Abstract
A support for an electron microscopy sample, the support comprising a metallic foil having one or more holes therethrough wherein thickness of the metallic foil is less than 50 nm and/or the mean linear intercept grain size is 50 nm or less, wherein the ratio of the diameter of each hole to the thickness of the metallic foil is 15:1 or less, and wherein the metallic foil consists of either (a) one or more metals selected from transition metals, aluminium and beryllium, or an alloy thereof; or (b) degenerately doped silicon wherein the dopant element is selected from boron, aluminium, boron and arsenic at a concentration of 10.sup.20 atoms/cm.sup.3 or higher.
Claims
1. A support for an electron microscopy sample, the support comprising a metallic foil having one or more holes therethrough wherein thickness of the metallic foil is less than 50 nm and/or the mean linear intercept grain size is 50 nm or less, wherein the ratio of the diameter of each hole to the thickness of the metallic foil is 15:1 or less, and wherein the metallic foil consists of either (a) one or more metals selected from transition metals, aluminium and beryllium, or an alloy thereof; or (b) degenerately doped silicon wherein the dopant element is selected from boron, aluminium, boron and arsenic at a concentration of 10.sup.20 atoms/cm.sup.3 or higher.
2. A support according to claim 1 wherein thickness of the metallic foil is less than 50 nm and the mean linear intercept grain size is 50 nm or less.
3. A support according to claim 1 wherein the edge roughness of each hole is 20 nm or less as measured by the root mean square deviation from the expected theoretical hole edge profile.
4. A support according to claim 1 wherein the diameter of each hole is 750 nm or less.
5. A support according to claim 1 wherein the support has a light wavelength transmittance maximum of from 650 to 800 nm.
6. A support according to claim 1 wherein the holes are arranged in a hexagonal array or a square pattern array.
7. A support according to claim 1 wherein the metallic foil is suspended across holes in an electron microscopy grid.
8. A support according to claim 7 wherein the metallic foil and the grid are integrally formed.
9. A support according to claim 7 wherein the grid comprises a mesh having a mean hole size that is on a micrometre scale and the mesh holes are tessellating hexagons or tessellating squares.
10. A support according to claim 1 wherein the metallic foil consists of one or more of gold, palladium and platinum or an alloy thereof, optionally wherein the metallic foil consists of gold or an alloy thereof.
11. A support according to claim 1 wherein the support consists of one or more of gold, palladium and platinum or an alloy thereof, optionally wherein the support consists of gold or an alloy thereof.
12. Use of the support according to claim 1 in transmission electron cryo-microscopy.
13. A method of manufacturing a metallic foil for a support according to claim 1, the method comprising the steps of depositing a metallic layer onto a patterned substrate that is cooled to 200K or less to form a layer having a thickness of 50 nm or less and having one or more holes therethrough; removing the deposited metallic layer; and forming the metallic layer into a support for an electron microscopy sample, wherein the metallic foil consists of either (a) one or more metals selected from transition metals, aluminium and beryllium, or an alloy thereof; or (b) degenerately doped silicon wherein the dopant element is selected from boron, aluminium, boron and arsenic at a concentration of 10.sup.20 atoms/cm.sup.3 or higher.
14. A metallic foil for a support formed by a method according to claim 13.
15. A method of electron microscopy imaging comprising a step of sequentially imaging a sample suspended in a hole of a support according to claim 1, wherein each image encompass at least a part of the edge of the hole and the electron beam encompasses the hole and the complete edge of the hole.
16. A method of electron microscopy imaging according to claim 15 wherein at least a part of the edge of the hole in each image is compared to the other images to remove any relative shift between sequential images and/or wherein the sequential images of the specimen in the hole are weighted to account for damage to the specimen.
17. A support according to claim 1 wherein the metallic foil and the support each independently consists of one or more of gold, palladium and platinum or an alloy thereof.
18. A support according to claim 1 wherein the metallic foil and the support both consist of the same material selected from one or more of gold, palladium and platinum or an alloy thereof.
19. A support according to claim 1 wherein the metallic foil and the support both consist of gold or an alloy thereof.
Description
SUMMARY OF THE FIGURES
[0069] So that the invention may be understood, and so that further aspects and features thereof may be appreciated, embodiments illustrating the principles of the invention will now be discussed in further detail with reference to the accompanying figures, in which:
[0070] FIG. 1 shows movement of gold nanoparticles in vitreous ice in a range of foil hole sizes.
[0071] FIG. 1A shows typical drift-corrected electron micrograph used for tracking gold particles in vitreous water on all-gold supports; scale bar is 0.5 μm. The inset (20 nm×20 nm) shows an overlay of the initial and final positions of a gold nanoparticle at the beginning of irradiation and after a fluence of 60e.sup.−/Å.sup.2.
[0072] FIGS. 1B and 1C show the root mean of the squared displacements of 200 to 2000 particles from 10 to 50 movies of different diameter holes (UltrAuFoil R2/2-1.9 μm, UltrAuFoil R1.2/1.3-1.2 μm, UltrAuFoil R 0.6/1-0.8 μm, and custom made grids with 0.3 μm, and 0.2 μm holes) are plotted as a function of cumulative electron fluence for 0° (B) and 30° tilt (C). Error bars show standard error in the mean. All exposures were under the same illumination condition (300 kV, 2.4 μm beam diameter, 8 e.sup.−/Å.sup.2/s). The ice thickness in all imaged holes was 300±50 Å.
[0073] FIG. 1D shows that thin films of ice used in cryoEM buckle during vitrification if the compressive stress (N) exceeds a critical value (N.sub.0) determined by the aspect ratio (2a/h) of the film. Electron irradiation causes the film to move in response to additional stresses in it, as evident from the correlated particle movement at the beginning of irradiation.
[0074] FIG. 2A shows a model of the stress accumulation in thin films of amorphous ice during cryoplunging and their response to electron irradiation. The density of liquid and amorphous water is plotted (stars) as a function of temperature, with a solid line to guide the eye. During cryoplunging into liquid ethane, water is rapidly cooled from, typically from 277K to 91K (arrow at bottom). The largest specific volume change experienced by water below its homogeneous nucleation point is (ΔV/V).sub.max≈5.5%. The thin film can only withstand compression of up to (ΔV/V).sub.crit before it buckles (critical stress buildup range corresponds to a 300 Å thick layer in a 1 μm hole).
[0075] FIG. 2B shows the diffusivity of water molecules in liquid and amorphous ice is plotted (crosses) as a function of temperature. The solid line is a fit to these values. The extrapolated diffusivity in amorphous ice at 84K is vanishingly low, ˜10.sup.−46 Å.sup.2/s. The shaded region in the bottom left indicates the range of diffusivity in amorphous ice at temperatures in the 0-100K range, where it is stable indefinitely. During imaging with 300 keV electrons, water molecules move pseudo-diffusively by 1 Å.sup.2/(e.sup.−/Å.sup.2). At typical imaging fluxes of 0.1-10 e.sup.−/Å.sup.2/s, this is equivalent to 0.1 to 10 Å.sup.2/s (shaded band between the top and middle) and corresponds to an instantaneous local temperature of 147K.
[0076] FIG. 3 shows a foil that is an all-gold specimen support designed for movement-free cryoEM imaging. FIGS. 3A and 3B show optical micrographs, in (A) reflected and (B) transmitted unpolarized white illumination of the patterned gold foil (hexagonal array of 200 nm diameter holes with 600 nm pitch) on a 600-mesh thin-bar gold grid. The scale and the corresponding area is the same for (A) and (B). The foil is blue in colour in transmitted light is due to a strong red absorption enhancement by the periodic hole pattern.
[0077] FIG. 3C shows a transmission electron micrograph of a single grid square on one of these grids. A 3 mm grid contains about 800 of these hexagons, each of which includes more than 5,000 holes in a regular pattern. The circle encloses more than 800 holes, which can all be imaged at high magnification without moving the stage during high-speed data collection.
[0078] FIG. 3D shows a transmission electron micrograph of the holey gold foil. The arrows show the pitch of the regular hexagonal pattern.
[0079] FIG. 3E shows a transmission electron micrograph of a single hole in the nanocrystalline foil.
[0080] The roundness of the 200 nm hole has been improved by reducing the gold grain size to 10 nm.
[0081] FIG. 3F shows a low-dose transmission electron micrograph of the protein DPS (220 kDa) vitrified on a grid of the present invention with 260 nm holes.
[0082] FIG. 4 shows the structure of DPS determined at <2 Å resolution and 1 e.sup.−/Å.sup.2 fluence using a 260 nm hole support.
[0083] FIG. 4A shows plot of the mean squared particle displacement during irradiation (positive slope) for the ensemble of all DPS particles used in the reconstruction, and plot of the relative B-factor for each frame with respect to the first (negative slope) with a linear fit to the B-factor decay which agrees with the expected slope from radiation damage alone. The mean squared displacement of the particles is linear with the fluence, in agreement with purely diffusional movement corresponding to an effective diffusion constant of 0.02 Å.sup.2/(e.sup.−/Å.sup.2).
[0084] FIG. 4B shows selected side chains (His51, Glu82, Asp156) and a water molecule from per-frame DPS reconstructions show the progression of radiation damage. The residues from the refined model are shaded by atom, and the contoured density map is shown as a mesh.
[0085] FIG. 4C shows the real (triangles) and imaginary (squares) parts of selected Fourier pixels at 2.2, 3.1, 4.5, and 7 Å resolution, plotted as a function of total fluence. The structure factors can be extrapolated to their values before the onset of irradiation, corresponding to the undamaged structure (filled symbols at 0 fluence).
[0086] FIG. 5 shows the optical transmission spectra of a support of the present invention, a continuous gold foil, a commercial UltrAuFoil, and a bare grid for comparison. The peak at 508 nm is characteristic of all thin gold films. Only the present support, due to its holes with a diameter comparable to the wavelength of light, produces a characteristic minimum at around 645 nm and a maximum at 714 nm. These are due to a resonance corresponding to a localized surface plasmon at the hole circumference.
[0087] FIG. 6 shows how a microscopy support of the present invention practically achieves the theoretical pseudo-diffusion limit in comparison to five known specimen support designs. Specimen supports of amorphous carbon on amorphous carbon in FIG. 6A, suspended ice in FIG. 6B, graphene on carbon in FIG. 6C, gold in FIG. 6D and graphene on gold in FIG. 6E all show an RMS displacement value of ribosomes with Mw 2 MDa that is greater than the specimen support of the present invention shown in FIG. 6F which demonstrates an RMS displacement value virtually identical to that of the theoretical pseudo-diffusion limit shown in FIG. 6G.
[0088] FIG. 7 shows root mean squared displacements of all tracked particles. FIG. 7A to 7J show the root mean squared movement of gold nanoparticles embedded in a suspended ice film in different hole diameters as a function of cumulative fluence, at angles of 0° or 30° tilt. Stage drift has been subtracted from these displacements. Error bars show standard error in the mean. The dots show the displacements of individual particles (200 to 2,000 particles per plot). All exposures were under the same illumination condition (300 kV, 2.4 μm beam diameter, 8 e.sup.−/Å.sup.2/s). The ice thickness in all imaged holes is 300±50 nm.
[0089] FIG. 8 shows movement tracking on the same grid with varying hole sizes. Mean squared displacements of particles in holes smaller than 300 nm, imaged at 0° (A) and 30° (C.) tilt, and in holes with diameters in the 500 to 560 nm range, imaged under identical conditions (B and D). The movement of the particles in the smaller holes appears to be fully diffusive, whereas the particles in the larger holes move as expected from the buckling model.
[0090] FIG. 9 shows optimal aspect ratio determination for the stability of suspended ice films. The darkest shaded region indicates the range of hole diameter and ice thickness combinations which are fully expected to be stable when vitrified in liquid ethane at about 90K and imaged with electrons at liquid nitrogen temperature. The combinations of hole diameters and ice thicknesses which lie in the white region are expected to be unstable due to buckling during vitrification. The dashed line shows the largest stable hole diameter for a given ice thickness, and the dotted line is a more conservative estimate of the same threshold. These lines are only indicative limits. There is some variability in the slopes due to ice thickness variations within the holes, hole shape variations, and uncertainty in the Poisson ratio of amorphous water. The different hole sizes and the corresponding ice thickness, in which gold nanoparticles were tracked in this work, are shown with black markers. The hole diameter and ice thickness for the DPS dataset in particular is labelled.
[0091] FIG. 10 shows controlling the shape of sub-micrometer (nanometer) holes in a nanocrystalline gold foil. Transmission electron micrographs of typical holes in a gold foil produced by evaporation onto a silicon template (210 nm holes) at ambient temperature (A) and at 85K, achieved by liquid nitrogen cooling of the substrate (B). The gold was evaporated at the same rate (1 Å/s) in both cases. Reducing the temperature reduces the gold grain size by a factor the order of 10× by reducing the surface diffusivity of the deposited gold. This allows for the formation of more regular and rounder holes.
[0092] FIGS. 11A to 11E illustrate the improvements of the present electron microscopy supports by detailing and comparing the defects in currently known supports.
[0093] FIG. 12 shows scanning electron micrographs of a HexAuFoil grid, fully fabricated on a holey wafer, and still attached to the wafer. All micrographs are acquired at 30° tilt, with 30 kV acceleration voltage using an Everhart-Thornley detector.
[0094] FIG. 12A shows one full HexAuFoil grid that has a diameter of 3 mm and is separated from the neighbouring grids on the wafer. The darkest areas correspond to the exposed silicon surface of the templating wafer. Arrow 1 points to one of the four fiducial markers on the grid, which label each of the four quadrants. The grid also has two rim marks, which are visible by eye. The largest quadrant mark (arrow 2) is clear of foil, and this location can be conveniently used to perform electron microscope alignments and flux measurement. Arrow 3 points to the thin gold foil connection strips between the grids which provide continuous electrical contact for electroplating. The dashed boxes indicate the magnified areas in FIGS. 12B, 12C, and 12D (from top to bottom).
[0095] FIG. 12B shows that each grid contains a center mark. The writing appears mirrored by design; when the grid is separated from the wafer and viewed from the flat foil side, it will be flipped to the correct orientation.
[0096] FIG. 12C shows that each hexagon is 50 micrometres wide, and contains 8000-9000 holes. In designs with alternative pitches, this size hexagon might have 3000-5000 holes. For smaller hexagons, the number of holes per hexagon is reduced in proportion to the open area. The grid bars are formed of electroplated gold, and are 10 micrometres wide and 10 micrometres thick in this example. The preferred thickness is from 5 to 20 micrometers. The aspect ratio of the bar (thickness/width) is preferably in the range from 0.25 to 4, and in most cases 0.5 to 2, with this example equal to 1.
[0097] FIG. 12D shows that each grid has a clear rim mark, which is also visible by eye (requiring dimensions of at least 0.2 mm). The radial direction from the center of the grid toward the rim mark is indicated with a line going across the middle of the hexagons. This line is visible in the electron microscope, and can be used, along with the other alignment features, to map the orientation of the grid in the microscope, relative to its orientation during specimen preparation, for example.
[0098] FIG. 13 shows HexAuFoil grids with 200-300 nm hole diameters. FIGS. 13A and 13B show scanning electron micrographs of two HexAuFoil gold foils, still attached to the templating silicon holey wafer via the sacrificial copper underlayer. Both micrographs are acquired at 0° tilt, with 30 kV acceleration voltage using an Everhart-Thornley detector. The scale is set by the center-to-center hole spacing, which is 600 nm for both. The foils in FIGS. 13A and 13B differ only by the templating hole diameter, which is 300 nm for FIG. 13A and 200 nm for FIG. 13B, respectively. Discs of gold foil can be seen at the bottom of each hole in the silicon wafer, with a shadowing angle dependent on the viewing angle from the gold source during electron beam evaporation of the foil towards the given point on the wafer. If the holes are insufficiently deep, these discs can remain attached to the foil and obstruct the holes when the foil is released from the wafer. A depth of 500 nm is sufficient to avoid this for 200-300 nm holes and 300 Å thick copper and gold foils.
[0099] FIG. 14 shows HexAuFoil grids released from the wafer post-fabrication.
[0100] FIG. 14A shows a scanning electron micrograph (45° tilt, with 2 kV acceleration voltage using an Everhart-Thornley detector) of the bar side of the grid after release from the wafer and removal of the copper adhesion layer. The grid is clipped in a standard clip-ring/clip holder used for transmission electron microscopy.
[0101] FIG. 14B shows a scanning electron micrograph (45° tilt, with 30 kV acceleration voltage using an Everhart-Thornley detector) of one of the hexagons on the same grid, acquired from the flat foil side of the grid, i.e. after the grid is flipped over relative to its orientation in FIG. 14A. This is the side of the grid that was originally covered with the sacrificial copper layer, making contact to the silicon template. The holey foil spans each grid hexagon and remains intact.
[0102] FIG. 14C shows a scanning electron micrograph (45° tilt, with 30 kV acceleration voltage using an Everhart-Thornley detector) of the holey gold foil demonstrates the edge flatness of each hole and surface flatness of the foil. These characteristics help with the formation of a thin, flat ice layer when the grids are used for cryoEM sample preparation.
[0103] FIG. 14D shows a transmission electron micrograph of the suspended gold foil on the grid after release from the wafer, which can be used as a sample support for transmission electron microscopy. The spacing between the holes is 600 nm. The dashed box delineates the area magnified in FIG. 14E.
[0104] FIG. 14E shows a transmission electron micrograph of one hole in the gold foil of the free-standing HexAuFoil grid. The hole diameter is 300 nm as indicated. The edge roughness is limited by the grain size of the gold foil, in this case approximately 20 nm for gold deposited at 85-90 Kelvin substrate temperature. Dashed black circle is exactly round, for comparison with the edge of the hole.
[0105] FIG. 14F shows in-plane movement statistics of gold nanoparticles in the HexAuFoil grids produced by the wafer-scale method (right) indicate the performance of these grids in terms of reducing specimen movement is equivalent to that of the HexAuFoil grids produced by the small scale method in the previous publication (Naydenova, Jia & Russo 2020) (left).
DETAILED DESCRIPTION
[0106] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
[0107] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention.
[0108] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors are not bound by any of these theoretical explanations.
[0109] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0110] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words “have”, “comprise”, and “include”, and variations such as “having”, “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[0111] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means, for example, +/−10%.
[0112] The words “preferred” and “preferably” are used herein refer to embodiments of the invention that may provide certain benefits under some circumstances. It is to be appreciated, however, that other embodiments may also be preferred under the same or different circumstances. The recitation of one or more preferred embodiments therefore does not mean or imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, or from the scope of the claims.
[0113] As used herein, the term “metallic” is used to refer to a material or component (such as a foil) displaying properties of a metal. In particular they display high electrical and thermal conductivity. In many cases electrical conductivity in metallic materials is higher than 10.sup.4 S/m.
Electron Microscopy Support
[0114] A support for electron microscopy is an apparatus which allows the carriage of the sample to be examined by electron microscopy into and out of the electron microscope. A degree of mechanical strength is provided to the support by a peripheral wall or rim inside which is typically arranged a mesh of members (such as grid bars). The sample to be examined is mounted onto the support within an area defined by the periphery of the grid bars. In cryoEM, the sample itself is suspended in a film (such as in a vitreous ice film) which is suspended in the holes or pores of a foil that is suspended between the grid bars. Foils are also commonly referred to as supporting films.
[0115] The foil part of the support typically has a mesh or “holey film” structure. These foils are typically described in the art with two numbers, for example “2/1”—this means a foil with two micrometre pores at a one micrometre spacing. Similarly, a foil designated 2/4 would have holes or pores of two micrometres, at a spacing of four micrometres, and so on. The term “support” includes instances where the foil is provided with and without a grid.
Example 1—High Resolution cryoEM Structural Determination of DNA Protection During Starvation Protein (DPS)
[0116] To demonstrate the use of movement-free specimen supports for high-resolution cryoEM the structure of the 220 kDa DNA protection during starvation protein (DPS) was determined. DPS was plunge frozen on grids with 280 Å thick gold foil with 260 nm holes. The average resolution from an initial reconstruction from about 9 hours of automated data collection on a modern 300 keV microscope, easily reached <2 Å and the total particle displacement was 0.86 Å RMS in 35 e.sup.−/Å.sup.2 of irradiation. The absence of buckling also ensured no significant rotation of the particles during imaging. In contrast to all previous single particle cryoEM datasets to date, maps reconstructed from each frame show that the first frame (1 e.sup.−/Å.sup.2 or 3 MGy) contained the most structural information and the quality (B-factor) of sequential frames decays linearly with dose/fluence. A linear decay in B-factor with dose is expected from studies of radiation damage in X-ray and electron crystallography, but has never previously been observed for single particle cryoEM due to movement at the onset of irradiation.
Example 2—Gold Foil Characterisation
[0117] The mean linear intercept grain size of some gold foils fabricated as described herein were measured by TEM and found to be 100±10 Å . This is approximately 20 times smaller than the grain size in gold foils fabricated under similar conditions but at room temperature. The small grain size allows for both thinner foils and smoother hole edges. The typical edge roughness of 200 to 300 nm holes (deviation from a circular shape) is less than 10 nm.
Comparative Examples—Previous Foils are Unfit for Purpose
[0118] The following examples illustrate the improvements of the present electron microscopy supports by detailing and comparing the defects in currently known supports.
[0119] Defect type 1: Malformed holes due to increased grain size due to increased evaporation rate
[0120] The gold film shown in FIG. 11A was evaporated at a rate of 6 Å/s onto the patterned substrate held at about 90K. The sacrificial copper layer (not shown) was evaporated at 27 Å/s. These evaporation rates resulted in malformed holes due to the increased grain size. The hole diameters vary between 50 and 250 nm. Compare with the foil in FIG. 11D, which was produced by evaporation onto the same template at the same temperature, but at a lower rate (1 Å/s for both copper and gold), and has the same thickness. In that foil, the typical hole deviation from roundness is 10 nm or less.
[0121] Defect type 2: Malformed holes due to increased grain size due to increased evaporation temperature are shown in FIG. 10A.
[0122] Defect type 3: Malformed holes due to over-etching
[0123] The gold foil shown in FIG. 11B was evaporated in a way identical to the one from FIG. 11E, onto the same substrate. The release (etching of the sacrificial Cu layer in ferric chloride) was 2 times slower for this film than for the one in FIG. 11E (30 min vs 15 min). This resulted in irregular enlargement of the holes (by about 50 nm) due to etching of the gold by the ferric chloride. The distance between holes (pitch) is 600 nm.
[0124] Defect Type 4: Porosity
[0125] The gold foil shown in FIG. 11C was manufactured by the method in Russo 2014 where the gold evaporation is at room temperature. The foil thickness is 326 Å, and the holes are 2 μm in diameter. This is thicker than the present foils. Due to the larger grain size of about 200 nm the foil remains porous at this thickness and is unstable. This was demonstrated using in the paper Russo 2014 which shows that even a 397 Å thick foil becomes unstable due to this porosity and discontinuous metal foil. The typical pore dimensions are 200 nm long×10 nm wide and 30 to 40/μm.sup.2.
[0126] Gold foils produced by the sputtering method in Janbroers et al. 2009 also suffer from this porosity, besides not being made from pure gold. The pores are clear in FIG. 1C and FIG. 5. This is in contrast to the present foils which do not have such pores.
[0127] Although the data provided herein relates to gold, similar improvements are expected to be seen in materials having similar structural and electrical properties such as transition metals, aluminium, beryllium and degenerately doped silicon having a second element selected from boron, aluminium, phosphorus, and arsenic at a concentration of 10.sup.20 atoms/cm.sup.3 or higher
References
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[0130] 3. Russo, C. J. & Passmore, L. A. Ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377-1380 (2014).
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