High-speed photoelectron microscopes for biological and other applications

Abstract

The imaging system includes a light source configured to emit radiation onto a biological sample, the light source being optimized for high-speed imaging of the biological sample using the photoelectric effect. The system also includes a sample holder configured to hold the biological sample in place during imaging. The system also includes a stage assembly to which the sample holder is mounted, the stage assembly moving the biological sample during imaging. The system further includes a detector configured to receive electrons emitted from the biological sample in response to radiation from the light source.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority based on the U.S. Provisional Patent Application No. 63 / 470,042 filed on 31 May 2023, the full disclosure of said application is incorporated herein by reference. [Background technology]

[0002] An electron microscope is a type of microscope that uses a beam of electrons (which have short wavelength and wave-like properties) to create a magnified image of a sample. This is in contrast to conventional optical microscopes, which use visible light to create magnified images of objects. There are several different types of electron microscopes. For example, transmission electron microscopes (TEMs) are commonly used to observe thin samples by passing electrons through the sample to generate a projection image. Scanning electron microscopes are generally used to observe samples by scanning the surface of the sample with an electron beam and generating an image based on local changes in electron emission of various energies resulting from the initial interaction between the electron beam and the sample. Another type of electron microscope is the photoelectron microscope (PEEM). PEEMs use photons to cause electrons to be emitted from a sample and use local changes in electron emission to generate image contrast. [Overview of the Initiative] [Means for solving the problem]

[0003] An exemplary imaging system includes a light source configured to emit radiation onto a biological sample, the light source being optimized for high-speed imaging of the biological sample using the photoelectric effect. The system includes a sample holder configured to hold the biological sample in place during imaging. The system also includes a stage assembly to which the sample holder is mounted, the stage assembly moving the biological sample during imaging. The system further includes a detector configured to receive electrons emitted from the biological sample in response to radiation from the light source.

[0004] In one embodiment, the light source includes a continuous-wave (CW) laser. In another embodiment, the light source includes a frequency quadrupled neodymium-doped yttrium aluminum garnet (Nd:YAG) laser having a wavelength in the range of 244 nanometers to 254 nanometers. In another embodiment, the stage assembly includes a piezo-driven flexure stage optimized for high-speed imaging. In yet another embodiment, the detector includes a time-delay integral sensor optimized for high-speed imaging. In one embodiment, the biological sample is thinly sliced, and the sample holder includes a wafer adjusted to a size that can hold the biological sample. In some embodiments, the sample holder is coated with a reflective coating to mitigate the adverse effects of heat accumulation during imaging.

[0005] In one embodiment, the system includes one or more mirrors that direct light from a light source onto a biological sample. The system may also include an active alignment mechanism configured to align the light source with the biological sample. In another embodiment, the system includes a fiber optic cable connected to the light source, which is also connected to a vacuum chamber where imaging takes place. The system may also include an objective lens configured to focus radiation from the light source onto a region of interest (ROI) of the biological sample.

[0006] In exemplary embodiments, the stage assembly comprises multiple stages assembled and combined, providing both speed and vibration-free movement over a range of motion exceeding the size of the biological sample. In one embodiment, the first stage of the multiple stages is a cross-roller bearing stage, and the second stage of the multiple stages is a piezo-driven flexure stage or a magnetic bearing stage. The system may also include one or more sensors that monitor the Z-stage of the stage assembly moving in the Z-direction, and one or more sensors that identify any movement error in the Z-direction. In another embodiment, a vacuum aperture is placed between the biological sample and the stage assembly so that the stages of the stage assembly and the biological sample can be pumped separately.

[0007] In another embodiment, the detector is configured to perform continuous imaging while the sample is moving. The system may also include an electric or electromagnetic deflector in the path of electrons emitted from the biological sample, which can deflect the electrons using a sawtooth deflection amplitude.

[0008] In another embodiment, the sample holder includes a wafer immersed in water, the biological sample is received above the water, and the biological sample is placed on the wafer as the water is removed by drainage or evaporation. In yet another embodiment, the biological sample is fused with a block of magnetic material so that the position of the biological sample received above the water can be manipulated with a magnet.

[0009] In another embodiment, a stain is introduced into a biological sample to highlight one or more areas of the biological sample, and the stain has an altered electron yield under photoemission conditions. In one embodiment, the biological sample is stained with osmium tetroxide and embedded in epoxy resin, and the biological sample stained with osmium tetroxide has an increased electron yield compared to the epoxy resin. In another embodiment, slices are collected on a gold-coated surface. In another embodiment, the sample holder is treated with laser radiation to prevent the generation of gas emission during imaging. In one embodiment, the sample holder is treated with ion radiation to remove a portion of the sample once or multiple times during imaging. In another embodiment, the sample holder is made of copper with high thermal conductivity to help reduce heat during imaging. In one embodiment, the sample holder is treated to minimize light absorption, and this treatment makes the sample holder transparent to light of wavelengths used to extract electrons. In another embodiment, the surface of the sample holder is changed to a Bragg mirror to make the sample holder reflective, thereby minimizing light absorption.

[0010] Other key features and advantages of the present invention will become apparent to those skilled in the art by examining the following drawings, detailed description, and appended claims. [Brief explanation of the drawing]

[0011] Exemplary embodiments of the present invention are described below with reference to the accompanying drawings, where the same reference numerals indicate the same elements. [Figure 1] An overview of a photoelectron microscope for imaging biological samples is provided according to an exemplary embodiment. [Figure 2] An exemplary embodiment shows the arrangement of a stage assembly that enables high-speed and / or continuous movement of a sample during imaging. [Figure 3] An imaging strategy for successful imaging during stage transitions is shown according to an exemplary embodiment. [Figure 4] An exemplary embodiment shows a sample holder optimized for high-speed imaging of a large number of thin samples (e.g., thin biological tissue slices). [Figure 5] The present invention illustrates a computer system programmed or otherwise configured to implement a method for acquiring PEEM images of biomedical specimens at accelerated throughput, according to an exemplary embodiment. [Figure 6] This image shows a sample section with a thickness of 80 nm irradiated with a mercury lamp, according to an exemplary embodiment. Additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, and only exemplary embodiments of the present disclosure are shown and described. As will be understood, other different embodiments of the present disclosure are possible, and some of their details can be modified in various obvious ways without all departing from the present disclosure. Therefore, the drawings and description should be considered exemplary and not limiting. [Modes for carrying out the invention]

[0012] Transmission electron microscopy and scanning electron microscopy are tools frequently used for imaging and / or high-throughput image acquisition of biomaterials. Photoelectron microscopy (PEEM) is a promising alternative to these types of imaging scenarios, offering the ability to improve acquisition speed, ease of sample preparation, and the amount of information that can be extracted per image. Photoelectron microscopy has been successfully used in a variety of materials science applications. However, conventional PEEM systems have been used only sporadically in imaging biological / medical specimens because neither the microscope speed nor the contrast mechanism is optimized for biomedical imaging.

[0013] This specification describes a PEEM optimized for biomedical imaging, hereafter referred to as "BioPEEM" or the proposed system. In exemplary embodiments, the proposed system may include an imaging column, a stage mechanism, a light source, a detector, customized automation software, an analytical pipeline, and various sample preparation steps specifically tailored for improved performance in imaging biomedical specimens. In alternative implementations, the proposed system may include fewer, additional, and / or different elements.

[0014] Another aspect of this disclosure provides a method for extracting multiple grayscale images for each sample. For example, multiple grayscale images can be extracted for each sample by using chemical or material stains having different work functions, ionization potentials, electron densities, or oxidation states. Alternatively, the sample material can be probed using photons of different energies to obtain differential contrast or sample information from different depths. Furthermore, the use of energy filters that enable the acquisition of additional channels in the assembled composite image is described herein.

[0015] Another aspect of this disclosure is directed to a non-temporary computer-readable medium having machine-executable code that implements any of the methods described herein when executed by one or more computer processors. Another aspect of this disclosure is directed to a system including one or more computer processors and computer memory coupled thereto. The computer memory includes machine-executable code that implements any of the methods described herein when executed by one or more computer processors.

[0016] As stated, this disclosure is directed toward an improved photoelectron microscope (PEEM) system and method optimized for biomedical imaging. The proposed microscope operates by emitting photons onto a sample and causing those photons to generate photoelectrons to be emitted from the sample. Figure 1 shows an overview of a photoelectron microscope imaging a biological sample according to an exemplary embodiment. In the shown embodiment, the sample to be imaged is a brain slice having a cell membrane, placed on a silicon carrier. In alternative scenarios, any other type of biomaterial can be used as the sample.

[0017] In an exemplary embodiment, a light source is used to emit a beam of photons that excite the imaged sample. In certain embodiments, the photons used for excitation can have a wavelength of 157 nanometers (nm). Alternatively, the wavelength can be less than 157 nm, greater than 157 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, etc. The light source can be a laser or a mercury lamp in certain embodiments. Alternatively, different types of light sources can be used. In the embodiment of FIG. 1, the light source emits high-intensity ultraviolet (UV) light and irradiates the sample. In alternative implementations, different types of high-intensity excitation light can be used. Due to this high intensity of the light source, the image acquisition time can be very short, for example, less than 10 minutes (min), less than 1 minute, less than 30 seconds (s), less than 10 s, less than 5 s, less than 1 s, less than 100 milliseconds (ms), less than 10 ms, less than 5 ms, less than 1 ms, etc. To enable such high-speed imaging, the microscope is operated to image while the sample is moving, realizing a scenario with little or no time loss due to stage movement.

[0018] In response to being excited by photons from the light source, electrons are emitted from the sample. The electrons leave the sample due to their kinetic energy and are then accelerated towards the objective lens of the camera or detector. In certain embodiments, the acceleration voltage of the emitted electrons can be 10 kilovolts (kV). Alternatively, the acceleration voltage can be less than 10 kV, greater than 10 kV, greater than 15 kV, greater than 20 kV, greater than 25 kV, etc. The image formed by the emitted electrons is magnified by one or more electrostatic or electromagnetic lenses and projected onto the detector. The detector can be a direct electron detector in certain embodiments. Alternatively, the detector can utilize a microchannel plate and / or a scintillator.

[0019] FIG. 2 shows the arrangement of a stage assembly that enables fast and / or continuous movement of a sample during imaging. In some embodiments, the order of the stages in the stage assembly can be shuffled. The stage assembly includes an XY stage 201 that moves in both the X and Y directions with a movement range sufficient to cover the entire sample. Such a stage can have a range of 1 inch in each of the X and Y directions in one embodiment. Alternatively, the movement range in the XY directions may be less than 1 inch, more than 1 inch, more than 2 inches, more than 3 inches, more than 4 inches, more than 5 inches, more than 6 inches, etc. In some embodiments, the XY stage 201 may be a direct-drive cross-roller bearing stage.

[0020] The stage assembly also includes a high-precision short-distance Z stage 202 used for Z error compensation of the XY stage 201 and / or focusing of the microscope. The Z stage 202 can have a movement range of 100 micrometers (um) in the Z direction in one embodiment. Alternatively, the movement range of the Z stage 202 may be more than 100 um, less than 100 um, less than 10 um, less than 1 um, etc. The stage assembly also includes a first measurement device 203 and a second measurement device 204 that together form a non-contact Z distance measurement device. The non-contact Z distance measurement device is used to measure the Z error of the XY stage 201 and issue movement commands to the Z stage 202 to correct any measured error. Specifically, when the stage moves in the X and Y directions, the stage rocks up and down, thereby erroneously changing the Z position of the sample. This Z-direction variation is detected by a non-contact Z measurement device, which may be in the form of a capacitance sensor or an interferometer sensor in one embodiment. The non-contact Z distance measurement device 203 / 204 includes a holder 205 to which the measurement device is attached. In one embodiment, the holder 205 is made of a material having a low coefficient of thermal expansion such as invar.

[0021] The stage assembly in Figure 2 also includes an X-stage 206 that moves in the X direction and a Y-stage 207 that moves in the Y direction. The movement of the X-stage 206 and Y-stage 207 is optimized to avoid generating high-frequency movement noise present in many other types of stages. For example, the XY stage 201 may have a large range of movement that includes undesirable movements in the form of vibration, shear, etc. Conversely, the X-stage 206 and Y-stage 207 may have a small range of movement that is very smooth and avoids vibration, etc., of the XY stage 201. The root mean square (RMS) movement error of these stages may be less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, and less than 2 nm in the frequency range above 500 Hz. In some embodiments, the X-stage 206 and Y-stage 207 may be combined into a single XY stage. In exemplary embodiments, the X-stage 206 and Y-stage 207 may be piezoelectric-driven flexure stages or magnetic bearing stages. In one embodiment, the X stage 206 and the Y stage 207 may each have a travel range of 100 μm. Alternatively, the travel range of the stages may be less than 100 μm, greater than 100 μm, greater than 500 μm, greater than 1 mm, greater than 2 mm, greater than 4 mm, etc.

[0022] The stage assembly further includes a sample holder 208. The sample holder 208 can hold the sample mechanically, magnetically, or using adhesive. In some embodiments, the sample holder 208 may be part of a wafer loading and handling system. Figure 2 also shows a sample 209 placed in the sample holder 208. In some embodiments, the sample 209 may be a wafer whose weight is optimized to allow for high-speed movement of the stage. In some embodiments, the sample 209 may be a wafer whose back surface is structured to reduce weight while maintaining rigidity, a wafer made of a material with a good rigidity-to-weight ratio such as titanium, or a sandwich structure of a thin wafer and a holder structure made of another material and / or structured to support the thin wafer. In some embodiments, the sample 209 may be optimized for laser heat dissipation. In another embodiment, a vacuum aperture may be installed to divide the sample chamber into two separate volumes. The vacuum aperture may include a vacuum gate valve which can be placed between two vacuum chambers in embodiments where two chambers are required for analysis. This allows the stage area to be, for example, 10 -11 Less than 10 -10 Less than 10 -9 Less than 10 -8 Less than 10 -7 Less than 10 -6 Less than 10 -5 Less than 10 -4 Low vacuum levels, such as below Toll, can be achieved. In exemplary embodiments, the stage does not stop while image acquisition is taking place. In some embodiments, the space charge effect of induced electrons prevents imaging with sufficiently fast pulses (like a stroboscope).

[0023] Figure 3 shows an imaging strategy for successfully imaging during stage movement according to an exemplary embodiment. Figure 3 shows an example of stroke trajectories 301 that the sample can take during imaging. Depending on the desired imaging to be performed, there may be one or more such strokes represented by trajectories 301, such as 5, 10, 50, 100, 500, or more. The strokes may overlap by a value of less than 1%, greater than 1%, greater than 5%, or greater than 10% of the stroke width 302. While the sample is moving, the illumination area 306 does not move, which means that in the microscope's reference system, the emitted electrons 303 always originate from the same area. The electrons 303 are flying through the column, and with respect to a given source point, the X and Z positions are correlated by the movement of the stage. Therefore, the detector assembly must ensure that the electrons are correctly attributed to the source point.

[0024] Ensuring that received electrons are correctly attributed to their source point can be achieved by reading out the camera 305 at a very high frame rate and algorithmically compensating for motion. In another embodiment, electron assignment can be achieved by using a time-delay integral (TDI) detector, a type of charge-coupled device (CCD) detector specifically built for this scenario. In yet another embodiment, electron assignment can be achieved by deflecting the electrons using an electrostatic or electromagnetic beam deflection device 304, thereby compensating for sample movement. In yet another embodiment, the detector can be physically moved to counteract continuous sample movement.

[0025] The proposed microscope can be used to image a wide variety of samples, including biological and other materials. Figure 4 shows a sample holder 401 optimized for high-speed imaging of numerous thin samples (e.g., thin biological tissue slices) according to an exemplary embodiment. In some embodiments, the sample holder 401 may be circular and / or wafer. The wafer material can be optimized for thermal conductivity and / or transparency. In some embodiments, the wafer can be made from silver, copper, fused silica, or calcium fluoride. In some embodiments, the wafer can be coated with a material that enhances reflectivity, such as modified aluminum or a Bragg mirror structure. The wafer can also be coated, such as gold plating or silane treatment, to improve the interaction between the sample and the sample holder 401.

[0026] In exemplary embodiments, the sample material 402 is of biological origin and can be treated with osmium tetroxide, lead aspartate, uranyl acetate, or a similar substance as a staining agent. In some embodiments, the sample material 402 can be embedded in an epoxy resin and / or optimized to have low absorption to the wavelength of incoming photons and / or optimized to minimize damage to the sample by UV radiation. In exemplary embodiments, the sample material 402 can be fused with a magnetic material 403. Wafers can be loaded by immersing the wafer in a liquid to float the sample on the liquid, and then draining or evaporating the liquid. In these embodiments, the magnetic material can be used to move, manipulate, and / or orient the sample material 402. In some embodiments, the sample material 402 can be treated with ion emission 404 to remove material. Ion emission treatment can be performed before and / or during imaging. For example, ion emission treatment can be performed one, two, three, four, five, six, seven, eight, nine, ten, 100, or more times during an imaging run. In another embodiment, a control system can ensure a uniform milling effect by predicting the amount of ion milling removed based on previous imaging data and adjusting the residence time accordingly.

[0027] In exemplary embodiments, ion emission treatment can be used to achieve a three-dimensional (3D) imaging effect of the sample material 402. In some embodiments, the sample can be treated with laser radiation before imaging 405 is performed. Laser radiation can be used to degas the sample and / or to remove improperly attached materials so that they do not distort the electric field or burn in the imaging vacuum. In some embodiments, the magnetic material 403 can be treated with heat-inducing radiation (e.g., laser light) and heated above the Curie temperature to remove magnetism so that the magnetism does not adversely affect imaging.

[0028] In another exemplary embodiment, the proposed system may be partially implemented using one or more computer systems programmed or otherwise configured to implement the method described herein for acquiring PEEM images. Figure 5 shows a computer system 501 programmed or otherwise configured to implement a method for acquiring PEEM images of a biomedical sample at accelerated throughput, according to an exemplary embodiment. The computer system may be configured to provide autofocus and / or other image optimization functions. In some embodiments, the computer system may perform preliminary imaging runs to discover optimized imaging parameters in one or more, 10, 100, or any other number of regions of interest (ROIs) and interpolate optimized imaging conditions for the entire wafer from those ROIs.

[0029] In one embodiment, the computer system 501 can generate commands or signals to operate a device or mechanism for reading sensors when the stage setup is in the appropriate position. The computer system 501 may be a computer system located remotely from a user's electronic device or an electronic device. The electronic device may be a mobile electronic device. The computer system 501 may include a central processing unit (CPU, also referred to herein as “processor” and “computer processor”) 505, which may be a single-core or multi-core processor or multiple processors for parallel processing. The computer system 501 also includes memory or memory locations 510 (e.g., random-access memory, read-only memory, flash memory), an electronic storage unit 515 (e.g., a hard disk), a communication interface 520 for communicating with one or more other systems (e.g., a network adapter), and peripherals 525 such as cache, other memory, data storage, and / or an electronic display adapter.

[0030] The memory 510, storage unit 515, interface 520, and peripheral devices 525 communicate with the CPU 505 via a communication bus (solid wire) such as a motherboard. The storage unit 515 may be a data storage unit (or data repository) for storing data. The computer system 501 can be operably connected to a computer network ("network") 530 via the communication interface 520. The network 530 may be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, the network 530 is a telecommunications and / or data network. The network 530 may include one or more computer servers that enable distributed computing such as cloud computing. In some cases, the network 530 may implement a peer-to-peer network via the computer system 501, so that devices connected to the computer system 501 can act as clients or servers.

[0031] The CPU 505 can execute a sequence of machine-readable instructions embodied in a program or software. Instructions can be stored in a memory location such as memory 510. Instructions can be directed to the CPU 505 so that it implements the methods of this disclosure. Examples of operations performed by the CPU 505 include fetching, decoding, executing, and writing back. The CPU 505 may be part of a circuit, such as an integrated circuit. One or more other components of the system 501 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0032] The storage unit 515 can store files such as drivers, libraries, and stored programs. The storage unit 515 can also store user data such as user settings or user programs. In some cases, the computer system 501 may include one or more additional data storage units located outside the computer system 501 (for example, on a remote server communicating with the computer system 501 via an intranet or the internet). The computer system 501 can communicate with one or more remote computer systems via the network 530. For example, the computer system 501 can communicate with remote computer systems. Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., Apple® iPad®, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone®, Android®-enabled devices, Blackberry®), or personal digital assistants.

[0033] A user can access the computer system 501 via the network 530. Methods such as those described herein can be implemented by machine-executable code (e.g., a computer processor) stored in an electronic storage location of the computer system 501, such as memory 510 or an electronic storage unit 515. The machine-executable code or machine-readable code can be provided in software form. During use, the code can be executed by the processor 505. In some cases, the code can be retrieved from the storage unit 515 and stored in memory 510 for quick access by the processor 505. In some situations, the electronic storage unit 515 can be omitted, and machine-executable instructions can be stored in memory 510. The code may be pre-compiled and configured for use in a machine having a processor adapted to execute the code, or it may be compiled during execution.

[0034] The code may be provided in a programming language that can be selected to allow the code to be executed in a pre-compiled or running-compiled format. Various aspects of the systems and methods provided herein, such as computer system 501, can be embodied in programming. Various aspects of the technology can be considered as “products” or “manufactured goods” in the form of machine (or processor) executable code and / or associated data, usually placed on or embodied on some kind of machine-readable medium. Machine-executable code can be stored in memory (e.g., read-only memory, random-access memory, flash memory) or electronic storage units such as hard disks. “Storage” type media can include any or all tangible memories of a computer, processor, or associated modules such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage for software programming at any time.

[0035] All or part of the software may be transmitted from time to time via the Internet or various other communication networks. Such communications can, for example, enable the loading of software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may contain software elements include optical, electrical, and electromagnetic waves used, for example, through physical interfaces between local devices, through wired and optical terrestrial communication networks, and even through various wireless links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as media containing software. As used herein, unless limited to non-temporary, tangible “storage” media, terms such as computer or machine “readable media” refer to any medium involved in providing instructions for execution to a processor. Thus, machine-readable media, such as computer executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media.

[0036] Non-volatile storage media, such as optical disks or magnetic disks, or any storage device such as any computer, can be used to implement databases as shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wires, and optical fibers, and include wiring that constitutes buses in computer systems. Carrier transmission media can take the form of electrical or electromagnetic signals, or sound waves or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card paper tapes, any other physical storage media having a pattern of holes, RAM, ROMs, PROMs and EPROMs, FLASH®-EPROMs, any other memory chips or cartridges, carriers that carry data or instructions, cables or links that carry such carriers, or any other media from which a computer can read programming code and / or data. Many of these computer-readable media can be involved in transporting one or more instructions, one or more sequences, to a processor for execution.

[0037] The computer system 501 includes an electronic display 535 having a user interface (UI) 540, or can communicate with the electronic display 535. The portal may be provided through an application programming interface (API). Users or entities can interact with various elements within the portal via the UI. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0038] Imaging by the proposed system was tested by the inventors on a biological sample in the form of a mouse brain slice. The mouse brain sample was stained using a standard staining protocol for biological electron microscopy and embedded in epoxy resin. Subsequently, the polymer block was ultrathin sectioned into 40 nm to 80 nm thin slices on an air table to avoid chattering. A silicon substrate (7×7 mm 2 ) coated with polycrystalline gold (poly-Au) below 50 nm was used to pick up the sample sections cut from the water bath. The gold layer is used for several purposes, including improving the conductivity of the substrate and better adhering the biological sample to the substrate because gold tends to bond with sulfur. The gold layer also enables the user to quickly identify the biological section based on the distinct photoelectron emission intensity from the gold and the sample section. This method has proven to bring a significant improvement in image resolution and greatly reduce the arc effect that can occur due to poor contact between the sample section and the native oxide layer on bare silicon.

[0039] In the proposed system, careful selection of the light source is important to obtain images with sharp intracellular resolution. First, the photon energy needs to exceed the work function of the bio-section so that photoelectrons can be emitted, detected, and form an image. Second, the photon flux not only has to be high enough to increase the S / N per unit time and speed up data acquisition, but also has to be maintained below the space charge limit. Furthermore, using too high power can adversely affect the sample by thermal heating.

[0040] The inventors verified the feasibility of bioPEEM using a continuous-wave (CW) mercury (Hg) lamp at room temperature. The Hg lamp used for testing has a shortest wavelength of approximately 250 nm (4.96 eV) and a broadband spectrum with a UV portion brightness of less than 566 cd / mm². The initial operation of imaging involves driving the sample stage and finding a section of the surface. Poly-Au has a lower work function and a higher density of states (DoS) than biological samples and epoxy resins. At this photon energy, Poly-Au exhibits much stronger photoelectron emission than the sample section. Therefore, the sample section can be easily identified in a 100 s μm field of view (FoV) by looking for a remarkably dark rectangular region with sharp edges. Unlike conventional imaging of homogeneous material systems, cellular structures are barely observable until high magnification is reached by gradually increasing the extraction voltage up to 12 kV. This requires continuous zooming in until the region of interest (RoIs) drops below 20 μm FoV. Typically, smaller apertures (150 μm) are used to further improve image resolution. Both conditions limit the total amount of photoelectrons reaching the detector, so unless the camera exposure time is several seconds, the imaging intensity is usually very low. This makes it very difficult to perform optimization of the focus and astigmatism correction device in live mode. Figure 6 is a typical image of an 80 nm thick sample section irradiated with a mercury lamp according to an exemplary embodiment. The spatial resolution is determined by calculating the width between 16% and 84% of the error function, which is 40 nm to 50 nm.

[0041] Accordingly, various embodiments of photoelectron microscopes (PEEMs) used for imaging biomaterial samples and / or optimized for high-speed imaging, which, through a series of methods detailed herein, are orders of magnitude faster than the prior art. In some embodiments, one or more components of the PEEM are optimized for current tasks, such as a faster detector, brighter illumination, a mobile device to reduce overhead, and optimized sample preparation and presentation. In some embodiments, this setup is optimized for rapid acquisition of 3D image stacks of biological tissues such as brain material or cancerous tissue, enabling imaging of much larger volumes at high resolution.

[0042] In exemplary embodiments, any of the operations described herein can be performed by a computing system including a processor, memory, a user interface, transceivers, etc. The memory may be used to store computer-readable instructions that cause the computing system to perform the operations described herein when executed by the processor. All or part of the computing system may be incorporated into a robot unit. For example, the computing system may be incorporated into the printed circuit board of an individual robot unit. All or part of the computing system may also exist as a system separate from the robot unit and may be used to monitor and / or control the robot unit.

[0043] While various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Numerous variations, modifications, and substitutions can be made to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted. When the terms “at least,” “greater than,” or “greater than or equal to” precede the first number of a series of two or more numbers, “at least,” “greater than,” or “greater than or equal to” applies to each number in that series. For example, “greater than or equal to” is equivalent to “greater than or equal to” 1, “greater than or equal to” 2, or “greater than or equal to” 3. When the terms “not greater than,” “less than,” or “less than or equal to” precede the first number of a series of two or more numbers, “not greater than,” “less than,” or “less than or equal to” applies to each number in that series. For example, “less than or equal to” 3, “2,” or “less than or equal to” is equivalent to “less than or equal to” 3, “less than or equal to” 2, or “less than or equal to” 1.

[0044] The terms “real-time” or “real-time” as used interchangeably herein generally refer to events (e.g., actions, processes, methods, techniques, computations, calculations, analyses, visualizations, optimizations, etc.) performed using recently acquired (e.g., collected or received) data. In some cases, real-time events can be performed almost immediately after an initial event or within a sufficiently short time span after such an initial event, for example, within 1 second, 0.5 seconds, 0.1 seconds, 0.05 seconds, 0.01 seconds, 5 milliseconds, 1 millisecond, 0.5 milliseconds, 0.1 milliseconds, 0.05 milliseconds, 0.01 milliseconds, 0.005 milliseconds, 0.001 milliseconds, 0.0005 milliseconds, 0.0001 milliseconds, or less than 0.0001 milliseconds.

[0045] As used herein, the term “exemplary” means an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not to be construed as being preferable or more advantageous than any other aspect or design. Furthermore, for the purposes of this disclosure, unless otherwise specified, “a” or “an” means “one or more.”

[0046] The foregoing description of exemplary embodiments of the present invention has been presented for illustrative and explanatory purposes. The foregoing description is not intended to be exhaustive or to limit the invention to the exact forms disclosed, and modifications and variations are possible in consideration of the foregoing teachings or can be obtained from the practice of the present invention. The embodiments are selected and described to illustrate the principles of the present invention and, as practical applications of the present invention, to enable those skilled in the art to utilize the invention in various embodiments and with various modifications to suit specific conceivable uses. The scope of the present invention is intended to be defined by the claims appended herein and their equivalents.

Claims

1. A light source configured to emit light onto biological samples and optimized for high-speed imaging of biological samples using the photoelectric effect, A sample holder configured to fix the biological sample during imaging, A stage assembly to which the sample holder is attached and to which the biological sample is moved during imaging, A detector configured to receive electrons emitted from the biological sample in response to radiation from the light source, An imaging system characterized by including

2. The imaging system according to claim 1, wherein the light source includes a continuous wave (CW) laser or a quasi-CW laser.

3. The imaging system according to claim 1, wherein the stage assembly includes a piezo-driven flexure stage optimized for high-speed imaging.

4. The imaging system according to claim 1, wherein the detector includes a time-delay integral sensor optimized for high-speed imaging.

5. The imaging system according to claim 1, wherein the biological sample is cut into thin slices, and the sample holder includes a wafer adjusted to a size capable of holding the biological sample.

6. The imaging system according to claim 1, further comprising a reflective coating on the sample holder for mitigating the adverse effects of heat accumulation during imaging.

7. The imaging system according to claim 1, wherein the light source includes a frequency quadrupled neodymium-doped yttrium aluminum garnet (Nd:YAG) laser having a wavelength in the range of 244 nanometers to 254 nanometers.

8. The imaging system according to claim 1, further comprising one or more mirrors for directing light from the light source onto the biological sample.

9. The imaging system according to claim 1, further comprising an active alignment mechanism configured to align the light source with respect to the biological sample.

10. The light source further includes a fiber optic cable connected to the light source, The imaging system according to claim 1, wherein the fiber optic cable is also connected to a vacuum chamber in which imaging is performed.

11. The imaging system according to claim 1, further comprising an objective lens configured to focus radiation from the light source onto a region of interest (ROI) of the biological sample.

12. The imaging system according to claim 1, wherein the stage assembly comprises a collection and combination of multiple stages, providing both speed and vibration-free movement over a range of motion exceeding the size of the biological sample.

13. The imaging system according to claim 12, wherein the first stage among the plurality of stages is a piezo-driven flexure stage or a magnetic bearing stage, and the second stage among the plurality of stages is a cross-roller bearing stage.

14. The system further includes one or more sensors that monitor the Z-stage of the stage assembly as it moves in the Z-direction. The imaging system according to claim 1, wherein one or more sensors identify any movement error in the Z direction.

15. The imaging system according to claim 1, further comprising a vacuum aperture positioned between the biological sample and the stage assembly so that the stage of the stage assembly and the biological sample can be evacuated separately.

16. The imaging system according to claim 1, wherein the detector is configured to perform continuous imaging while the biological sample is in motion.

17. The imaging system according to claim 16, further comprising an electric deflector or an electromagnetic deflector in the path of the electrons emitted from the biological sample, and deflecting the electrons using a sawtooth deflection amplitude.

18. The imaging system according to claim 1, wherein the sample holder includes a wafer immersed in water, the biological sample is received on the water, and the biological sample is placed on the wafer by draining or evaporation.

19. The imaging system according to claim 18, wherein the biological sample is fused with a block of magnetic material, and the position of the biological sample, received on water, can be manipulated by a magnet.

20. The present invention further comprises a staining agent introduced into the biological sample to highlight one or more regions of the biological sample, The imaging system according to claim 1, wherein the staining agent has an electron yield that changes under photoelectron emission conditions.

21. The imaging system according to claim 1, wherein the biological sample is stained with osmium tetroxide and embedded in epoxy resin, and the biological sample stained with osmium tetroxide has an increased electron yield compared to epoxy resin.

22. The imaging system according to claim 1, wherein the sample holder is treated with laser radiation to prevent the generation of gas emission during imaging.

23. The imaging system according to claim 1, wherein the sample holder is treated with ion emission to remove a portion of the biological sample once or multiple times during imaging.

24. The imaging system according to claim 1, wherein the sample holder is made of copper with high thermal conductivity to help reduce heat during imaging.

25. The process further includes applying a treatment to the sample holder to minimize light absorption, The imaging system according to claim 1, wherein the process makes the sample holder transparent to light of a wavelength used to extract the electrons.

26. The imaging system according to claim 1, wherein the surface of the sample holder is changed to a Bragg mirror, making the sample holder reflective and thereby minimizing light absorption.

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