Transmission electron microscopy of streams of IONS and molecules

Abstract

A method of analyzing a sample by an electron microscope comprises: generating ions, molecules, or other particles, such as droplets, from a sample of interest; introducing the ions, molecules or other particles into an evacuated chamber of a Transmission Electron Microscope (TEM) in a direction that intersects the path of a pulsed electron beam; on each pulse of the electron beam, recording an electron diffraction pattern pertaining to ions through which the said pulse passes; and calculating a three-dimensional (3D) structure of an ion species from the recorded electron diffraction patterns.

Description

TRANSMISSION ELECTRON MICROSCOPY OFSTREAMS OF IONS AND MOLECULESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of the filing date of commonly-assigned United States Provisional Patent Application number 63 / 590,757, titled "Transmission Electron Microscopy of Streams of Ions and Molecules" which was filed on 16-October-2023, the disclosure of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present application relates to electron microscopy and electron microscopes. More particularly, the present invention relates to electron microscopy of sub-micron particles of both organic and inorganic materials, including, for example, biological molecules, such as proteins and peptides as well as various other molecules, molecular clusters, and microcrystalline materials.BACKGROUND

[0003] The study of the three-dimensional inter-atomic and molecular structures of sub-microscopic particles is an active area of research. For example, the study of the three-dimensional structures of proteins in their native state is of great interest to the field of biochemistry. Protein molecular structures are often resolved by various techniques such as Scintillation Proximity Assay (SPA), Ultrafast Electron Diffraction (UED), Nuclear Magnetic Resonance (NMR) and X-Ray Diffraction (XRD). Unfortunately, the current electron-microscopy-related technologies for structural determinations require time-consuming complex sample preparation and sample transport steps. Specifically, to preserve the native protein structure in the harsh environment of an electron microscope, i.e., in vacuum and at high radiation levels, it is often necessary to cool sample material to a cryogenic temperature. Since the sample is usually provided in an aqueous solution and since the formation of crystalline ice can destroy the nativeprotein structure, it is generally necessary to flash freeze the sample so that the protein molecules become embedded in vitreous ice. The vitreous ice is formed by cooling the aqueous solution at a cooling rate that is on the order of 105K / s to a temperature of less than the glass transition temperature of approximately 165 K. So-called High Pressure Freezing (HPF) is an alternative to ambient-pressure flash freezing. In this alternative procedure, vitrification may be performed at a pressure of approximately 2100 bar at a cooling rate of "only" approximately 600 K / s. As the cooling rate is lower than ambient-pressure flash freezing, thicker vitrified samples can be formed.

[0004] In practice, a cryogenic sample in vitrified ice is formed, for example, by applying a thin layer of an aqueous solution on a carrier, for example a TEM grid. Excess water is then removed by blotting, and the remaining carrier with the aqueous solution is then plunged in a cryogenic fluid, such as ethane or liquid nitrogen. In another method that is described by White et al. ("A second generation apparatus for time- resolved electron cryo-microscopy using stepper motors and electrospray", H. D. White et al., J. Struct. Biol 144 (2003), pp 246-252), an aqueous solution comprising sample material is sprayed on the carrier while the carrier (still at room temperature) is plunged into the cryogenic fluid.

[0005] Generally, additional preparation steps are required before the sample proteins can be examined by electron microscopy and electron diffraction techniques. For example, if the thickness of the vitreous ice layer is greater than approximately 500 nm, then the sample must be thinned using either an ultramicrotome or an ion beam milling apparatus. All the while, the sample must be transported in and out of the thinning apparatus while maintaining it in a frozen state and while taking care to not expose it to water vapor or other gaseous substances that could otherwise condense onto the sample.

[0006] Once the sample, on its grid support, has been introduced into an electron microscope, it may be necessary to survey the sample material in the grid coordinates of regions of interest. The protein molecules in each such region of interest willpresumably be randomly oriented. Once the regions of interest have been established, electron images are taken at a plurality of locations, in sufficient quantity to ensure that electron images are acquired from most of the molecular protein orientations. Once good quality images have been obtained from a sufficient number of protein orientations, the three-dimensional (3D) structure of the protein of interest may be derived by mathematical analysis. Typically, such 3D structural reconstructions rely on data averaged over thousands of individual particles.

[0007] The various sample preparation steps and data acquisition steps that are required to generate a three-dimensional protein structure are tedious. In practice, it is found that the full protein structural determination procedure may require several weeks of laboratory work. Accordingly, there is a need in the art of proteomics studies, as well as in the art of general biochemistry, to be able to experimentally determine and experimentally verify protein structures and structures of other macromolecules with greater efficiency.

[0008] There is a body of work devoted to creating electron diffraction patterns from supercooled molecules (which also could be embedded into helium droplets) and oriented using one or more laser pulses [US9, 279,778, J. C. H. Spence, R. B. Doak. Single Molecule Diffraction, PhysRevLett. 92 (2004) 198102; Ma Z. et al. Ultrafast isolated molecule imaging without crystallization. PNAS, 2022, Vol. 119, No. 15, e2122793119],

[0009] However, the extent of orientation was never shown to approach the required one within better than tens of degrees which might be sufficient for small molecules but not for complex protein molecules. Further, complexity of the setup is likely to preclude its widespread use in analytical electron microscopy.SUMMARY

[0010] Against this prior art, the current invention proposes the use of protein molecules in their native state (preferably, with their solvation spheres preserved) to be produced by reduced-desolvation native mass spectrometry. Instead of laser alignment, pulsed exposure to intense electron pulse allows to create diffraction pattern at aparticular, although unknown, phase of molecular rotation over time frames that are negligible comparing to the period of rotation. Following acquisitions of thousands to millions noisy images of individual particles, a consensus high-resolution image will be generated from all these different projections as established in cryo-electron microscopy.

[0011] The present inventors provide solutions to the above-noted need in the art by describing methods and apparatus by which a flow of particles, comprising either neutral molecules or ions that are optionally preselected, in accordance with their mass- to-charge (m / z) ratios, by mass spectrometric filtration, is introduced directly into the objective lens gap of a Transmission Electron Microscope (TEM) for further analyses using an electron beam. In embodiments, the particles may comprise or may be derived from biological compounds, such as proteins, peptides, etc. In some embodiments, the flow of particles may, after passing through the TEM, be introduced into a mass spectrometer and mass analyzed therein in order to supplement the data derived from electron beam analyses.

[0012] According to some embodiments, the present disclosure describes methods and apparatus that include introduction of a directed beam of charged particles comprising ions of proteins, peptides, or other biological or non-biological analytes directly into an objective lens gap of a transmission electron microscope (TEM) and analyzing those particles using an electron-beam that is oriented substantially at a right angle relative to the ion particle beam. The duration of exposure of each particle to the electron beam must be sufficiently short so that an electron diffraction exposure may be obtained before the structure of the particle is altered by the interaction with the beam. One means of achieving this result is to pulse the electron beam such that each pulse has a duration of a few picoseconds or less. Alternatively, the ion particles may be accelerated to a velocity that causes them to pass through a continuous electron beam at a defined speed that limits the duration of beam exposure of each particle to the picosecond or sub-picosecond range. Although electron-beam pulse durations are generally specified in the picosecond range in this document, it should be kept in mindthat the actual pulse length that may be endured by any sample without structural change will generally depend on the composition of the sample and, accordingly, will need to be determined for each sample and each compound. Accordingly, a first exemplary method of the present disclosure may comprise some portion of the following procedural steps:1. A flow of particles comprising ions of a compound of interest is generated by conventional atmospheric pressure ionization method, such as those that are used in Mass Spectrometry - e.g., electrospray ionization (ESI), heated electrospray ionization (HESI), thermospray ionization, atmospheric pressure chemical ionization (APCI), matrix assisted laser desorption ionization (MALDI), etc. The compound of interest may be provided in solution in a purified form. Various sample preparation and purification procedures may include such steps as dissolving powder followed by a buffer exchange, protein expression followed by concentration / purification protocols, size-exclusion chromatography, capillary electrophoresis, solid-phase extraction, liquid chromatography, affinity separations, or other liquid-phase separations. In general, it is desirable that the initial preparation process provide the sample supply in solution and that the sample supply not have undergone agglomeration and / or denaturization so that the proteins are in a native-like state. In other embodiments, the sample supply could be also directly prepared from intact cells or cell membranes, where membrane proteins and complexes could be stabilized by membranes, nanodiscs, nanorafts, nanocontainers and other methods known in the art.2. The particles are introduced into an ion conditioning module in which they are separated from gas molecules and solvent so as to generate free ions, and in which the ions are focused and collimated into an ion beam, preferably filtered according to mass-to-charge ratio, optionally cooled, and optionally mass analyzed to verify that primarily ions of interest remain subsequent to the filtering. According to some exemplary embodiments, the particles may becooled to room temperature or even frozen and / or covered or coated by vitrified water as required by the nature of the particle.3. The filtered ions are transported through an ion transport module, preferably through multiple chambers of progressively increasing vacuum, into an analysis chamber of a charged particle beam microscope.4. The ions are illuminated by picosecond or sub-picosecond pulses (i.e ., pulse widths not greater than 100 picoseconds, or, more preferably, less than 10 picoseconds or, even more preferably, not greater than one picosecond) of the electron beam and diffraction data is gathered using a fast camera or other imaging detector, such as a direct electron detector, that is synchronized to the pulses. Each randomly oriented particle generates own electron diffraction pattern. Alternatively, the ion-containing particles may be accelerated to a velocity that causes them to pass through a continuous electron beam at a defined speed that limits the duration of beam exposure of each particle. The main condition is that rotational period of an ion is substantially (preferably, tens to thousands of times) longer than the duration of exposure, thus limiting smearing of the diffraction pattern below resolution of each image. Rotational period could be extended by controlling and reducing energy of ions, primarily by thermalizing them to room or even cryogenic temperatures prior to exposure to the electron pulse.5. Optionally, part of the flow of the introduced particles continues to a mass spectrometer for mass and charge analysis.6. Mathematical reconstruction of the structure of particles as determined by electron diffraction which could be complemented by detection of another signal and / or additional information from other physico-chemical methods.

[0013] According to one aspect of the present teachings, a method of analyzing a sample by an electron microscope comprises: generating ions from a sample of interest;introducing the ions into an evacuated chamber of a Transmission Electron Microscope (TEM) in a direction that intersects the path of a pulsed electron beam; recording a series of electron diffraction patterns pertaining to ions that pass through and interact with the pulsed electron beam; and calculating a three-dimensional (3D) structure of an ion species from the recorded electron diffraction patterns.

[0014] According to another aspect of the present teachings, a method of analyzing a sample by an electron microscope comprises: introducing gaseous molecules or liquid droplets comprising molecules into an evacuated chamber of a Transmission Electron Microscope (TEM) in a direction that intersects the path of a pulsed electron beam; recording a series of electron diffraction patterns pertaining to ions that pass through and interact with the pulsed electron beam; and calculating a three-dimensional (3D) structure of a molecular species from the recorded electron diffraction patterns, wherein the gaseous molecules or liquid droplets pass through the evacuated chamber at a temperature above the boiling point of liquid helium.

[0015] According to yet another aspect of the present teachings, an analysis system is provided, the system comprising: an electron microscope sub system comprising: an electron source configured to generate a pulsed electron beam; an evacuated chamber of a Transmission Electron Microscope (TEM); an objective lens within the TEM column; and an electron detector; an ion source configured to generate ions from a sample; andan ion transport module configured to direct a beam of ions generated by the ion source into the evacuated electron microscope column so as to intersect therein with an electron beam generated by the electron source; wherein the objective lens is configured to project an electron diffraction pattern of ions that pass through the electron beam onto the electron detector.

[0016] According to still yet another aspect of the present teachings, an analysis system is provided, the system comprising: an electron microscope sub system comprising: an electron source configured to generate a pulsed electron beam; an evacuated chamber of Transmission Electron Microscope (TEM); an objective lens within the TEM column; and an electron detector; a fluid emitter apparatus configured to direct a stream of gaseous, liquid or other molecule-bearing particles into the evacuated TEM column so as to intersect therein with an electron beam generated by the electron source; and a fluidic sample transport apparatus configured to transport a fluid sample from a sample source to the fluid emitter apparatus; wherein the objective lens is configured to project, onto the electron detector, an electron diffraction pattern of molecules of the stream of gaseous, liquid or other molecule-bearing particles that pass through the electron beam.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above noted and various other aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings, not necessarily drawn to scale, in which:

[0018] FIG. 1 is a schematic cross-sectional depiction of a of a portion of a first embodiment of a particle beam analysis system, such as a Transmission Electron Microscope (TEM) system, in accordance with the present teachings;

[0019] FIG. 2A is a schematic cross-sectional depiction of a of a portion of a second embodiment of a particle beam analysis system in accordance with the present teachings;

[0020] FIG. 2B is a detailed view of an ion beam positioning, deflecting and focusing system as may be employed in the particle beam analysis system of FIG. 2A;

[0021] FIG. 3 is a schematic cross-sectional depiction of a of a portion of a third embodiment of a particle beam analysis system in accordance with the present teachings;

[0022] FIG. 4 is a schematic cross-sectional depiction of a portion of a fourth embodiment of a particle beam analysis system in accordance with the present teachings;

[0023] FIG. 5A is a plan cross-sectional view of a first Micro-Electromechanical System (MEMS) device that may be employed within the particle beam analysis system of FIG. 4 to inlet ions, molecules, or other particles into the particle beam analysis system;

[0024] FIG. 5B is a plan cross-sectional view of a second MEMS device that may be employed within the particle beam analysis system of FIG. 4 to inlet ions, molecules, or other particles into the particle beam analysis system;

[0025] FIG. 6A is a longitudinal elevation cross-sectional view of the MEMS device of FIG. 5B;

[0026] FIG. 6B is a first transverse elevation cross-sectional view of the MEMS device of FIG. 5B;

[0027] FIG. 6C is a second transverse elevation cross-sectional view of the MEMS device of FIG. 5B;

[0028] FIG. 6D is a portion of a plan cross-sectional view of a modified version of the second MEMS device of FIGS. 6A through 6C;

[0029] FIG. 7 is a flow diagram of a method in accordance with the present teachings for operating a charged particle beam analysis system to investigate the structures of neutral molecules and other particles; and

[0030] FIG. 8 is a flow diagram of a method in accordance with the present teachings for operating a charged particle beam analysis system to investigate the structures of ions.DETAILED DESCRIPTION

[0031] The following description is presented to enable any person skilled in the art to make and use the invention and is provided in the context of a particular application and its requirements. Various modifications to the described embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments and examples shown but is to be accorded the widest possible scope in accordance with the features and principles shown and described.

[0032] In the description of the invention herein, it is understood that a word appearing in the singular encompasses its plural counterpart, and that a word appearing in the plural encompasses its singular counterpart, unless implicitly or explicitly understood or stated otherwise. Furthermore, it is understood that, for any given component or embodiment described herein, any of the possible candidates or alternatives listed for that component may generally be used individually or in combination with one another, unless implicitly or explicitly understood or stated otherwise. Moreover, it is to be appreciated that the figures, as shown herein, are not necessarily drawn to scale, wherein some of the elements may be drawn merely for clarity of the invention. Also, reference numerals may be repeated among the various figures to show corresponding or analogous elements. Additionally, it will beunderstood that any list of candidates or alternatives is merely illustrative, not limiting, unless implicitly or explicitly understood or stated otherwise.

[0033] Unless otherwise defined, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. It will be appreciated that there is an implied "about" prior to any quantitative terms mentioned in the present description, such that slight and insubstantial deviations are within the scope of the present teachings. In addition, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. As used herein, "a" or "an" also may refer to "at least one" or "one or more." Also, the use of "or" is inclusive, such that the phrase "A or B" is true when "A" is true, "B" is true, or both "A" and "B" are true.

[0034] In a first aspect, the present disclosure provides methods and apparatus in which a stream either organic or inorganic particles is repeatedly imaged by an electron beam while the particles "fly" through the electron beam as they are inlet into a TEM column. Such methods and apparatus may advantageously be employed in several disciplines, such as in the biological and biochemical sciences, for studies of macromolecular biological particles, such as proteins, peptides, nucleic acids, etc., or ions of such compounds. More broadly, the particles may comprise any type of particle, either charged or uncharged, that can be penetrated by electrons of a certain energy. The images may be images of the particles themselves or may be images of electron diffraction patterns generated as electrons pass through the particles. Assuming that the various particles in the stream will have random orientations, then, with a sufficiently large number of images, it is possible to reconstruct a 3D image of each species of particle. The orientation of each particle relative to the electron beam needs to be inferred from the dataset, as is currently done in the SPA technique.

[0035] FIG. 1 is a schematic cross-sectional depiction of a of a portion of a first charged particle beam analysis system, such as a Transmission Electron Microscope(TEM) system, in accordance with the present teachings. In particular, FIG. 1 illustrates a portion of a TEM column. The illustrated TEM system 10 comprises an electron source (in a portion of the TEM column that is not shown in the drawings), an electron beam condenser lens (not shown), a first objective pole piece 12 of a first objective lens, a second objective pole piece 14 of a second objective lens, and a detector 18 housed within an interior volume 13 of a vacuum chamber 11. Other components of the first and second objective lenses are omitted for clarity. In addition to the illustrated components, the TEM system generally comprises other beam-shaping and beamcontrolling components for manipulating a pulsed electron beam 16. Such other components may include a controllable beam blocker (i.e., a beam blanker) and one or more controllable beam shifter lenses. In operation, the various TEM components are in electrical communication with a control environment (not shown) that may be configured with computer readable instructions that automatically cause the various components of the TEM system 10 to operate in a coordinated manner so as to, at controlled times, generate the pulses of electrons that propagate, according to a beamlike geometry, parallel to an elongate axis 19 of the TEM column, in a direction from the top to the bottom of the drawing.

[0036] The TEM system 10 may further comprises an ion conditioning module 202 that is configured to receive ions and / or charged droplets 23 from a nozzle or other emitter portion of an ionization source 21, the ions and / or charged droplets 23 being generated from a sample of interest 22. For example, the atmospheric pressure ionization source 21 may comprise an atmospheric pressure ion source such as an electrospray (ESI) ion source, as is illustrated in FIG. 1. Preferably, ESI operates in native mode when ions are sprayed at around neutral pH in a buffer-exchanged solution as known in the art. As another example the ionization source may comprise any other known atmospheric pressure or non-atmospheric-pressure ionization source, such as a thermospray ionization source, an atmospheric pressure chemical ionization (APCI) ionization source, a matrix assisted laser desorption ionization (MALDI) ionization source, and the like. Some of such ion sources can generate either negatively chargedor positively charged ions and analyses of both positive and negative ions are contemplated by this disclosure.

[0037] The plume of generated ions and / or charged droplets 23 are intercepted by an ion inlet 25 of an ion conditioning module 202. In the illustrated embodiment, the ion inlet 25 comprises an ion transfer tube that comprises a lumen that is of sufficiently small diameter to effectively prevent most atmospheric gases from entering the ion conditioning module 202. In the illustrated configuration, the ion source (i.e., an electrospray nozzle 21) and the ion transfer tube 25 are both electrodes. In operation, a power supply (not shown) provides an electrical potential difference between the nozzle 21 and the ion transfer tube 25 that causes a portion of the ions to enter a lumen of the ion transfer tube.

[0038] Preferably, the sample 22 comprises a solution of a known purified compound of interest (or a small number of known compounds of interest) in a solution. In addition to the compound of interest and a suitable solvent, the sample solution may comprise additional components, such as buffers for controlling pH and salts for facilitating ionization. In more-complex experiments, the sample may comprise a plurality of compounds, some of which may be of interest. In such situations, a liquid chromatograph or other chemical separation apparatus (not shown) may be interposed between the sample source 22 and the ion source 21 for the purpose of fractionating the various compounds so that the compounds are introduced, in sequence and essentially one-at-a-time, into the ion source 21. Preferably, the ion transfer tube 25 is in thermal communication with a heater (not shown) for the purpose of, during operation, volatilizing droplets as well as any molecules of solvent that remain adducted to the ionized sample (a process known as desolvation).

[0039] The main functions of the ion conditioning module 202 are to separate and remove any residual solvent or ambient atmospheric gas from the ion stream, focus the ion stream into a collimated beam, and select a particular ion species of interest (i.e., an ion species having a particular mass-to-charge ratio) by filtering the ion stream so as to eliminate other ion species. Accordingly, the ion conditioning module 202 maycomprise various components usually found in mass spectrometer systems, such as an ion focusing and gas separation apparatus 203, such as, for example, an ion funnel, and a mass filtering apparatus 210, such as a quadrupole mass filter. Additional ion beam collimation may be performed by conventional ion lenses (not specifically illustrated) of the ion conditioning module. The ion conditioning module 202 may optionally comprise an ion cooling apparatus 212 to dampen ion thermal kinetic energy and an optional mass analyzer 214 to verify the identity / identities of samples of the mass selected ions. Preferably, the ion conditioning module 202 comprises two or more differentially- pumped compartments (not illustrated) that are evacuated by separate or multi-port vacuum pumps (not illustrated) and that are separated from one another by one or more partitions (not illustrated) having gas conductance limiting apertures therein. Such differentially-pumped compartments permit the internal gas pressure to progressively decrease in a direction towards the TEM vacuum chamber 11.

[0040] An ion transport module 206 may be interposed between the ion conditioning module 202 and an ion inlet aperture 207 of the TEM vacuum chamber 11. The main functions of the ion transport module 206 are to deliver the beam of selected ions into the ion inlet aperture 207 as well as to further reduce the internal gas pressure to the working pressure of the TEM. In conjunction with the gas-pressure- reduction function, the ion transport module 206, in similarity to the ion conditioning module 202, comprises two or more differentially-pumped compartments (not illustrated) that are evacuated by separate or multi-port vacuum pumps (not illustrated) and that are separated from one another by one or more partitions (not illustrated) having gas conductance limiting apertures therein. Together, the differentially-pumped compartments of the ion conditioning module 202 and the ion transport module 206 serve to reduce the internal pressure from approximately 10'5torr within the ion conditioning module 202 to approximately 10 to 10 torr within the interior of theTEM vacuum chamber 11.

[0041] In operation of the system 10, the interior volume 13 of the vacuum chamber 11, the ion transport module 206 and the ion conditioning module 202 are all evacuated by not-illustrated vacuum pumps, as noted previously. In operation of the TEM, while the chamber is under vacuum, the electron source (not shown) and various lenses of the TEM column are configured to cause a pulsed beam 16 of electrons to propagate within the interior volume 13 parallel to the column axis 19 in a direction from the electron source (not shown) towards the first objective lens (represented by pole piece 12) and, therefrom, towards the second objective lens (represented by pole piece 14). Simultaneously, ions of the sample of interest are introduced into the interior volume 13 through the ion inlet aperture 207 of the TEM chamber 11. A set of collimating and focusing lenses 31 may be provided to cause the introduced ions to propagate as a beam 26 that intersects the path of the pulsed electron beam 16 the vicinity of the TEM column axis. The ion inlet aperture 207 and ion optical components 31, 32 and 33 may be configured such that the ion beam 26 and the pulsed electron beam 16 may intersect at essentially a right angle to one another within a beam intersection region 24.However, in other embodiments, as discussed below, the path of the ion beam may be oriented such that the ion beam and the pulsed electron beam intersect at some point angle other than ninety degrees, so as to maximize the probability of interactions between ions and electron beam pulses.

[0042] Depending on the relative fluxes of electrons and ions within the beam intersection region 24, a proportion of the ions will collide and interact with electrons of the pulsed beam 16. Any ions that do not interact with electrons pass through the beam intersection region 24 towards a receiving electrode 32. Preferably, a common electrical potential is applied to the receiving electrode 32 and to the electrode of the collimating and focusing lenses 31 (or to another electrode) that is closest to the ion beam axis 16 so that there is no electric field component that is perpendicular to the TEM column axis 16 within the beam intersection region 24. Nonetheless, small electric field components that might otherwise perturb the electron beam may be corrected by beam-correcting lenses in the TEM column. Accordingly, under such conditions, theportion of the ion beam 26 between the ion optical components 21, 32 is an ion drift region. This configuration helps to avoid perturbation of the electron beam by the ion optical components that guide the introduced ions. The ions that pass through the receiving electrode 32 may be neutralized by a subsequent grounding electrode (not shown) or, optionally, may be focused, by ion optics 33, into an optional tube lens 34 that passes through an ion outlet aperture 208 of the wall of the chamber 11 for the purpose of transporting the ions to an optional mass analyzer or mass spectrometer 35. As an example, the ion optics 33 may comprise an ion funnel.

[0043] Depending upon the type of and the configuration of the ion source, the ions that enter the beam intersection region 24 may be either positively charged or negatively charged. Depending on the relative fluxes of electrons and ions within the beam intersection region 24, there is a statistical probability that any particular ion will collide with and interact with one or more electrons. If the ions are positively charged, then, depending on the energy of the electrons involved in the collision as well as an additional statistical probability factor, the nature of the ion / electron interaction may be one of: (a) simple elastic scattering without any other reaction; (b) reduction in charge state or complete neutralization of charge; (c) initiation of ion fragmentation without a change in total charge; (d) ion fragmentation with a change in total charge state; and (e) transmission of the electrons through the ion without charge-state reduction or fragmentation and with electron diffraction, including zero order diffraction.

[0044] As a result of the combined simultaneous occurrences of the above-listed interactions, some electrons, comprising electrons that did not interact with any ions and electrons that were transmitted through ions, will pass through the second objective lens 14 to the detector 18. Other elastically scattered electrons or electrons that reacted with ions will generally not reach the detector. Also, as a result of the combined simultaneous occurrences of the above-listed interactions, some ions, will continue along ion beam path 26 to be either neutralized at the receiving electrode 32 or else detected by optional mass spectrometer 35, if present. The ions that continuealong a straight-line path towards the receiving electrode 32 or mass spectrometer 35 may comprise: those ions that did not interact or react with electrons; ions for which the charge state was reduced; and fragment ions or other ions for which spatial scattering is insignificant. Other ions will generally be neutralized by collision with objects within the chamber.

[0045] The detector 18 that is depicted in FIG. 1 is an imaging detector and, for purposes of sensitivity, is preferably a direct electron detector. Hypothetically, the TEM lenses can be configured to generate images of individual ions or of collections of ions as each such ion or collection of ions passes through the electron beam 16. However, since images of individual ion particles may not be particularly informative and may be recorded with very low signal-to-noise ratio, it is preferable to configure the lenses of the TEM column to project an electron diffraction pattern onto the detector 18. Each randomly oriented ion particle generates its own electron diffraction pattern. Since many ion particles are interrogated by the electron beam, the individual electron diffraction patterns from particles of a same ion species that are similarly oriented may be co-added, during subsequent computational analysis, to obtain improved signal-to- noise characteristics. Following acquisitions of thousands to millions noisy images of individual particles, a consensus high-resolution image will be generated from all these different projections as known in cryo-electron microscopy. To accelerate the convergence of the algorithm, known sequence or subsequences of the investigated particle could be used to predict its structure using AlphaFold or similar software utilizing AI / ML. This predicted structure could then be used as the first iteration for the reconstruction algorithm.

[0046] Electron diffraction is presently the most convenient analysis mode as a result of the low number of electrons that are needed (i.e., ~0.01 e / A2or more) as well as because single-particle imaging using randomly oriented diffraction images is well known from the domain of X-rays. For imaging of individual particles, this minimum dose of >0.01 e / A2, or preferably >0.05 e / A2, needs to be delivered per every successfuldiffraction image, i.e. every electron pulse should carry sufficient number of electrons to guarantee such dose upon interaction.

[0047] FIG. 2A is a schematic cross-sectional depiction of a of a portion of a second embodiment of a particle beam analysis system in accordance with the present teachings. In similarity to the particle beam analysis system 100 shown in FIG. 1, the particle beam analysis system 200 of FIG. 2A utilizes an ion conditioning module 202 for preparing ions to be introduced into the interior volume 13 of analysis chamber 11. However, in contrast to the system 100, in which ions are introduced into the analysis chamber volume 13 as a continuous stream, the system 200 is configured to introduce discrete packets of ions into the analysis chamber as separate pulses. As described further below, the particle beam analysis system 200 may also comprise a laser 90 that is capable of generating pulses 91 of el I iptica lly polarized laser light that are transmitted to the region of intersection 24 between the ion pulse propagation trajectory 26 and the electron beam 16.

[0048] In operation of the particle beam analysis system 200, the ions of each ion packet are temporarily accumulated, thermalized and stored in an ion storage apparatus 215 which, in this embodiment, is a curvilinear multipole ion trap that is known as a "C- trap". Such C-trap devices are utilized in the mass spectrometry arts to inject packets of ions into ORBITRAP™ mass analyzers. Various mass spectrometer systems which that utilize ORBITRAP mass analyzers are commercially available from Thermo Fisher Scientific of Waltham Massachusetts USA. In brief, an ORBITRAP mass analyzer is a type of electrostatic trap mass analyzer. Structural and operational details of ORBITRAP mass analyzers and mass spectrometers employing such mass analyzers are described in Makarov, Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis, Anal. Chem., 72(6), 2000, pp. 1156-1162 and in U.S. Pat. No. 5,886,346 in the name of inventor Makarov and in U.S. Pat. No. 6,872,938 in the names of inventors Makarov et al. For details of the curved trap, or C-trap, apparatus and its coupling to an electrostatic trap, the interested reader is referred U.S. Pat. Nos. 6,872,938; 7,498,571; 7,714,283; 7,728,288; and 8,017,909 each of which is herebyincorporated herein by reference in its entirety. Any other panoramic mass analyzer could be employed, for example, an Astral™ analyzer as described in WO2013110587A2, WO2013110588A2, US2023118221A1, US10699888B2, each of which is incorporated herein in its entirety.

[0049] In operation of the system 200, a packet of ions that is to be injected into the interior volume 13 of the vacuum chamber 11 is initially trapped within the C-trap 217 by: (a) opening electrostatic ion gate 215 while electrostatic ion gate 216 remains closed, and (b) subsequently closing ion gate 215. The ions of the trapped ion packet are then allowed to equilibrate at their thermal velocities in about 1 mtorr pressure of nitrogen within the C-trap 217. Subsequently, the electrical potential of the electrodes of the C-trap 215 is raised from ground potential to an appropriate voltage relative to the vacuum chamber 11 that causes the ejection of the ions of the ion packet towards and past the electrodes 219 and then through the ion inlet aperture 207 of the chamber 11.

[0050] En route to the ion inlet aperture 207, the ions pass through several electrostatic lenses, some of which are necessary for differential pumping concerns, while others attempt to shape and direct the beam itself. FIG. 2B schematically depicts a common lens configuration. The purpose of lens 233, which comprises individual electrodes 233a, 233b, 233c and 233d, is to offset the ion trajectory 231 by an offset distance, A / inj, generally 3 mm in practice, which helps to prevent neutral gas molecules from entering the vacuum chamber interior through ion inlet aperture 207. Generally, electrodes 233a and 233d are maintained at ground potential during ion packet injection while electrodes 233b and 233c are maintained at a same voltage, o, the polarity of which depends on the polarity of the ions being injected. For example, assuming that the injected ions are positively charged, the voltage, Vo may be approximately -300 V. Provided that the field strength that is experienced by ions between the electrode pair 233a, 233b is identical to the field strength the that the ionsexperience between electrode pair 233c and 233d, then the ions enter and exit lens 233 without a change in direction.

[0051] As also shown in FIG. 2B, an Einzel lens 237 (Szilagyi, M., Electron and Ion Optics, Plenum Press, 1988), which comprises apertured electrode plates 237a, 237b and 237c, focuses the beam towards the ion inlet aperture 207. Generally, electrode plate 237b may be maintained at approximately 1200 V (depending on specific configurations) while electrode plates 237a and 237c are maintained at ground potential. A second function of lens 237 is to counteract any beam expansion that might have occurred between the exit of lens 233 and the entrance of lens 237. Finally, a deflector electrode 234, which is mounted adjacent to the ion inlet aperture, may be employed to cause the trajectory 26 of each packet of ions that is introduced into the vacuum chamber to intersect the trajectory of the ion beam 16 at a non-perpendicular angle (i.e., at an angle other than what is depicted in the example of FIG. 2A). By appropriate control of the pulsing of packets of ions by the C-trap 217 as well as of the lenses 237 and 234, a spatially focused set of ions may be introduced into the electron beam 16 along a trajectory that overlaps the electron beam over a greater distance than would be otherwise possible using a ninety-degree intersection angle between the ion beam 26 and the electron beam 16.

[0052] As noted above, a pulsed laser 90 may be employed within the particle beam analysis system 200. United States patent number 9,279,778, in the names of Beckman et al., describes that intense electrical fields generated by elliptically polarized infrared laser light may be employed to precisely orient thousands of sample ions simultaneously without otherwise heating or exciting the sample ions. Patent 9,279,778 further describes that such alignment may persist for as long as 50 microseconds. Such alignment greatly facilitates the interpretation of electron diffraction patterns that may be obtained as the ions propagate through the electron beam. An Nd-YAG laser that generates wavelength of 1064 nm may be used for the purpose of orienting molecules or ions. This wavelength is found to not significantly heat or perturb the structure of the sample ions. It has also been found that the elliptical polarization of the laser may bechanged by a selected amount to rotate sample particles to obtain diffraction from multiple orientations. In operation of the system 200, the laser 90 may be pulsed in synchronization with the pulsing of the electron beam as well as with the passage of ion packets through the electron beam such that the packets, electron pulses and laser pulses simultaneously intersect along the axis 19. It should be noted that the laser 90 may also be optionally employed within the particle beam analysis system 100 shown in FIG. 1.

[0053] FIG. 3 is a schematic cross-sectional depiction of a of a portion of another embodiment of a particle beam analysis system in accordance with the present teachings. The system 300 that is depicted in FIG. 3 provides a particle beam or molecular beam 26m that intersects an electron beam 16 within a vacuum chamber 13 of an electron microscope. According to the particular non-limiting example that is depicted in FIG. 3, a liquid sample 322 volatilizes to release gas molecules that are introduced into the interior 13 of the vacuum chamber 11. The driving force for generating the particle or molecular beam is a pressure differential between a sample 322 held in a sealed container and the vacuum chamber interior 13. A set 305 of differentially evacuated chambers, exemplified by the three illustrated evacuated chambers 5a, 5b and 5c having flow-restricting apertures 6a, 6b between them, may be interposed between the sample and the internal environment of the vacuum chamber 11. As is well known, the chamber pressure may be progressively reduced by each flowrestricting aperture, with each of the chambers evacuated chambers 5a, 5b and 5c being coupled to a respective vacuum pump (not shown) by a respective vacuum conduit 7a, 7b, 7c.

[0054] The particle or molecular beam 26m is introduced into the vacuum chamber by a Micro-Electromechanical System (MEMS) emitter device 301e that comprises an internal gas channel and that is fluidica I ly coupled to the evacuated chambers 305 by a capillary 301. Either the emitter device 301e or the capillary 301 or a portion of either or both may be oriented at an angle that is not perpendicular to the axis 19 of the electron beam 16 (e.g., hypothetical tilted beam 26t) such that the total length ofoverlap and interaction between the tilted molecular beam 26t and the electron beam16 is increased. As described previously, a pulsed laser 90 may be employed to align molecules of the particle or molecular beam 26m, 26t.

[0055] FIG. 4 is a schematic cross-sectional depiction of a portion of a fourth embodiment of a particle beam analysis system in accordance with the present teachings. In the particular embodiment that is depicted in FIG. 4, a liquid sample is contained within an ampoule 401 that is completely sealed except for a connection to a capillary. A flow of liquid is directed into the capillary 402 by capillary action and is thereby directed into a MEMS emitter device 403. The liquid may itself be an analyte. Alternatively, the liquid may comprise an analyte that is dissolved in the liquid.

[0056] FIG 5A depicts a plan view of a first exemplary emitter device 403a as may be employed in the system 400 (FIG. 4). The emitter device 403a comprises a wafer 451 having an opening 452 therethough for passage therethrough of an electron beam 16. The wafer 451 also comprises a channel (not shown) into which the capillary 402 is affixed. The capillary delivers a flow of the liquid into an internal channel of a nozzle 462 which emits a spray of droplets 2 into the cutout 452 such that the droplets pass through the electron beam 16 for analysis as described previously herein. As taught in patent publications WO2023023231A1, WO2022226279A1, WO2021217076A1 as well as others, to enable reliable spraying into vacuum, a diameter or height (for non-circular cross-section) of the tip of the nozzle 462 does not exceed 1 urn, preferably does not exceed 100 nm and, most preferably, does not exceed 50 nm. In an alternative embodiment, the ampoule 401 may be replaced by a sealed cavity (not shown) within or on the wafer 451 such that the liquid sample is provided directly to the emitter nozzle 462.

[0057] An alternative emitter device 403b, as may be employed in the system 400 (FIG. 4) is depicted in plan view in FIG. 5B. Additional cross-sectional views through the alternative emitter device 403b corresponding to sections A-A', B-B' and C-C' are shown in FIGS. 6A, 6B and 6C, respectively. In contrast to the emitter device 403a of FIG. 5A,the emitter device 403b of FIG. 5B comprises a wafer 461 having a single aperture 455 therethrough for passage of the electron beam 16, the aperture being just large enough to temporarily stabilize the position of a single droplet 2' of a plurality droplets 2 as the various droplets are introduced from capillary 402 into and through the aperture 455.

[0058] FIG. 6D is a portion of a plan cross-sectional view of a modified version 403c of the MEMS device 403b that has been discussed above in reference to FIG. 5B and FIGS. 6A through 6C. In contrast to the previously-discussed MEMS device, the device 403c further includes at least one of two possible windows 463a, 463b that provide partial containment for a droplet or particle within the aperture 455. The window or windows is / are preferably less than 100 nm thick so as to be transparent to electrons of the electron beam 16. The skilled artisan would recognize that there are variety of materials from which such windows may be constructed.

[0059] Because of the possible generation of a plurality of various differing ion species within the beam intersection region 24 (i.e., as a result of charge-state modification and / or ion fragmentation), the diffraction patterns of different ion species may overlap one another. One means of preventing this is to control electron beam pulses (in the case of a pulsed electron beam) or the sparsity of particles (corresponding to the average distance between particles) that pass through a continuous beam such that, preferably, each diffraction image corresponds to a single particle. In case of overlapping diffraction patterns, it may be necessary to separate (i.e., "deconvolute") the individual overlapping diffraction patterns from one another. This deconvolution may be aided by making use of supplemental ion-species mass and charge state data derived from the mass spectrometer 35, if present. Such mass spectrometric data may be obtained both when the electron beam is present and when it is blanked, in order to recognize and distinguish instances of ion fragmentation and charge state reduction. The deconvolution may also be performed by comparison to synthetic diffraction patterns calculated from hypothetical molecular structural models that are generated from either previously-experimentally-determined structures or are generated by trained artificial intelligence classifiers. Once a diffraction pattern of a particular ionspecies has been extracted from the electron diffraction data, it may then be used to generate or validate a three-dimensional (3D) model of an ion species or a neutral molecule.

[0060] In order to achieve the goal of efficiently capturing a plurality of electron diffractograms of adequate quality with high duty cycle and without causing significant changes in or damage to (e.g., fragmentation) of ions as they pass through the pulsed electron beam, it is necessary to employ a suitable pulsed electron source. Recently, Kim et al. (Kim, H. Y., M. Garg, S. Mandal, L. Seiffert, T. Fennel, and E. Goulielmakis. "Attosecond field emission." Nature 613, no. 7945 (2023): 662-666) have described a suitable electron source in which sub-cycle light transients are employed to induce optical field emission of electron pulses from tungsten nanotips. The laser-driven pulse repetition rate of this electron source is 1 Megahertz. Importantly, the width of the pulses is in the sub-picosecond range (e.g., 400 femtoseconds), which implies that each electron diffractogram can be acquired in a time increment that is less than the time within which radiation damage can occur. Further each pulse comprises approximately 1000 electrons with an energy spread of approximately 250 eV, which is suitable for obtaining diffractograms of adequate quality.

[0061] In view of the above discussions, FIG. 7 is a flow diagram of a first method, method 70, for operating a charged particle beam analysis system, in accordance with the present teachings, so as to analyze the structures of neutral molecules and, possibly, other uncharged particles. The first step, step 71, of the method 70 comprises delivering a liquid or gaseous sample to a Micro-Electromechanical System (MEMS) emitter device that is disposed within an evacuated chamber of an electron microscope. For example, the sample may be a gas sample, that is delivered to a MEMS emitter 301e that comprises an internal gas channel and that is f luidica Uy coupled to external differentially pumped evacuated chambers 305 by a capillary 301. In this case, a gas pressure difference between the laboratory environment and the interior of the evacuated electron microscope chamber drives the flow of gas. As another example, the sample may be a liquid sample that is delivered to a MEMS emitter nozzle through acapillary under the influence of capillary action. In this latter case, the MEMS emitter device may be constructed as shown in either FIG. 5A or 5B.

[0062] In step 72 of the method 70 (FIG. 7), molecules or other particles of interest are delivered from the MEMS emitter along a trajectory that intersects an electron beam path along which a pulsed electron beam is propagated. At each pulse, a respective diffraction pattern is recorded that pertains to the molecules / particles through which the electron pulse passes. All or a portion of the MEMS emitter device may be oriented at an angle that is not perpendicular to the axis 19 of the pulsed electron beam 16 such that the total length of overlap and interaction between the path of the molecules / particles and the electron beam 16 is increased. Preferably, the pulses of the electron beam are sufficiently short duration (e.g., 10-100 picoseconds or shorter) such that the interactions between the electrons and the molecules / particles do not cause changes to the molecular structures of the molecules / particles of interest and molecules do not have time to rotate noticeably and smear the diffraction pattern. Under such circumstances, there is not a need to encapsulate the ions within a cryogenic liquid, as is frequently done to prevent structural damage. Also in step 72, a pulsed laser may be disposed so as to deliver elliptically polarized laser light pulses to the ions as they pass through the pulsed electron beam, where the laser pulses are delivered, in synchronicity with the electron beam pulses, to the intersection region bean electron beam and particle beam pathways. The laser pulses can serve to mutually align the ions as they pass through the electron beam, thereby facilitating subsequent interpretation of electron diffraction patterns. The laser should comprise a wavelength that does not alter the structures of the molecules or particles.

[0063] In operation, some electrons of each electron beam pulse will encounter and pass through molecules or other particles of interest within the region of intersection of the electron and ion beams. Such electrons are then caused to pass through an objective lens of the microscope that is focused such that an image of an electron diffraction pattern is captured on an imaging detector. After acquiring such data, the laser polarization may be rotated, if appropriate, in step 73, so as to modify thealignment of subsequent packets of molecules as they pass through the electron beam and while electron diffraction data relating to the subsequent packets is recorded. These data may be later co-added and / or otherwise analyzed in subsequent steps 74 and 75.

[0064] FIG. 8 is a flow diagram of a method, method 80, for operating a charged particle beam analysis system, in accordance with the present teachings, for analyzing the structures of ions. The first step, step 81, of the method 80 comprises generating ions and possibly other particles from a sample of interest. Any one of several well- known atmospheric pressure ionization techniques and other ionization techniques may be employed for carrying out this step. Especially preferable are so-called "soft" ionization techniques that generate ionized intact molecules, without significant insource fragmentation, such ionized intact molecules often referred to as "molecular ions". Such ionization techniques include, without limitation, electrospray ionization (ESI), heated electrospray ionization (HESI), thermospray ionization, paper spray ionization, atmospheric pressure chemical ionization (APCI), matrix assisted laser desorption ionization (MALDI), as well as others. Generally, the sample that is to be ionized by atmospheric pressure ionization comprises one or more compounds of interest dissolved in a suitable solvent or, in the case of MALDI, embedded in a suitable matrix material.

[0065] In the next step, step 82 of the method 80, a particle stream that is emitted from the ion source is manipulated in various ways in preparation for transporting ions into an electron microscope column. In the above-described apparatus embodiments, such manipulations may be performed in an ion conditioning module 202 and / or an ion transport module 206. For example, the particle stream may be entrained in a fluid transport medium comprising molecules of ambient laboratory gas as well as molecules of residual solvent from which the ions of interest must be separated or liberated. An ion transfer tube 25, which may be heated, and an ion focusing and gas separation apparatus 203 may be provided for this purpose. The lumen of the ion transfer tube serves as a gas restricting aperture, thereby allowing a vacuum to be maintained withinthe ion conditioning module 202. The heater, if present, aids in volatilizing residual solvent and in desolvating ions. The ion focusing and gas separation apparatus 203 removes most remaining gas and may also serve, in conjunction with not-illustrated ion lenses, to focus the ions into a collimated beam. The mass filtering apparatus 210 may be used to select ions of particular interest by eliminating ions, in accordance with their mass-to-charge ratios, that are not of interest (or that are not of immediate interest). The ions that are eliminated may be ions generated from compounds in a sample 22 that are co-dissolved with a particular compound of immediate interest. The eliminated ions may also include different ion species, all from the compound of immediate interest, that are other than (e.g., have different charge states than) a particular ion species of immediate interest. Further, an optional mass analyzer 214 may be employed to verify the identity (i.e., mass-to-charge ratio or ratios) of the selected ions. Finally, the ion conditioning module 202 and / or ion transport module 206 may be employed to reduce the pressure within which the ions are carried to a desired operating pressure of an electron microscope column.

[0066] In the next step, step 83, of the method 80, the ions are introduced into a Transmission Electron Microscope column along a direction that crosses the path of an electron beam which may be a pulsed beam or a continuous beam. The ions may be introduced into the TEM column through an ion entrance aperture of an evacuated chamber of the electron microscope. The introduction of ions may be facilitated by pulsing packets of ions out from an ion trap, such as a C-trap 217 of the ion conditioning module 202 that temporally accumulates the ions into the various packets. Additional steering and focusing electrodes between the ion conditioning module and the TEM column may be employed to focus and direct the packets of ions along a desired trajectory that maximizes their interaction with an electron beam.

[0067] Inside the TEM vacuum chamber, additional ion lenses may be employed to cause the ions to be focused into a beam that crosses the beam path of the pulses of a pulsed electron beam. Preferably, the pulses of the electron beam are sufficiently short duration (e.g., 10-100 picoseconds or shorter) such that the interactions between theelectrons and the ions do not cause changes to the ions' structure. Under such circumstances, there is not a need to encapsulate the ions within a cryogenic liquid. Optionally, a pulsed laser may be disposed so as to deliver, in synchronicity with the electron beam pulses, elliptically polarized laser light pulses to the ions as they pass through the pulsed electron beam. The laser pulses can serve to mutually align the ions as they pass through the electron beam, thereby facilitating subsequent interpretation of electron diffraction pattern.

[0068] In operation, some electrons of each pulse will encounter and pass through free ions and / or neutral molecules within the region of intersection of the electron and ion beams. Such electrons are then caused to pass through an objective lens of the microscope that is focused such that an image of an electron diffraction pattern is captured on an imaging detector. If the electron beam is pulsed, the imaging detector separately captures electron diffraction pattern data of each pulse such that there is a corresponding image for each respective pulse, where each pulse preferably corresponds to passage of a single particle through the electron beam pulse.Alternatively, if the beam is not pulsed, the sparsity of particles that pass through the beam and their velocity through the beam may be adjusted so that, preferably, each electron diffraction exposure corresponds to passage of a single particle through the continuous electron beam. After acquiring such data, the laser polarization may be rotated, if appropriate, in step 87, so as to modify the alignment of subsequent packets of ions as they pass through the electron beam and while additional electron diffraction data relating to the subsequent packets is recorded. These data may be later co-added and / or otherwise analyzed in subsequent steps 85 and 86.

[0069] In optional step 84 of the method 80, ions that pass through the region of intersection of the electron and ion beams may be re-focused and passed out of the TEM column through a tube lens to an ion inlet port of a mass spectrometer or a simple mass analyzer. The mass spectrometer or mass analyzer may then generate one or more mass spectra of the ions to provide information that enables identification of the various ion species that may be present in the ion beam. For comparison purposes,such mass spectra may be obtained from ions that pass through the region of intersection during electron beam pulses as well as from ions that pass through the region of intersection between electron beam pulses. Mass analyses may be performed either prior to the entry of particles into the TEM vacuum chamber 11 by mass analyzer 214, if present, and / or after passage of the particles out of the TEM vacuum chamber 11 by mass spectrometer or mass analyzer 35, if present.

[0070] Regardless of whether a structural investigation relates to neutral molecules / particles (e.g., FIG. 7) or to ions (e.g., FIG. 8), there will be instances in which a sample 22 may comprise more than one compound of interest. As but one example, if proteins are being analyzed and if the sample 22 is an extract from a biological sample, then, in the absence of extensive pre-purification, the sample may comprise a plurality of protein components as well as non-protein components. To prevent multiple different protein (or other) molecules from being simultaneously delivered to the ion source 25, a chemical component fractionation device (not specifically depicted in FIG. 1), such as a chromatography column, a capillary electrophoresis device, a solid-phase extraction device, etc., may be interposed between the sample 22 and the ion source 21. In such configurations, a series of different compounds may elute from the fractionation device at different respective times. Accordingly, if there is more than one compound of interest, the analysis system may optionally be configured to analyze another compound of interest by repeating the either steps 71-74 (FIG. 7) or steps 81-83 and, optionally, step 84 (FIG. 8) during the time that the other compound elutes.

[0071] After data acquisition, in a first optional data analysis step, e.g., step 74 of the method 70 or step 85 of the method 80, any potentially overlapping electron diffraction patterns that may result from the presence of multiple different ion or molecule species are mathematically deconvolved into their various component patterns, thereby generating a plurality of diffraction patterns, each of which corresponds to a respective ion species, possibly making use of mass spectral data as possibly acquired in optional step 84. In the final step, e.g., step 75 or step 86, one or more three-dimensional (3D) structures of ions or molecules are calculated from the deconvoluted electrondiffraction patterns. This step may also use constraints derived from the mass spectral data.

[0072] The discussion included in this application is intended to serve as a basic description. The present invention is not intended to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention. Functionally equivalent methods and components are within the scope of the invention, as defined by the claims. Various embodiments of methods and apparatuses that are in accordance with the present teachings may be described by one or more of the following listed items.

[0073] Item 1: A method of analyzing a sample by an electron microscope comprising: generating ions from a sample of interest; introducing the ions into an evacuated chamber of a Transmission Electron Microscope (TEM) in a direction that intersects the path of a pulsed electron beam; recording a series of electron diffraction patterns pertaining to ions that pass through and interact with the pulsed electron beam; and calculating a three-dimensional (3D) structure of an ion species from the recorded electron diffraction patterns.

[0074] Item 2: A method as recited in item 1, wherein the ions are preferably not encapsulated within liquid helium, liquid nitrogen or liquid argon or, more preferably, are not encapsulated within any other liquid or frozen material.

[0075] Item 3: A method as recited in item 1, wherein the ions are not encapsulated in vitreous water ice.

[0076] Item 3: A method as recited in item 1, wherein the ions that pass through and interact with the pulsed electron beam are not supported on a solid substrate.

[0077] Item 5: A method as recited in item 1, wherein the ions that are introduced into the evacuated chamber are introduced into the chamber as separated packets of ions.

[0078] Item 6: A method as recited in item 5, wherein the packets of ions are generated by: accumulating and trapping a quantity of ions within an ion trap; and pulsing the accumulated quantity of ions out of the trap by changing an electrical potential applied to electrodes of the ion trap.

[0079] Item 7: A method as recited in item 6, wherein the ion packets are directed, by one or more ion lenses, through an aperture in the evacuated chamber so as to intersect the path of the pulsed electron beam at an angle other than ninety degrees.

[0080] Item 8: A method as recited in item 1, wherein the ions that are introduced into the evacuated chamber are pre-selected, prior to their introduction into the evacuated chamber, in accordance with their mass-to-charge ratio, by a mass filtering apparatus.

[0081] Item 9: A method as recited in item 1, further comprising conducting a mass spectral analysis of ions that pass completely through the evacuated chamber to a mass spectrometer through an ion outlet aperture.

[0082] Item 10: A method as recited in item 1, further comprising directing laser pulses to a region at which the ions pass through the path of the electron beam, wherein the laser pulses are synchronized to the electron-beam pulses.

[0083] Item 11: A method of analyzing a sample by an electron microscope that comprises: introducing gaseous molecules or liquid droplets comprising molecules into an evacuated chamber of a Transmission Electron Microscope (TEM) in a direction that intersects the path of a pulsed electron beam; recording a series of electron diffraction patterns pertaining to ions that pass through and interact with the pulsed electron beam; and calculating a three-dimensional (3D) structure of a molecular species from the recorded electron diffraction patterns, wherein the gaseous molecules or liquid droplets pass through the evacuated chamber at a temperature above the boiling point of liquid heliumor, preferably, above the boiling point of liquid nitrogen or, more preferably, above the boiling point of liquid argon.

[0084] Item 12: A method of analyzing a sample as recited in item 11, wherein the gaseous molecules or liquid droplets are introduced into the evacuated column using a Micro-Electromechanical System (MEMS) device comprising a nozzle.

[0085] Item 13: A method of analyzing a sample as recited in item 12, wherein a gas comprising the gaseous molecules or a liquid from which the liquid droplets are generated is delivered to the MEMS device through a capillary.

[0086] Item 14: A method of analyzing a sample as recited in item 12, wherein a source of the gas comprising the gaseous molecules or a source of the liquid from which the liquid droplets are generated is contained within a sealed chamber of the MEMS device.

[0087] Item 15: A method of analyzing a sample as recited in item 12, further comprising directing laser pulses to a region at which the gaseous molecules or the liquid droplets pass through the path of the electron beam, wherein the laser pulses are synchronized to the electron-beam pulses.

[0088] Item 16: An analysis system comprising: an electron microscope sub system comprising: an electron source configured to generate a pulsed electron beam; an evacuated chamber of a Transmission Electron Microscope (TEM); an objective lens within the TEM column; and an electron detector; an ion source configured to generate ions from a sample; and an ion transport module configured to direct a beam of ions generated by the ion source into the evacuated electron microscope column so as to intersect therein with an electron beam generated by the electron source;wherein the objective lens is configured to project an electron diffraction pattern of ions that pass through the electron beam onto the electron detector.

[0089] Item 17: An analysis system as recited in item 16, further comprising an ion conditioning module that is configured to receive the ions and / or charged droplets from an ionization source and to process the ions prior to introducing them into the ion transport module.

[0090] Item 18: An analysis system as recited in item 17, wherein the ion conditioning module comprises and ion focusing and gas separation apparatus;

[0091] Item 19: An analysis system as recited in item 17, wherein the ion conditioning module comprises a mass filtering apparatus.

[0092] Item 20: An analysis system as recited in item 17, wherein the ion conditioning module comprises an ion cooling apparatus.

[0093] Item 21: An analysis system as recited in item 17, wherein the ion conditioning module comprises a mass analyzer.

[0094] Item 22: An analysis system as recited in item 16, further comprising a laser configured to direct laser pulses to a region at which the ions pass through the path of the pulsed electron beam.

[0095] Item 23: An analysis system as recited in item 16, further comprising a C-trap configured to repeatedly accumulate and temporarily store ions and to periodically deliver stored ions to the ion transport module as separate packets of ions.

[0096] Item 24: An analysis system comprising: an electron microscope sub system comprising: an electron source configured to generate a pulsed electron beam; an evacuated chamber of a Transmission Electron Microscope (TEM); an objective lens within the TEM column; and an electron detector;a fluid emitter apparatus configured to direct a stream of gaseous, liquid or other molecule-bearing particles into the evacuated TEM column so as to intersect therein with an electron beam generated by the electron source; and a fluidic sample transport apparatus configured to transport a fluid sample from a sample source to the fluid emitter apparatus; wherein the objective lens is configured to project, onto the electron detector, an electron diffraction pattern of molecules of the stream of gaseous, liquid or other molecule-bearing particles that pass through the electron beam.

[0097] Item 25: An analysis system as recited in item 24, wherein the fluid emitter apparatus comprises a Micro-Electromechanical System (MEMS) device.

[0098] Item 26: An analysis system as recited in item 25, wherein the MEMS device comprises a nozzle.

[0099] Item 27: An analysis system as recited in item 25, wherein the MEMS device comprises an internal chamber that is configured to temporarily contain a single fluid droplet.

[0100] Item 28: An analysis system as recited in item 1 , wherein the internal chamber of the MEMS device comprises at least one window that is transparent to electrons.

[0101] Item 29: An analysis system as recited in item 25, wherein a source of the gaseous, liquid or other molecule-bearing particles is contained within a sealed chamber of the MEMS device.

[0102] Item 30: An analysis system as recited in item 24, further comprising a laser configured to direct laser pulses to a region at which the ions pass through the path of the pulsed electron beam.

[0103] Item 31: A method of analyzing a sample as recited in item 1, wherein the duration of each electron pulse is such that a rotational period of an ion is between 10 times and 3000 times longer than the electron pulse duration.

[0104] Item 32: : A method of analyzing a sample as recited in item 11, wherein the duration of each electron pulse is such that a rotational period of an ion is between 10 times and 3000 times longer than the electron pulse duration.

Claims

CLAIMSWhat is claimed is:

1. A method of analyzing a sample by an electron microscope comprising: generating ions from a sample of interest; introducing the ions into an evacuated Transmission Electron Microscope (TEM) column in a direction that intersects the path of an electron beam; recording a series of electron diffraction patterns pertaining to ions that pass through and interact with the electron beam and calculating a three-dimensional (3D) structure of an ion species from the recorded electron diffraction patterns.

2. A method of analyzing a sample as recited in claim 1, wherein the ions are generated from one or more compounds of the sample of interest that are separated from one another by fractionation.

3. A method of analyzing a sample as recited in claim 1, further comprising: selecting the ions that are introduced into the evacuated TEM column by means of a mass filtering apparatus.

4. A method of analyzing a sample as recited in claim 1, further comprising: transporting the ions from the TEM column into a mass analyzer; and mass analyzing the transported ions using the mass analyzer.

5. A method of analyzing a sample as recited in claim 1, further comprising cooling the ions prior to introducing them into the evacuated TEM column.

6. A method of analyzing a sample as recited in claim 1, wherein the electron beam is a pulsed electron beam and the recording of each electron diffraction pattern corresponds to ions that pass through and interact with a single pulse.

7. A method of analyzing a sample as recited in claim 6, wherein the ions are introduced into the TEM column either as separate particles or within separated particles and each electron diffraction pattern corresponds to a single such particle.

8. A method of analyzing a sample as recited in claim 6, wherein the durations of the pulses are adjusted to be less than the minimum interaction time required for the structures of the particles to be altered by the interactions.

9. A method of analyzing a sample as recited in claim 1, wherein the electron beam is a continuous electron beam, wherein the ions are introduced into the TEM column either as separate particles or within separated particles, and wherein the average distance between particles that pass through the TEM column is adjusted such that each electron diffraction pattern corresponds to a single such particle.

10. A method of analyzing a sample as recited in claim 9, wherein the velocity of the particles through the TEM column is adjusted such that the interaction time of the particles with the electron beam is less than the minimum time required for the structures of the particles to be altered by the interaction.

11. An analysis system comprising: an electron microscope sub system comprising: an electron source; an evacuated Transmission Electron Microscope (TEM) column; an objective lens within the TEM column; and an electron detector; an ion source configured to generate ions received from a sample; andan ion transport module configured to direct a beam of ions generated by the ion source into the evacuated electron microscope column so as to intersect therein with an electron beam generated by the electron source; wherein the objective lens is configured to project an electron diffraction pattern of ions that pass through the electron beam onto the electron detector.

12. An analysis system as recited in claim 11, further comprising: an ion conditioning module configured to receive the ions from the ion source and to deliver either conditioned ions or a subset of the ions to the ion transport module.

13. An analysis system as recited in claim 12, wherein the ion conditioning module comprises one or more of: an ion focusing and gas separation apparatus, a mass analyzer, a mass filtering apparatus, and an ion cooling apparatus.

14. An analysis system as recited in claim 11, further comprising: a mass analyzer or mass spectrometer configured to receive the ions from the evacuated TEM column and mass analyze the received ions.

15. An analysis system as recited in claim 11, further comprising: a chemical component fractionation device configured to receive compounds from the sample of interest and to deliver a series of fractionated compounds to the ion source.

16. An analysis system as recited in claim 11, wherein the electron source is configured to generate a pulsed electron beam with pulses having pulse widths that are not greater than 10 picoseconds in duration.

17. An analysis system as recited in claim 16, wherein the pulsed electron source is configured to generate the pulsed electron beam with pulses having pulse widths that are not greater than one picosecond in duration.

18. An analysis system as recited in claim 16, wherein the pulsed electron source is configured to generate the pulsed electron beam with a pulse repetition rate of at least one Megahertz.

19. An analysis system as recited in claim 11, wherein the electron source is configured to generate a continuous electron beam, and the ion transport module is configured to direct the beam of ions and through the evacuated electron microscope column so as to intersect and interact with the electron beam with a velocity such that interaction between each ion and the electron beam is not greater than 10 picoseconds in duration.

20. An analysis system as recited in claim 19, the ion transport module is configured to direct the beam of ions and through the evacuated electron microscope column so as to intersect and interact with the electron beam with a velocity such that interaction between each ion and the electron beam is not greater than 1 picosecond in duration.

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