Method for temperature monitoring in cryo-electron microscopy

Abstract

To provide a method for monitoring temperature in cryo-electron microscopy.SOLUTION: Temperatures of cryo-electron microscopic samples are assessed based on portions of images associated with high temperature superconductor (HTSC) areas or other thermal sensor materials that are thermally coupled to or thermally proximate the samples. Such thermal areas can be provided on sample mounts such as metallic grids, carbon films, or on sample stages. In examples using HTSCs, HTSCs having critical temperatures between -175°C and -135°C are typically used.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to the evaluation of sample temperature in cryo-electron microscopy.

Background Art

[0002] Cryo-electron tomography (cryo-ET) is based on a freezing technique that traps cellular water in an amorphous (glass-like) layer in which all cellular components are embedded. This process is known as vitrification. Samples typically need to be maintained at a temperature of less than about -135 °C to avoid devitrification and degradation, but in most cases a temperature of less than -150 °C is preferred to provide a margin of error. In many cases, it is difficult to evaluate the temperature of such small samples and to determine whether they have been properly maintained at low temperature. This is particularly difficult after the sample has been placed on the electron microscope substrate stage. If the sample is not properly maintained at low temperature, the sample will no longer exhibit its original structure. In some cases, it may later be found that the sample was not properly maintained at the appropriate temperature, despite the fact that a large amount of instrument time was required to process the sample. For this reason, improved methods and approaches for determining, evaluating, and assessing sample temperature in electron microscopy would be useful.

Summary of the Invention

[0003] The method includes disposing a thermal sensor in thermal proximity to a sample, imaging at least a portion of the thermal sensor, and evaluating the temperature of the sample based on the brightness of the image or a current associated with the thermal sensor. According to some examples, the thermal sensor is a superconducting material, and evaluating the temperature of the sample includes determining whether the sample is at a temperature lower than or higher than a critical temperature associated with the superconducting material. In a typical example, the thermal sensor is disposed on a sample holder, and the sample holder includes at least one of a metal grid and a perforated carbon film. The sample holder can include a conductive surrounding ring, and the thermal sensor can be disposed on the conductive surrounding ring. The brightness of the thermal sensor can be determined by exposing the thermal sensor to a charged particle beam (CPB) such as an electron beam. In a representative example, based on the exposure of at least a portion of the thermal sensor to the charged particle beam (CPB), a CPB image of the exposed portion is formed, and the temperature associated with the temperature sensor is determined based on the brightness of the thermal sensor in the CPB image. In some cases, the thermal sensor is in contact with the sample or within the sample.

[0004] A sample holder for CPB microscopy includes a sample support defining a sample region for holding a sample and at least one temperature sensor disposed in thermal proximity to the sample region. In some examples, the sample support is a carbon film, and the temperature sensor contacts or is thermally coupled to the carbon film. The sample holder can include a metal grid in contact with the carbon film, and the temperature sensor contacts or is thermally coupled to the metal grid. The sample holder can also include a conductive ring disposed around at least a portion of the sample support defining the sample region, and the temperature sensor contacts or is thermally coupled to the surrounding ring. The thermal sensor can be a high temperature superconductor (HTSC) having a critical temperature of -135 °C to -175 °C, or a plurality of particles of a thermal sensor material fixed to or in contact with the sample support.

[0005] The CPB system includes a sample stage operable to hold a sample holder at a sample position and a thermal sensor disposed thermally proximate to the sample position, the thermal sensor being configured to generate a thermal response signal based on a temperature associated with the sample position. A processor is coupled to the thermal sensor and is operable to indicate the temperature of the sample holder based on the thermal response signal. The system can include at least one computer-readable medium storing a calibration of the thermal response signal as a function of temperature, e.g., as a look-up table. In some examples, the thermal response signal is one of the image brightness, resistance, resistivity, or current in response to CPB exposure of a CPB image. According to some examples, the thermal sensor is fixed to or in contact with the sample holder. In a typical example, the brightness of an image of the thermal sensor is used to estimate the temperature of the sample holder or the sample.

[0006] A method for evaluating sample temperature by cryo-electron microscopy includes placing a thermal sensor in thermal contact with a vitrified sample, directing an electron beam at the thermal sensor, and detecting backscattered electrons (BSE) or secondary electrons (SE) associated with the thermal sensor in response to the electron beam. At least a partial image of the thermal sensor is generated based on the detected SE or BSE, and the temperature of the vitrified sample is determined based on the image portion.

[0007] The foregoing and other objects, features, and advantages of the disclosed technology will become apparent from the following detailed description, which proceeds with reference to the accompanying figures.

Brief Description of the Drawings

[0008]

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DETAILED DESCRIPTION OF THE INVENTION

[0009] In the examples described below, a so-called high-temperature superconductor (HTSC) is used because the high-temperature superconductor exhibits a sharp change in resistivity at or near the critical temperature in relation to the change from the normal conductor state to the superconducting state. Since the HTSC can have a critical temperature in a range closer to the normal sample temperature of the low-temperature electron microscopy method than the critical temperature of the low-temperature superconductor (LTSC), it is convenient for the applications disclosed herein. However, LTSC and other materials can also be used as shown in some of the examples below. Any material having a variable electron microscope brightness dependent on temperature can be used as a thermal sensor.

[0010] The brightness of the image portion is generally related to the secondary electrons detected in response to exposure to the electron beam or the detected backscattered electrons. In the case of typical thermal sensor materials (such as HTSC), in a representative HTSC, as shown in FIG. 2, the thermal sensor material has a lower brightness when the conductivity is high and a higher brightness when the conductivity is low.

[0011] By establishing the brightness of the material image as a function of temperature, the sample temperature can be evaluated using a material that is in thermal contact with the sample or is placed near it. The large changes available in LTSC and HTSC can be convenient because of the large changes in the brightness of the images they can present in a specific temperature range. The brightness of the image can be based on the response of the material to any kind of CPB (usually an electron beam) or electromagnetic radiation such as visible light, infrared (IR), far-infrared, ultraviolet (UV), extreme UV, X-rays, gamma-ray radiation, or other radiation. In some examples, the temperature can be evaluated using the response spectrum or one or more wavelengths of the response spectrum. In such examples, the change in the spectrum indicates a temperature change, and a specific spectrum can be calibrated to enable temperature estimation based on the spectrum.

[0012] As used herein, the critical temperature T Crefers to the temperature associated with the conductor / superconductor transition, and T* refers to the temperature at which the conductivity of the material exhibits a non-linear variation as a function of temperature. In the case of HTSCs such as YBCO, such non-linear variations can occur at temperatures T* C significantly higher than the conventional HTSC critical temperature T. Thus, both these temperatures are related to the variation of conductivity and, in some cases, to the variation of the secondary emission yield. These properties of HTSCs can be used to evaluate the sample temperature by electron microscopy, as will be described in detail below. However, other materials can also be used, such as LTSCs or other materials where the conductivity is a non-linear function of temperature, and HTSCs are simply a convenient example. Other materials that exhibit linear or other variations in resistivity can also be used. HTSCs and LTSCs exhibit large variations at the critical temperature associated with the conductor-superconductor transition. For other materials, such as those that vary gradually or linearly, calibrations can be performed so that the temperature can be evaluated using measurements of resistance, resistivity, image brightness, or other material-dependent properties.

[0013] The examples generally refer to electron microscopy, but other charged particle beam systems can be arranged similarly. Further, the disclosed systems, methods, and apparatuses can be used in transmission electron microscopy, scanning electron microscopy, ion beam microscopy, and other CPB systems, as well as in optical microscopy, confocal microscopy, fluorescence microscopy using a laser or other light source, X-ray imaging systems, or systems using other types of electromagnetic radiation. Some examples will be described with reference to HTSC temperature sensors, but this is for illustrative convenience and other sensor materials can be used.

[0014] A portion of the HTSC or other thermal sensor material (regardless of whether it has other characteristics that are a function of linear or non-linear resistivity or temperature) is arranged such that an evaluation of the temperature of that portion enables an estimation of the sample temperature. The sample and the sensor material portion are said to be thermally coupled if there is a heat conduction path between the sample and the sensor material portion, regardless of whether they are coupled via one or more different materials or components. The sample and the sensor material are said to be in thermal proximity if the temperatures of the sample and the sensor material are correlated such that the sample temperature can be evaluated based on the temperature of the sensor material portion. Typically, the sensor material portion is both thermally and physically proximate and is conveniently thermally coupled. Temperatures in the range of 15 K to 300 K or above or below can be estimated.

[0015] Example 1 Referring to FIG. 1, a representative electron microscope system 100 includes an electron emitter 104 operable to emit electrons from a tip 106. A condenser lens 110 is arranged to generate an electron beam from the emitted electrons and generally direct the electron beam along an axis 119. A beam deflector 111 is arranged along the axis 119 and can selectively deflect or scan the electron beam with respect to a sample S to generate a deflected beam 130. The sample S is placed on a sample stage 140 that can enable translational and tilt adjustments of the sample S. The sample S is typically fixed or formed on a sample holder 144 that can include a sensing region 142 formed from HTSC or other sensor material. An objective lens 132 is arranged to direct the electron beam from the sample S to a detector 146. In some examples, one or more detectors, such as a detector 148, are arranged to detect the electron beam or radiation responsive to the electron beam, such as secondary electrons, X-rays, or other charged particles or electromagnetic radiation, generally as indicated by 130.

[0016] In a typical example, the sample holder 144 includes a perforated support film of carbon, for example, having a plurality of circular holes. These holes are often necessary for the rapid freezing process. Layers or individual regions of frozen cells, proteins, or other samples of interest are placed on the perforated support film embedded in a freezing liquid such as water. In a CPB system such as an electron microscope, a coating of a conductor such as one or more metals, carbon, or other materials is applied to reduce the charging effect. As will be described in detail below, the perforated carbon support film can be placed on a metal grid.

[0017] The system controller 150 is coupled to an emitter driver 152, a condenser lens controller 154, and an objective lens controller 156 (usually a voltage or current source) to establish the operating conditions for electron microscopy. The system controller 150 is also coupled to a beam deflector 111 to control electron beam deflection and scanning. The system controller 150 is further coupled to detectors 146, 148 and can generate an image based on the signals received from the detectors 146, 148. The ammeter 160 is coupled to the sensing region 142 and to the system controller 150 operable to evaluate the sample temperature based on the measured current from the ammeter 160. Alternatively, the relative brightness of the sensing region 142 in an electron beam image or other CPB image can be used by the system controller 150 or by an operator of the device using the display image.

[0018] In some examples, a light radiation source 170, such as one or more lasers, LEDs, or other electromagnetic radiation sources, is arranged to irradiate the sample S. An X-ray source 172 can also be provided, and the radiation returning from the sample S can be directed to a spectrometer 174, such as a charged particle or electromagnetic radiation spectrometer. In some examples, the system is configured for X-ray photoelectron spectroscopy or Raman spectroscopy. Generally, the detector can be arranged to receive forward-scattered charged particles and / or electromagnetic radiation, as well as backward-scattered charged particles and / or electromagnetic radiation. Such additional sensors, sources, and detectors are generally coupled to the system controller 150.

[0019] Referring to FIG. 2, the relative brightness of various sensing regions as a function of temperature is shown for HTSC YBCO with various Gd doping levels and a conventional conductor (Cu). The following table is a list of some HTSCs and some conventional low-temperature superconductors (LTSCs) (shown in italics). At temperatures below Tc, the resistivity of the material is zero. [Table 1]

[0020] Other HTSCs generally include the bismuth strontium calcium copper oxide (BSSCCO) family (including Bi-2223 in the table above).

[0021] Materials such as any of those listed above can be deposited by one or more of sputtering, evaporation, electroplating, chemical vapor deposition, or other processes. Alternatively, such materials can be formed into strips, disks, particles, or other shapes and fixed or placed in thermal proximity to the sample as needed. Such HTSC portions are arranged to be thermally coupled to the sample so that the sample temperature can be estimated based on the HTSC characteristics, whether formed in a predetermined position or as separate components.

[0022] Example 2 A representative sample holder 900 for use in the system of FIG. 1 is shown in FIGS. 9A - 9B. The perforated carbon film 904 is arranged to support the sample 903 (shown as one or more cells 902) and is then placed on the metal grid 906. The metal ring 908 is bonded around the metal grid 906. The temperature sensor material 910 is placed on or above the metal ring 908, but in other cases, it can be arranged as described below.

[0023] Example 3 Referring to FIG. 3A, a representative sample holder 380 typically includes a conductive grid 382 typically formed with a conductive surrounding ring 384 made of Cu, Rh, Au, or other metals. The region between the grid lines can include a thin film such as an amorphous carbon film having a plurality of holes or pores. Such a film is generally referred to as a "perforated carbon film", and the sample under investigation can usually be fixed to the film by instant freezing. The temperature sensor 386 is placed on the surrounding ring 384, and additional sensors can be provided on the surrounding ring 384, the conductive grid 382, or the region between the grid lines. The following examples generally relate to the configuration of the temperature sensor, and details of the sample stage such as the carbon film may be omitted for the sake of convenience of explanation.

[0024] Example 4 Referring to FIG. 3B, a representative sample holder 302 includes a sample placement area 306 which, in the case of TEM applications, typically includes a metal grid and a thin film that allow electron beam transmission. The sample holder 302 also includes a surrounding area 308 that can be used to secure the sample holder 302 to the sample stage for one or more tilts or translations as needed. The surrounding area 308 generally need not be transmissive to the electron beam like the sample placement area 306, but it may be convenient to provide both the sample placement area 306 and the surrounding area 308 in an integral substrate such as a single grid or film. A transmission aperture 304 can be provided as well. One or more temperature sensors 310A - 310D can be disposed in the surrounding area 308 and thermally coupled to the sample placement area 306. The temperature sensors 310A - 310D can be formed of the same or different HTSC or other materials that exhibit a change in resistivity or other properties at or near the temperature of interest. In many applications, it is necessary to maintain the sample temperature below - 135°C to avoid devitrification of the sample, and a target temperature of - 150°C is used to provide a safety margin. In this temperature range, Bi - 2223 and Ti - Ba - Cu - oxide HTSCs are convenient choices. The temperature sensors 310A - 310D are arranged in a specific array in FIG. 3B, but other arrays of fewer or more sensors can be used. In some examples, only a single temperature sensor is provided.

[0025] For applications such as SEM, the sample stage need not exhibit a high electron beam transmission rate, and the sample placement area 306 and the surrounding area 308 can be defined, for example, on the surface of mounting posts such as metal mounting posts. The sensors can then be provided on the sides of such posts or elsewhere, as well as on the end faces that can function as the sample placement surface.

[0026] Example 5 Referring to FIG. 3C, a representative sample holder 322 typically includes a sample placement area 326 that includes a metal grid and a thin film. The sample holder 322 also includes a surrounding area 328 that can be used to secure the sample holder 322 to the sample stage. The surrounding area 328 and the sample placement area 326 can be provided as areas of a single integrated substrate, such as a single grid or film. The temperature sensor 330 can be disposed in the surrounding area 308 and is thermally coupled to the sample placement area 326. In this example, the temperature sensor 330 is an annular ring disposed around the sample placement area 326.

[0027] Example 6 In an alternative configuration shown in FIG. 3D, a representative sample holder 342 includes a sample placement area 346 disposed within a surrounding area 348 that can be used to secure the sample holder 342 to the sample stage. The surrounding area 348 and the sample placement area 346 can be provided as areas of an integrated substrate. The annular sensors 350, 352 are disposed around the sample placement area 346 and can be made from different sensor materials, such as HTSCs, having different critical temperatures. Additional annular sensors (or sensors of other shapes) can be provided and disposed around or near the surrounding area 348, or near the sample location on the sample placement area 346.

[0028] Example 7 In another alternative configuration shown in FIG. 3E, a representative sample holder 352 includes a sample placement area 356 disposed within a surrounding area 358 that can be used to secure the sample holder 352 to the sample stage. The surrounding area 358 and the sample placement area 356 can be provided as areas of an integrated substrate. The annular region around the sample placement area 356 includes sensors 360A, 360B that can be HTSCs having different critical temperatures or other materials having different temperature responses.

[0029] Example 8 Referring to FIG. 3F, the sample holder 390 includes a conductive grid 393, a carbon or other film 394, and a conductive surrounding ring 392. The sensor 396 is disposed on a part of the film 394 and the conductive grid 393.

[0030] Example 9 Referring to FIG. 3G, the sample holder 370 includes a conductive grid 372, a carbon or other film 374, and a conductive surrounding ring 376. The sensor region 378 defined on the film 374 and the grid 372 includes one or more sensor particles 371. A representative sample cell 375 is disposed on the film 374, and a representative sample cell 377 includes a temperature sensor particle 379 therein. The temperature sensor particle 379 is disposed in the cell, for example, by enabling the cell to incorporate the temperature sensor particle 379.

[0031] Example 10 Referring to FIG. 4A, the substrate stage 402 includes a sample holder placement area 404 defined on the placement surface 406. Typically, the substrate stage 402 includes other fasteners such as fasteners 428, 429 that can secure a sample holder such as one or more clips, screws, or a metal grid. The substrate stage 402 is generally arranged such that the sample can be translated and tilted during a sample preparation process such as image acquisition and / or ion milling. In some cases, a portion of the substrate stage 402 is tiltable for imaging or sample preparation, and such a portion is schematically shown as 421. The coolant ports 416, 418 are fluidly coupled to a coolant chamber such as coolant tube 414 around the substrate stage 402 to allow for the flow of a coolant such as liquid nitrogen (LN2). One or more sensors such as sensors 410, 412 are disposed on the placement surface 406 and thermally coupled to the sample placement area 404. Electrical connections 422 such as cables, wires, or conductive layers can provide electrical connections to one or more of the substrate stage 402, the sample placement area 404, and the sensors 410, 412. An electron beam transmission aperture 420 can also be provided.

[0032] Example 11 In another example shown in FIG. 4B, the substrate stage 452 includes a sample holder placement area 454 defined on the placement surface 456. Typically, the substrate stage 452 includes one or more fasteners (not shown in FIG. 4B) that can secure the sample holder. The substrate stage 452 is generally arranged such that the sample can be translated and tilted during image acquisition, and the tiltable portion is indicated by 471. The coolant ports 466, 468 are fluidly coupled to a coolant chamber, such as a coolant tube or bore, defined in the stage body 470 to allow for the flow of a coolant, such as liquid nitrogen (LN2). The sensor 462 can be formed as a square, rectangular, or circular ring on the placement surface 456 and can be thermally coupled to the sample placement area 454. As shown, the sensor 462 is disposed on the tiltable portion 471. Optionally, an additional sensor 473 can be disposed on the fixed portion. An electrical connection 472, such as a cable, wire, or conductive layer, provides an electrical connection to the substrate stage 452 and the sample placement area 454, and to one or both of the sensors 460, 473. An electron beam transmission aperture 460 can be provided as well.

[0033] Example 12 A typical method 500 for evaluating temperature based on the sensor region as described above involves identifying, at 502, the sensor region location on a sample holder or sample stage, and storing, at 504, the coordinates associated with the identified location. At 506, part or all of the available sensor region is irradiated and the associated current is measured. Typically, an electron beam is deflected to be incident on the sensor region or otherwise formed so as to irradiate the sensor region, either sequentially or simultaneously. An image or image portion associated with the sensor region is evaluated at 508 to determine a relative brightness based on the detected current associated with the sensor region. In some examples, a single sensor region is used and the sensor region image can be a single pixel or multiple pixels for part or all of the sensor region. For sensors positioned within a normal field of view, no deflection or steering of the electron beam to the sensor region is required. At 510, it can be determined whether to compare the brightness of the sensor region with the brightness or calibration value of one or more reference regions. If so, at 514, the brightness of one or more reference regions is determined, which may or may not require additional electron beam steering or deflection. The brightness of the reference region can be based on a previously stored value and no additional measurement is required. At 516, it is possible to confirm a sample temperature or temperature range, such as above or below a temperature based on a critical temperature associated with the sensor material used. More generally, the measured brightness can be compared with a calibration curve or calibration data that enables a temperature estimate based on the measured brightness. For example, the temperature of the sensor region can be varied, the associated brightness measured, and sensor calibration established using the resulting temperature-brightness data. In some examples, since the calibration curve fits the measurement data, the temperature can be determined from the calibration curve. Alternatively, the measurement data can be used with an interpolation procedure to calculate the sensor temperature. In other examples, the current in the sensor can be measured without the display or formation of an image.

[0034] The brightness of the image portion can be determined using backscattered CPB portion or secondary emission. In some examples, an image of the sample and / or the sensor area is displayed, and the operator can evaluate the sample temperature by inspecting one or more of the displayed images. For convenience, one or more reference images of one or more pixels can be displayed with brightness corresponding to one or more temperature values. Next, the operator can estimate the sample temperature by visual comparison. When multiple sensor materials are used, the temperature or temperature range of the sample can be further evaluated by image inspection. For example, one sensor area can have a brightness associated with being below the first critical temperature of a first HTSC, while another sensor area indicates a temperature above a second critical temperature, thereby allowing the sample temperature to be evaluated as being between the first and second critical temperatures.

[0035] Example 13 Using HTSCs and other materials that exhibit linear, non-linear, or other changes in resistance, a temperature profile can be determined, thereby further evaluating the temperature of the sample and the stage. Referring to FIG. 6, the sample holder 602 includes a sample placement area 604 surrounded by a sensor material 606. By directing an electron beam at various portions of the sensor material 606, the temperature across the sample holder 602 can be evaluated. In some cases, a temperature profile of the sample holder can be obtained. If necessary, temperature profiles for multiple cooling conditions such as various sample stages, various coolants, and various coolant flow rates can be obtained. In some cases, the sensor material can be placed in the sample placement area 604 (or the sensor material can be provided on the entire surface of the sample holder 602), enabling the evaluation of the temperature offset between more peripheral sensors and the sample area 602. Such a temperature profile can be stored in a computer-readable memory. As described previously, for sensor areas within a standard field of view, no additional electron beam deflection is required for imaging or measuring the sensor.

[0036] Referring to FIG. 7, the sample stage 700 includes a sample holder placement area 704 surrounded by a first sensor 706 and a second sensor 708 that substantially cover the placement surface 710. The coolant couplings 718, 720 are coupled to cooling tubes or cavities not shown in FIG. 7. Examination of the sensors 706, 708 enables evaluation of the temperature across the placement surface 710, and such evaluation can be made as a function of cooling conditions such as coolant flow rate or temperature or other variables.

[0037] Example 14

[0038] FIG. 8 and the following discussion are intended to provide a brief and general description of an exemplary computing environment in which the disclosed technology may be implemented. Although not required, the disclosed technology is described in the general context of computer-executable instructions, such as program modules, being executed by a personal computer (PC). Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Further, the disclosed technology may be implemented in other computer system configurations including, but not limited to, portable devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, etc. The disclosed technology may also be practiced in distributed computer environments where tasks are performed by remote processing devices linked through a communications network. In a distributed computer environment, program modules may be located in both local and remote memory storage devices.

[0039] Referring to FIG. 8, an exemplary system implementing the disclosed technology includes a general - purpose computing device in the form of an exemplary conventional PC 800, including one or more processing units 802, a system memory 804, and a system bus 806 that couples various system components including the system memory 804 to the one or more processing units 802. The system bus 806 may be any of several bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures. Exemplary system memory 804 includes read - only memory (ROM) 808 and random access memory (RAM) 810. The ROM 808 stores a basic input / output system (BIOS) 812 that contains basic routines that help transfer information between elements within the PC 800.

[0040] The exemplary PC 800 further includes one or more storage devices 830 such as a hard disk drive for reading from and writing to a hard disk, a magnetic disk drive for reading from and writing to a removable magnetic disk, and an optical disk drive for reading from and writing to a removable optical disk (such as a CD - ROM or other optical medium). Such storage devices can each be connected to the system bus 806 by a hard disk drive interface, a magnetic disk drive interface, and an optical drive interface. The drives and associated computer - readable media provide non - volatile storage of computer - readable instructions, data structures, program modules, and other data for the PC 800. Other types of computer - readable media can store data accessible by a PC, such as magnetic cassettes, flash memory cards, digital video disks, CDs, DVDs, RAM, ROM, etc., and can also be used in the exemplary operating environment.

[0041] Some program modules can be stored in a storage device 830 including an operating system, one or more application programs, other program modules, and program data. A user can input commands and information into the PC 800 via one or more input devices 840 such as a keyboard and a pointing device such as a mouse. Other input devices can include a digital camera, a microphone, a joystick, a game pad, a satellite broadcast receiver, a scanner, and the like. These and other input devices are often connected to one or more processing units 802 via a serial port interface connected to the system bus 806, but may also be connected by other interfaces such as a parallel port, a game port, or a universal serial bus (USB). A monitor 846 or other type of display device is also connected to the system bus 806 via an interface such as a video adapter. Other peripheral output devices such as speakers and printers (not shown) may be included.

[0042] The PC 800 can operate in a network environment using a logical connection to one or more remote computers such as a remote computer 860. In some examples, one or more networks or communication connections 850 are included. The remote computer 860 may be another PC, a server, a router, a network PC, or a peer device or other common network node, and typically includes many or all of the elements described above with respect to the PC 800, but only the memory storage device 862 is illustrated in FIG. 8. The personal computer 800 and / or the remote computer 860 can be connected to a local area network (LAN) and a wide area network (WAN). Such networking environments are common in offices, enterprise-scale computer networks, intranets, and the Internet.

[0043] When used in a LAN networking environment, the PC800 is connected to the LAN via a network interface. When used in a WAN networking environment, the PC800 typically includes a modem or other means for establishing communication via a WAN such as the Internet. In a network environment, program modules or portions thereof illustrated in connection with the personal computer 800 may be stored in a remote memory storage device or elsewhere on a LAN or WAN. The network connections shown are exemplary, and other means of establishing a communication link between computers may be used.

[0044] The PC800 is coupled to communicate with and / or control the CPB system. The memory 804 can include a memory portion 804A that stores sensor temperature characteristics or calibration data and sensor region positions, a memory portion 804B that provides CPB defect control, a memory portion 804C that stores temperature profiles of the stage or sample holder, a memory portion 804D that stores reference values such as current values or image brightness values from non-sensor regions, and a memory portion 804E that includes computer-executable instructions for comparing sensor values (current or brightness), calculating an expected sample temperature based on the stored temperature profile, and / or obtaining a temperature profile.

[0045] General Considerations The above example relates to electron microscopy for the sake of convenience of explanation, but other CPB systems, or systems based on electromagnetic radiation such as optical microscopes or X-ray systems can also be used. The temperature evaluation of the sample can be performed using the brightness of the sensor region in the image for the sake of convenience. As used herein, brightness refers to the apparent visual brightness or related brightness value of the displayed image in a stored or storable representation of an image or a portion of an image, such as JPEG, TIFF, bitmap, or other representation. Brightness can be obtained as a function of wavelength, charged particle energy, or other parameters, as well as a function of temperature. Brightness can be normalized to a range from dark (0) to bright (1) or other values.

[0046] In this specification, the region where the sensor material is provided is referred to as a sensor, sensor region, sensor patch, or temperature sensor for the sake of convenience. An image refers to a displayed image presented for visual observation by an operator of a device, and stored values that can be used to provide a visual image.

[0047] Referring to FIG. 10, a representative calibration method 1000 includes setting the sensor temperature at 1002 and recording the brightness (or other parameter) of the sensor at 1004. The set temperature and brightness are stored in a computer-readable memory at 1006. If there is interest in additional temperatures as determined at 1008, the additional temperatures are set at 1002 and the brightness measurements and data storage are repeated. If no additional temperatures are used, a calibration curve or table can be created at 1010. In some cases, an analytical fit can be generated such that the temperature T = T(B), where B is the brightness.

[0048] As used herein, a sample refers to a sample to be investigated, such as a biological sample, and not a sample support, metal grid, carbon film, etc.

[0049] The change in resistivity as a function of temperature or the change in other sensors can be examined by measuring the current in response to CPB irradiation such as secondary emission or backscattered CPB portions related to the sensor region. Such currents can be used to form an image of the thermal sensor. As used herein, higher brightness is associated with a larger detected current, as in a typical CPB image, but inverse contrast images can also be used. In some cases, one or more reference regions (i.e., non-sensor regions) are also examined for comparison with the sensor region for temperature evaluation. The disclosed approach is particularly suitable for cryo-electron tomography using an appropriate sample tilting mechanism and image reconstruction method.

[0050] For convenience, the sensor material has a large difference in resistivity in the target temperature range. Since HTSC has zero resistance below the critical temperature, the ratio of resistivity above and below the critical temperature becomes very large (theoretically infinite). For convenience, other materials that exhibit a resistivity ratio of 2.5:1, 5:1, 10:1, 20:1, 50:1 or more within the selected temperature range (often -175 °C to -135 °C in the case of cryo-electron microscopy) can be used. Generally, any material can be used along with calibration as a function of temperature. Material properties such as light reflectivity and transmittance, secondary emission ratio, optical spectrum, resistivity, etc. can also be used.

[0051] As used in this application and the claims, the singular forms "a", "an", and "the" include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the term "comprising" means "including". Further, the term "coupled" does not exclude the presence of intermediate elements between the coupled items.

[0052] The systems, devices, and methods described in this specification should not be construed as being in any way limiting. Instead, this disclosure is directed to all novel and non-obvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with each other. The disclosed systems, methods, and devices are not limited to any particular aspect or feature or combination thereof, and the disclosed systems, methods, and devices are not required to have any one or more particular advantages or to solve any particular problems. Although any theory of operation is for ease of explanation, the disclosed systems, methods, and devices are not limited to such theory of operation.

[0053] Some of the operations of the disclosed methods are described in a particular order for convenient presentation, but it should be understood that this presentation style encompasses rearrangements unless a particular order is required by the specific terms described below. For example, operations described in sequence may in some cases be rearranged or performed simultaneously. Further, for simplicity, the accompanying figures may not show the various ways in which the disclosed systems, methods, and devices can be used with other systems, methods, and devices. In addition, the description sometimes uses terms such as "generate" and "provide" to describe the disclosed methods. These terms are high-level abstractions of the actual operations being performed. The actual operations corresponding to these terms will vary depending on the particular implementation and will be readily recognizable to those of ordinary skill in the art.

[0054] In some instances, values, procedures, or devices are referred to as "lowest," "best," "minimum," etc. Such descriptions are intended to indicate that a selection can be made from among a number of available functional alternatives, and it should be understood that such a selection need not be superior, smaller, or otherwise preferable to other selections.

[0055] Examples are described with reference to directions indicated as "above", "below", "upper", "lower", etc. These terms are used for convenience of explanation and do not imply any specific spatial orientation. As used herein, thermal contact does not require direct physical contact, but only a heat conduction path.

[0056] From the perspective of a number of possible embodiments to which the principles of the technology of the present disclosure can be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be construed as limiting the scope of the present disclosure. Accordingly, we claim all that falls within the scope and spirit of the appended claims.

Claims

1. placing a thermal sensor in thermal proximity to a sample; imaging at least a portion of the thermal sensor; evaluating the temperature of the sample based on the brightness of the imaged portion of the thermal sensor or a current associated with the imaged portion of the thermal sensor; comprising; wherein the thermal sensor is a superconducting material, and evaluating the temperature of the sample comprises determining whether the sample is at a temperature lower than or higher than a critical temperature associated with the superconducting material; a method.

2. The method according to claim 1, wherein the superconducting material is a high temperature superconductor (HTSC).

3. The method according to claim 1, wherein the thermal sensor is disposed on a sample holder.

4. The method according to claim 3, wherein the sample holder includes at least one of a metal grid and a perforated carbon film, and the thermal sensor is disposed on at least one of the metal grid and the perforated carbon film.

5. The method according to claim 3, wherein the sample holder includes a conductive surrounding ring, and the thermal sensor is disposed on the conductive surrounding ring.

6. placing a thermal sensor in thermal proximity to a sample; imaging at least a portion of the thermal sensor; evaluating the temperature of the sample based on the brightness of the imaged portion of the thermal sensor or a current associated with the imaged portion of the thermal sensor; determining the brightness of the thermal sensor by exposing the thermal sensor to a charged particle beam (CPB); a method comprising.

7. placing a thermal sensor in thermal proximity to a sample; imaging at least a portion of the thermal sensor; evaluating the temperature of the sample based on the brightness of the imaged portion of the thermal sensor or a current associated with the imaged portion of the thermal sensor; forming a CPB image of an exposed portion based on exposure of at least a portion of the thermal sensor to a charged particle beam (CPB), and determining the temperature associated with the thermal sensor based on the brightness of the thermal sensor in the CPB image; a method comprising.

8. The method according to claim 1, wherein the thermal sensor is in contact with the sample or arranged within the sample. **Claim 9**: A sample holder for a CPB microscopy method, comprising: a sample support defining a sample area for holding a sample, wherein the sample support is a carbon film or a metal grid; a conductive peripheral ring disposed around at least a part of the sample support defining the sample area; at least one temperature sensor disposed thermally close to the sample area on the conductive peripheral ring, wherein the at least one temperature sensor includes a plurality of high-temperature superconducting (HTSC) materials having different critical temperatures, and each HTSC material is disposed at a respective position on the conductive peripheral ring; a sample holder including the above components. **Claim 10**: The sample holder according to claim 9, wherein the sample support is a carbon film, and the temperature sensor is in contact with or thermally coupled to the carbon film. **Claim 11**: The sample holder according to claim 10, further including a metal grid in contact with the carbon film, and the temperature sensor is in contact with or thermally coupled to the metal grid. **Claim 12**: The sample holder according to claim 9, wherein the temperature sensor is in contact with or thermally coupled to the peripheral ring. **Claim 13**: The sample holder according to claim 12, wherein the temperature sensor is a high-temperature superconductor (HTSC) having a critical temperature between -135°C and -175°C. **Claim 14**: The sample holder according to claim 9, wherein the temperature sensor includes a plurality of particles of a temperature sensor material fixed to or in contact with the sample support. **Claim 15**: A system configured to perform the method according to any one of claims 1 to 8, comprising: a sample stage operable to hold a CPB sample holder at a sample position; a thermal sensor operable to contact the CPB sample holder and coupled to the CPB sample holder, the thermal sensor being configured to generate a thermal response signal based on a temperature associated with the sample position; a processor coupled to the thermal sensor and operable to generate a display of the temperature of the CPB sample holder based on the thermal response signal; a system including the above components. **Claim 16**: The system of claim 15, further comprising at least one computer-readable medium storing a calibration of a thermal response signal as a function of temperature.

17. The system of claim 16, wherein the calibration is stored as a look-up table.

18. The system of claim 16, wherein the thermal response signal is one of image brightness, resistance, resistivity, or current in a CPB image.

19. The system of claim 15, further comprising a CPB-permeable sample holder, wherein the thermal sensor is fixed to the CPB-permeable sample holder.

20. A method for evaluating the temperature of a sample in cryo-electron microscopy, comprising: placing a thermal sensor in thermal contact with a vitrified sample; directing an electron beam at the thermal sensor and detecting one or more of backscattered electron intensity, secondary emission intensity, or conductivity associated with the thermal sensor in response to the electron beam; generating at least a partial image of the thermal sensor based on the detected current; determining the temperature of the vitrified sample based on the partial image; and a method including the above.

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