Comparing holographic imaging

Abstract

Disclosed herein are apparatus and methods for comparing holographic imaging to improve reconstructed structural and molecular information. An example method includes at least: acquiring a plurality of holograms of a sample, wherein each of the plurality of holograms is acquired at a different electron beam energy; and determining atomic and structural information of the sample based at least on a comparison of at least two of the plurality of holograms.

Description

Technical Field

[0001] The present invention relates generally to holography, and in particular to enhanced holography-based analytics. Background Art

[0002] Holographic techniques have been known for some time for imaging and studying materials. However, while developments in imaging hardware have improved, the quality and quantity of information that can be obtained from holograms has not similarly improved. In the field of biology, the study of soft materials, such as proteins, is extremely important, but the properties of soft materials limit analytical techniques. Although holography provides a less destructive analytical and imaging technique, the limited information that can be obtained from the resulting holograms and / or reconstructions limits the usefulness of holography. Thus, it is desirable to improve the information obtained from holography, especially low-energy holography, at least in the field of biological research. Summary of the invention

[0003] Disclosed herein are apparatus and methods for comparing holographic imaging to improve reconstructed structural and molecular information. An example method includes at least: acquiring a plurality of holograms of a sample, wherein each of the plurality of holograms is acquired at a different electron beam energy; and determining atomic and structural information of the sample based at least on a comparison of at least two of the plurality of holograms.

[0004] The example apparatus includes at least: an emitter coupled to emit an electron beam toward a sample; a detector coupled to receive the electron beam after interacting with the sample; and a controller coupled to the emitter and the detector. The controller includes code that, when executed, causes the controller to: acquire a plurality of holograms of the sample with the electron beam, wherein each of the plurality of holograms is acquired at a different electron beam energy; and determine atomic and structural information of the sample based at least on a comparison of at least two of the plurality of holograms. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 is an example sample preparation and analysis system according to an embodiment of the present disclosure.

[0006] Figure 2 Detailed description is given of an exemplary analysis subsystem for acquiring a hologram of a sample according to an embodiment of the present disclosure.

[0007] Figure 3 Includes sample images.

[0008] Figure 4 An example method for acquiring a hologram and determining structural and atomic information of a sample according to an embodiment of the present disclosure.

[0009] Figure 5is an example image sequence according to an embodiment of the present disclosure.

[0010] Figure 6 is an exemplary functional block diagram according to an embodiment of the present disclosure.

[0011] Like reference numerals refer to corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION

[0012] Embodiments of the invention are described below in the context of an electron holographic system. In particular, embodiments are directed to an electron holographic system capable of acquiring multiple holograms of a sample to determine sample molecular and / or structural information based on a comparison of at least two of the holograms. In such embodiments, each hologram is acquired at a different illumination energy. Because elastic and inelastic scattering are energy dependent, holograms at different illumination energies provide different holographic information that, when compared, provide more detailed structural and molecular information of the sample.

[0013] As used in this application and in the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. In addition, the term "comprising" means "including." Furthermore, the term "coupled" does not exclude the presence of intervening elements between the coupled items.

[0014] The systems, devices, and methods described herein should not be construed as limiting in any way. Indeed, the present disclosure is directed to all novel and non-obvious features and aspects of the various disclosed embodiments, either individually or in various combinations and sub-combinations formed with each other. The disclosed systems, methods, and devices are not limited to any specific aspect or feature or combination thereof, nor do the disclosed systems, methods, and devices require the existence of any one or more specific advantages or the resolution of any one or more specific problems. Any theory of operation is for ease of explanation, but the systems, methods, and devices of the present disclosure are not limited to such theory of operation.

[0015] Although the operations of some of the disclosed methods are described in a particular sequential order for ease of presentation, it should be understood that unless the specific language set forth below requires a specific ordering, this description encompasses rearrangement. For example, in some cases, the operations described sequentially may be rearranged or performed simultaneously. In addition, for simplicity, the accompanying drawings may not show the various ways in which the disclosed systems, methods, and devices can be used in conjunction with other systems, methods, and devices. In addition, this specification sometimes uses terms like "generating" and "providing" to describe the disclosed methods. These terms are high-level abstractions of the actual operations performed. The actual operations corresponding to these terms will vary depending on the specific implementation and are easily discernible by those of ordinary skill in the art.

[0016] In some instances, values, procedures, or devices are referred to as "lowest," "optimal," "minimum," etc. It should be understood that such descriptions are intended to indicate that a selection may be made among a number of functional alternatives used, and that such selection is not necessarily better, lesser, or otherwise preferred than other selections.

[0017] Direct electron imaging using an electron beam can be used to obtain a hologram of a sample. The electrons in the electron beam form reference waves and scattered waves from the object of interest (e.g., a protein) that will interfere. The interference pattern can then be captured on a detector further down the optical axis. This arrangement is also known as coaxial holography. Known digital holographic techniques can be used to numerically reconstruct the outgoing wave from the detected hologram, thereby producing a kind of "silhouette" of the object. Both the elastic and inelastic scattering cross sections of atoms exhibit energy dependencies that can be exploited to obtain a deeper understanding of the sample being imaged. However, a single hologram may not provide the deeper understanding desired.

[0018] By capturing holograms at two or more wavelengths (e.g., energies), it is possible to infer information about the object by comparing the holograms and / or their reconstructions; more information can be inferred than from just the individual holograms / reconstructions. For example, higher resolution can be obtained by applying a type of deconvolution, and / or atomic differences can be detected. At some energies, the object will be more opaque to the coherent electron beam because of the energy-dependent scattering cross section. As a result, different depths of the object will be probed by different energies, where "depth" is in the same direction as the optical axis. Known deconvolution techniques applied to differences in holograms or reconstructions should reveal z-depth information for the atoms that form the sample. In addition, the elastic scattering cross sections of atoms are energy-dependent, and there are some differences between known substances (e.g., H and C), which can allow identification of individual atoms that form the sample. By comparing the reconstructed intensities of the holograms, it is possible that material differences can be observed.

[0019] Figure 1is an example sample preparation and analysis system 100 according to an embodiment of the present disclosure. The system 100 is capable of preparing various samples for subsequent analysis including holographic analysis techniques. Although at least the analysis aspect can be used on any desired sample type, the sample preparation and delivery aspects may be more applicable to soft sample types that tend to be fragile, such as biological materials. In some embodiments, the sample is a protein or biomolecule that is desired to be analyzed in a native state. In such embodiments, the system 100 ionizes the protein, filters the desired protein from the ionized sample supply, and transports the ionized protein to an analysis area, in which the protein is deposited onto a substrate for analysis. The deposition can be performed to reduce or eliminate any damage to the sample material. Although the discussion uses biological materials as example samples, other sample types are also within the scope of the present disclosure.

[0020] The system 100 may include at least a sample preparation and selection subsystem 102, a sample transport and deposition subsystem 106, an analysis subsystem 104, and a controller 107. Although not shown, various vacuum pumps and power supplies may be coupled to at least some of the subsystems and components to provide a desired operating environment. In some embodiments, the three subsystems 102, 104, and 106 may form an overall sample preparation and analysis system for imaging a sample, wherein the overall system includes a continuous path from sample introduction to imaging / analysis. However, in other embodiments, the subsystems 102 and 106 may form an overall system for preparing a sample and delivering the sample to a substrate, which may then be delivered to a selected analysis tool by any desired means (e.g., a stand-alone holographic system). Regardless of the embodiment, the holographic techniques discussed herein may be implemented to obtain more detailed sample information than general holographic analysis.

[0021] Subsystem 102 performs various functions on an input sample supply to provide a desired sample. In some embodiments, the sample is a protein, such as lysozyme, calmodulin, protein A / G, OmpF porin (an outer membrane protein of E. coli (1)), monoclonal antibody immunoglobulin (IgG), C-reactive protein (CRP), streptavidin, and human serum albumin, to name a few. Of course, the previous list of proteins is included as an example and is not limiting of the present disclosure. The various functions provided by subsystem 102 include at least ionizing the input sample supply and filtering or selecting a target sample type from the sample supply. An example subsystem 102 may be a Thermo Scientific™ Q Exactive™ mass spectrometer. In some embodiments, the filtered, ionized sample may be kinetically cooled to reduce the total kinetic energy of the sample. After cooling, the sample is provided to subsystem 106. In embodiments where the sample is a protein, it may be desirable for the protein to be in a desired native state after preparation and filtration, which may be demonstrated in some embodiments. Of course, in other embodiments, the protein need not be in a desired native state and the sample need not be a protein. Discussion of samples composed of proteins is for illustrative purposes only and is not intended to be limiting in any way.

[0022] Subsystem 104 may include a direct imaging system, such as a coaxial or reflection holographic imaging system, which includes at least an emitter and a detector. The direct imaging system may include a charged particle beam emitter, such as an electron emitter or a light emitter such as a laser. Regardless of the type of emitter, subsystem 104 is capable of adjusting the emitted energy so that the resulting illumination beam includes charged particles or photons of the desired energy / wavelength / frequency. The detector may be a pixel-based solid-state detector or a microchannel phase plate detector, but any detector compatible with the emitter is contemplated herein. In some embodiments, the sample may be placed between the emitter and the detector for coaxial holography. In other embodiments, the emitter, sample, and detector may be arranged for reflection holography. Of course, other arrangements are also contemplated herein and are within the scope of the present disclosure.

[0023] In some embodiments, subsystem 104 is a direct electron imaging system that generates an electron interference image of a sample. In such embodiments, the electron interference image, such as a hologram, can be used to form a reconstructed image of the sample. Subsystem 104 generates a hologram using illumination of different energies, so that multiple holograms of the sample, each at different energies, can be obtained. Due to the nature of the interaction of the illumination energy with the sample, each hologram can provide information about different qualities of the sample. As a result, once there are two or more holograms of the sample acquired, the holograms (or reconstructed images based thereon) can be compared to obtain more detailed information about the sample, such as structural and / or atomic information of the sample that may not be available based on a single hologram. Structural information can be the depth of atoms and complexes that form the sample, the depth being in the illumination direction, wherein the atomic information may include the identification and / or binding energy of the atoms that form the sample. In general, subsystem 104 is not limited by other subsystems, and subsystems 102 and 106 can be used to select samples for any final analysis technique and deposit the samples.

[0024] Subsystem 106 provides sample transport and deposition processes for the prepared sample provided by subsystem 102. In some embodiments, the sample may be ionized by subsystem 102, and in such embodiments, subsystem 106 may include a plurality of ion transport stages coupled together, the transport stages terminating at the deposition location. The terminal of the transport stage may include an ion optical device configured to slow down the speed of the sample ions so that they can be deposited on the substrate without destruction or at least with minimal destruction. For example, in some embodiments, it may be necessary to deposit proteins for subsequent analysis at an energy that reduces or prevents protein denaturation. Preferably, the ion energy does not exceed 30 eV / charge or more preferably 10 eV / charge when deposited.

[0025] Additionally or alternatively, some embodiments of system 100 may not have subsystem 102 coupled to subsystem 104 via subsystem 106. In such embodiments, the sample may be transported from subsystem 106 to subsystem 104 by a user, for example manually. For example, an ionized protein sample is deposited onto a substrate, which is then removed from subsystem 106 before insertion into subsystem 104. In some embodiments, the substrate of subsystem 106 is located in an analysis position. In such embodiments, the sample is deposited onto the substrate and is ready for analysis in the position. However, in other embodiments, the substrate is translated from the deposition position to the analysis position.

[0026] Controller 107 is coupled to other subsystems of system 100 to control and / or initiate their individual and carefully designed operations. Controller 107 may be one or more processing cores or microcontrollers and associated memory and storage devices for storing executable code. In addition to controlling the operation of system 100, controller 107 may also process data generated by analysis subsystem 104. Of course, the processing of this data may be performed alternatively or additionally by an offline computer. The processing of data may include generating a reconstructed image from one or more holograms acquired by subsystem 104, and / or comparing two or more holograms or reconstructions of a sample to determine the structure and / or atomic information of the sample. The structural information may be depth and / or position information of the components (e.g., atoms and molecules) that form the sample, and the atomic information may include identification of individual atoms and / or binding information of the components that form the sample. The comparison may include various mathematical operations, such as deconvolution, convolution, addition, subtraction, division, multiplication, averaging, and combinations thereof. In general, obtaining holograms of different energies and then comparing those holograms may provide more detailed sample information than a single hologram can provide.

[0027] In operation, a sample supply is provided to subsystem 102. The sample supply, which may be in solid or liquid form, includes, for example, a target protein together with other carrier substances and / or impurities. Subsystem 102 may initially ionize the sample supply, for example, by electrospray ionization, in preparation for filtration. Filtration may then be performed to extract the target protein from the sample supply. For example, the target protein may be filtered using a mass-to-charge ratio technique, for example, by a mass spectrometer. Once the ionized protein has been prepared, the ionized protein is kinetically cooled and then transported to a substrate via subsystem 106. In order to ensure that the ionized protein remains in its native state, a soft landing technique is used, for example, to deposit the ionized protein onto the substrate. After deposition, one or more holograms of the sample may be formed by subsystem 104. For example, multiple holograms of the sample may be formed, each of which is formed using a different irradiation energy. Once at least two holograms are obtained, the controller 107 may compare the holograms to obtain more detailed structural and / or atomic information about the sample, such as the depth and atomic identification of the components that form the sample. Acquiring multiple holograms, each at a different energy, and then comparing the holograms provides more detailed sample information than a single hologram can provide. As mentioned, the energy dependence of elastic and inelastic scattering of atoms allows differences between holograms at different energies to provide a deep understanding of the internal structure and composition of the sample.

[0028] Figure 22 is an example analysis subsystem 204 for acquiring a hologram of a sample according to an embodiment of the present disclosure. The analysis subsystem 204 (only one example of the analysis subsystem 104) can be used to obtain a hologram of the sample under different illumination energies. As mentioned, holograms are obtained under different illumination energies, and structural and atomic information about the sample can be determined by comparing different holograms. The comparison may involve deconvolution, convolution, multiplication, division, addition, subtraction averaging, and combinations thereof. The structural and atomic information may include at least depth information, atomic identification, and binding information of the components that form the sample. The components that form the sample include atoms, molecules, and / or other complexes that make up the sample.

[0029] Subsystem 204 includes at least an emitter 208 and a detector 210. Sample 212 may be located between emitter 208 and detector 210 for forming hologram 220 on the surface of the detector. Such an arrangement forms a coaxial holographic system, but the present disclosure covers other arrangements. Emitter 208 may be any desired type of emitter, such as a charged particle emitter (e.g., electron or ion), or a photon-based emitter (e.g., laser). In some embodiments, emitter 208 is an electron emitter capable of providing a coherent electron beam with an energy from 50 to 500 eV. When referring to the energy of the electron beam, it should be understood that each electron forming the electron beam has the desired energy. Regardless of the type of emitter, the irradiation wave front 214 propagates toward sample 212. When wave front 214 reaches sample 212, elastic and inelastic interactions occur with the sample (e.g., with the components forming the sample), so that the un-diffracted wave 216 and the diffracted wave 218 interact at the detector 210 to form a hologram 220. The hologram 220 may also be referred to as an interference pattern.

[0030] In some embodiments, the emitter 208 can be adjusted to provide an irradiation beam of different energies. For example, the emitter 208 can provide an electron beam of electrons of any desired energy, such as 50 eV, 75 eV, 100 eV, etc. However, the energy level may be limited by the material type of the sample 212. For example, biological materials (such as proteins) can limit the electron energy to 250 eV or less. In the case where each irradiation is at a different energy, the resulting hologram 220 may be different. However, the difference may allow the structure and / or atomic information of the sample to be determined. This is at least partially due to the energy dependence of the inelastic and elastic scattering cross sections of the atoms / molecules that form the sample 212. By acquiring multiple holograms 220, each at different irradiation energies, and comparing the multiple holograms 220, the determination of structural and atomic information can be derived from the hologram, thereby generating deeper / refined information of the sample 212.

[0031] Figure 3Example images 301 and 303 are included. Image 301 is an example hologram as acquired by a detector (e.g., detector 210), and image 303 is an example reconstruction of the hologram in image 301. Image 301 may have been acquired using subsystem 204 and represents hologram 220 of a protein-based sample. A controller (not shown) coupled to or included with subsystem 204 provides a reconstructed image 303 based on hologram 301. A controller, such as controller 107, generates the reconstruction using any known reconstruction technique. The dark areas in image 303 show the locations of the components that form the imaged sample. Since this is a single hologram / reconstruction, the information provided by the hologram or reconstruction is limited, such as relative two-dimensional information of the components that form the sample. However, such information can be improved using the techniques disclosed herein, that is, additional depth information (in the direction of the illumination beam) and atomic information can be obtained.

[0032] Figure 4 4 is an example method 405 for acquiring a hologram and determining the structure and atomic information of a sample according to an embodiment of the present disclosure. Method 405 can be performed by any direct imaging system. In some examples, a direct electron holography system such as system 100 can be used to implement method 405.

[0033] Method 405 may begin at process block 407, which includes acquiring a plurality of holograms of a sample, wherein each hologram is acquired using a different illumination energy. In some embodiments, the illumination is an electron beam that may be provided at varying energies, for example, from 50 eV to 500 eV or more. In other embodiments, the illumination energy is a laser.

[0034] Process block 407 can be decomposed into two separate processes, such as process blocks 415 and 417. Process block 415 includes exposing the sample to the irradiation energy multiple times, wherein each exposure has a different irradiation energy. Process block 415 is followed by process 417, which includes detecting each exposure of the sample to obtain each hologram in the plurality of holograms.

[0035] Process block 407 may be followed by process block 411, which includes determining molecular and atomic information of the sample based at least on a comparison of holograms in the plurality of holograms. The comparison may be performed using any known mathematical operation as indicated in process block 413, such as deconvolution, convolution, addition, subtraction, multiplication, division, averaging, and combinations thereof. The final result of the comparison may provide depth information of the atoms or complexes forming the sample, the depth being in the direction of illumination propagation. Additionally or alternatively, the comparison may identify specific atoms forming the material due to their different elastic and inelastic scattering cross sections, and / or information about their binding energies.

[0036] In some embodiments, process block 407 may optionally, alternatively, or additionally be followed by process block 409, which includes forming a reconstructed image of the sample for each of the plurality of holograms. Reconstruction of each hologram may be performed using any known reconstruction algorithm, such as non-iterative reconstruction using a paraxial approximation, non-iterative reconstruction using a non-paraxial algorithm.

[0037] Process block 409 may be followed by process block 419 , which includes determining molecular and atomic information of the sample based at least on the comparison of each of the reconstructed images. The comparison may be performed using any one or combination of the operations shown in process block 413 .

[0038] Figure 5 501, 503 and 404, which depict a series of holograms that are subtracted to determine structural information of a sample. Image 501 is a first hologram acquired at a first energy, image 503 is a second hologram acquired at a second energy, and image 505 is a hologram based on the subtraction of image 501 from image 503. All images are of the same sample, and as can be seen, images 501 and 503 provide information on changes in the sample and their differences shown in image 505 provide additional details that are not visible in images 501 and 505. For example, each point in image 505 shows a ring around a central feature that is not seen in the other two images.

[0039] Figure 6 is an example functional block diagram 600 according to an embodiment of the present disclosure. Figure 6 1 is a block diagram illustrating a computer system 600 that may be included in embodiments of the present invention. Computer system 600 may be an example of computing hardware included with system 100, such as controller 107, subsystems 102, 104, 106, and / or coupled servers (not shown). Computer system 600 includes at least a bus 640 or other communication mechanism for communicating information, and a hardware processor 642 coupled to bus 640 for processing information. Hardware processor 642 may be, for example, a general purpose microprocessor. Computing system 600 may be used to implement the methods and techniques disclosed herein, such as methods 301 and 401, and may also be used to obtain an image and segment the image using one or more categories.

[0040] The computer system 600 also includes a main memory 644, such as a random access memory (RAM) or other dynamic storage device, coupled to the bus 640 for storing information and instructions to be executed by the processor 642. The main memory 644 may also be used to store temporary variables or other intermediate information during execution of instructions to be executed by the processor 642. Such instructions, when stored in a non-transitory storage medium accessible to the processor 642, make the computer system 600 a special-purpose machine customized to perform the operations specified in the instructions.

[0041] Computer system 600 further includes a read only memory (ROM) 646 or other static storage device coupled to bus 640 for storing static information and instructions for processor 642. A storage device 648, such as a magnetic or optical disk, is provided and coupled to bus 640 for storing information and instructions.

[0042] The computer system 600 may be coupled to a display 650, such as a cathode ray tube (CRT), via bus 640 for displaying information to a computer user. An input device 652, including alphanumeric and other keys, may be coupled to bus 640 to communicate information and command selections to processor 642. Another type of user input device is a cursor control 654, such as a mouse, trackball, or cursor direction keys, for communicating direction information and command selections to processor 642 and for controlling cursor movement on display 650. This input device typically has two degrees of freedom in two axes, a first axis (e.g., x) and a second axis (e.g., y), which allow the device to specify a position in a plane.

[0043] The computer system 600 may implement the techniques described herein using customized hard-wired logic, one or more ASICs or FPGAs, firmware, and / or program logic that is combined with the computer system to make or program the computer system 600 into a special-purpose machine. According to one embodiment, the techniques herein are performed by the computer system 600 in response to the processor 642 executing one or more sequences of one or more instructions contained in the main memory 644. Such instructions may be read into the main memory 644 from another storage medium (e.g., storage device 648). Executing the sequence of instructions contained in the main memory 644 causes the processor 642 to perform the process steps described herein. In alternative embodiments, hard-wired circuitry may be used in place of or in combination with software instructions.

[0044] The term "storage medium" as used herein refers to any non-temporary medium that stores data and / or instructions that cause a machine to operate in a specific manner. Such storage media may include non-volatile media and / or volatile media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 648. Volatile media include dynamic memory, such as main memory 644. Common forms of storage media include, for example, floppy disks, floppy disks, hard disks, solid-state drives, magnetic tapes or any other magnetic data storage media, CD-ROMs, any other optical data storage media, any physical medium with a pattern of holes, RAM, PROM and EPROM, Flash-EPROM, NVRAM, any other memory chip or cartridge memory, content addressable memory (CAM) and ternary content addressable memory (TCAM).

[0045] Storage media are distinct from, but may be used in conjunction with, transmission media. Transmission media participate in the transfer of information between storage media. For example, transmission media include coaxial cables, copper wire, and optical fiber, including the wires that comprise bus 640. Transmission media may also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications.

[0046] Various forms of media may be involved in carrying one or more sequences of one or more instructions to the processor 642 for execution. For example, the instructions may initially be carried on a disk or solid-state drive of a remote computer. The remote computer may load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to the computer system 600 may receive the data on the telephone line and convert the data into an infrared signal using an infrared transmitter. An infrared detector may receive the data carried in the infrared signal, and appropriate circuitry may place the data on the bus 640. The bus 640 carries the data to the main memory 644, from which the processor 642 retrieves and executes the instructions. The instructions received by the main memory 644 may optionally be stored on the storage device 648 before or after execution by the processor 642.

[0047] The computer system 600 also includes a communication interface 656 coupled to the bus 640. The communication interface 656 provides a two-way data communication coupled to a network link 658, which is connected to a local network 660. For example, the communication interface 656 can be an integrated services digital network (ISDN) card, a cable modem, a satellite modem, or a modem for providing a data communication connection to a corresponding type of telephone line. As another example, the communication interface 656 can be a local area network (LAN) card for providing a data communication connection connected to a compatible LAN. A wireless link can also be implemented. In any such embodiment, the communication interface 656 sends and receives electrical signals, electromagnetic signals, or optical signals that carry digital data streams representing various types of information.

[0048] The network link 658 typically provides data communication to other data devices through one or more networks. For example, the network link 658 may provide a connection through a local network 660 to a host computer 662 or data equipment operated by an Internet Service Provider (ISP) 664. The ISP 664 in turn provides data communication services through the global packet data communication network now commonly referred to as the "Internet" 666. Both the local network 660 and the Internet 666 use electrical, electromagnetic, or optical signals that carry digital data streams. The signals through the various networks and the signals on the network link 658 and the signals through the communication interface 656 are example forms of transmission media that carry digital data to and from the computer system 600.

[0049] Computer system 600 can send messages and receive data including program code through one or more networks, network link 658 and communication interface 656. In the Internet example, server 668 can transmit the requested code for an application through Internet 666, ISP 664, local network 660 and communication interface 656.

[0050] The received code may be executed by processor 642 as it is received, and / or stored in storage device 648 or other non-volatile storage for later execution.

[0051] The embodiments discussed herein to illustrate the disclosed technology should not be considered limiting and merely provide examples of implementation. Those skilled in the art will appreciate the countless other ways in which the disclosed technology can be implemented, which are contemplated herein and are within the scope of the present disclosure.

[0052] An example method for performing comparative holographic imaging includes at least: acquiring multiple holograms of a sample using an electron beam, wherein each of the multiple holograms is acquired at a different electron beam energy; and determining atomic and structural information of the sample based on at least a comparison of at least two of the multiple holograms.

[0053] The above example method is further characterized in that the atomic information provides information about atoms forming the sample as well as bonding information.

[0054] The above example method is further characterized in that the structural information includes depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

[0055] The above exemplary method is further characterized in that the composition of the sample includes atoms and composites forming the sample.

[0056] The above example method further includes forming a reconstructed image of the sample for each hologram of the plurality of holograms to obtain a plurality of reconstructed images.

[0057] The above example method further includes determining atomic and structural information of the sample based at least on a comparison of two of the plurality of reconstructed images.

[0058] The above example method is further characterized in that the atomic information provides information about atoms forming the sample as well as bonding information.

[0059] The above example method is further characterized in that the structural information includes depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

[0060] The above example method further includes adjusting the electron emitter to vary the energy of the electron beam between acquiring each of the plurality of holograms.

[0061] The above example method is further characterized in that: acquiring multiple holograms of the sample includes at least exposing the sample to an electron beam multiple times, wherein the energy of the electron beam is different for each acquisition of a hologram in the multiple holograms; and detecting an interference pattern by a detector in response to each exposure.

[0062] The above exemplary method is further characterized in that the energy of the electron beam is varied in the range of 50 eV to 250 eV.

[0063] The above example method is further characterized in that the detector is a direct electron detector or a microchannel plate detector.

[0064] The above example method is further characterized in that determining atomic and structural information of the sample based at least on comparison of holograms in multiple holograms includes averaging, convolving, deconvolving, subtracting, adding, multiplying, dividing, or a combination thereof, the multiple holograms to determine the atomic and structural information.

[0065] An example apparatus for implementing differential holographic imaging may include at least: an emitter coupled to emit an electron beam toward a sample; a detector coupled to receive the electron beam after interacting with the sample; and a controller coupled to the emitter and the detector. The controller includes code that, when executed, causes the controller to: acquire a plurality of holograms of the sample with the electron beam, wherein each of the plurality of holograms is acquired at a different electron beam energy; and determine atomic and structural information of the sample based at least on a comparison of at least two of the plurality of holograms.

[0066] The above example apparatus is further characterized in that the atomic information provides information about atoms forming the sample as well as bonding information.

[0067] The above example apparatus is further characterized in that the structural information comprises depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

[0068] An example device for determining atomic and structural information of a sample based at least on a comparison of holograms in a plurality of holograms further includes code that, when executed by a controller, causes the controller to perform the following operations: averaging, convolving, deconvolving, subtracting, adding, multiplying, dividing, or a combination thereof, at least two of the plurality of holograms to determine the atomic and structural information.

[0069] The above example apparatus further includes code that, when executed by the controller, causes the controller to perform the following operations: forming a reconstructed image of the sample for each of the plurality of holograms to obtain a plurality of reconstructed images.

[0070] The above example apparatus further includes code that, when executed by the controller, causes the controller to: determine atomic and structural information of the sample based at least on a comparison of two of the plurality of reconstructed images.

[0071] The above example apparatus further includes code that, when executed by the controller, causes the controller to: expose the sample to the electron beam multiple times, wherein the energy of the electron beam is different for each acquisition of a hologram in the plurality of holograms; and detect an interference pattern in response to each exposure.

Claims

1. A method for holography, comprising: acquiring a plurality of holograms of the sample using an electron beam, wherein each of the plurality of holograms is acquired at a different electron beam energy; as well as Atomic and structural information of the sample is determined based at least on a comparison of at least two holograms of the plurality of holograms, wherein the atomic information provides information about the atoms forming the sample as well as binding information, and wherein identification of the atoms forming the sample is based on different elastic and inelastic scattering cross sections of the atoms. 2 . The method of claim 1 , wherein the structural information comprises depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

3. The method of claim 2, wherein the components of the sample include atoms and compounds forming the sample. 4 . The method of claim 1 , further comprising forming a reconstructed image of the sample for each of the plurality of holograms to obtain a plurality of reconstructed images. 5 . The method of claim 4 , further comprising determining atomic and structural information of the sample based at least on a comparison of two of the plurality of reconstructed images. The method of claim 5 , wherein the atomic information provides information about the atoms forming the sample and bonding information.

7. The method of claim 5, wherein the structural information comprises depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

8. The method of claim 1, further comprising adjusting an electron emitter to vary the energy of the electron beam between acquiring each of the plurality of holograms.

9. The method of claim 1, wherein acquiring a plurality of holograms of the sample comprises at least: exposing the sample to the electron beam a plurality of times, wherein the energy of the electron beam is different for each acquisition of a hologram in the plurality of holograms; as well as In response to each exposure, an interference pattern is detected by a detector.

10. The method of claim 9, wherein the energy of the electron beam varies from 50 eV to 250 eV.

11. The method of claim 9, wherein the detector is a direct electron detector or a microchannel plate detector.

12. The method of claim 1, wherein determining atomic and structural information of the sample based at least on a comparison of the holograms in the plurality of holograms comprises: The plurality of holograms are averaged, convolved, deconvolved, subtracted, added, multiplied, divided, or a combination thereof to determine the atomic and structural information.

13. An apparatus for holography, comprising: an emitter coupled to emit an electron beam toward the sample; a detector coupled to receive the electron beam after interacting with the sample; as well as A controller coupled to the emitter and the detector, the controller comprising code that, when executed, causes the controller to: acquiring a plurality of holograms of the sample using an electron beam, wherein each of the plurality of holograms is acquired at a different electron beam energy; as well as Atomic and structural information of the sample is determined based at least on a comparison of at least two holograms of the plurality of holograms, wherein the atomic information provides information about the atoms forming the sample as well as binding information, and wherein identification of the atoms forming the sample is based on different elastic and inelastic scattering cross sections of the atoms.

14. The apparatus of claim 13, wherein the structural information comprises depth information of the composition of the sample, the depth information being in the direction of the electron beam path.

15. The apparatus of claim 13, wherein the code to determine atomic and structural information of the sample based at least on a comparison of the holograms in the plurality of holograms further comprises code that, when executed by the controller, causes the controller to: The at least two holograms of the plurality of holograms are averaged, convolved, deconvolved, subtracted, added, multiplied, divided, or a combination thereof to determine the atomic and structural information.

16. The apparatus of claim 13, further comprising code that, when executed by the controller, causes the controller to: A reconstructed image of the sample is formed for each of the plurality of holograms to obtain a plurality of reconstructed images.

17. The apparatus of claim 16, further comprising code that, when executed by the controller, causes the controller to: Atomic and structural information of the sample is determined based on at least a comparison of two of the plurality of reconstructed images.

18. The apparatus of claim 13, further comprising code that, when executed by the controller, causes the controller to: exposing the sample to the electron beam multiple times, wherein the energy of the electron beam is different for each acquisition of a hologram in the plurality of holograms; and An interference pattern is detected in response to each exposure.

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