Method for studying nanoscale biological samples in electron beam apparatus with reduced radiation damage

Abstract

Method for investigating at least one nano-scale biological sample (1), said method comprising a step a) (100) of preparing an embedding liquid (3) containing a plurality of nano-scale biological samples (1) wherein the nano-scale biological samples (1) have a maximum diameter MD wherein MD < = 30 nm, a step b) (200) of preparing a film (2) of the embedding liquid (3) on an electrically conductive substrate (4) in an application zone (22), the substrate (4) is wetted by the embedding liquid (3) in the application zone (22), and wherein the film (2) of the embedding liquid (3) has an average thickness AT, where AT < = 30 nm; step c) (300) adjusting the film (2) on the substrate (4) to a measurement temperature MT, wherein-100 DEG C < = MT < =-1 DEG C; step d) (400) measures at least one of the nanoscale biological samples (1) within the film (2) on the substrate (4) in an electron beam device (32) at a measurement temperature MT, where the at least one nanoscale biological sample (1) is exposed to an electron beam (41, 44). The present invention provides a method for studying nanoscale biological samples, wherein single nanoscale biological samples can be studied in a simple manner at high spatial resolution.

Description

Technical Field

[0001] The present invention relates to a method for studying biological samples at nanoscale in an electron beam device. Background Art

[0002] Such a process is known, for example, from US Pat. No. 7,413,872.

[0003] Electron beams are frequently used to examine the structure of nanoscale biological objects such as proteins or virus particles, either via direct microscopy or via diffraction methods. The typical instrument for studying nanoscale biological objects is the transmission electron microscope (=TEM); it should be noted that the investigated nanoscale biological objects are also called nanoscale biological specimens.

[0004] In order to achieve a spatial resolution in the sub-nanometer range, a significant electron dose of the electron beam is required. There are samples that can tolerate even high electron doses, such as metal nanoparticles. However, a wide range of samples are quickly damaged in the electron beam, such as polymers, certain battery materials involving lithium, and most biological samples such as proteins and virus particles. Typically, the electron dose limit for proteins and virus particles is 10 electrons per 100 nm. For this purpose, a sufficient signal-to-noise ratio (=SNR) is obtained for a spatial resolution of about 5 nm under standard conditions. Better resolution will require a higher electron dose, whereby the dose is proportional to at least the square of the spatial resolution (see textbook: L. Reimer, H. Kohl, Transmission Electron Microscopy: Physics of Image Formation, Springer, 2008, p. 479, formula 11.17), but stronger ratios up to the fourth power have also been reported under certain conditions (N. de Jonge, Ultramicroscopy 187 (2019) 113-125). In order to distinguish the structure of a wide range of biological nanoscale objects, a spatial resolution of at least 0.5 nm will be required, and it is highly desirable to mitigate radiation damage.

[0005] A key issue in electron microscopy of biological samples is the occurrence of radiation damage via inelastic electron scattering (RF Egerton, Micron 119 (2019) 72-87). The damage process includes the generation of excited states of molecules and the generation of charge pairs (ionization). If charge pairs are generated on the sample surface, secondary electron emission can occur, causing the sample to be charged. Structural damage to the sample occurs mainly in the case of atomic bond breakage following the inelastic scattering process. Heating of the sample is irrelevant to typical conditions involving biological samples. Another process (so-called impact damage) is generally of a much smaller magnitude than damage via charging effects, and it can be overcome by reducing the beam energy to below the impact threshold, but the energy level to prevent hydrogen damage is so low that only limited spatial resolution can be achieved.

[0006] The first partial compensation for radiation damage is to reduce the electron inflow (also referred to as electron flux) to reduce the number of excited states and charge pairs generated per unit time (EE Fill, F. Krausz and MG Raizen, New J. Phys. 10 (2008) 93015-1-7). Under typical experimental conditions in electron microscopy, multiple adjacent charge pairs are generated so that the net Coulomb force is directed outward, promoting beam damage. Below the threshold inflow, there is only one adjacent charge pair at a time point, so that the net Coulomb force is directed toward recombination. Mitigating beam damage by operating the electron beam at low electron flux has been reported in the open literature, for example, S. Keskin and N. de Jonge, Nano Lett. 18 (2018) 7435-7440. Using low electron beam flux (less than ) for transmission electron microscopy examination of biological samples, thereby reducing electron beam damage caused by charging, but causing radiation damage to the same samples at higher fluxes.

[0007] In order to achieve balance in charge separation in organic molecules, an electron transfer process needs to occur. The rate constant of electron transfer between two fixed redox states is provided by (RA Marcus, N. Sutin, Biochim. Biophys. Acta 811 (1985) 265-322):

[0008]

[0009] The driving force is -ΔG 0 , temperature is T, Boltzmann constant is k b , the reorganization energy is λ, and the electronic coupling is H ab In one model, H ab Provided by (CC Moser et al., Nature 355 (1992) 796-802):

[0010]

[0011] Where the distance between the donor and the acceptor is R, the tunneling factor is β, and the maximum electron coupling is V0. The recombination time varies depending on how far the electrons produced in the ionization event have traveled, but it also depends on the precise electron transfer pathway in the molecule (DN Beratan et al., Science 252 (1991) 1285-1288). Electron transfer in proteins generally occurs at about room temperature and over distances less than a few nanometers.

[0012] It is also important that the guest is placed on a conductive support that is connected to the electrical neutrality of the electron beam apparatus (usually "ground" potential). The latter is generally achieved by placing the sample on a conductive foil, such as a carbon foil, which is placed in the apparatus via a sample holder.

[0013] The second partial compensation for radiation damage is by freezing samples that are usually liquid samples (such as most biological samples). Primary damage occurs when electrons directly collide with the observed object, and secondary damage occurs after the medium surrounding the object is collided and chemical free radicals and free electrons diffuse to the object, causing damage. In addition, the molecular structure of the internal bond breakage remains fixed in the solid ice matrix to reduce structural degradation. The frozen medium protects the sample against secondary radiation damage due to reduced diffusion and increased structural stability compared to the situation in liquid (LF Kourkoutise al., Annu. Rev. Mater. Res. 42 (2012) 33-58).

[0014] A practical problem is that freezing biological samples can lead to ice crystals which damage the morphology of the sample being studied. The current state of the art method used in structural biology is to freeze the sample to liquid nitrogen temperature quickly enough to obtain amorphous ice, for example. The sample generally consists of a layer of protein-containing saline placed on a porous carbon membrane. After removal of excess liquid and rapid freezing, the resulting free-standing amorphous ice layer in the pores in the carbon membrane has a thickness of about 50-70 nm. During the examination of proteins in this free-standing ice layer, the temperature is kept below the so-called glass transition temperature of ice (generally about 100°K). Ice crystals are thereby prevented from forming.

[0015] However, ice at these low temperatures is an insulator. Pure ice exhibits σ = 10 at its freezing point. -7 Ω -1 m -1 The poor static conductivity (see GW Gross et al., J. Glaciol. 21 (1978) 143) and decreases to less than 10 for temperatures below 200 K. -12 Ω-1 m -1 At the latter temperature, ice remains an insulator even when ions are mixed with ice (see Stillman and Grimm, Lunar and Planetary Science XXXIX (2008) 2277). It is thus impossible to compensate the sample for the charge from the neutral position of the device during secondary electron emission. Above a certain electron dose, the sample is charged so much that the electric field in the sample exceeds 8×10 7 Vm -1 The dielectric strength of ice is reduced (T. Kohno et al., IEEE Transactions on Electrical Insulation, EI-15 (1980) 27-32), and sample damage occurs, such as the formation of hydrogen.

[0016] In addition, the charge transfer rate in the guest becomes highly limited. To illustrate the above temperature effect on electron transfer, a typical value of ΔG of Equation 1 can be considered in proteins. 0 = -1.5 eV, and λ = 0.75 eV, and the temperature changes from 293 K to 77 K. This temperature change causes the electron transfer rate to increase by 6 × 10 8 This would essentially block electron transfer in the protein. However, if -ΔG 0 =λ, electron transfer is possible in proteins (H. Wang et al., J. Phys. Chem. B 113 (2009) 818-824), which is only a rare case in practice. Therefore, at low temperatures, it is not possible to mitigate radiation damage by operating at low electron fluxes to enable charge pairs to recombine. Low temperature sample damage is thus limited at a certain threshold electron dose (usually ) or above occurs.

[0017] Thus, the above-described first partial compensation for radiation damage cannot be combined with the above-described second partial compensation for radiation damage, which limits the effectiveness of achievable radiation damage mitigation strategies.

[0018] The current approach to overcome the limitation of radiation damage of frozen biological samples is via so-called single-particle tomography ("Single-particle reconstruction of biological molecules-story in a sample", Nobel Lecture, December 8, 2017 by Joachim Frank). In this method, images of many similar objects, such as a single type of purified protein or virus particles, are recorded via transmission electron microscopy (TEM). The objects are embedded in a thin layer of ice and deposited in random orientations. The electron dose is limited to 10 electrons per gram. The value below the damage threshold of , and as a result, each image exhibits a poor signal-to-noise ratio (SNR). The three-dimensional structure is then calculated by averaging tens of thousands of images, thereby increasing the SNR. However, in this case, unique structural information of a single object such as a protein cannot be obtained, and the difference information between structural subtypes cannot be obtained when the number of objects in a type is less than the number of object images that need to be averaged to achieve the required SNR, which seriously limits the study of protein function.

[0019] In US 7,659,510, it is proposed to use ions such as Na + Cl - Doping ice cooled at liquid nitrogen temperatures is intended to make the ice conductive at liquid nitrogen temperatures. The doping changes the band gap of the insulating ice, and then places a potential on the sample such as to charge the sample. Similar approaches are described in US 2012 / 0220046 A, US 2010 / 0243482 A and Chih-Yu Chao, Chinese Journal of Physics Vol. 45, No. 6-1, December 2007, 557-578.

[0020] However, in general, the ion mobility in ice at liquid nitrogen temperatures approaches zero. In addition, amorphous ice does not contain crystalline regions known in typical semiconductors such as silicon crystals. Therefore, there is no continuous conductive path required for the sample to become conductive.

[0021] US 7,413,872 describes a device for preparing samples for cryo-electron microscopy. In an environmental chamber, a support for the sample or carrier and at least one blotting element to which a medium for absorbing the liquid is attached are arranged. The grid is immersed in the particle suspension to be examined. As a result of the blotting, the film thickness in the grid openings is reduced, for example, from 3-4 μm to 100 nm. The sample is cooled very quickly so that the fluid becomes vitreous. The sample is examined by cryo-microscopy, for example at -170°C.

[0022] Furthermore, US Pat. No. 7,420,184 describes a sample holder for electron microscopy having a temperature switch, so that the sample can be switched between liquid nitrogen temperature and room temperature.

[0023] Purpose of the Invention

[0024] It is an object of the present invention to provide a method for studying biological samples on the nanoscale, wherein individual biological samples on the nanoscale can be studied in a simple manner with high spatial resolution. SUMMARY OF THE INVENTION

[0026] According to the present invention, this object is achieved by a method for studying at least one biological sample at nanoscale, said method comprising

[0027] Step a) preparing an embedding liquid containing a plurality of nanoscale biological samples, wherein the nanoscale biological samples have a maximum diameter MD, wherein MD is ≤30 nm,

[0028] Step b) preparing a thin film of embedded liquid on a conductive substrate in the application area, thereby placing a nanoscale biological sample on the substrate,

[0029] wherein the substrate is wettable by the embedding liquid in the application area,

[0030] and the film in which the liquid is embedded has an average thickness AT, wherein AT ≤ 30 nm;

[0031] Step c) adjusting the thin film on the substrate to a measurement temperature MT, wherein -100°C≤MT≤-1°C;

[0032] step d) measuring at least one nanoscale biological sample in a thin film on a substrate in an electron beam device at a measuring temperature MT,

[0033] Wherein the at least one nanoscale biological sample is exposed to an electron beam.

[0034] The method of the invention proposes to prepare the nanoscale biological sample to be studied in a thin film on a substrate according to step b), wherein the thin film has a low average thickness AT, wherein AT≤30 nm. The nanoscale biological sample has a maximum diameter MD, wherein MD≤30 nm.

[0035] The advantage of a low film thickness is that in step c) the film can be conditioned (usually cooled) to a moderately low temperature, selected between -1°C and -100°C, preferably between -1°C and -50°C, without forming ice crystals that could damage nanoscale biological samples.

[0036] In step d), the film which has been adjusted to said temperature is subjected to measurement in an electron beam apparatus, generally a TEM (Transmission Electron Microscope); said temperature is therefore also referred to as measurement temperature MT.

[0037] By conditioning the film to a moderately low measurement temperature MT in step c), the film or its embedding liquid becomes immobilized or stabilized (or at least less mobile and more stable than at the preparation temperature of step b) (generally room temperature)). Typically, the film applied from the embedding liquid is not liquid at said measurement temperature MT. First, the film is able to resist the vacuum present in the electron beam device during step d) for a longer period of time; the film will show low and generally negligible evaporation in the vacuum (over the measurement time). Second, the diffusion of chemical free radicals and free electrons is reduced, which reduces radiation damage (due to "secondary damage") under electron beam irradiation during the measurement of at least one nanoscale biological sample. Third, structural stability is provided to the sample.

[0038] It is also important with regard to radiation damage that the measuring temperature MT between -100°C and -1°C and preferably between -50°C and -1°C is still relatively high compared to current state-of-the-art studies at about liquid nitrogen temperature (-196°C = 77K). This relatively high measuring temperature improves the electron transfer in the respective biological sample and between the respective biological sample and the (conductive) substrate and its sample carrier. In addition, the improved electron transfer and the improved conductivity, especially within the immobilized embedding liquid, improve the charge pair recombination, so that charge accumulation only occurs at relatively high electron fluxes.

[0039] Therefore, the sample preparation and measurement using the method of the present invention can tolerate higher electron doses (especially 10 electrons per nanometer) during the measurement of at least one nanoscale biological sample. or more, and usually 50 electrons per or more) and higher electron fluxes (usually 5 electrons per per second or more) without significant radiation damage. Accordingly, relatively high spatial resolution in electron beam measurements (especially TEM measurements) can be achieved in relatively short measurement times or with a relatively small number of objects that need to be averaged to obtain sufficient SNR.

[0040] It should be noted, however, that the electron flux should still be low enough to allow charge recombination without charge accumulation. In general, for typical electron beam energies of 30 to 300 keV used in high-resolution transmission electron microscopy, this is at or below 50 electrons per gram. per second and preferably about 10 electrons per The electron flux per second is sufficient in the method of the present invention; it should be noted that lower electron energies in the range of 50 eV-30 keV may also be used, as used in other electron beam devices.

[0041] The nanoscale biological samples to be studied are generally proteins, but can also be peptides, virus particles, DNA sequences or other substances that can exist in the human or animal body or in plants or fungi.

[0042] To facilitate the preparation of the film, the substrate is wettable by the embedding liquid at least in the application area (in particular, the contact angle α is <90°, preferably <45°). In this way, the embedding liquid itself spreads over the application area. To achieve a low thickness, the liquid can be removed carefully (for example by suction or evaporation), and / or only a few femtoliters of embedding liquid are initially placed on the substrate (see also below).

[0043] It should be noted that the measurement of at least one nanoscale biological sample with an electron beam in step d) is generally performed in order to examine the structure of the nanoscale biological sample. The measurement may include phase contrast (including bright field imaging and dark field imaging), diffraction, holography or laminography, generally performed in a transmission electron microscope.

[0044] Preferred variations of the invention

[0045] A preferred variant of the method according to the invention is characterized in that the measuring temperature MT is less than the solidification temperature of the bulk phase of the embedding liquid. By selecting a measuring temperature below the solidification point of the bulk phase of the embedding liquid, the thin film of the embedding liquid is reliably fixed or stabilized. It should be noted that due to the low film thickness, the film can be prevented from forming ice crystals, even below the solidification point of the bulk phase of the embedding liquid. The measuring temperature can be kept above the solidification temperature of the thin film of the embedding liquid (if feasible), the latter temperature can be lower than the solidification temperature of the bulk phase of the embedding liquid due to boundary layer effects. It should be noted that the thin film of the embedding liquid can be in particular in a gel-like state or an amorphous state or a solidified state or some different state at the measuring temperature. The solidification temperature of the bulk phase of the embedding liquid can be easily determined experimentally, for example with a macroscopic sample of the embedding liquid in a test tube and slowly reducing the test tube temperature; the freezing of the embedding liquid is sufficiently visible to the naked eye or can be easily detected with a sensor. If desired, the film can also be cooled below the solidification temperature of the thin film of the embedding liquid, and here AT should be less than MD to avoid the formation of ice crystals. Whether ice crystals are forming can be easily checked via transmission electron microscopy images, and the AT reduced if necessary.

[0046] The ice nucleation rate is 10 for a very thin layer of liquid. 14 m -3 s -1, but for the nanodroplets with a radius of 2.4 nm, the latter value decreases by eight orders of magnitude to 10 6 m -3 s -1 , thereby obtaining supercooled water microdroplets at the nanoscale. For the nanodroplets placed on a plane, ice nucleation is completely avoided. Thus, ice crystals will not form in the water layer with a thickness of about 3 nm on the surface of the graphene film serving as a sample carrier. In practice, in the case of using brine (e.g., water mixed with, for example, NaCl) instead of pure water (which reduces nucleation crystallization compared to ice), the liquid layer can be thicker (e.g., 5 nm), so that the thicker layer still does not crystallize.

[0047] By adjusting the thin film embedding the liquid on the substrate and the nanoscale biological sample (or only the sample) in the embedding liquid to a measurement temperature below the solidification temperature of the bulk embedding liquid, the diffusion of chemical free radicals and free electrons is reduced, and the evaporation of the thin film of the embedding liquid in the vacuum of the electron microscope is prevented.

[0048] In a preferred variant, AT ≤ MD is selected. In the case of adjusting the thin film to the measurement temperature, this helps to avoid the formation of ice crystals in the embedding liquid of the thin film.

[0049] Another condition of the preferred variant is that in step d), the electron beam energy of the electron beam is 50 eV to 300 keV,

[0050] preferably 30 keV to 300 keV,

[0051] and the electron flux of the electron beam is 0.1 electron per per second to 50 electrons per per second,

[0052] preferably 1 electron per per second to 10 electrons per per second. These parameters have been proven in practice. In particular, for typical conditions in high-resolution TEM equipment, using the above electron flux can support reducing radiation damage through charge recombination.

[0053] In a preferred variant, steps a) and b) are completed at a preparation temperature PT, where PT > 0 °C. This simplifies the implementation of steps a) and b). It should be noted that the preparation temperature generally ranges from 0 °C < PT ≤ 40 °C, and usually PT is at room temperature (20 °C).

[0054] Highly preferred is a variant which comprises during step b) placing an initial large amount of embedding liquid on the substrate, wherein the large amount is greater than required for the thin film, and then reducing the amount of embedding liquid on the substrate until only the thin film remains. This process is particularly convenient to put into practice, and the application areas can be loaded quickly and reliably with embedding liquid. Furthermore, if desired, it is easy to load a large number of application areas (especially 5 or more, or even 20 or more) with embedding liquid at one time.

[0055] Preferably, this variant is further developed, characterized in that reducing the amount of embedding liquid comprises placing a liquid absorbing medium in contact with the embedding liquid,

[0056] Especially wherein the liquid absorbing medium comprises filter paper or brushed metal wire,

[0057] And / or characterized in that reducing the amount of embedding liquid comprises evaporating some of the embedding liquid. Reducing the amount of embedding liquid by contact with a liquid absorption medium is also referred to as suction. By suction, particularly large amounts of embedding liquid can be removed in a rapid manner. In particular, a TEM grid carrying a substrate, wherein the substrate, such as a graphene foil or a carbon foil, has perforations, such as microcracks, can be squeezed onto a filter paper. For evaporating some of the embedding liquid, generally only some time is waited, preferably a defined time in a defined atmosphere. Generally, evaporation is performed at room temperature and / or at a normal atmosphere; alternatively, it can be performed under set conditions, especially at a temperature elevated compared to room temperature, or in an atmosphere surrounding the substrate at a reduced humidity, especially after the embedding liquid has been applied to the substrate. The film thickness can be set very accurately by evaporation, and additionally the nanoscale biological sample density that can be achieved in the film is particularly high.

[0058] A further variant is advantageous in which, during step b), in order to prepare a thin film of embedding liquid in an application zone with an application zone area AZA, an initial small volume SV of embedding liquid is placed on the substrate in the application zone, wherein SV≤AT*AZA. After having been applied to the application zone, the desired average thickness of the thin film can be achieved "by itself" in the case of the initial small volume of embedding liquid spreading over the application zone of the substrate, which is wettable by the embedding liquid there. In this variant, it should be noted that it is generally not necessary to reduce the amount of embedding liquid applied in step b); however, sometimes some evaporation of the embedding liquid also takes place in this variant.

[0059] Also preferred is a variant in which in step b) an initial amount of embedding liquid is placed on the substrate in the application zone as follows: first an application tip, in particular an atomic force microscopy (= AFM) tip, is immersed in a supply reservoir containing an embedding liquid for a nanoscale biological sample and then the application zone is contacted with the application tip. In the sense of the present invention, contacting the application zone with the tip includes both: the application tip directly contacts the application zone, or a drop of embedding liquid attached to the tip contacts the application zone. This is the actual procedure of placing a small amount of embedding liquid (in particular an embedding liquid of a small volume SV as mentioned above) on the substrate or its application zone. The material of the application tip should be wettable by the embedding liquid (contact angle β<90°, preferably β<45°), but the wetting is preferably not as good as on the substrate in the application zone (i.e. preferably α<β, where α is the contact angle of the embedding liquid on the substrate in the application zone, and β is the contact angle of the embedding liquid on the tip material). The amount of liquid attached to the application tip can be adjusted via the immersion depth of the application tip into the surface of the embedding liquid in the supply reservoir.

[0060] Also preferred is a variant in which in step b) an initial amount of embedding liquid is placed on the substrate in the application zone with an application tip which has microchannels through which the initial amount of embedding liquid is discharged. This is also a practical way to place small amounts of embedding liquid, especially small volumes SV as mentioned above, on the substrate or its application zone.

[0061] Also preferred is a variant in which the initial volume of embedding liquid placed on the substrate during step b) is 1 fL to 2 μL. It should be noted that a typical initial volume of embedding liquid in case of a subsequent reduction (e.g. by aspiration or evaporation) of the amount of embedding liquid is 0.2 μL-2 μL. According to the invention, a typical amount of embedding liquid corresponding to a small volume SV as mentioned above is usually of the order of 1 fL to 100 fL (e.g. for a typical 200 mesh 3 mm TEM grid (corresponding to a typical TEM grid window of length 60 μm), with AZA=3600 μm 2 and AT = 10nm, then the result is SV = 36fL (3.6*10 -17 m 3 ).

[0062] In an advantageous variant, the experimental parameters applied during step b) for producing a film of average thickness AT have been previously determined in a calibration experiment.

[0063] In particular, the experimental parameters include at least one of the following: the temperature of the atmosphere surrounding the substrate, the pressure of the atmosphere surrounding the substrate, the humidity of the atmosphere surrounding the substrate, the composition of the atmosphere surrounding the substrate, the temperature of the substrate, the type of liquid absorption medium, the contact time with the liquid absorption medium, the contact pressure with the liquid absorption medium, the evaporation time, the initial large amount of embedding liquid placed on the substrate, the initial small volume SV of the embedding liquid placed on the substrate in the application area, the initial amount of embedding liquid placed on the substrate, the initial volume of embedding liquid placed on the substrate, the downward pressure of the application tip, the discharge flow rate of the embedding liquid through the microchannel. By means of calibration experiments, samples can be prepared from nanoscale biological samples in a reproducible manner and the samples can be studied in a standardized and optimized procedure, wherein the results of different samples can be compared with a high information content. It should be noted that the calibration experiments each generally include determining the obtained average thickness of the obtained thin film, which is generally measured by electron microscopy in a frozen (or immobilized) state. The experimental parameters in the calibration experiments can be adjusted until the desired average film thickness is obtained.

[0064] Particularly preferred is the variant in which the embedding liquid is chosen to be water with added salts,

[0065] Preferably, the added salt is NaCl,

[0066] Particularly preferred is the salt added in the form of NaCl in a concentration of 10 mM. By adding salt, a high conductivity of the (immobilized) liquid-embedded film can be achieved.

[0067] In a preferred variation, the substrate is a graphene foil or a carbon foil. For providing a good electron transfer from the film (or the sample in the film) via the substrate to the substrate holder / sample holder and finally to the ground, the graphene foil or the carbon foil provides a good conductivity, and therefore helps to minimize radiation damage. In addition, the graphene foil or the carbon foil is fully suitable for TEM research, and is particularly suitable for electron beam transmission without distorting the TEM image of the imaging sample too much. It should be noted that the graphene foil or the carbon foil is generally very thin, such as a thickness of 5nm or lower, and especially wherein the graphene foil can be a monoatomic film. Generally, the above-mentioned very thin graphene or carbon foil is loaded on a more solid porous film (e.g., a porous film with a thickness of 50nm or higher), such as a porous carbon film or a porous silicon nitride film.

[0068] Also advantageous is a variant in which, before step b), the substrate is treated with plasma at least in the region of one or more application zones,

[0069] Preferably wherein the plasma is formed from oxygen and / or argon,

[0070] Most preferably, the plasma is at 10 -3The pressure of millibars is formed from oxygen and / or argon. The plasma is able to render the substrate hydrophilic, at least in one or more application zones of the substrate to which the embedding liquid is to be applied. Furthermore, the plasma treatment cleans the substrate surface.

[0071] In an advantageous variant, after step b) and before step c), the thin film of the embedding liquid is covered with a cover substrate, in particular wherein the cover substrate is a graphene foil or a carbon foil. The cover substrate can contribute to minimizing the evaporation of the embedding liquid (fraction) of the thin film and / or obtaining small liquid pockets in the vacuum of the electron beam device in step d). Furthermore, in the case of a conductive cover substrate, the cover substrate can improve the electron transfer from the thin film to the substrate holder / sample holder and ultimately to the ground, and thereby contribute to minimizing radiation damage.

[0072] In yet another advantageous variant, the embedding liquid contains a conductive polymer and / or a conductive protein. The conductive polymer and / or conductive protein improves the electron transfer from the studied nanoscale biological sample to the substrate and substrate holder / sample holder and ultimately to ground, and thus helps to minimize radiation damage in step d). It should be noted that the conductive polymer (or protein) can also serve as a linker molecule.

[0073] Preferred is a variant in which the embedding liquid contains a negative stain,

[0074] Especially wherein negative stain is selected as α-D-glucose 1-phosphate dipotassium salt hydrate, preferably applied with 2mM concentration. Chemical stain can preserve the structure of nanometer scale biological sample, especially by keeping water molecule layer around sample. Stain can further provide conductivity to substrate or substrate holder / sample holder and finally to ground, thereby minimizing radiation damage. By applying negative stain, stable film in the vacuum of electron beam equipment can be obtained without using covering substrate. Generally, film is gel-like at measuring temperature MT. In this modification, generally in step b), (especially at least at the end of reducing program), evaporation is applied to reduce the amount of embedding liquid.

[0075] Particularly preferred is a variant in which, in step c), the film on the substrate is first cooled to a low temperature LT, wherein LT ≤ -196°C, and then warmed again to the measuring temperature MT. Generally, the first cooling is performed at a high cooling rate, such as within 240 s or less or even within 120 s or less (assuming a preparation temperature PT of 0°C to 40°C). By first cooling (at a sufficiently high cooling rate), the embedding liquid of the film can be converted into amorphous ice without forming ice crystals. The subsequent warming to the measuring temperature MT, even where MT is above the glass transition temperature of the embedding liquid, will not result in crystallization of the embedding liquid of the film due to its low film thickness. It should be noted that the low temperature LT is at or below the boiling temperature of liquid nitrogen (LN2) at 1 bar.

[0076] In an alternative variant, the temperature of the film is always kept above or at said measuring temperature MT during steps b) to d). This simplifies and accelerates the sample preparation procedure. In particular, only moderately low temperatures need to be provided, which can usually be provided without handling cryogen liquids such as LN2 or LHe and using low-cost equipment.

[0077] Also preferred is a variation in which the substrate is placed on a TEM grid, wherein the TEM grid has a plurality of intersecting strips defining windows between the intersecting strips. Each window can be used as an application area. The TEM grid simplifies sample handling in the TEM. Further, the TEM grid (generally made of copper) provides a good conductive path.

[0078] A preferred further development of the above-mentioned variant provides that during step b), the embedding liquid is initially applied as droplets spread over a plurality of screen windows, in particular all screen windows of a TEM grid. Typically, some of the embedding liquid is then removed, for example by suction, preferably with the TEM grid being pressed onto a filter paper. This wets all screen windows or the respective application area with the embedding liquid particularly quickly and reliably.

[0079] A particularly preferred variant provides that the substrate is placed on a TEM grid, wherein the TEM grid has a plurality of intersecting strips defining windows between the intersecting strips; and during step b), an embedding liquid is applied between the strips in at least one of the windows,

[0080] In particular, the embedding liquid is carefully applied between the mesh strips in at least one mesh window. The mesh strips can form a (maximum) limitation of the application area. The mesh window or the respective application area is easily recognizable in an electron beam device, and the nanoscale biological samples therein are accordingly easily studied. The TEM grid (generally composed of copper) further provides a good conductive path along the mesh strips. In the case of applying an embedding liquid of a small volume SV, the respective small volume SV of the embedding liquid can be easily placed on the respective mesh window (especially in the center).

[0081] In a preferred variant, the substrate comprises at least one topical coating defining at least one application zone, wherein the topical coating makes the substrate wettable with an embedding liquid in the application zone. The application zone, in particular its area or extension, can be clearly defined by the topical coating. In addition, substrate materials that were initially not suitable for being wetted by the embedding liquid can become suitable by the topical coating. This expands the scope of substrate materials for the present invention. The topical coating can be prepared especially from linker molecules (see below).

[0082] The conditions for favorable variations are

[0083] The substrate is at least partially coated with linker molecules for connecting the nanoscale biological sample to the substrate,

[0084] In particular, wherein the nanoscale biological sample is a protein and the substrate comprises a graphene foil,

[0085] The linker molecule is attached to the substrate on one side via one or more non-polar chemical groups, in particular wherein the one or more non-polar chemical groups are based on pyrene, pyridine, porphyrin or amine,

[0086] The invention relates to a method for preparing a nanometer biological sample. ...

[0087] In a preferred further development of the above variant, the linker molecule contains a conductive chain, in particular wherein the conductive chain is based on carbon atoms or carotenoids with unsaturated carbon bonds or chains of carbon atoms and oxygen atoms. In this way, the electron transfer from the thin film or nanoscale biological sample to the substrate can be improved and radiation damage can thereby be minimized.

[0088] Also preferred is a further development of the above variant, wherein the linker molecule comprises a molecule that specifically binds to the nanoscale biological sample to be studied, in particular wherein the nanoscale biological sample comprises a protein, an antibody, a binding peptide, a DNA sequence, a ligand of a receptor, a chelated metal or histidine. Said molecule has a high affinity for binding to the nanoscale biological sample to be studied, also referred to herein as a ligand molecule. Depending on the biological substance to be studied (i.e. the nanoscale biological sample), there are various compounds (containing the ligand molecule and the nanoscale biological sample). For example, the linker molecule may contain biotin as a ligand for the protein streptavidin, and the compound with the attached linker molecule is amine-PEG2-biotin.

[0089] Also within the scope of the present invention is a method for determining an electron flux threshold for measuring a nanoscale biological sample using an electron beam, the method comprising the steps of:

[0090] Step α) studying a first nanoscale biological sample according to the method of the invention described above, wherein in step d) the first nanoscale biological sample is exposed to a preselected electron dose at a first electron flux, and the structure of the first nanoscale biological sample is detected,

[0091] step β) studying a further nanoscale biological sample according to the method of the invention described above, wherein in step d) the further nanoscale biological sample is exposed to a preselected electron dose at a further electron flux higher than the previously applied electron flux, and a further structure of the further nanoscale biological sample is detected,

[0092] Step γ) Repeat the procedure of step β) until the structure of the nanoscale biological sample becomes distorted, thereby determining the electron flux threshold.

[0093] In the course of the method it is determined what electron flux a selected nanoscale biological sample can just tolerate at a selected electron dose in an electron beam device before the molecular structure becomes distorted (as visible for example as changes in the initial TEM image or in the diffraction pattern). It should be noted that several series of measurements according to steps α) to γ) can be carried out for a correspondingly several different electron doses. Studying the nanoscale biological sample just below the determined flux threshold will result in an optimal spatial resolution for the study of the sample in the electron beam device. The flux at which the structure becomes distorted can be identified by e.g. the disappearance of diffraction peaks at higher spatial frequencies, or by a becoming fainter TEM image, indicating a mass loss of the (diffracting) protein. When a specific spatial resolution e.g. Identification of structural damage when the diffraction signal disappears above a certain spatial frequency, e.g. The goal is In case the relative measure of quality decreases by, for example, 20% or more compared to the first image in the image series, a conclusion about structural damage can be drawn. The relative measure of quality is the integrated signal at the image pixel at the location of the protein minus the integrated signal at the image pixels surrounding the protein. A suitable electron flux to start with is 0.5 electrons per per second. For example, the throughput can be doubled with each step.

[0094] In a preferred variant of the above method for determining the flux threshold, the preselected electron dose is at least 50 electrons per Preferably at least 200 electrons per These electron doses can be adequately tolerated without significant radiation damage according to the present invention and generally result in good resolution of the nanoscale biological specimens being studied.

[0095] Particularly preferred is the method according to the invention for studying at least one nanoscale biological sample as described above, wherein in step d) the nanoscale biological sample is exposed to an electron dose and an electron flux, wherein the electron flux is lower than the flux threshold determined for the electron dose serving as a preselected electron dose in the method according to the invention for determining the flux threshold as described above. The study of the nanoscale biological sample then results in excellent spatial resolution, in particular TEM image resolution (typically 0.5 nm or better (lower)), and precise structural analysis can be obtained in a short time without significant radiation damage to the sample. A typical flux threshold for reduced radiation damage is 10 electrons per 10 nm. per second.

[0096] Also within the scope of the present invention is a sample for studying at least one nanoscale biological specimen in an electron beam device, wherein the sample comprises:

[0097] - a conductive substrate, in particular a graphene foil or a carbon foil, arranged on a TEM grid,

[0098] - a thin film of immobilized embedding liquid on the substrate in the application area,

[0099] The immobilized embedding liquid contains a plurality of nanoscale biological samples, wherein MD is the maximum diameter of the nanoscale biological sample and MD is ≤ 30 nm,

[0100] The film has an average thickness AT, where AT ≤ 30 nm,

[0101] wherein the substrate is wettable by the embedding liquid in the application area,

[0102] and wherein the sample is at the measurement temperature MT,

[0103] Among them, -100℃≤MT≤-1℃,

[0104] In particular, the sample is prepared according to steps a) to c) of the method of the present invention for studying at least one nanoscale biological sample as described above. The sample prepared in this way can withstand higher radiation doses / electron doses without being damaged. The measuring temperature is high enough to allow improved electron transfer within the nanoscale biological sample and through the film, and at the same time the measuring temperature is low enough to reduce the diffusion of chemical free radicals and free electrons through the film to the nanoscale biological sample. The embedding liquid of the film is immobilized by adjusting the film or its embedding liquid to the measuring temperature MT. Generally, the measuring temperature is below the solidification temperature of the embedding liquid bulk phase, but no ice crystals are formed in the film due to its low thickness. It should also be noted that generally AT≤MD.

[0105] The scope of the present invention also includes the use of a sample preparation device in the method according to the invention for investigating at least one biological sample at the nanoscale as described above, wherein the sample preparation device is suitable for automatically carrying out at least step b) and preferably also step c). With the sample preparation device used according to the invention, simple sample preparation as well as rapid and precise sample investigation can be achieved.

[0106] Further advantages can be understood from the description and the drawings. The features mentioned above and below can be used according to the invention individually or together in any combination. The embodiments mentioned should not be understood as an exhaustive list but rather as having exemplary character for the description of the invention.

[0107] DETAILED DESCRIPTION AND DRAWINGS

[0108] The invention is illustrated in the accompanying drawings.

[0109] Figure 1 A schematic overview showing the process of the method of the present invention;

[0110] Figure 2 Schematically shows a cross-sectional view of a portion of a film after step c) of the method according to the invention in an exemplary variant, wherein the film is placed on a substrate;

[0111] Figure 3 Schematically shows a cross-sectional view of a portion of a film after step c) of the method according to the invention in an exemplary variant, wherein the film has a covering substrate;

[0112] Figure 4 A diagram showing conductivity (upward axis) plotted against temperature (rightward axis) for water and various concentrations of brine;

[0113] Figure 5 TEM images showing several hemocyanin proteins as nanoscale biological specimens prepared in a film containing a negative stain, prepared and measured according to a variation of the method of the present invention, wherein the measurement temperature is -14°C;

[0114] Figure 6 In a schematic perspective view, in a variant of the method according to the invention, the thin film is produced in step b), a drop of an embedding liquid is placed on the sample using a tip with a microchannel (left-hand partial image (a)), and some of the embedding liquid is removed by suction, wherein the TEM grid is pressed onto a filter paper (right-hand partial image (b));

[0115] Figure 7 The following partial images (a) to (d) show diagrammatically the Figure 6 Preparation of

[0116] Figure 8The schematic cross-sectional view shows the production of the film in step b) in a variant of the method according to the invention, in which some of the embedding liquid is evaporated;

[0117] Fig. 9 The three subsequent partial images (a) to (c) show in schematic cross-section the production of the film in step b) in a variant of the method according to the invention, in which a small volume of embedding liquid is carefully placed in the application area defined by the TEM grid strips;

[0118] Fig.10 Schematically illustrates an exemplary measurement setup used in the method according to the invention, in particular in steps c) and d) of the method according to the invention;

[0119] Fig.11 Transmission electron microscopy images showing hemocyanin in films prepared according to steps a) and b) and conditioned and measured at different measuring temperatures, selected from up to 1000 electrons per The dosage series were carried out according to the invention at -14°C (above), at room temperature (middle), and at -180°C (lower).

[0120] Fig.12 shows a diagram of the normalized TEM image intensity (upward axis) of samples prepared according to steps a) and b) and conditioned and measured at three different measurement temperatures, plotted against the accumulated electron dose (rightward axis);

[0121] Fig.13 Schematically shows a cross-sectional view of a portion of a thin film after step c) of the method according to the invention in an exemplary variant, wherein a nanoscale biological sample, here a protein, is immobilized on a substrate via a linker molecule;

[0122] Fig.14 In the three subsequent partial images (a) to (c) it is shown in schematic cross-section that in the variant using a partial coating, a thin film is produced on a substrate in step b) of the method according to the invention;

[0123] Fig.15 The two subsequent partial images (a) and (b) show in schematic cross-section the production of a thin film on a substrate in step b) of the method according to the invention in the variant using an application tip such as an AFM tip and a supply reservoir for the embedding liquid.

[0124] As from Figure 1As can be seen from the schematic diagram of the present invention, the present invention proposes a method for studying nanoscale biological samples using a four-step process. In the first step a), see number 100, an embedding liquid containing a nanoscale biological sample is prepared. For this purpose, for example, the sample can be placed in a solvent, or the solvent can be placed on the biological sample. A typical solvent is saline. The biological sample to be studied has a maximum diameter MD of 30nm or less, usually 20nm or less. The solvent is generally based on water. In the second step b), a thin film of the embedding liquid is prepared on a conductive substrate, see number 200. The embedding liquid wets the substrate. The substrate can contain, for example, graphene. Typically, a large initial amount / volume (larger than required for the film) of the embedding liquid can first be deposited on the substrate, and then the excess liquid can be removed to obtain a thin film, or a small initial amount / volume of the embedding liquid can be deposited to directly obtain a thin film. At least at the end of step b), the thin film of the embedding liquid has an average thickness AT of 30nm or less and usually 20nm or less. In a subsequent step c), see numeral 300, the film is adjusted to a measuring temperature MT, wherein -100°C ≤ MT ≤ -1°C and preferably -50°C ≤ MT ≤ -1°C. Typically, the temperature is set directly by cooling to MT; alternatively, the temperature can be set by first cooling the sample below MT and warming to MT again. Finally, in step d), see numeral 400, the film is studied at temperature MT in an electron beam device such as a TEM.

[0125] In one aspect of the invention it is proposed that nanoscale organic objects such as proteins or viral particles are loaded onto a sample carrier in such a way that electron transfer between the object and the sample carrier is possible, wherein it is proposed to first apply the object in a liquid (also called embedding liquid), then reduce the thickness of the liquid to a very thin liquid layer (also called film) at the sample carrier, and then freeze (cool) the sample to a temperature below the freezing point of the embedding liquid bulk phase, whereby the thickness of the liquid layer is below the minimum thickness required for ice crystal formation, and the temperature of the layer is high enough so that electron transfer still occurs. The sample is then examined in the vacuum environment of an electron beam device at an electron flux below the ionizing radiation damage threshold, whereby the total dose can be increased to above

[0126] The sample carrier is loaded into a sample holder with a device for cooling and measuring the temperature, which is commercially available as a so-called TEM sample cryo-holder. The sample is prepared from an ultra-thin layer of liquid and the guest is immobilized by embedding on the surface of a film (e.g. graphene or carbon). The guest is, for example, a protein.

[0127] Figure 2A cross-sectional view of a film 2 after step c) of the method according to the invention is shown in an exemplary variant. A nanoscale biological sample 1 (here a protein 1a) is embedded in a thin film 2 (also referred to as a thin layer) of an immobilized embedding liquid 3. The immobilized embedding liquid 3 is here cooled water and a negative stain 24 of a low atomic number mixed therewith; the film 2 is, for example, at a temperature of -14°C (measuring temperature). The film 2 is loaded on a substrate 4 belonging to a sample carrier, wherein the substrate 4 is here selected as a graphene film (also referred to as graphene foil) 4a. The immobilized embedding liquid 3 covers the protein 1a, see area 8a, and covers the substrate 4 in the vicinity of the protein 1a, see area 8b. In the example shown, the layer thickness 5 of the substrate 4 in area 8b is less than 5 nm in order to prevent the formation of ice crystals. The protein 1a is here located at a close distance 6 of 1-2 nm from the substrate 4 so that the electron transfer between the protein 1a and the substrate 4 occurs at a sufficient rate to allow the sample to be measured by a low electron flux (<10 electrons per per second) electron beam (the electron beam is not shown, but the Fig.10 ) Charge compensation during irradiation. It should also be noted that the maximum diameter MD of the nanoscale biological sample 1 and the average thickness AT of the thin film 2 are both 30 nm or less, where AT≤MD, and in the example shown, approximately MD=10 nm and AT=5 nm. It should be noted that the thin film 2 on the substrate 4 can be used as a sample 9 for measuring at least one nanoscale biological sample 1 in an electron beam device.

[0128] The preparation protocol, wetting behavior of the film, and chemical composition and thickness of the liquid layer are selected so that 1) the liquid forms a thin layer on the guest to protect the structure of the guest after exposing the sample to a vacuum, 2) the liquid provides electrical conductivity between the guest and the sample carrier, and 3) no ice crystallization occurs upon freezing despite temperatures above the glass transition temperature.

[0129] In one possible embodiment of the method, the sample is prepared as follows: a droplet of 0.2 μl volume of saline containing dissolved proteins, such as hemocyanin, is placed on a support membrane consisting of a graphene monolayer on a holey carbon film mounted in a standard 3 mm TEM gold grid. After a waiting time of several minutes, the main fraction of the excess liquid is removed by wicking with filter paper until the surface appears dry but with a shiny (reflective) appearance, indicating the presence of a thin liquid film. The sample is then covered with a thin film consisting of a graphene monolayer.

[0130] Figure 3A cross section of a film 2 is shown after step c) of the method according to the invention in a corresponding exemplary variant. A nanoscale biological sample 1 (here a protein 1a) is embedded in a film (or thin layer) 2 of an immobilized embedding liquid 3, which is here cooled water mixed with NaCl; the film 2 is, for example, at a temperature of -14°C (measuring temperature). The film 2 is loaded on a substrate 4 belonging to a sample carrier, wherein the substrate 4 is selected here as a graphene film (graphene foil) 4a. The sample 9 is covered by a covering substrate 7 (here a second layer of graphene (or graphene foil) 7a). The layer thickness 5 of the cooling water layer (or immobilized embedding liquid 3) in the region 8b adjacent to the protein 1a is about 10 nm here. The protein 1a is located here at a close distance 6 of 1-2 nm from the substrate 4 so that the electron transfer between the protein 1a and the substrate 4 occurs at a sufficient rate to allow the sample to be measured by a low electron flux (<10 electrons per per second) electron beam (the electron beam is not shown, but the Fig.10 ) Charge compensation during irradiation. The maximum diameter MD of the nanoscale biological sample 1 and the average thickness AT of the thin film 2 are both 30 nm or less, where AT≤MD, and in the example shown are approximately MD=10 nm and AT=10 nm.

[0131] After having been covered with the second graphene layer, the sample is inserted into the vacuum chamber of the electron beam equipment via the TEM sample holder. On exposure to the vacuum, some of the excess liquid evaporates but the liquid layer remains closed between the two graphene films. It should be noted that after the final thickness of the film has been reached, the sample is cooled to the measurement temperature so that the embedding liquid becomes immobilized.

[0132] In particular, the absorption and / or evaporation is done under calibration conditions, preferably such that the (average) liquid layer thickness becomes thinner than the protein size and a surface layer is formed at the carrier film, whereby the layer becomes so thin that no ice crystals are formed upon freezing. The calibration conditions involve the ambient air humidity, the absorption time, the absorption pressure, the type of liquid absorption medium. The calibration is done as follows: the absorption is performed for a set of conditions and the samples are examined via electron microscopy to measure the sample thickness and to check for the presence of ice crystals. Considering pure water, the nucleation rate of ice nucleation is 10 14 m -3 s -1 A nanodroplet with a radius of 2.4 nm will show 10 6 m -3 s -1The ice nucleation rate is reduced (T. Li et al, Nat. Commun. 4 (2013) 1887-1-6). For nanodroplets placed on a flat surface, ice nucleation is completely avoided (Xiang-Xiong Zhang et al., J. Chem. Phys. 141 (2014) 124709). As a result, ice crystals will not form in the water layer with a thickness of about 3 nm on the surface of the graphene film as a sample carrier. In practice, if the embedding liquid is not pure water but salt water (e.g. water with Na + Cl - Mixing), which reduces nucleation crystallization compared to ice, the liquid layer can be thicker (e.g. 5nm), so that thicker layers still do not crystallize. In addition, since the liquid is enclosed between two sheets of graphene, providing a mechanical barrier against ice crystal formation, the ice nucleation rate is reduced.

[0133] The sample is then slowly brought to a temperature just below the freezing point of the sample (the measurement temperature), i.e. below the freezing point of the bulk phase of the embedding liquid. It should be noted that this state is sometimes referred to as "frozen", although no ice crystals are formed due to the low thickness of the film formed by the embedding liquid on the substrate.

[0134] The temperature of the conditioned / cooled sample (measurement temperature) needs to be chosen based on three criteria. 1) The liquid surrounding the guest must be immobilized, typically by choosing a measurement temperature below the freezing point of the bulk phase of the embedding liquid. 2) The temperature needs to be high enough to allow electron transfer to occur in the guest of interest, such as a protein. 3) The temperature needs to be high enough to allow electron transfer between the guest and the sample carrier.

[0135] To achieve criterion 1, a temperature below 273°K (=0°C) would be required to ensure freezing of a thin aqueous liquid layer at ambient pressure. The temperature depends on the presence and concentration of saline. For a liquid containing a physiological concentration of 100 mol / l (6% by weight) of NaCl, the freezing temperature would be about 269°K (Wikipedia saline page, https: / / en.wikipedia.org / wiki / Saline_water, downloaded on 22.09.2023). In order to allow some tolerance for the exact freezing point, a temperature of 260°K can be chosen (measurement temperature MT). For a typical value of the driving force ΔG 0 = -1.5 eV and λ = 0.75 eV, the latter temperature would still satisfy criterion 2 since it would allow electron transfer in the protein, the electron transfer rate at this temperature being reduced by a factor of only 2 compared to room temperature.

[0136] For charge transfer between a nanoscale biological sample and a sample carrier, one approach is to load the sample as close to the sample carrier as possible and select a temperature such that electron transfer occurs between the guest and the sample carrier. For example, one can consider a diameter of The globular protein receives electron beam The electron flux and thus the current are:

[0137] Corresponding to about 3×10 -15 A.

[0138] For example, we can assume an ideal situation: the distance between the free electrons generated on the protein surface and the carrier layer providing electroneutrality is The rate of electron transfer between a donor and an acceptor separated by this distance in a protein is of the order of 10 9 s -1 (CC Moser et al., Nature 355 (1992) 796), sufficient for charge compensation of 5 orders of magnitude, thus achieving criterion 3.

[0139] To further enhance the conductivity of the sample, a second method for charge transfer between the guest and the sample carrier is to include ions in the liquid surrounding the guest. The measurement temperature is then chosen so that the film with ions is still conductive, achieving criterion 3. Compared to pure ice, doping ice with 7 μM NaCl increases the conductivity to σ = 2 × 10 -5 Ω -1 m -1 (GW Gross et al., J. Glaciol. 21 (1978) 143, or see also Stillman and Grimm, Lunar and Planetary Science XXXIX (2008) 2277).

[0140] Cited from GW Gross et al., J. Glaciol. 21 (1978) 143 Figure 4 Illustration of the variation of static conductivity of ice grown from different NaCl solutions with temperature, compared with pure ice. At the freezing point (0°C, see the scale in the upper part of the figure) pure ice (cross x) exhibits a conductivity σ = 4×10 -9 Ω -1 cm -1 Ice grown from a 0.1 mM NaCl aqueous solution obtained a NaCl concentration of 7 μM in the ice, showing an increased conductivity of σ = 2 × 10 -7 Ω -1 cm -1 .

[0141] The effect of doping on charge transfer can be estimated as follows. Assuming that the protein is covered with a thickness of The mixed ice layer is surrounded by a neutral conductive carrier. It is assumed that the current from the protein to the carrier will take the shortest path and pass through the area Thus, a resistance path of resistance R = t / (Aσ) is obtained. For ice mixed with NaCl, R = 5×10 12 Ω. The secondary electrons emitted from the sample during electron beam irradiation can be considered as a power source, which is connected via the resistor presented by the mixed ice layer between the protein and the carrier connected to the electron neutrality. The electron flux accepted by the protein Lower I=3×10 -15 A, the voltage drop on the mixed ice layer reaches U = IR = 1.6 × 10 -2 V. The corresponding electric field strength will then reach F = 8 × 10 6 Vm -1 , whose dielectric strength in ice is 8×10 7 Vm -1 The following (T. Kohno et al., IEEE Transactions on Electrical Insulation, EI-15 (1980) 27) will allow the circuit to function. On the other hand, if no doping is used, the system will achieve F = 10 9 Vm -1 , which will be above the dielectric strength, and sample damage (generally the generation of hydrogen bubbles) will occur.

[0142] The sample conditions thus achieved provide the guest embedded in a thin cryoimmobilized layer while being electrically conductive to optimize radiation damage mitigation.

[0143] For obtaining structural information of the sample, electron beam exposure in transmission mode is preferred, enabling the application of electron dose-efficient contrast mechanisms such as phase contrast, diffraction, holography or lithography.

[0144] The method of the present invention reduces the electron beam damage problem to a large extent for a wide class of radiation sensitive samples such as proteins, viral particles, (bio)minerals, polymers, soft matter nanoparticles, electrolytes, etc. Due to the increased tolerance to radiation damage, better spatial resolution may be achieved for these samples compared to the current state of the art.

[0145] Various other embodiments of the present method are possible, examples of which are described below.

[0146] The desorbed sample can be slowly frozen (cooled) to a temperature of 260° K in a low water vapor content atmosphere provided by anhydrous nitrogen, so that freezing does not result in condensation of ice on the sample. The sample is then inserted into a vacuum.

[0147] Alternatively, the aspirated sample can be frozen (cooled) quickly enough to a sufficiently low temperature (also called low temperature LT, generally where LT ≤ -196°C) to obtain amorphous ice, and then the sample is inserted into a vacuum and carefully heated to a temperature of, for example, 260° K (measurement temperature MT). The aspiration step prior to freezing should be performed in a manner as described above, so as to maintain a liquid layer only a few nanometers thick and thus prevent ice crystals from forming once the sample is heated above the glass transition temperature.

[0148] The sample carrier may include carbon, graphene oxide, silicon nitride or any other material that provides mechanically firm support and provides charge transfer to the conductive substrate. In the case of electron beam transmission, the substrate should be thin enough to allow electron transmission. Sufficiently thin means that the thickness of the substrate material is at least less than one-tenth of the mean free path length of elastic electron scattering at the set electron energy, and its amount is about 0.12 μm of carbon at 200 keV beam energy (K. Iakoubovskii and K. Mitsuishi, J. Phys. Condens. Matter 21 (2009) 155402). The substrate does not necessarily need to be a planar sheet, but can also be a curved, such as a carbon nanotube, or a tip shape.

[0149] Accordingly, the method can also be used for thick substrates, in which case the backscattered signal or the amplified secondary emission signal is used for detection, for example in scanning electron microscopy.

[0150] In yet another embodiment, the substrate is first placed in a controlled environment, such as a pure argon chamber or a vacuum chamber, and the sample is placed on the substrate in the chamber to minimize contamination.

[0151] In yet another embodiment, instead of saline, a chemical stain is surrounded by the object to preserve the structure of a biological sample such as a protein or a virus particle, and to provide conductivity via the presence of ions. An example is a stain of a low atomic number substance, such as provided by applying water containing dissolved potassium salt of glucose-1-phosphate. In addition, the sample is not covered by a graphene sheet. Upon exposure to vacuum, some of the excess liquid evaporates but the liquid layer transforms into a gel that protects the structure of the object from damage in the vacuum, so that the layer of water molecules remains stable at the protein (see above). Figure 2 ). It was experimentally demonstrated that this embodiment allows obtaining excellent TEM images (see below Figure 5 ), where ice crystals are absent from the frozen sample and the sample tolerates high electron doses (for up to No sample deformation was observed within the measurement range of electron dose).

[0152] Figure 5Transmission electron microscopy (TEM) image showing several hemocyanin proteins in tubular form in a thin layer of low atomic number negative stain placed on a graphene layer supported on a holey carbon film. The proteins can be seen as circles or rectangles, depending on their orientation on the support graphene. The edges of the holes in the carbon film are visible at the bottom right. The electron flux is 3.5 electrons per per second, the exposure time is 4 seconds, providing 14 electrons per The sample prepared and measured according to the invention was kept at a measurement temperature of -14° C. during imaging.

[0153] The embedding liquid used may contain conductive polymers or conductive proteins to provide a transmission pathway for electrons.

[0154] The embedding liquid used may contain heavy water (for additional radiation protection) or an ionic liquid with a very low vapor pressure (to protect the sample from evaporation). Another type of liquid other than water or saline can be used, such as oils or electrolytes.

[0155] The sample preparation step is preferably performed in an automated manner by a sample preparation device 25, such as Figure 6 As shown. Figure 6 The left picture (a) illustrates that a small liquid droplet 10 containing protein and saline is precisely pipetted at a defined position on a standard 3 mm grid for transmission electron microscopy (TEM grid) 11 by a micropipette device, the micropipette tip (or application tip with microchannel) 12 of the device being positioned above the 3 mm grid 11. It should be noted that the liquid droplet 10 here represents an initial large amount 15 of embedding liquid, which is larger than required to prepare the desired film on the TEM grid 11. The 3 mm grid 11 contains a copper frame supporting a porous carbon film, and the graphene film covers the central surface of the carbon film (see also below). The grid 11 is kept in place by a micro clamp 13. In addition, as shown in FIG. Figure 6 The right-hand image (b) illustrates that in order to remove excess liquid from a liquid droplet 10 located on a TEM grid 11, the 3 mm grid 11 is carefully pressed against a piece of liquid absorbing medium 14 (here a piece of filter paper 14a) by a micro-gripper 13. The micropipette tip 12 and micro-gripper 13 are automatically operated in a sample preparation device 25 by one or more robotic arms and electronic controllers (not shown in further detail).

[0156] Figure 7The aspiration process is illustrated in somewhat more detail in schematic cross-sectional views in four subsequent partial images (a) to (d). As shown in partial image (a), a substrate 4 (here a porous carbon foil 17 containing a graphene foil 4a) is loaded onto a mesh strip 16 of a 3 mm TEM grid 11. A micropipette tip 12 applies a liquid droplet 10 of an embedding liquid 3 (here containing protein and saline), see partial image (b). The micropipette tip 12 is lowered onto the carbon foil 17 so that the liquid droplet 10 contacts the carbon foil 17. The tip 12 is then withdrawn, leaving some of the embedding liquid 3 at the carbon foil 17 containing the graphene foil 4a, wherein the substrate surface is wettable by the embedding liquid 3, see partial image (c). The 3 mm TEM grid 11 is then placed on a piece of filter paper 14a. Some of the embedding liquid 3 passes through the small holes in the carbon foil 17 and the graphene foil 4a (in Figure 7 Not shown, but see below Figure 8 ) flows to the bottom of the 3 mm grid 11, and from there flows to the mesh strips 16, whereby it is absorbed in the filter paper 14a. The filter paper 14a is then removed, see partial image (d), thereby removing the liquid 3a absorbed in the filter paper 14a, so that a thinner liquid layer than that initially applied, i.e., a film 2 embedding the liquid 3, is retained on the porous carbon foil 17 containing the graphene foil 4a.

[0157] A subset of automated sample preparation for use in the present invention can be achieved with commercially available equipment, for example using the equipment described in US 2004 / 157284 (= US 7,413,872).

[0158] Figure 8 The typical structure of the substrate 4 used for the present invention, as well as a variant of the thin film preparation based on evaporation, is illustrated in a little more detail. A carbon foil 17 with holes 18 carries a graphene film (or graphene foil) 4a and is loaded on a mesh strip 16 of a 3 mm TEM grid 11. Here, an area of ​​the carbon foil 17 with a diameter 19 is covered with an initial large amount 15 of embedding liquid 3, forming a contact angle α with the carbon foil 17 carrying the graphene foil 4a. Since the substrate 4 (or carbon foil 17 and graphene foil 4a) is wettable by the embedding liquid 3, the wetting angle (or contact angle) α is less than 90° and generally also less than 45°. In the illustrated variant, the (maximum) thickness 21 of the embedding liquid 3 decreases over time due to evaporation. The embedding liquid 3 here contains proteins 1a and saline. The proteins 1a deposited from the embedding liquid accumulate at the carbon foil 17, where they are in the final film (the final film is not shown here, but for example, the comparison Figure 2 ) becomes fixed.

[0159] Fig. 9A further variant of the film preparation according to the invention is illustrated in the subsequent partial images (a) to (c). As can be seen in partial image (a), a porous carbon foil 17 containing graphene foil 4a is loaded on the strips 16 of a 3 mm TEM grid 11. A micropipette tip 12 applies a liquid droplet 10 of an embedding liquid 3 (here containing protein and saline). The tip 12 consists of a stretched glass capillary, wherein the tip diameter is smaller than the distance between the strips 16 of the 3 mm TEM grid 11 (typically 60 μm), i.e., smaller than the grid window 16a to be carefully filled with the embedding liquid 3. The tip 12 is lowered to the carbon foil 17 so that the liquid droplet 10 contacts the foil 17, see partial image (b). The tip 12 is then withdrawn, leaving some embedding liquid 3 at the graphene-containing carbon foil 17, which is wetted by the embedding liquid 3 (e.g. due to plasma treatment). The embedding liquid 3 spreads in the application zone 22 defined by the mesh strips 16 limiting the mesh windows 16a, leaving a liquid film 2 of uniform thickness 23, see partial image (c). In the variant shown, the droplet 10 of the embedding liquid 3 has an initial small volume SV, which is selected so that for the application zone area AZA of the application zone 22 on which the embedding liquid 3 of the small volume SV spreads, the uniform layer thickness 23 (which is here at the same time the average thickness AT of the film 2) is 30nm or less, preferably 20nm or less. In this case, the film 2 is completed and it is not necessary to further reduce the embedding liquid 3 before carrying out the conditioning step. For example, for a typical 3mm TEM grid 11, the SV can be selected to be 36fL to achieve an AT of 10nm.

[0160] Other automation that can be performed includes automated loading of the 3 mm grid into the sample holder tip for electron microscopy. The sample holder is first loaded into an adapter in the sample preparation device. The 3 mm grid is positioned in the tip with a mechanical manipulator.

[0161] The sample preparation device may also contain one or more additional micropipette or cantilever with microchannels for adding fluids, such as chemical stains or liquid droplets containing conductive polymers.

[0162] A further addition could be to enclose the sample in a controlled environment such as pure argon, or also to load the device into a vacuum chamber.

[0163] In yet another embodiment, the device contains freezing means (cooling means), which can be of the rapid cooling type.

[0164] The sample is placed in an electron beam device for studying the nanoscale structure of the sample. Various types of equipment that can be used according to the present invention are commercially available, such as a transmission electron microscope, a scanning transmission electron microscope, a scanning electron microscope, or an electron diffraction device. The substrate containing the sample loaded on, for example, a 3mm grid is placed in a vacuum chamber of an electron beam device, for example, by cooling the sample holder, such as the cryotransfer holder described in US2019 / 131106 or US2012 / 024086. The temperature can also be controlled with a modified electron beam device, such as described in US 2007 / 252090. In the case of using a scanning electron microscope, the sample can be loaded on a cooling Peltier stage.

[0165] Fig.10 An exemplary measurement setup is described, which is used according to the present invention for measuring nanoscale biological samples in thin films in an electron beam device 32. A 3 mm grid 11 containing a porous carbon foil loaded with a graphene film (in which proteins are embedded in a cooled liquid) is loaded in the tip 30 of the TEM sample holder, wherein the rod body 31 is placed in the vacuum chamber of the electron beam device 32. The rod body 31 extends to the outside of the vacuum 33, where it is connected to the control function part. A reservoir 34 containing liquid nitrogen at a temperature of -196°C is connected via a copper wire 35, which transfers heat away from the tip 30 through the rod body 31 for the purpose of cooling the tip 30. A heating coil 36 is loaded in the tip 30 for the purpose of heating the tip 30 against cooling, so that the desired temperature (i.e., the measurement temperature MT for preparing and performing the measurement) can be set. The heating coil 36 is connected to an electric wire 36a which is in turn connected to a DC regulating device 37 outside the electron beam device 32. For temperature regulation, the temperature is measured with a thermocouple 38 in the tip 30, which is connected via a further electrical wire 38a to a measuring device 39 outside the electron beam apparatus 32. An electron source 40 (at a potential of e.g. 200 kV) generates an electron beam 41, which is shaped by e.g. two condenser mirrors 42, causing a collision zone of the electron beam at a sample (or specimen) 43. The transmitted electron beam 44, which contains information about the proteins in the sample 43, is shaped by e.g. two projection mirrors 45 and projected onto a camera 46. The camera 46 is controlled and read by a computer 47 for data acquisition, storage and initial data processing.

[0166] Fig.11 and 12 Experimental results are presented, pointing out the advantages of the invention, especially the tolerance of the sample to an increased electron dose during the measurement. Fig.11 Transmission electron microscopy images depicting hemocyanin, selected from up to 1000 electrons per Dose series were performed according to the invention at -14°C (upper), and for comparison at room temperature (middle), and at -180°C (lower). A high-throughput filter was applied to filter out features larger than the protein size and a Gaussian filter (σ=2 pixels) was applied to reduce pixel noise in each image. (first image) to Only small changes in protein structure were observed between room temperature RT and -180C. The protein structure has changed dramatically compared to the first image.

[0167] Fig.12 Measurements of radiation tolerance are shown to illustrate the benefits of the method according to the invention for studying biological samples at nanoscale. A series of TEM images were recorded at different measurement temperatures, i.e. at room temperature (RT), -14°C and -180°C, and subsequently analyzed. TEM images similar to Figure 5 For analysis, select the image area showing circular proteins (compare Fig.11 ), and the regional image intensity is repeatedly measured after a specific electron dose has been accumulated in each case. To obtain the regional image intensity, the radially integrated pixel intensity is calculated from the center of the protein, where the maximum pixel intensity corresponds to the densest area of ​​the protein. Normalization is applied as follows: the pixel intensity in the protein area is divided by the pixel intensity in the stained area adjacent to the protein. The image intensity of each selected area in the image series is divided by the intensity in this selected area of ​​the first image in the image series, thereby obtaining a standardized intensity, which can be used as an indication of the "remaining integrity" of the protein structure. The standardized intensity is plotted against the cumulative electron dose received by the sample. For analyzing radiation tolerance, a trend line (polynomial fit curve, see dotted line) is fitted to the experimental curve and the intercept is determined with a relative intensity of 0.8. The maximum cumulative electron dose for which the relative intensity remains above 0.8 is used as the criterion for the maximum electron dose that the sample can tolerate. A maximum of 300 electrons per gram was determined for samples at -14°C. The samples measured at the two other temperatures (RT and -180 °C) only tolerated a maximum dose of 60 electrons per According to the present invention, the sample measurement temperature is set to -14°C in order to increase the electron dose tolerance by a factor of 5 compared to the two other temperatures.

[0168] Similar to the dose series of TEM images described above, a flux series of TEM images can be recorded according to the present invention for a specific pre-selected electron dose. In each TEM image, a different electron flux is applied, wherein the pre-selected electron dose is distributed with different irradiation / measurement times. For each flux, a different location on the sample (and thus a different nanoscale biological sample / protein) is selected. A typical flux series starts with a low electron flux such as and multiply the flux from image to image in the series, e.g. to And so on. The sample image that has maintained its protein structure (ie, the structure is still not distorted) measured with the highest throughput is determined. It should be noted that the series can be ended as soon as the first structural distortion is identified.

[0169] Structural distortion can be determined, for example, using the above-mentioned normalized strength criterion of 0.8 (see Fig.12 ) is determined. Structural distortions are directly visible in TEM images; for example, in the above dose series (see Fig.11 ), in the -180℃ series Images and The structural distortion can be clearly observed in the image (compared to the image comparison); structural distortions in one flux series can be visualized in the same way. It should be noted that various ways of identifying structural distortions can be applied, such as analyzing structural contrast, structural diameter, and many other ways.

[0170] For future measurements of this type of sample and a pre-selected electron dose, the highest flux that has preserved the protein structure can be chosen to efficiently obtain good quality TEM images.

[0171] Fig.13 Shown in an exemplary variant is a portion of a film 2 after step c) of the method according to the invention, in which a linker molecule 55 is applied. Fig.13 Film 2 is similar to that shown in Figure 2 The film is shown in FIG. Fig.13In a variation of , the protein 1a attached to the graphene 4a carrier film is covered by an ultrathin cooling layer of the embedding liquid 3. A conductive polymer 50 having unsaturated carbon bonds 51a is connected to the carrier graphene foil 4a via a non-polar group (here an aromatic group) 52, and has a polar group (here a carboxyl group) 53 for attaching to the protein 1a by forming an H-bond 54 with the protein 1a side chain. The conductive polymer 50 acts as a linker molecule 55 here, wherein the linker molecule 55 improves the attachment of the protein 1a to the substrate 4 by mediating between the non-polar substrate 4 and the polar protein 1a (and the polar aqueous embedding liquid 3). The unsaturated carbon bonds 51a form conductive chains 51 that provide some conductivity, so that the electron transfer between the substrate 4 and the protein 1a is enhanced, which helps to reduce radiation damage.

[0172] It should be noted that according to the present invention, a local coating containing linker molecules can be used to define the application area 22 of the film 2 to be prepared, such as Fig.14 As shown. In partial picture (a), a substrate 4 is shown, where it carries two partial coatings 60 consisting of linker molecules. The substrate 4 is non-polar, for example, having a graphene upper surface. In each coating 60, the lower end of the linker molecule is attached to the non-polar surface of the substrate 4 containing non-polar groups, and the polar group is exposed at its upper end. An initial large amount 15 of an aqueous embedding liquid 3 containing a nanoscale biological sample (not shown) is now deposited on the substrate 4, see partial picture (b). Subsequently, excess embedding liquid 3 is removed, for example, by evaporation or absorption (not shown in detail). The remaining embedding liquid 3 then forms a thin film 2 on the partial coating 60 on the substrate 4, while it defines the application area 22 of the thin film 2, see partial picture (c). The partial coating 60 is wettable by the aqueous (and therefore polar) embedding liquid 3, while the remaining surface of the substrate 4 is not wettable, so that during the reduction of the embedding liquid 3, a thin film 2 is formed by a self-organizing process due to the wetting behavior.

[0173] Fig.15 The application of a thin film of an embedding liquid 3 on a substrate 4 is illustrated schematically, wherein the embedding liquid 3 containing a nanoscale biological sample is taken from a supply reservoir 71 by dipping an application tip 70 into the embedding liquid 3 contained in the supply reservoir 71, see partial picture (a). The application tip 70 is then moved to the substrate 4, wherein a droplet 10 of the embedding liquid 3 adheres to the tip 70. The tip 70 is then lowered toward the substrate 4, see partial picture (b). When the droplet 10 contacts the surface of the substrate 4, the embedding liquid 3 flows down from the tip 70 and wets the substrate 4, forming a thin film on the substrate (not shown in detail); the film thickness can be adjusted, for example, by suction or evaporation, if necessary. It should be noted that the application tip 70 can be selected as an atomic force microscope (=AFM) tip.

[0174] 1. A method for studying at least one nanoscale biological sample (1), the method comprising:

[0175] Step a) (100) prepares an embedding liquid (3) containing a plurality of nanoscale biological samples (1), wherein the nanoscale biological samples (1) have a maximum diameter MD, wherein MD is ≤ 30 nm,

[0176] Step b) (200) prepares a thin film (2) containing a liquid (3) on a conductive substrate (4) in the application area (22), thereby placing a nanoscale biological sample (1) on the substrate (4),

[0177] wherein the substrate (4) is wettable by the embedding liquid (3) in the application area (22),

[0178] and the film (2) in which the liquid (3) is embedded has an average thickness AT, wherein AT ≤ 30 nm;

[0179] Step c) (300) adjusting the film (2) on the substrate (4) to a measurement temperature MT, wherein -100°C ≤ MT ≤ -1°C;

[0180] Step d) (400) measures at least one of the nanoscale biological samples (1) in the thin film (2) on the substrate (4) in the electron beam device (32) at the measurement temperature MT,

[0181] At least one nanoscale biological sample (1) is exposed to an electron beam (41, 44).

[0182] 2. The method according to embodiment 1, characterized in that the measuring temperature MT is lower than the solidification temperature of the bulk phase of the embedding liquid (3).

[0183] 3. The method according to any of the preceding embodiments, characterized in that AT≤MD.

[0184] 4. The method according to embodiment 4, characterized in that

[0185] In step d) (400), the electron beam energy of the electron beam (41, 44) is 50 eV to 300 keV,

[0186] Preferably 30keV to 300keV,

[0187] and the electron flux of the electron beam (41, 44) is 0.1 electron per Up to 50 electrons per second Every second,

[0188] Preferably 1 electron per Up to 10 electrons per second per second.

[0189] 5. The method according to one of the preceding embodiments, characterized in that step a) (100) and step b) (200) are performed at a preparation temperature PT, wherein PT>0°C.

[0190] 6. A method according to one of the preceding embodiments, characterized in that during step b) (200), an initial large amount (15) of embedding liquid (3) is placed on the substrate (4), wherein the large amount (15) is greater than required for the film (2), and then the amount of embedding liquid (3) on the substrate (4) is reduced until only the film (2) remains.

[0191] 7. The method according to embodiment 6, characterized in that reducing the amount of embedding liquid (3) comprises placing a liquid absorption medium (14) in contact with the embedding liquid (3),

[0192] In particular, wherein the liquid absorbing medium (14) comprises filter paper (14a) or brushed metal wire,

[0193] And / or characterized in that reducing the amount of embedding liquid (3) comprises evaporating some of the embedding liquid (3).

[0194] 8. A method according to one of the preceding embodiments, characterized in that during step b) (200), in order to prepare a film (2) of embedded liquid (3) in an application area (22) having an application area area AZA, an initial small volume SV of embedded liquid (3) is placed on a substrate (4) in the application area (22), wherein SV ≤ AT*AZA.

[0195] 9. A method according to one of the preceding embodiments, characterized in that, in step b) (200), an initial amount of embedding liquid (3) is placed on the substrate (4) in the application area (22) as follows: first, an application tip (70), in particular an atomic force microscopy (=AFM) tip, is immersed in a supply tank (71) of embedding liquid (3) containing a nanoscale biological sample (1), and then the application area (22) is contacted with the application tip (70).

[0196] 10. A method according to one of the preceding embodiments, characterized in that in step b) (200), an initial amount of embedding liquid (3) is placed on the substrate (4) in the application area (22) using an application tip (12), wherein the application tip has a microchannel through which the initial amount of embedding liquid (3) is discharged.

[0197] 11. Method according to one of the preceding embodiments, characterized in that during step b) (200), the initial volume of the embedding liquid (3) placed on the substrate is 1 fL to 2 μL.

[0198] 12. A method according to the preceding embodiment, characterized in that the experimental parameters applied during step b) (200) for producing a film (2) having an average thickness ΔT have been predetermined in a calibration experiment,

[0199] In particular, the experimental parameters include at least one of the following: the temperature of the atmosphere surrounding the substrate (4), the pressure of the atmosphere surrounding the substrate (4), the humidity of the atmosphere surrounding the substrate (4), the composition of the atmosphere surrounding the substrate (4), the temperature of the substrate (4), the type of liquid absorption medium (14), the contact time with the liquid absorption medium (14), the contact pressure with the liquid absorption medium (14), the evaporation time, the initial large volume (15) of the embedding liquid (3) placed on the substrate (4), the initial small volume SV of the embedding liquid (3) placed on the substrate (4) in the application area (22), the initial amount of the embedding liquid (3) placed on the substrate (4), the initial volume of the embedding liquid (3) placed on the substrate (4), the downward pressure of the application tip (70), and the discharge flow rate of the embedding liquid (3) through the microchannel.

[0200] 13. The method according to one of the preceding embodiments, characterized in that the embedding liquid (3) is selected as water with added salt,

[0201] Preferably, the added salt is NaCl,

[0202] It is particularly preferred that the added salt is NaCl at a concentration of 10 mM.

[0203] 14. The method according to one of the preceding embodiments, characterized in that the substrate (4) is a graphene foil (4a) or a carbon foil (17).

[0204] 15. A method according to one of the preceding embodiments, characterized in that, after step b) (200) and before step c) (300), the film (2) encapsulating the liquid (3) is covered with a covering substrate (7), in particular wherein the covering substrate (7) is a graphene foil (7a) or a carbon foil.

[0205] 16. The method according to one of the preceding embodiments, characterized in that the embedding liquid (3) contains an electrically conductive polymer (50) and / or an electrically conductive protein.

[0206] 17. The method according to one of the preceding embodiments, characterized in that the embedding liquid (3) contains a negative stain (24),

[0207] In particular, the negative stain (24) is selected as α-D-glucose 1-phosphate dipotassium salt hydrate, preferably applied at a concentration of 2 mM.

[0208] 18. The method according to one of the preceding embodiments, characterized in that in step c) (300) the film (2) on the substrate (4) is first cooled to a low temperature LT, wherein LT≤-196°C, and then warmed again to a measuring temperature MT.

[0209] 19. A method according to any of the preceding embodiments, characterized in that the substrate (4) is placed on a TEM grid (11), wherein the TEM grid (11) has a plurality of intersecting strips (16) defining windows (16a) between the intersecting strips (16), and characterized in that during step b), embedding liquid (3) is applied between the strips (16) in at least one of the windows (16a),

[0210] In particular, the embedding liquid (3) is carefully applied between the mesh strips (16) into at least one mesh window (16a).

[0211] 20. A method according to one of the preceding embodiments, characterized in that the substrate (4) comprises at least one local coating (60) defining at least one application area (22), wherein the local coating (22) makes the substrate (4) wettable by the embedding liquid (3) in the application area (22).

[0212] 21. The method according to one of the preceding embodiments, characterized in that

[0213] The substrate (4) is at least partially coated with a linker molecule (55) for connecting the nanoscale biological sample (1) to the substrate (4),

[0214] In particular, wherein the nanoscale biological sample (1) is a protein (1a) and the substrate (4) comprises a graphene foil (4a),

[0215] Characterized in that the linker molecule (55) is attached to the substrate (4) on one side via one or more non-polar chemical groups (52), in particular wherein the one or more non-polar chemical groups (52) are based on pyrene, pyridine, porphyrin or amine,

[0216] And it is characterized in that the linker molecule (55) has one or more polar chemical groups (53) on its other side, especially wherein the one or more polar chemical groups (53) contain butyric acid-succinimidyl ester, pyrenebutyric acid (PBA) or carboxyl group.

[0217] 22. The method according to embodiment 21, characterized in that the linker molecule (55) contains a conductive chain (51), in particular wherein the conductive chain (51) is based on a carbon atom (51a) with an unsaturated carbon bond or a carotenoid or a chain of carbon atoms and oxygen atoms.

[0218] 23. The method according to one of embodiments 21 or 22, characterized in that

[0219] The linker molecule (55) comprises a molecule that specifically binds to the nanoscale biological sample (1) to be studied, in particular wherein the nanoscale biological sample (1) comprises a protein (1a), an antibody, a binding peptide, a DNA sequence, a ligand of a receptor, a chelated metal or histidine.

[0220] 24. A method for determining an electron flux threshold for measuring a nanoscale biological sample (1) using an electron beam (41, 44), comprising the steps of:

[0221] Step α) studying a first nanoscale biological sample (1) according to one of the preceding embodiments, wherein in step d) (400) the first nanoscale biological sample (1) is exposed to a preselected electron dose at a first electron flux and the structure of the first nanoscale biological sample (1) is detected,

[0222] Step β) studying a further nanoscale biological sample (1) according to one of the aforementioned embodiments, wherein in step d) (400) the further nanoscale biological sample (1) is exposed to a preselected electron dose at a further electron flux higher than the previously applied electron flux, and a further structure of the further nanoscale biological sample (1) is detected,

[0223] Step γ) Repeat the procedure of step β) until the structure of the nanoscale biological sample (1) becomes distorted, thereby determining the electron flux threshold.

[0224] 25. The method according to embodiment 24, characterized in that the preselected electron dose is at least 50 electrons per Preferably at least 200 electrons per

[0225] 26. The method for studying at least one nanoscale biological sample (1) according to one of embodiments 1 to 23,

[0226] Wherein in step d)(400) the nanoscale biological sample (1) is exposed to an electron dose and an electron flux, wherein the electron flux is lower than a flux threshold determined for the electron dose serving as a preselected electron dose in the method according to embodiment 24 or 25.

[0227] 27. A sample (9; 43) for studying at least one nanoscale biological sample (1) in an electron beam device (32), wherein the sample (9; 43) comprises:

[0228] - a conductive substrate (4), in particular a graphene foil (4a) or a carbon foil (17), which is arranged on a TEM grid (11),

[0229] - an immobilized film (2) of embedding liquid (3) on a substrate (4) in the application zone (22),

[0230] The immobilized embedding liquid (3) contains a plurality of nanoscale biological samples (1), wherein MD is the maximum diameter of the nanoscale biological samples (1) and MD is ≤30 nm,

[0231] wherein the film (2) has an average thickness AT, wherein AT≤30 nm,

[0232] wherein the substrate (4) is wettable by the embedding liquid (3) in the application area (22),

[0233] and wherein the sample (9; 43) is at a measuring temperature MT,

[0234] Among them, -100℃≤MT≤-1℃,

[0235] In particular, the sample (9; 43) is prepared according to steps a) to c) (100-300) of the method according to one of embodiments 1 to 23.

[0236] 28. Use of a sample preparation device (25) in a method according to one of embodiments 1 to 23, wherein the sample preparation device (25) is suitable for automatically performing at least step b) (200) and preferably also step c) (300).

[0237] Reference number list

[0238] 1 Nanoscale biological samples

[0239] 1a Protein

[0240] 2 Thin film (thin layer)

[0241] 3 (fluid or fixed) embedding liquid

[0242] 3a The embedded liquid portion is removed using a liquid absorption medium

[0243] 4 Base

[0244] 4a Graphene film (graphene foil)

[0245] 5 Film thickness (in area 8b)

[0246] 6 Close distance (below the protein)

[0247] 7 Covering the base

[0248] 7a Second / covering graphene film (graphene foil)

[0249] 8a Thin film region at nanoscale biological samples

[0250] 8b The film region adjacent to the nanoscale biological sample

[0251] 9 samples

[0252] 10 Microdroplets (containing embedding fluid of nanoscale biological samples)

[0253] 11 TEM grid

[0254] 12 Application tip with microchannels

[0255] 13 Micro Clip

[0256] 14 Liquid Absorption Medium

[0257] 14a Filter paper

[0258] 15 Initial volume of embedding fluid

[0259] 16 TEM grid stripes

[0260] 16a Net window

[0261] 17. Porous carbon film (carbon foil)

[0262] 18 holes

[0263] 19 Diameter

[0264] 21 Maximum thickness of embedding liquid on substrate

[0265] 22 Application Area

[0266] 23 Uniform layer thickness

[0267] 24 Negative stain

[0268] 25 Sample preparation equipment

[0269] 30 Sample holder tip

[0270] 31 Rod body

[0271] 32 Electron beam equipment

[0272] 33 Vacuum outer area

[0273] 34 LN2 bath

[0274] 35 Copper wire

[0275] 36 Heating coil

[0276] 36a wire (for heating coil)

[0277] 37 DC regulator

[0278] 38 Thermocouple

[0279] 38a wire (for thermocouple)

[0280] 39 Measuring device

[0281] 40 Electron Source

[0282] 41 (generated) electron beam

[0283] 42 electron condenser

[0284] 43 samples / samples

[0285] 44 (Transmitted) electron beam

[0286] 45 Electronic Projection Mirror

[0287] 46 Camera

[0288] 47 Computer

[0289] 50 Conductive polymer

[0290] 51 Conductive chain

[0291] 51a Unsaturated carbon bond

[0292] 52 Non-polar group (here aromatic group)

[0293] 53 Polar group (here carboxyl)

[0294] 54 Hydrogen Bond

[0295] 55 Linker molecules

[0296] 60 Partial coating

[0297] 70 Application tip (for supplying the pool)

[0298] 71 Supply tank for embedding liquid

[0299] 100 Step a) Preparing embedding liquid

[0300] 200 Step b) Preparation of thin film

[0301] 300 Step c) Adjusting the film

[0302] 400 Step d) Measuring the Nanoscale Biological Sample

[0303] AT Average thickness of the film encapsulating the liquid

[0304] AZA application area

[0305] MD Maximum diameter of nanoscale biological samples

[0306] SV Small size

[0307] α Wetting angle

[0308] List of cited references

[0309] 1 US 7,413,872=US 2004 / 157284

[0310] 2 L.Reimer,H.Kohl,Transmission Electron Microscopy:Physics of ImageFormation,Springer,2008,page 479,equation 11.17

[0311] 3 N.de Jonge, Ultramicroscopy 187(2019)113-125

[0312] 4 RFEgerton,Micron 119(2019)72-87

[0313] 5 EEFill,F.Krausz and MGRaizen,New J.Phys.10(2008)93015-1-7

[0314] 6 S.Keskin and N.de Jonge,Nano Lett.18(2018)7435-7440

[0315] 7 RAMarcus,N.Sutin,Biochim.Biophys.Acta 811(1985)265-322

[0316] 8 CCMoser et al., Nature 355(1992)796-802

[0317] 9 DNBeratan et al.,Science 252(1991)1285-1288

[0318] 10 LFKourkoutis et al.,Annu.Rev.Mater.Res.42(2012)33-58

[0319] 11 G.W.Gross et al.,J.Glaciol.21(1978)143

[0320] 12 Stillman and Grimm,Lunar and Planetary Science XXXIX(2008)2277

[0321] 13 T.Kohno et al.,IEEE Transactions on Electrical Insulation,EI-15(1980)27-32

[0322] 14 H.Wang et al.,J.Phys.Chem.B 113(2009)818-824

[0323] 15 Joachim Frank,"Single-particle reconstruction of biologicalmolecules-story in a sample",Nobel Lecture,December 8,2017

[0324] 16 US 7,659,510=US 2009 / 242795

[0325] 17 US 2012 / 0220046 A

[0326] 18 US 2010 / 0243482 A

[0327] 19 Chih-Yu Chao,Chinese Journal of Physics Vol.45,No.6-I,December2007,557-578

[0328] 20 US 7,420,184=US 2007 / 252090

[0329] 21 T.Li et al,Nat.Commun.4(2013)1887-1-6

[0330] 22 Xiang-Xiong Zhang et al.,J.Chem.Phys.141(2014)124709

[0331] 23 Wikipedia page"saline water",https: / / en.wikipedia.org / wiki / Saline_water downloaded on 22.09.2023

[0332] 24 K.Iakoubovskii and K.Mitsuishi,J.Phys.Condens.Matter 21(2009)155402

[0333] 25 US 2019 / 131106

[0334] 26 US 2012 / 024086

Claims

1. A method for studying at least one nanoscale biological sample (1), the method comprising step a) (100) preparing an embedding liquid (3) containing a plurality of nanoscale biological samples (1), wherein the nanoscale biological samples (1) have a maximum diameter MD, where MD ≤ 30 nm, step b) (200) preparing a thin film (2) of the embedding liquid (3) on a conductive substrate (4) in an application area (22), thereby placing the nanoscale biological samples (1) on the substrate (4), wherein in the application area (22) the substrate (4) is wettable by the embedding liquid (3), and wherein the thin film (2) of the embedding liquid (3) has an average thickness AT, where AT ≤ 30 nm; wherein step a) (100) and step b) (200) are completed at a preparation temperature PT, where PT > 0 °C; step c) (300) adjusting the thin film (2) on the substrate (4) to a measurement temperature MT, where -100 °C ≤ MT ≤ -1 °C; step d) (400) measuring at least one in the nanoscale biological samples (1) in the thin film (2) on the substrate (4) in an electron beam device (32) at the measurement temperature MT, wherein at least one nanoscale biological sample (1) is exposed to an electron beam (41, 44).

2. The method according to claim 1, characterized in that The measurement temperature MT is less than the solidification temperature of the bulk of the embedding liquid (3).

3. The method according to claim 1, characterized in that AT ≤ MD.

4. The method according to any one of the preceding claims, characterized in that in step d) (400), the electron beam energy of the electron beam (41, 44) is 50 eV to 300 keV, preferably 30 keV to 300 keV, and the electron flux of the electron beam (41, 44) is 0.1 electron per Up to 50 electrons per second Every second, Preferably 1 electron per Up to 10 electrons per second per second.

5. The method according to claim 1, characterized in that the preparation temperature PT is selected in the range 0 °C < PT ≤ 40 °C.

6. The method according to claim 1, characterized in that During step b) (200), an initial large amount (15) of the embedding liquid (3) is placed on the substrate (4), where the large amount (15) is greater than that required for the thin film (2), and then the amount of the embedding liquid (3) on the substrate (4) is reduced until only the thin film (2) remains.

7. The method according to claim 6, characterized in that Reducing the amount of the embedding liquid (3) includes placing a liquid absorption medium (14) in contact with the embedding liquid (3), especially where the liquid absorption medium (14) comprises filter paper (14a) or drawn metal wire, and / or characterized in that reducing the amount of the embedding liquid (3) includes evaporating some of the embedding liquid (3).

8. The method according to any one of claims 1 to 5, characterized in that During step b) (200), in order to prepare a thin film (2) of the embedding liquid (3) in an application area (22) having an application area area AZA, an initial small volume SV of the embedding liquid (3) is placed on the substrate (4) in the application area (22), where SV ≤ AT * AZA.

9. The method according to claim 1, characterized in that In step b) (200), the initial amount of the embedding liquid (3) is placed on the substrate (4) in the application area (22) as follows: First, the application tip (70), especially an atomic force microscopy (= AFM) tip, is dipped into a supply pool (71) of the embedding liquid (3) containing the nanoscale biological samples (1), and then the application tip (70) is brought into contact with the application area (22).

10. The method according to claim 1, characterized in that In step b) (200), an initial amount of embedding liquid (3) is placed on the substrate (4) in the application area (22) using an application tip (12), which has microchannels through which the initial amount of embedding liquid (3) is discharged.

11. The method according to claim 1, characterized in that During step b) (200), the initial volume of embedding liquid (3) placed on the substrate is 1 fL to 2 μL.

12. The method according to claim 1, characterized in that The experimental parameters applied during step b) (200) for producing a film (2) having an average thickness AT have been previously determined in a calibration experiment, In particular, the experimental parameters include at least one of the following: the temperature of the atmosphere surrounding the substrate (4), the pressure of the atmosphere surrounding the substrate (4), the humidity of the atmosphere surrounding the substrate (4), the composition of the atmosphere surrounding the substrate (4), the temperature of the substrate (4), the type of liquid absorption medium (14), the contact time with the liquid absorption medium (14), the contact pressure with the liquid absorption medium (14), the evaporation time, the initial large volume (15) of the embedding liquid (3) placed on the substrate (4), the initial small volume SV of the embedding liquid (3) placed on the substrate (4) in the application area (22), the initial amount of the embedding liquid (3) placed on the substrate (4), the initial volume of the embedding liquid (3) placed on the substrate (4), the downward pressure of the application tip (70), and the discharge flow rate of the embedding liquid (3) through the microchannel.

13. The method according to one of the preceding claims, characterized in that The embedding liquid (3) is chosen to be water with added salt, Preferably, the added salt is NaCl, It is particularly preferred that the added salt is NaCl at a concentration of 10 mM.

14. The method according to one of the preceding claims, characterized in that The substrate (4) is a graphene foil (4a) or a carbon foil (17).

15. The method according to one of the preceding claims, characterized in that After step b) (200) and before step c) (300), the thin film (2) embedding the liquid (3) is covered with a covering substrate (7), in particular wherein the covering substrate (7) is a graphene foil (7a) or a carbon foil.

16. The method according to one of the preceding claims, characterized in that The embedding liquid (3) contains a conductive polymer (50) and / or a conductive protein.

17. The method according to one of the preceding claims, characterized in that The embedding liquid (3) contains a negative stain (24), In particular, the negative stain (24) is selected as α-D-glucose 1-phosphate dipotassium salt hydrate, preferably applied at a concentration of 2 mM.

18. The method according to one of the preceding claims, characterized in that In step c) (300) the thin film (2) on the substrate (4) is first cooled to a low temperature LT, wherein LT ≤ -196°C, and then warmed again to a measuring temperature MT.

19. The method according to any of the preceding claims, characterized in that The substrate (4) is placed on a TEM grid (11), wherein the TEM grid (11) has a plurality of intersecting strips (16) defining screen windows (16a) between the intersecting strips (16), and characterized in that during step b), an embedding liquid (3) is applied between the strips (16) in at least one screen window (16a), In particular, the embedding liquid (3) is carefully applied between the mesh strips (16) into at least one mesh window (16a).

20. The method according to one of the preceding claims, characterized in that The substrate (4) comprises at least one partial coating (60) defining at least one application zone (22), wherein the partial coating (22) renders the substrate (4) wettable by the embedding liquid (3) in the application zone (22).

21. The method according to one of the preceding claims, characterized in that The substrate (4) is at least partially coated with a linker molecule (55) for connecting the nanoscale biological sample (1) to the substrate (4), In particular, wherein the nanoscale biological sample (1) is a protein (1a) and the substrate (4) comprises a graphene foil (4a), Characterized in that the linker molecule (55) is attached to the substrate (4) on one side via one or more non-polar chemical groups (52), in particular wherein the one or more non-polar chemical groups (52) are based on pyrene, pyridine, porphyrin or amine, And it is characterized in that the linker molecule (55) has one or more polar chemical groups (53) on its other side, especially wherein the one or more polar chemical groups (53) contain butyric acid-succinimidyl ester, pyrenebutyric acid (PBA) or carboxyl group.

22. The method according to claim 21, characterized in that The linker molecule (55) contains a conductive chain (51), in particular wherein the conductive chain (51) is based on a carbon atom (51a) having an unsaturated carbon bond or a carotenoid or a chain of carbon atoms and oxygen atoms.

23. The method according to claim 21 or 22, characterized in that The linker molecule (55) comprises a molecule that specifically binds to the nanoscale biological sample (1) to be studied, in particular wherein the nanoscale biological sample (1) comprises a protein (1a), an antibody, a binding peptide, a DNA sequence, a ligand of a receptor, a chelated metal or histidine.

24. The method according to one of the preceding claims, characterized in that The measurement temperature MT is selected to be -50°C ≤ MT ≤ -1°C.

25. A method for determining an electron flux threshold for measuring a nanoscale biological sample (1) using an electron beam (41, 44), comprising the following steps: Step α) studying a first nanoscale biological sample (1) according to one of the preceding claims, wherein in step d) (400) the first nanoscale biological sample (1) is exposed to a preselected electron dose at a first electron flux and the structure of the first nanoscale biological sample (1) is detected, step β) studying a further nanoscale biological sample (1) according to one of the preceding claims, wherein in step d) (400) the further nanoscale biological sample (1) is exposed to a preselected electron dose at a further electron flux higher than the previously applied electron flux, and a further structure of the further nanoscale biological sample (1) is detected, Step γ) Repeat the procedure of step β) until the structure of the nanoscale biological sample (1) becomes distorted, thereby determining the electron flux threshold.

26. The method according to claim 25, characterized in that The preselected electron dose is at least 50 electrons per Preferably at least 200 electrons per 27. Method for studying at least one biological sample (1) at the nanoscale according to one of claims 1 to 24, Wherein in step d)(400) the nanoscale biological sample (1) is exposed to an electron dose and an electron flux, wherein the electron flux is lower than a flux threshold determined for the electron dose serving as a preselected electron dose in the method according to claim 25 or 26.

28. Use of a sample preparation device (25) in a method according to one of claims 1 to 24, wherein the sample preparation device (25) is adapted to automatically perform at least step b) (200) and preferably also step c) (300).

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