the illuminating source wavelengths, the only photons transmitting through an optical path from the source 101 through imaging optics to a detector are those that have passed through the sample holder window 202. In the case of biological imaging in the water window, for example, it may be advantageous to have a 1 pm tungsten or other high atomic number, highly absorptive film on the sample holder 201 , or that the sample holder 201 be made largely of a thin film of tungsten.

The window membrane can provide a well in which a sample can sit, and this may be particularly advantageous for liquid based samples. Note that while for simplicity in Figure 1 , the window 202 is shown as being rectangular, the window can have any suitable shape such as circular.

For the purpose of illumination at multiple angles simultaneuosly it is important that the sample holder 201 is sufficiently thin that it does not occlude the illumination of the sample at higher illumination angles. So keeping the total thickness of the sample holder 201 to less than about half of the diameter/side length of the window 202 can improve system performance.

In variations of the above embodiment, the window membrane 202 could also be coated with a filter for specific wavelength band selection from the source 101 for imaging.

For imaging small samples, of the order of 10 pm in diameter, the window membrane 202 can be sufficiently strong to support many orders of magnitude vacuum differential. This can be required where the source side of the sample holder 201 is at a vacuum level appropriate for x-ray production, and the imaging side of the sample holder 201 is at atmospheric pressure. In some implementations, the sample could be maintained in an atmospheric pressure of air, while a remaining portion of the path length to the imaging optics and/or detector, described below, could comprise atmospheric pressure helium or hydrogen separated from the air with a thin window (not shown).

In variations of this embodiment, the window 202 could also be completely void, such that the window serves only to restrict direct illumination of the imaging optics by x-rays which have not passed through the sample.

In still further variations of the illustrated embodiment, the sample holder may comprise a capillary tube inserted into an appropriate receiver, but in any case the sample holder should have a geometry allowing illumination of a sample at multiple angles simultaneuosly. Referring back to Figure 1 , in the embodiment 3 arrays 3020, 3010 and 3030 of imaging optics (only two of which 30101 , 30102 are labelled) are placed in front of the window 202, in such a manner that each of the individual optics, such as the optics 30101 and 30102, are illuminated by the x-ray source 101 through the window 202 and any sample disposed in front of the window. In one implementation, the total optical path length from the source 101 through the imaging optics to a detector could be of the order of 100 mm. The system is typically configured to provide magnifications of the order of between 100 and 1000 and so the great majority of the optical path length is between the imaging optics 30101 , 30102 and their respective detector.

The optics can be reflective, refractive, or diffractive or a mixture of these appropriate for the wavelength being used, the sample being imaged and the resolution required.

In the illustrated embodiment, each imaging optic 30101 , 30102 comprises a Fresnel or other type of zoneplate. Arrays of zoneplates on a planar substrate such as the substrates 3010, 3020 and 3030 can be manufactured relatively easily and the mutual focus of each zoneplate can be controlled by accurate lithographic

positioning. Each of the zoneplates 30101 , 30102 is capable of producing a respective image of a sample on an associated detector, and these can be

processed in a conventional manner to produce a tomographic 3D image of the sample.

For example, when imaging with a 2.5 nm wavelength source 101 , a Rayleigh resolution of about 40 nm is possible with optics 30101 , 30102 that have a full capture angle of only 4 degrees. Over 1000 of such optics 30101 , 30102 could fit into the 2p steradians of a hemisphere extending around the imaging side of the sample holder 201.

A number of factors dictate the maximum incidence angle at which a zoneplate can be used. At angles higher than a few degrees, elliptical, rather than circular, zones can be used, and at higher angles again zones that have a 3D or stacked 2D structure, such as the ones developed for high efficiency by the David Group in Paul Scherrer Institut (PSI), Switzerland, and by the Schneider Group in BESSY, Berlin, can be used. In Figure 1 , three separate arrays 3010, 3020, 3030 of imaging optics, each comprising 7x7=49 zoneplates are depicted. A central array 3020 is disposed at an angle normal to the chief ray extending normally through the sample holder 201 , while arrays 3010 and 3030 are disposed towards either side of the array 3020 and are inclined at angles to the array 3020 towards the sample holder 201 in order to collect and image at higher angles than is possible from a single planar array. In this example, the three arrays 3010, 3020, 3030 extend around an arc of approximately 65 degrees.

In this case, the focal distances for each zoneplate would be of the order of 40- 200 pm for wide bandwidth zoneplates, so the total frame width for each array would ideally be of the order of 40-200 pm, but could be much longer in the other direction. So for example, similar results could also be achieved using only two arrays inclined relative to one another. These could wider than the arrays 3010, 3020 and 3030 of Figure 2, but it will be appreciated that two arrays are more limited in their angular range than greater numbers of arrays.

In other implementations, an array of zoneplates could be provided around an internal hemispherical surface with zoneplates positioned like pits in a golf ball. This is particularly advantageous for volume zoneplates, where zones comprising relatively thick 3D structured micro-optics can be tilted towards the optical axis of the zoneplate. These volume zoneplates are particularly advantageous for X-ray imaging with energies above 1 keV, with optimum zoneplate thicknesses of greater than 1 pm up to greater than 100 pm.

In any case, it is expected that using one or more arrays of zoneplates, potentially over 100 individual imaging optics 30101 , 30102 would simultaneously produce the images required for tomography.

Referring to Figure 2, in the embodiment, each of the arrays 3010, 3020 and 3030 image the sample object 500 onto a respective imaging detector 5010, 5020, and 5030. It will be appreciated that using a single or small number of detectors for each array 3010, 3020, 3030 is of course advantageous from the point of view of cost and complexity of operation. As such, while each individual optic 30101 , 30102 will have an associated detector, any given detector may be arranged to receive image information from a plurality of optics, possibly even all of the optics for a given array 3010, 3020, 3030. In some cases, it may even be possible for a detector to image information from optics located on different arrays, but because of the relative angles between adjacent arrays, this may be less practical.

The requirement for the imaging detectors is that the images produced by the respective imaging optics can be recorded with sufficient spatial frequency and dynamic range and with sufficiently low background noise to produce high quality 2D images which can form the basis of 3D tomograms.

The detectors 5010, 5020, 5030 can be direct imaging CCD, CMOS, CMOS-Hybrid (such as described at https://medipix.web.cern.ch/collaboration/medipix3- collaboration), microchannel plate-phosphor, or indirect phosphor/CCD/CMOS, or other pixelated imaging x-ray detectors. The size of the pixels, their photon capacity, their inherent noise levels, and other detector paramaters will dictate the optimum magnification for a given imaging application. In the soft x-ray region of the spectrum, CCD or CMOS cameras are the detectors of choice.

CCD detectors generally provide low noise over a long acquisition time with uniform sensor response.

CMOS cameras are pixelated cameras where each pixel has a respective amplifier. They are relatively low cost, high spatial resolution devices and form the vast majority of imaging devices in consumer electronics. They are also widely used in industrial imaging. To improve sensitivity, back-illuminated CMOS cameras are now being sold commercially, and in this configuration the cameras have a high x-ray and soft x-ray sensitivity. The pixel size in these cameras can be as small as 1 pm, which reduces the magnification required for high spatial resolution imaging, compared to CCDs with pixel sizes that are usually greater than 10 pm. The small pixel size comes at a cost of lower dynamic range than the larger CCD and CMOS pixel devices, and there is a trade-off between magnification, dynamic range and exposure time in microscopy and imaging applications that needs to be optimised for each imaging situation and modality. In the case of imaging at 2.5 nm, a 2.4 pm pixel with a 15k electron pixel depth has a depth of about 100 of these 2.5 nm photons, which will be sufficient for many applications. Pixel binning such a camera to 2X2 pixels would allow for a depth of 400 photons. In some embodiments, it may be advantageous to have some elements of a detector array optimised for dynamic range, and others optimised for spatial resolution.

While they have poor dynamic range, poor sensitivity and need chemical

development and complex readout procedures, PMMA and photoresist based detectors could also be used to record images of x-rays down to a few nanometre resolution.

In biological cell imaging for example, the sample 500 will typically be less than 10 pm wide and will typically need to be imaged with a 3D spatial resolution of about 50 nm. (Applications will typically involve illumination of samples of about the thickness of the depth of field of the zoneplates, and for water window wavelengths this is ~5pm.) The Nyquist sampling limit means that the effective object pixel size will have to be somewhat smaller than 25 nm and so this corresponds to about 400 pixels across the 10 pm image. With a single 4096x4096 pixel detector, about 100 of these images could be projected onto the detector, with appropriate baffling between images acquired from adjacent optics, with the requirement for subsequent image flattening as the relative angle of the various individual optics 30101 , 30102 to the sample will vary.

T urning back to the zoneplates: the zoneplates 30101 , 30102 will in general be formed by concentric elliptical rings where the ellipse radii intersect alternate positive and negative Fresnel zones in the wavefront centred at the centre of the object to be imaged and with their second focus at a respective imaging detector. In Figure 1 , zoneplate 30101 is illuminated with the chief ray at an angle normal to both the vertical and horizontal axes of the zoneplate substrate, and so the major and minor axes of the zoneplate Fresnel zones are equal and so the zoneplate 30101 can comprise circular rings of radially increasing diameter and decreasing thickness. On the other hand, zoneplate 30102 has a chief ray with the largest angle Q with respect to the zoneplate substrate normal. This means that for zoneplate 30102 the Fresnel ellipsoids describing rings of constant phase will be sliced at an angle other than normal to the ellipsoid major axis, and the zoneplate rings will need to be elliptical to avoid astigmatism/aberration above a certain chief-ray to zoneplate-normal angle as disclosed in“Astigmatism correction in x-ray scanning photoemission microscope with use of elliptical zone plate,” FI Ade et al, Appl. Phys. Lett. 60, 1040 (1992). It may also be advantageous to modify the zoneplate zones in order to bend the image off axis, as disclosed in Mochi, Goldberg, Nualleau and Huh,“Improving the performance of the actinic inspection tool with an optimized alignment procedure”, SPIE Advanced Lithography, Proceedings Volume 7271 , Alternative Lithographic Technologies; 727123 (2009). This configuration produces images off axis, with different wavelengths appearing at different angles. For a narrow band line emitting plasma source, it may be that an off axis shift will separate wavelengths sufficiently to produce a better signal to noise ratio in the image. The other benefit of off axis operation is that the zero order x-rays are not directed onto the same place on the imaging detector where the required image is being formed.

As mentioned above, in order to preserve a constant phase difference in phase zoneplates, it may also be necessary to modify the 3D shape of the zones to maintain a constant pathlength through the zoneplate material. For example, as well as the elliptical shape, the zones may need to be tilted, or structured in such a way to approximate this tilt, in the direction of illumination, such as zone plates available from Paul Scherrer Institut, Switzerland.

Further, to make maximum use of the x-rays emitted from an incoherent source in tomographic imaging, it is advantageous to use optics with as high a bandwidth as possible. The highest radiance incoherent x-ray sources emit into relatively wide bandwidths, and much of this photon flux could be used for absorption imaging, if the chromatic aberration of the optics 30101 , 30102 can be managed so that image quality is maintained.

The bandwidth over which a zoneplate can be made to image effectively is governed by the number of zones. For a zoneplate with N zones at central wavlength l, the zoneplate can image effectively over a bandwidth of Dl < l/N. While the optical quality of a zoneplate diminishes somewhat as N gets very small, in the present system, image quality can be maintained for N greater than approximately 10 zones, so that effective use can be made of bandwidths (l/Dl) of over 5% in imaging. In the case of incoherent x-ray sources, including laser plasmas, high emission over a bandwidth wider than 5% can be filtered effectively by thin metal foils and/or coatings over the window 202, and often to some extent by the transmission of the sample itself, and the detector response. Note that current zoneplate based microscopes work using a single zoneplate with a large number of zones (typically hundreds), and therefore require a narrow

bandwidth x-ray source. Such a high N zoneplate has a relatively long focal length which facilitates the rotation of the sample. This requires complex filtering or wavelength selection from incoherent sources, using gratings or multilayer mirrors and makes the imaging inefficient.

On the other hand, for many applications, absorption contrast is available in a sample over bandwidths of >25%, and so broadband optics allow for far more efficient use of available photons. This leads to a number of possibilities in terms of imaging with the multiple zoneplates 30101 , 30102 of the present application. Thus, at low N, a broadband source may be used, with the consequence that the focal length can be very short. In conventional zoneplate based tomography, this would require the zoneplate to be extremely close to a sample which would be required to rotate, making operation difficult, whereas embodiments of the present invention do not require the sample to rotate and so are not adversely affected by having a short focal length - indeed they benefit from this, as will be explained below.

Nonetheless, in embodiments of the present invention, there remains a trade off between the improvement in bandwidth from reducing zone number N, and increasing the background level from the zero and higher orders. While N could be as low as approximately 10, N of around 20 provides a good compromise for biological imaging with relatively thick samples.

For example, a zoneplate designed for a source 101 emitting at about 2.5 nm, with 10 zones and an outer zone width of 50 nm has a focal length of around 40 pm, whereas a zoneplate with 20 zones and an outer zone width of 50 nm has a focal length of around 80 pm. At these low numbers of zones, the intensity of the focused first order image with respect to the zero and higher orders is lower than for a zoneplate with a higher number of zones, but the simplicity of the imaging system enabled by the low N zoneplate means that overall imaging performance may be similar for many applications. There will in general be a trade off between image quality, imaging speed and system complexity and cost. For a zoneplate with 100 zones the imaging performance will be very high, but the allowed bandwidth will be only 1 %, although this may be acceptable for many applications. In the present system, because the imaging optics 30101 ,30102 and the sample 500 remain static during imaging, it is more practical for short focal length zoneplates to be deployed. This has the advantage that a large portion of the photon spectral bandwidth available from plasma or electron beam based sources can be utilised, greatly reducing the required imaging time. For example, 10 zone zoneplates can effectively image with a bandwidth of 2.5 nm ± 0.125 nm i.e. 2.37 nm to 2.63 nm, and 20 zone zoneplates can effectively image with a bandwidth of 2.5 nm ± 0.07 nm i.e. 2.43 nm to 2.57 nm. In this bandwidth range, it is possible to get a laser plasma 101 , Figure 3, to emit significantly greater than 1 % of the total input laser energy, representing greater than an order of magnitude improvement on line based emitters.

The individual zoneplates 30101 , 30102 in each array 3010, 3020, 3030 can also be designed in order to be optimised for different central wavelengths, and, imaging with different filter sets, so making it possible to image different spectral regions simultaneously, adding a multispectral component to the 3D tomogram image sets. This would be particularly attractive if objects in the sample have different contrast levels on either side of a filter.

For any imaging application at a particular wavelength, a combination of a filter with a sharp low wavelength cutoff, and an x-ray emission spectrum with a sharp longer wavelength feature which between them produce a relatively well defined region of x-ray illumination wavelengths will allow for optimal zoneplate illumination. For example, a palladium laser plasma with the correct laser parameters emits strongly below 2.7 nm, but very little between 2.7 nm and 3.4 nm. This fast fall off in emission, coupled with a 1 pm thick Cr filter, and the inherent spectral absorption of an aqueous sample > 5 pm thick, results in a spectrum that is sufficiently narrow (a bandwidth of about 0.2 nm) to allow high resolution imaging with zoneplates having < 20 zones, with a zoneplate with an outer zone of 50 nm and a focal length of about 80 pm.

Figure 3 shows schematically, a 10 pm diameter sample 500 being illuminated by a laser plasma source 101 for imaging in multiple different directions simultaneously. The plasma 101 is generated by focussing a laser source on a target 103 disposed adjacent the sample holder window 202 in which the sample 500 is located. The same type of geometry could be considered for a discharge plasma, with the sample holder 201 forming a disposable component of one of the discharge electrodes, or of an electron beam source with the anode in close proximity to or containing the sample.

The sample 500 is relatively thick, and forms an integral part of the protective layer that protects the imaging optics from damage induced by the x-ray production process. The sample 500 and sample holder 201 are disposable in this regard. The sample 500 can be imaged until either it or the sample holder 201 are damaged to the point where the image quality is affected.

The sample 500 is 3 dimensional and in order to produce a 3D tomogram using standard tomography techniques, multiple images must be taken of the sample at multiple angles. The greater the range of angles and the finer the step between angles, the more accurately a tomogram can be computed. The plasma source 101 is ideally larger than the sample 500 to be imaged, so that the sample is bright-field illuminated over a range of angles, as illustrated schematically in Figure 2, with ray lines 4011 and 4031.

The shape of the plasma source 101 can be optimised depending on the angles the sample is being imaged at and the optical setup used in the imaging, ideally keeping the size of the source to the minimum required to illuminate all imaging optics 30101 , 30102. The source 101 is positioned and the imaging optics are optimised so that the maximum of emitted soft x-ray flux is used in imaging.

For example, the source 101 being used to illuminate three zoneplate arrays 3020, 3010, 3030 of Figures 1 and 2 is more efficient because it is elongated in the same direction 102 of extension of the arrays. Thus, as shown in Figure 2, with ray lines 4011 and 4031 , different regions of the source 101 illuminate different regions of the zoneplate arrays 3020, 3010, 3030. In the case of a 10 pm diameter sample 500, for example, the source 100 could have a tubular shape of approximately of 15 pm diameter and somewhat over 30 pm long in order to illuminate the sample out to >

55 degrees.

It will be appreciated that in some embodiments, a portion of the plurality of imaging optics may not be directly illuminated by the source, but the detectors may still image x-rays scattered into relatively high angles by the sample. The regions of the sample with the highest spatial frequency, and therefore the smallest features, will scatter into the highest angles, so that imaging in this way allows further structural information to be gathered to improve the imaging performance of the system. In some imaging applications it may be advantageous to have a higher proportion of the imaging optics illuminated in dark field than in bright field, particularly where small features are the required imaging target. An example of this would be defect identification in semiconductors or nanoparticle location in cells.

In such cases there will be a trade off between the amount of light used from the emission solid angle of the source, versus the available dark-field imaging solid angle. Various embodiments can be implemented with either: a narrow-band line emitting source or source/filter/sample combination and a zoneplate with a large number of zones, where signal to background is to be optimised; or a broader band source, with a zoneplate with fewer zones where imaging speed is more important.

For many applications these will present a trade-off which the end user can prioritize.

The above described embodiment allows over 100 individual 2D images to be acquired simultaneously, at a wavelength of about 2.5 nm, which is a region suitable for biological cell imaging. By comparison to obtaining the same number of images sequentially by rotating the sample in front of a single optic, this allow up to 2 orders of magnitude improvement in source output solid angle use efficiency, with

concomitant imaging time reductions. In the case of biological imaging, this enables whole cells to be imaged with the soft x-ray output of a single laser plasma. These plasmas can produce x-ray pulses in pulses less than 1 ns long, and therefore could be used to illuminate and image a biological cell before the radiation damage to the cell has time to change it structurally, within the spatial resolution of the soft x-ray imaging mechanism. As such, microscopy systems according to the present application can be deployed to tomographically image cells that do not need to be cryo-frozen. The advantage of this is in sample preparation simplicity, reduction of sample preparation imaging artefacts, microscope throughput, and in reduction of manufacturing cost.

A limitation on the optical system is imposed by the field of view (FOV) of the optics being used. For many applications the laterial field of view of the zoneplates will be of the order of 10 pm, and the depth of focus will be a few microns. For example, in the 2.4 nm to 4.3 nm water window region of the spectrum, where aqueous samples can be imaged, the sample itself must be less than about 10 pm thick in order to be sufficiently transmissive to be imaged. These types of limits allow the dimensions of the window 202 and the zoneplate parameters including zone number, zone materials, and central stop diameter and thickness (if such a stop is used) to be optimised to image the largest sample size, with the minimum unwanted background signal.

As mentioned above, for most practical applications, a filter will be required to select the imaging wavelength. Thin film free standing filters are commonly used to select a narrow band of radiation from an x-ray source. For example, a 1 pm thick Cr or Ti filter can select a bandwidth of about 1 nm with peak transmission of ~ 15%.

Combinations of films (Cr and Al for example) can be used to eliminate unwanted radiation and restrict the operation bandwidth optimally.

The same film combinations could be used either: within the window 202 of the sample holder 201 ; within the imaging optics, for example, as a layer of the substrate on which the zoneplates 30101 , 30102 are formed; or deposited directly on the imaging detector(s) 5020, 5010, 5030.

The advantage of applying a filter film directly on the imaging detector or the zoneplate or the sample holder would be that it does not have to be free standing, so that the choice of materials becomes wider, and overall transmission can be improved by optimising filter thicknesses. For example, mixtures including materials which oxidise quickly such as Mg would be possible, by depositing the sensitive imaging layer first, and putting capping layers of other materials on top as a barrier layer. The specific advantage of putting the filter directly on the detector is that multiple wavelength regions within the zoneplate bandwidth can be imaged simultaneously on separate detectors or on different detector regions with different filters deposited.

It is important to note that the present application is concerned with 3D imaging of materials which, given that they have a substantial thickness, will also have a transmission spectrum in the x-ray region. It is the combination of the source spectral flux, the sample and filter transmissions, and the optic and detector efficiency, that will decide the imaging time for a particular setup. The source emission by the sample transmission spectrum multiplied by the filter transmission spectrum gives the effective spectrum seen by the imaging optic, and it is this spectrum for which the optics need to be optimised. Plasmas of many medium to high atomic number materials emit very strongly in the soft x-ray region of the spectrum, and the features are sufficiently broad to provide substantial illumination, and sufficiently narrow to allow specific materials to be used in specific imaging spectral windows in order to shape the effective illumination spectrum to allow optimal imaging. For example, materials such as Zr, Nb, Mo, Rh, Ru, Pa, S1₃N₄, BN or liquid nitrogen can be combined with filters such as Cr, V, Ti, Mg in the 2.3 nm to 4.3 nm‘water window’ region of the spectrum in order to deliver an effective spectrum suitable for imaging hydrated samples with zoneplates having > 10 zones and preferably 20 zones.

In variants of the above described embodiments, different filters can be used on different individual zoneplates 30101 , 30102 within the arrays 3010, 3020, 3030, thus allowing simultaneous multispectral tomographic imaging, which may be of interest in giving complex 3D elemental contrast in a sample.

For example, in biological cell imaging, a set of images taken with broadband illumination between 2 nm and 3 nm, with one set of zoneplates filtered by Cr and another group by Ti filters, would allow for the absorption imaging of both the cell structure and the definitive identification of Ti or T1O2 nanoparticles in the cell.

Separately, both detectors directly and not directly illuminated by the source can also be configured to detect x-ray induced flourescence of a sample.

As described above, the sample object 500 to be imaged is illuminated by a source 101 that emits into a large angle range. In the case of zoneplate and diffractive optics in general, there may be a zero order undiffracted beam which passes directly through the imaging optic and impinges an imaging detector, but only contributes an extra background to the recorded image signal. In order to remove or reduce this effect, a geometric zero order baffle can be built into the zoneplate 30101 , or placed before or after it, in order to geometrically block the direct illumination of the detector 5010 by the source 101. This way, only diffracted rays will be detected, and the signal to noise ratio can be improved. This is only possible where the zoneplate dimensions, and therefore the focal length are large with respect to the sample. The requirement is that the central stop on the zoneplate is at least twice the diameter of the sample being imaged. For a 5 pm diameter sample being imaged at 2.5 nm this would require a 10 pm diameter central stop, and a zoneplate with at least 100 zones, in order to capture and image with at least half the total solid angle of the zoneplate.

As mentioned, further baffling between the different detectors associated with respective zoneplates; or different portions of a detector common to a number of zoneplates such as the detectors 5010, 5020, 5030 will be required in order to ensure that the negative orders of diffraction from the zoneplates do not add unnecessary background noise to the images being recorded.

Referring now to Figure 4, in a further variant of the above described embodiment, radiation is collected simultaneously from many angles of a high divergence soft x- ray source 102, using multilayer mirrors 201 ...204 of appropriate materials (Mo/Si at 13.5 nm; Cr/V at 2.42 nm) between grazing and normal incidence, such as disclosed in“X-ray Fourier Ptychographic Microscopy (FPM)”, H. Simons,

https://arxiv.orq/pdf/1609.07513.pdf. In Figure 4, each mirror 201 -204, captures a small solid angle of the source and, although not essential, this angle can be further controlled by pupil pinholes or apertures, such that the illumination arriving at the sample 500 is spatially coherent.

A beam from each mirror impinges and is transmitted through or reflected from a sample (not shown) held within a holder 601 , with the beam being detected by a suitable pixelated detector located behind an array 801 of optics 701...704.

With appropriate baffling 101 to prevent scatter from adjacent illumination angles impinging adjacent detectors, it should be possible to illuminate the sample at hundreds of different angles simultaneously for these types of resolutions at this wavelength. With harder, lower wavelength x-rays, the angle becomes smaller for the same required resolution, so higher numbers of simultaneous diffraction patterns can be taken, or diffraction from higher spatial frequencies can be obtained with the same number of detectors. In still further variants of the embodiment of Figure 4, either a single spherical mirror or many flat (or spherical) mirror facets, at grazing angles, can also be used in place of the m irrors 201 ...204.

Note that in the examples provided above, numbers are quoted for wavelengths of 2.5nm and N=20 or in some cases N=10, however, it will be appreciated that these will changed based on imaging wavelength and the number of zones in each zoneplate.

Claims

Claims:

1. A microscopy system comprising: an x-ray source, having a first dimension along a first axis; a sample holder comprising a material opaque to wavelengths emitted by said x-ray source and defining a window transmissive to said wavelengths in which a sample to be imaged is located, said sample holder being configured to be located with said window adjacent said x-ray source so that radiation from said x-ray source passes through said window and said sample to radiate around a solid angle intersecting at least a plane incorporating said first axis; a plurality of optics disposed at respective locations around the intersection of said solid angle and said plane, each optic comprising a zoneplate comprising a number N of zones, where 10<N<200, wherein said first dimension of said source is large enough and said source is located close enough to said sample holder to provide a respective radiating path from said source through each of said optics; and at least one radiation detector intersecting each radiating path through each of said optics, wherein each of said source, sample, optic and detector are spaced along each respective radiative path and said source is configured to emit radiation for long enough and at sufficient intensity for a respective image be simultaneously produced on said at least one detector by each of said plurality of optics.

2. A microscopy system as claimed in claim 1 wherein said window extends substantially parallel to said first axis with a second dimension and wherein said second dimension is less than said first dimension.

3. A microscopy system as claimed in claim 1 wherein said source is operable to emit radiation for an image exposure time of approximately 1 ns.

4. A microscopy system as claimed in claim 1 comprising one or more arrays of optics, each array n comprising x[n]*y[n] zoneplates, with x zoneplates extending around said solid angle and y zoneplates extending perpendicularly outwards from said plane incorporating said first axis.

5. A microscopy system as claimed in claim 4 wherein for the or each array n, x[n]> 7 and y[n]>7.

6. A microscopy system as claimed in claim 1 wherein each zoneplate extends around a solid angle of approximately 4 degrees.

7. A microscopy system as claimed in claim 4 comprising three arrays of optics: a central array disposed parallel to said sample holder and extending around a chief radiative path extending perpendicularly from said source through said sample holder; and two side arrays extending from opposite sides of said central array and being disposed to approximate an arc extending around said solid angle.

8. A microscopy system as claimed in claim 7 comprising a respective detector for each array.

9. A microscopy system as claimed in claim 4 wherein an array of optics is defined on a hemispherical surface extending around said solid angle.

10. A microscopy system as claimed in claim 4 in which a central zoneplate of the or each array n comprises circular zones and wherein at least one other zoneplate of the or each array n comprise elliptical zones.

11. A microscopy system as claimed in claim 10 in which at least some of said at least one other zoneplates is structured to accommodate non-orthogonal radiation incident on said zoneplate.

12. A microscopy system as claimed in claim 1 wherein said x-ray source comprises a plasma or electron beam source.

13. A microscopy system as claimed in claim 12 where said plasma is induced by a laser focussing on a target disposed adjacent the window.

14. A microscopy system as claimed in claim 13 wherein said target comprises one of: Zr, Nb, Mo, Rh, Ru or Pa, Si3N4, BN or liquid nitrogen.

15. A microscopy system as claimed in claim 1 wherein said x-ray source comprises a discharge plasma and wherein the sample holder forms a component of a discharge electrode.

16. A microscopy system as claimed in claim 1 wherein said x-ray source emits with a bandwidth of great than approximately 5%.

17. A microscopy system as claimed in claim 1 wherein said radiation is filtered through a filter disposed either: between said source and the sample holder; within the sample holder; within the optics; or on the detector.

18. A microscopy system as claimed in claim 17 wherein said filter comprises one of a Cr, Ti or Mg.

19. A microscopy system as claimed in claim 17 wherein said plurality of optics are divided into sub-sets of optics, each sub-set being differently filtered.

20. A microscopy system as claimed in claim 1 wherein said x-ray source is configured to emit radiation in water window wavelengths and wherein said sample is biological.

21. A microscopy system as claimed in claim 1 wherein N=10 and an outer zone of said each zoneplate is approximately 50 nm in width with each zoneplate having a focal length of around 40 pm.

22. A microscopy system as claimed in claim 1 wherein N=20 and an outer zone of said each zoneplate is approximately 50 nm in width with each zoneplate having a focal length of around 80 pm.

23. A microscopy system as claimed in claim 1 wherein each zoneplate is absorptive and comprises a geometric zero order baffle.

24. A microscopy system as claimed in claim 1 wherein sample holder comprises one of silicon; tungsten; silicon coated with tungsten or other high atomic number elements.

25. A microscopy system as claimed in claim 2 wherein said sample holder has a thickness less than half said second dimension

26. A microscopy system as claimed in claim 25 wherein said second dimension is less than approximately 10 pm.

27. A microscopy system as claimed in claim 1 wherein said window comprises a well comprising a silicon nitride membrane.

28. A microscopy system as claimed in claim 27 wherein said membrane is coated with a filter material.

29. A microscopy system as claimed in claim 1 wherein said window is configured to receive a capilliary tube capable of being filled with a liquid transported sample.

30. A microscopy system as claimed in claim 1 wherein said system is configured to provide a vacuum between x-ray source and said sample holder.

31. A microscopy system as claimed in claim 30 wherein the remainder of the radiative path from said sample holder to said detector comprises atmospheric either: air, helium or hydrogen at atmospheric pressure.

32. A microscopy system as claimed in claim 1 wherein said source, sample, optic and detector are spaced along each respective radiative path to provide a magnification > 100.

33. A microscopy system as claimed in claim 1 wherein each of the at least one detector comprises either: CCD, CMOS, CMOS-hybrid, microchannel plate- phosphor, or indirect phosphor/CCD/CMOS detectors.

34. A microscopy system as claimed in claim 1 wherein said x-ray source comprises a plurality of mirrors arranged to reflect radiation from said source through said window.

35. A microscopy system as claimed in claim 34 wherein said mirrors comprise either: Mo/Si for an x-ray source emitting at 13.5 nm; or CrA/ at for an x-ray source emitting at 2.42 nm.

36. A microscopy system according to claim 1 further comprising a further plurality of optics disposed at respective locations outside said solid angle.

37. A microscopy system as claimed in claim 1 further comprising image processing software which when executed on a computing device is configured to produce a tomographic image of said sample from said plurality of simultaneously produced images.