Multimodal cryogenic-compatible GUID grid

Abstract

According to the present disclosure, a cryo-compatible sample grid having a multimodal cryo-EM compatible GUID includes: an external support structure that defines an area of the grid for holding one or more samples; and a plurality of internal support structures that define a plurality of apertures, each aperture being configured to hold a sample. The cryo-compatible sample grid further includes: a first identifier located on the external support structure; and a second identifier located within the area of the grid for holding the one or more samples. The first identifier can be read by an optical detector, while the second identifier can be read by an electronic detector (e.g., within an electron microscope). Specifically, the second identifier can be read by the electronic detector when one or more teeth and / or holes containing the second identifier are filled with ice from the vitrification process.

Description

Background Art

[0001] Cryo-electron microscopy (cryo-EM) is an electron microscopy technique for studying samples at low temperatures. Specifically, cryo-EM involves immersing a sample in an aqueous solution, placing the solution within a sample grid, and then the sample grid undergoes a vitrification process. During the vitrification process, the sample grid / solution(s) thereon is / are rapidly cooled so that the water molecules in the aqueous solution do not have time to crystallize, thereby forming an amorphous solid that causes little or no damage to the structure of the sample suspended therein. The sample is then evaluated with an electron microscope at low temperature.

[0002] Currently, scientists are working to use cryo-EM technology to identify the samples being evaluated by the electron microscope because once the vitrification process occurs, the current sample grids cannot be globally identified. This is because once the sample grids undergo the vitrification process, and / or they introduce an excessive thermal mass that interferes with the vitrification process, the current identification techniques are unreliable or ineffective.

[0003] This poses a significant tracking problem for the cryo-EM workflow because vitrification and evaluation are typically performed at different times, on different machines, and / or at different locations, resulting in the sample grids remaining vitrified for long periods. Additionally, since many samples / sample grids can be vitrified and / or stored simultaneously, there is a high chance of swapping out sample grids during the cryo-EM workflow. Currently, location-based record-keeping systems are used to identify sample grids, which quickly become complex and prone to misidentification. For example, in some laboratories, sample grids are placed in small storage boxes that can hold 4 to 6 sample grids, which are then stored in storage bottles that can hold 5 - 10 storage boxes, and the storage boxes can be placed in bottle racks that can hold 6 storage bottles. Since some laboratory drawers can hold 10 bottle racks, this means that a single such drawer can accommodate up to 1200 - 3600 sample grids. Therefore, scientists need a global way to identify and track sample grids that can be operated both before and after vitrification. Summary of the Invention

[0004] According to the present disclosure, a cryo-compatible sample grid having a multimodal cryo-EM compatible GUID includes: an external support structure that defines a region of the grid for holding one or more samples; and a plurality of internal support structures that define a plurality of apertures, each aperture being configured to hold a sample. Each of the individual apertures is configured to hold a sample. The cryo-compatible sample grid further includes: a first identifier located on the external support structure; and a second identifier located within the region of the grid for holding one or more samples, wherein the second identifier is readable by an electron microscope. The first identifier is readable by an optical detector, while the second identifier is readable by an electron detector (e.g., within an electron microscope). Specifically, the second identifier is readable by an electron detector when one or more teeth and / or holes containing the second identifier are filled with ice from the vitrification process.

[0005] According to the present disclosure, a method for identifying a cryo-compatible sample grid having a multimodal cryo-EM compatible GUID includes: using an electron microscope to generate an electron microscope image of a cryo-compatible sample grid that has undergone a vitrification process; identifying a region of the electron microscope image that includes a vitrification-compatible identifier; and determining the identity of the cryo-compatible sample grid based on the vitrification-compatible identifier. The method may further include: storing information related to the sample grid in a database location associated with the vitrification-compatible identifier; accessing the information stored in such a database location; and / or taking one or more actions based on the information stored in such a database location. The method may further include reading an additional identifier located on the sample grid with an optical sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The detailed description is described with reference to the accompanying drawings. In the drawings, the leftmost digit(s) of the reference numeral identifies the drawing in which the reference numeral first appears. The same reference numerals in different drawings indicate similar or identical items.

[0007] Figure 1 Illustrated are example sample grid(s) having a multimodal cryo-EM compatible GUID.

[0008] Figure 2 Illustrated are alternative example sample grid(s) having a multimodal cryo-EM compatible GUID.

[0009] Figure 3 Illustrated is an example cryo-EM environment for preparing, vitrifying, examining, and storing a sample grid having a multimodal cryo-EM compatible GUID.

[0010] Figure 4 Depicted is a sample process for evaluating a sample with a sample grid having a multimodal cryo-EM compatible GUID.

[0011] Figure 5 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including a non-linear arrangement of holes.

[0012] Figure 6 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including individual holes located at intersections of internal support structures.

[0013] Figure 7 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including a linear arrangement of holes along internal support structures.

[0014] Figure 8 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including multiple teeth protruding into the pore diameter.

[0015] Figure 9 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including notches protruding into the support structure.

[0016] Figure 10 A figure showing a portion of a cryo-EM compatible sample grid including a cryo-EM compatible GUID, the cryo-EM compatible GUID including a non-linear arrangement of holes on an identification surface.

[0017] Figure 11 A figure showing a cross-sectional view of a sample grid having a multi-modal cryo-EM compatible GUID, the cryo-EM compatible GUID illustrating the performance of a second mode cryo-EM GUID after vitrification of the sample grid.

[0018] Figure 12 A graphical representation of the 300 kV scattering angle of ice and / or dirt within the holes of a cryo-EM compatible GUID.

[0019] Figure 13 A graphical representation of the portion of incident electrons transmitted through ice and / or dirt within the holes of a cryo-EM compatible GUID at 300 kV.

[0020] In several views of the drawings, like reference numerals refer to corresponding parts. Generally, in the drawings, elements that may be included in a given instance are illustrated with solid lines, while elements that are optional for a given instance are illustrated with dashed lines. However, elements illustrated with solid lines are not required for all instances of the present disclosure, and an element illustrated with a solid line may be omitted from a particular instance without departing from the scope of the present disclosure. Detailed Description

[0021] Disclosed herein are sample grids having a multimodal cryo-EM compatible GUID. More specifically, the present disclosure includes improved cryo-EM sample grids that include a first optically-readable identifier and a second identifier that can be read by an electron microscope after the sample grid has undergone a vitrification process. Additionally, the present disclosure describes methods for identifying vitrified cryo-EM sample grids in an electron microscope. Such methods can include the electron microscope automatically storing data and / or performing one or more other options based on the second identifier.

[0022] Figure 1 FIG. is an illustration of a sample grid 100 having a multimodal cryo-EM compatible GUID. Figure 1 Illustrated is a sample grid 100 that includes an external support structure 102 that defines an area of the grid 104 for holding one or more samples. The sample grid 100 is shown as having a circular shape, however it may have other shapes that are avoided by the present invention. Specifically, the sample grid 100 may have any shape that allows it to be loaded into and / or operated within a vitrification device, an electron microscope, a sample grid holding device, or other device(s) of a sample evaluation workflow. For example, the sample grid 100 may be shaped to be compatible with one or more devices and / or setups for loading one or more samples onto the sample grid 100, undergoing a vitrification process in which one or more samples are vitrified, and / or facilitating examination of the vitrified samples with an electron microscope.

[0023] The area of the grid 104 for holding one or more samples includes one or more internal support structures 106 that define a plurality of apertures 108. The internal support structure(s) 106 are rigid support structures that are configured to support and / or hold a sample within one or more of the apertures 108. For example, Figure 1One or more internal support structures 106 are shown as a plurality of rods (e.g., copper rods) that form a plurality of square apertures 108 for holding a sample. Example internal support structure(s) 106 can be composed of many different materials capable of supporting and / or holding a sample, such as but not limited to copper, gold, molybdenum, silicon, silicon nitride, another crystalline material, another metal, or a combination thereof. In some embodiments, the internal support structure can be an integral part of the external support structure 102, a separate structure that is fastened, glued, welded, or otherwise attached to the external support structure 102, or a combination thereof. The sample can include a biological sample suspended in a solution. In this way, the solution can be placed in the apertures 108 within the sample grid 100 having a multimodal cryo-EM compatible GUID. In some embodiments, one or more internal support structures 106 comprise a single internal support structure. For example, such an internal support structure can comprise a grid-like structure defining a plurality of apertures 108. Alternatively, the internal support structure(s) 106 can be a single structure having regions of different thicknesses, and wherein the plurality of apertures 108 correspond to regions of the single structure having a thickness that allows incident electrons to pass through, such that these regions are transparent when viewed with a transmission electron microscope.

[0024] Although not shown in Figure 1 the apertures 108 can include structures configured to hold a sample / solution containing the solution within the apertures 108, such as a mesh, foil, etc. For example, a foil layer (e.g., a carbon foil) can be present on top of one or more of the internal support structures 106 and extend over one or more of the plurality of internal support structures 106. In this way, when the sample is placed on top of the foil, the foil allows the sample to be positioned above the plurality of internal support structures 106. In this way, since the foil allows electrons to pass through, the sample is visible with a transmission electron microscope.

[0025] As Figure 1 further shown, the sample grid 100 has a multimodal cryo-EM compatible GUID. Specifically, the sample grid 100 has one or more first mode cryo-EM compatible GUIDs 110 and one or more second mode cryo-EM compatible GUIDs 112. Each of the first mode cryo-EM compatible GUID 110 and the second mode cryo-EM compatible GUID 112 is vitrification compatible. This means that they (1) can be read before and after the sample grid 100 undergoes the vitrification process, and (2) do not introduce additional thermal mass to the sample grid that interferes with the vitrification process and / or interferes with the vitrification of the solution(s) held within the regions of the grid 104.

[0026] The first-mode cryo-EM compatible GUID 110 is located on the outer support structure 102 and is configured to be read by an optical detector. The first-mode cryo-EM compatible GUID 110 can be printed on the outer support structure 102, mechanically imprinted or scratched to deform the surface, additionally marked, laser-etched, roughened with a laser or ion beam, reconstructed with a laser or ion beam, chemically colored, chemically etched to remove surface material, electrical discharge machined (EDM) to remove surface material, drilled, or applied with a photoresist process. For example, the first-mode cryo-EM compatible GUID 110 can include a barcode 114, a QR code 116, an alphanumeric code 118, a data matrix, or another optically readable ID.

[0027] The second-mode cryo-EM compatible GUID 112 is located within the sample-holding region of the grid 104 and can be read by an electron microscope. That is, the second-mode cryo-EM compatible GUID 112 is configured to be read by an electron microscope after the sample grid 100 has been vitrified and during / while the sample is being examined. Figure 1 An expanded view 150 of a sub-region 152 that includes the sample-holding region. The second-mode cryo-EM compatible GUID 112 includes holes or cuts that allow electrons to pass through and / or travel to one or more detectors. That is, because the inner support structure 106 must have sufficient mechanical strength to support the sample, the thickness of the inner support structure 106 is such that they block electrons. Thus, when evaluated and / or analyzed with a transmission electron microscope, the inner support structures 106 appear as dark features (since they prevent electrons from reaching the microscope detector), and the individual holes or cuts of the second-mode cryo-EM compatible GUID 112 appear as bright features within the inner support structure 106 (because they allow electrons to pass through the inner support structure 106).

[0028] Although Figure 1The holes depicted are substantially circular in shape, but may have various other shapes in different embodiments, such as star-shaped, square, etc. Alternatively, the holes may also include semi-circular or square cutouts at the edges of the internal support structure 106. The pattern of holes in the second mode cryo-EM compatible GUID 112 is configured to allow for the global identification of the sample grid 100. For example, the size of the holes, the position of the holes, the shape of the holes, the spacing between the holes may be varied and / or otherwise patterned in order to encode one or more identifiers associated with the sample grid 100. In some embodiments, the second mode cryo-EM compatible GUID 112 may also encode orientation information related to the sample grid 100. For example, the second mode cryo-EM compatible GUID 112 may encode information regarding the position of the second mode cryo-EM compatible GUID 112 on the sample grid 100 and / or the orientation of the second mode cryo-EM compatible GUID 112 relative to the sample grid 100.

[0029] Figure 1 Shows a second mode cryo-EM compatible GUID 112, which includes a pattern of large cutouts 120 placed at the intersections of the internal support structure 106, a plurality of small cutouts 122 placed along a single internal support structure 106, and a non-linear array of holes 124. A single sample grid 100 may include multiple types of second mode cryo-EM compatible GUIDs 112, and / or a single type of second mode cryo-EM compatible GUID 112 may be repeated at multiple locations within the region of the grid 104 for holding one or more samples.

[0030] Figure 2 Is an illustration of an alternative sample grid 200 with a multimodal cryo-EM compatible GUID. Figure 2 Illustrates a sample grid 200, which includes an external support structure 202 that defines a semi-circular region of the grid 204 for holding one or more samples. The region of the grid 204 for holding one or more samples includes a plurality of internal support structures 206 that define a plurality of aperture sizes 208.

[0031] As Figure 2 Further shown, the sample grid 200 has one or more first mode cryo-EM compatible GUIDs 210 and one or more second mode cryo-EM compatible GUIDs 212. Each of the first mode cryo-EM compatible GUID 210 and the second mode cryo-EM compatible GUID 212 is vitrification compatible. Figure 2The first-mode cryo-EM GUID 210 is illustrated as being located within the identification region 214. Although each type of first-mode cryo-EM GUID is shown as occurring only once in the sample grid 200, in other embodiments, the GUIDs may be repeated at multiple locations on the sample grid 200. Additionally, Figure 2 The second-mode cryo-EM compatible GUID 212 is illustrated as being located in the non-central position 216. As shown in the enlarged inset 218 of the non-central position 216, the second-mode cryo-EM compatible GUID 212 includes a non-linear pattern of small holes 220 along two adjacent internal support structures, as well as a series of holes 222 of different sizes. Figure 2 An insert structure 224 is also illustrated, which is located within the aperture and includes a non-linear pattern of holes. The insert structure 224 is thermally isolated from the internal support structure.

[0032] Figure 3 FIG. is an illustration of an exemplary cryo-EM environment 300 for preparing, vitrifying, examining, and storing a sample grid 302 having a multimodal cryo-EM compatible GUID. Specifically, Figure 3 An exemplary cryo-EM environment 300 is shown, which includes an exemplary cryo-EM system(s) 304 for examining a sample grid 302 having a multimodal cryo-EM compatible GUID. The exemplary charged particle microscope system(s) 304 may include a cryogenic electron microscope 304. Figure 3 An exemplary charged particle microscope system(s) 304 is shown as a cryo-TEM system 306.

[0033] The exemplary charged particle microscope system(s) 304 includes a charged particle source 308 (e.g., a thermionic source, a Schottky emission source, a field emission source, etc.), which emits an electron beam 310 along an emission axis 312 and towards an accelerator lens 314. The emission axis 312 is a central axis that extends along the length of the exemplary charged particle microscope system(s) 304 from the charged particle source 108 and passes through the center of the sample grid 302. The accelerator lens 314 accelerates / decelerates, focuses, and / or directs the electron beam 310 towards a focusing column 316. The focusing column 316 focuses the electron beam 310 such that it is incident on a sample 318 within the sample grid 302. Additionally, the focusing column 316 may correct and / or tune the aberrations (e.g., geometric aberrations, chromatic aberrations) of the electron beam 310.

[0034] In some embodiments, the focusing column 316 may include one or more of an aperture 320 and an upper objective lens 322. The focusing column 316 focuses the electrons from the electron source 308 onto the sample 318.

[0035] The sample grid 302 can be held by the sample holder 324. Electrons or charged particles 326 passing through the sample 318 and / or emitted by the sample can enter the projector 328. In one embodiment, the projector 328 can be a part separate from the focusing column 316. In another embodiment, the projector 328 can be an extension of the lens field from a lens in the focusing column 316. The projector 328 can be adjusted by the computing device 350 such that the direct electrons or charged particles 326 pass through the sample 318 and impinge on the microscope detector system 330.

[0036] In the low magnification TEM (LM mode), when filled with a minimal amount of ice, the holes that constitute the second identifier of the sample grid 302 may become invisible. This phenomenon is thought to be due to the small aperture opening (pupil function) of the projection lens system in the LM mode, because the objective lens is turned off (i.e., since any material like ice will scatter electrons to angles larger than the acceptance angle in LM, hardly any electrons will reach the camera). However, when the example cryo-EM 304 is a TEM operating in the SA mode with the objective lens enabled, this phenomenon is overcome. However, it should be noted that the example cryo-EM 304 is not limited to the TEM in this disclosure.

[0037] Figure 3 An example cryo-EM environment 300 is also shown, which optionally includes a computing device(s) 350, a sample preparation device / setup(s) 352, an optical scanning device(s) 354, a vitrification device / setup 356, and a sample grid storage setup 358. Figure 3 The computing device(s) 350, the sample preparation device / setup(s) 352, the optical scanning device(s) 354, the vitrification device / setup 356, and the sample grid storage setup 358 are illustrated as separate, however, in various embodiments, one or more of these elements can be combined. For example, the computing device 350 and / or the optical scanning device 354 can be incorporated into a single device and / or other devices (e.g., the sample preparation device / setup(s) 352, the vitrification device / setup 356, the sample grid storage setup 358, etc.), and those skilled in the art will understand, Figure 3The computing device 350 depicted is merely illustrative and is not intended to limit the scope of the present disclosure. Computing systems and devices can include any combination of hardware or software that can perform the indicated functions, including computers, network devices, Internet appliances, PDAs, wireless telephones, controllers, oscilloscopes, amplifiers, and the like. The computing device 350 can also be connected to other devices not shown or, alternatively, can operate as a stand-alone system. Additionally, in some embodiments, the functionality provided by the illustrated components can be combined in fewer components or distributed among additional components. Similarly, in some embodiments, the functionality of some of the illustrated components may not be provided and / or other additional functionality may be available.

[0038] It should also be noted that the computing device(s) 350 can be a component of the example charged particle microscope system(s) 304, can be a device separate from the example charged particle microscope system(s) 304, and can communicate with the example charged particle microscope system(s) 304 via a network communication interface or a combination thereof. For example, the example charged particle microscope system(s) 304 can include a first computing device 350 that is an integral part of the example charged particle microscope system(s) 304 and serves as a controller that drives the operation of the example charged particle microscope system(s) 304 (e.g., by operating a scanning coil to adjust the scanning position on the sample grid 302, etc.). In such embodiments, the example charged particle microscope system(s) 304 can also include a second computing device 350 that is a desktop computer separate from the example charged particle microscope system(s) 304 and can execute to process data received from the microscope detector system 330 to generate an image of the sample(s) on the sample grid 302 and / or perform other types of analysis. The computing device 350 can receive user selections via a keyboard, mouse, touchpad, touchscreen, and the like.

[0039] The sample preparation device / setup(s) 352 includes a laboratory setup for preparing a sample and adding the sample to a sample grid 302 having a multi-modal cryo-EM compatible GUID. For example, the sample preparation device / setup(s) 352 can include a device for mixing a sample to be examined by the example cryo-EM system(s) 304 in a solution such that the sample is suspended in the solution, and / or a device for adding the solution / sample in which it is suspended to one or more apertures of the sample grid 302. In some embodiments, preparing the sample can include storing the positions of individual samples on the sample grid 302 using the computing device 350. For example, the computing device 350 can store a mapping of the individual samples stored in the sample grid 302 and / or the position of each sample on the sample grid 302.

[0040] The optical scanning device 354 can correspond to any device capable of optically reading a first identifier on a sample grid having a multi-modal cryo-EM compatible GUID. For example, the optical scanning device 354 can include a barcode scanner, a QR scanner, a camera, etc., which are capable of detecting the first identifier and performing an identification process to identify the sample grid 302 and / or interacting with the computing device 350 to identify the sample grid 302. Alternatively or additionally, the optical scanning device 354 can cause information to be stored in an information database associated with the sample grid 302 (e.g., sample information, the location of the sample on the sample grid, a timestamp, a laboratory identifier, research information, etc.). In some embodiments, the optical scanning device 354 can be a component sensor of the computing device 350.

[0041] The vitrification device / setup 356 includes a laboratory setup for subjecting the sample grid 302 to a vitrification process. The vitrification process causes the solution included on the sample grid 302 to vitrify, thereby allowing a sample suspended therein to be observed with an example cryo-EM system(s) 304. In some embodiments, the vitrification device / setup 356 can include the optical scanning device 354, which is positioned to scan the first identifier on the sample grid when the sample grid is in the vitrification device / setup 356. Based on the optical scanning device 354 detecting the first identifier, the vitrification device / setup 356 can access a database associated with the sample grid and take actions according to the information in the database (e.g., adjusting settings, performing vitrification, causing additional information to be stored in the database, causing information to be presented to a user on a graphical user interface on a display associated with the vitrification device / setup 356, etc.).

[0042] Once the sample grid 302 has undergone the vitrification process, it must be kept at a low temperature so that the solution remains vitrified. This inhibits the ability of the optical scanning device 354 to scan the first identifier of the sample grid 302 having a multi-modal cryo-EM compatible GUID. The sample storage device 358 can include a short-term storage unit (e.g., a transport flask), which allows the sample grid 302 to be transported between the vitrification device / setup 356 and an example cryo-EM system(s) 304. Alternatively, the sample storage device 358 can include a long-term storage unit, in which the sample grid 302 can be held before and / or after being examined by an example cryo-EM system(s) 304.

[0043] Figure 3It further includes a schematic diagram illustrating an example computing architecture 370 of the computing device 350. The example computing architecture 370 illustrates additional details of the hardware and software components that can be used to implement the techniques described in this disclosure. In the example computing architecture 370, the computing device includes one or more processors 372 and a memory 374 communicatively coupled to the one or more processors 372.

[0044] The example computing architecture 370 may include an identification module 376 and a control module 378 stored in the memory 374. The example computing architecture 370 is also illustrated as including an identification map 380 and an information database 382 stored on the memory 374. The identification map 380 is a data structure that maps together a first identifier, a second identifier, and identification information about the sample grid 302. The information database 382 is a database that includes information associated with the sample grid 302 and / or other sample grids. For example, the information database 382 may include information related to the sample(s) present on the sample grid 302, the location of the sample on the sample grid 302, the location of the sample grid 302, sensor data / image data generated by the sample grid and / or the sample thereon, EM microscope settings, EM protocols, and the like.

[0045] As used herein, the term "module" is intended to represent an example partitioning of executable instructions for discussion purposes and is not intended to represent any type of requirement or required method, manner, or organization. Thus, although various "modules" are described, their functionality and / or similar functionality may be arranged differently (e.g., combined into a smaller number of modules, broken down into a larger number of modules, etc.). Additionally, although certain functions and modules are described herein as being implemented by software and / or firmware executable on a processor, in other cases, any one or all of the modules may be implemented in whole or in part by hardware (e.g., a dedicated processing unit, etc.) to perform the described functions. In various embodiments, the modules described herein in connection with the example computing architecture 370 may be executed across multiple devices 350.

[0046] The identification module 376 can be executed by the processor 372 to determine the identity of the sample grid 302 based at least in part on the first identifier or the second identifier. The identification module 376 can also identify the orientation of the sample grid 302 in the instance cryo-EM system(s) 304 based on the second identifier. For example, the identification module 376 can access the identification map 380 and determine the identity of the sample grid 302 based on the first identifier or the second identifier. In response to the computing device 350 receiving sensor information from the instance cryo-EM system(s) 304 depicting the second identifier, the identification module 376 can determine the identity of the sample grid 302. In some embodiments, the identification module 376 can be configured to automatically detect that the second identifier is located within the sensor data of the instance cryo-EM system(s) 304 and can automatically identify the sample grid 302 based on the automatic detection of the sample grid 302.

[0047] In the case where the identification module 376 detects that a portion of the second identifier is located within the sensor data of the instance cryo-EM system(s) 304, the identification module 376 can cause the instance cryo-EM system(s) 304 to manipulate the sample grid 302 and / or the focusing column 316 such that the remainder of the second identifier is included in the sensor data. For example, to improve the visibility of the second identifier, the identification module 376 can cause an adjustment of the focusing column 316 that defocuses the electron beam 310 such that it is focused on the bottom surface of the sample grid 302 away from the electron source 308. In some embodiments, the identification module 376 can montage and / or stitch together the sensor data such that the entirety of the second identity is included and can perform the identification of the sample grid 302 based on the montaged and / or stitched sensor data. In another instance, in response to the computing device 350 receiving sensor information from the optical scanning device 354 depicting the first identifier, the identification module 376 can use the identification map 380 to determine the identity of the sample grid 302. The identification module 376 can also be executed to cause the identity of the sample grid 302 to be sent to and / or presented on the display 384 of the instance cryo-EM system(s) 304, the optical scanning device 354, and / or the computing device 350.

[0048] The control module 378 can be executed by the processor 372 to cause the computing device 250 and / or the instance(s) of cryo-EM system(s) 304 to take one or more actions and / or present information. In some embodiments, the control module 378 can be executed to adjust the settings of the instance(s) of cryo-EM system(s) 304 such that the instance(s) of cryo-EM system(s) 304 perform a particular operation, or a combination thereof. For example, the control module 378 can be executed to access a portion of the information database 382 associated with the sample grid 302, the portion identifying the microscope settings of the sample grid 302 and / or the sample thereon, and then cause the instance(s) of cryo-EM system(s) 304 to have the microscope settings identified in the information database 382. In another instance, the control module 378 can access a portion of the information database 382 that identifies the location of the sample on the sample grid 302 and can cause the instance(s) of cryo-EM system(s) 304 to automatically image the sample on the sample grid 302. In the case where the instance(s) of cryo-EM system(s) 304 include a focused ion beam (FIB), the control module 378 can also be executed to cause one or more of the sample grids to be moved and / or the FIB to be adjusted and then cause the cryo-EM system(s) 304 to automatically start a milling process. For example, the database can include instructions for a particular area to be milled, and the cryo-EM system(s) 304 can automatically mill those particular areas based on the instructions.

[0049] Alternatively or additionally, the control module 378 can cause the display 384 to present a protocol to be presented to the user, present information about the sample grid 302, etc. In some embodiments, the control module 378 can cause the display 384 to present a graphical user interface that includes selectable interfaces that allow the user to input and / or change data associated with the sample grid 302 and / or select protocol steps to be performed by the instance(s) of cryo-EM system(s) 304. The control module 378 can also be executed to cause information from the instance(s) of cryo-EM system(s) 304, the optical scanning device 354, and / or information input by the user into the computing device 350 to be stored in the information database 382. For example, the control module 378 can cause sensor data and / or images from the instance(s) of cryo-EM system(s) 304 to be stored in the information database 382 in association with a first identifier, a second identifier, and / or the sample grid 302.

[0050] The computing device 350 includes one or more processors configured to execute instructions, applications, or programs stored in the memory(ies) accessible by the one or more processors. In some instances, the one or more processors may include hardware processors including, but not limited to, a hardware central processing unit (CPU), a graphics processing unit (GPU), and the like. Although in many cases these techniques are described herein as being executed by one or more processors, in some cases these techniques may be implemented by one or more hardware logic components such as a field programmable gate array (FPGA), a complex programmable logic device (CPLD), an application specific integrated circuit (ASIC), a system on a chip (SoC), or a combination thereof.

[0051] The memory accessible by the one or more processors is an example of a computer-readable medium. A computer-readable medium may include two types of computer-readable media, namely computer storage media and communication media. Computer storage media may include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information, such as computer-readable instructions, data structures, program modules, or other data. Computer storage media includes, but is not limited to, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EEPROM), flash memory or other storage technology, compact disc read-only memory (CD-ROM), digital versatile disc (DVD), or other optical storage device, magnetic tape cartridges, magnetic tape, magnetic disk storage devices, or other magnetic storage devices, or any other non-transmission medium that can be used to store the desired information and is accessible by a computing device. Generally speaking, computer storage media may include computer-executable instructions that, when executed by one or more processing units, cause the various functions and / or operations described herein to be performed. In contrast, communication media embodies computer-readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave, or other transmission mechanism. As defined herein, computer storage media does not include communication media.

[0052] Those skilled in the art will also understand that, for the purposes of memory management and data integrity, items or portions thereof may be transferred between a memory and other storage devices. Alternatively, in other embodiments, some or all of the software components may be executed in a memory on another device and communicate with the computing device 350. Some or all of the system components or data structures may also be stored (e.g., as instructions or structured data) on a non-transitory computer-accessible medium or portable article for reading by an appropriate drive, various examples of which were described above. In some embodiments, instructions stored on a computer-accessible medium separate from the computing device 350 may be transmitted to the computing device 350 via a transmission medium or signal (such as an electronic, electromagnetic, or digital signal conveyed via a communication medium such as a wireless link). Various embodiments may also include receiving, sending, or storing instructions and / or data implemented in accordance with the foregoing description on a computer-accessible medium.

[0053] Figure 4 FIG. is a flowchart of an illustrative process that depicts a collection of blocks in a logical flow diagram, representing a series of operations that may be implemented in hardware, software, or a combination thereof. In the context of software, these blocks represent computer-executable instructions stored on one or more computer-readable storage media that, when executed by one or more processors, perform the operations. Generally, computer-executable instructions include routines, programs, objects, components, data structures, etc. that perform particular functions or implement particular abstract data types. The order in which the operations are described is not intended to be construed as a limitation, and any number of the described blocks may be combined in any order and / or in parallel to implement the processes.

[0054] Figure 4 Depicted is a sample process 400 for evaluating a sample using a sample grid having a multimodal cryo-EM compatible GUID. Process 400 may be implemented using either of sample grids 100 and 200, in any of the example cryo-EM environments 300, and / or by the computing device(s) 350 described above, or in other environments and computing devices.

[0055] At 402, a sample is prepared and placed on a sample grid having a multimodal cryo-EM compatible GUID. In some embodiments, preparing the sample includes mixing the sample to be evaluated on a cryo-EM in a solution such that the sample is suspended in the solution, and / or adding the solution / sample in which it is suspended to one or more apertures of a sample grid having a multimodal cryo-EM compatible GUID.

[0056] In step 404, scan the first mode GUID of the sample grid. Specifically, scan the first mode GUID of the sample grid with an optical scanning device that is capable of optically reading the first mode GUID on a sample grid having a multimodal cryo-EM compatible GUID. For example, the optical scanning device can be a barcode scanner, a QR scanner, a camera, or other types of optical scanners capable of detecting the first mode GUID. In some embodiments, scanning the sample grid includes identifying the sample grid based on the first mode GUID. In some embodiments, the first mode GUID of the sample grid is scanned before preparing the sample and / or placing the sample on the sample grid.

[0057] In step 406, store information about the sample grid and / or the sample thereon. Specifically, the information about the sample grid and / or the sample thereon is stored in a database location associated with the first mode GUID, the second mode GUID, and / or the sample grid. The information can include sample information, the location of the sample on the sample grid, a timestamp, a laboratory identifier, research information, or other information associated with the sample grid. The information can be entered by a user, or the information can be automatically generated by the optical scanner and / or another device. For example, in the case where the optical scanner is associated with a storage system, the storage system can cause the storage location of the sample grid to be included in the database.

[0058] In 408, the sample grid and the sample thereon undergo a vitrification process. The vitrification process causes the solution on the sample grid to vitrify, thereby allowing the sample suspended therein to be observable by cryo-EM. In some embodiments, the vitrification device can include an optical scanning device that is positioned to scan the first mode GUID on the sample grid, and then the vitrification device can access the database associated with the sample grid and take actions based on the information in the database (e.g., adjust settings, perform vitrification, cause additional information to be stored in the database, cause information to be presented to the user on a graphical user interface on a display associated with the vitrification device, etc.). Once the sample grid has undergone the vitrification process, it must be kept at a low temperature so that the solution remains vitrified.

[0059] At 410, the sample is identified with a second-mode GUID. Specifically, cryo-EM determines that the second-mode GUID is located within the region of the sample grid being evaluated and identifies the sample grid based on the second-mode GUID. For example, the second-mode GUID includes holes, teeth, or cuts that allow electrons to pass through the sample grid such that they are detected by one or more detectors of the cryo-EM. In this way, even when one or more of the teeth, cuts, and / or holes that contain the second identifier are filled with ice from the vitrification process, the cryo-EM can visualize the second-mode GUID. To improve the visibility of the second-mode GUID, the focus of the cryo-EM electron beam can be adjusted such that it is focused on the bottom side of the sample grid and / or the exit plane of the holes that make up the second-mode GUID. For example, in some embodiments, reading a cryo-EM-compatible GUID can include cryo-EM that automatically defocuses the electron beam of the electron microscope such that it is focused on the bottom side of the internal support structure of the sample grid.

[0060] In some embodiments, the cryo-EM can detect that a portion of the second-mode GUID is present in a first region of the sample grid and can cause a second region of the sample grid to be observed such that the remainder of the second-mode GUID is detected by the cryo-EM. In some embodiments, the sample grid is identified with a first portion of the second-mode GUID, and the orientation of the sample grid and / or the position of the second-mode GUID on the sample grid is identified with a second portion of the second-mode GUID.

[0061] At step 412, information associated with the second-mode GUID and / or the sample grid is optionally accessed. In some embodiments, information about the sample grid and / or the sample can be stored at a location in a database associated with the first-mode GUID, the second-mode GUID, and / or the sample grid. The information associated with the second-mode GUID and / or the sample grid is presented on a graphical user interface on a display of the cryo-EM microscope or an associated computing device. Alternatively or additionally, the accessed information can be used to cause the cryo-EM to perform one or more actions, such as changing the settings of the cryo-EM such that the cryo-EM automatically images the sample on the sample grid, causing one or more of the sample grids to be moved and / or the FIB to be adjusted, and then initiating a milling process.

[0062] At 414, the sample grid and / or the sample thereon is inspected with cryo-EM. For example, a sample grid with a multimodal cryo-EM compatible GUID can be inspected with a transmission electron microscope operating in SA mode with the objective lens enabled. In some embodiments, the settings of cryo-EM can be modified according to the stored operating conditions when the operating conditions are stored in a database in association with the second mode GUID and / or the sample grid. In this way, cryo-EM can automatically adjust its performance and / or evaluation process based on the information and / or settings previously stored in association with the second mode GUID and / or the sample grid.

[0063] At 416, information about the sample grid and / or the sample thereon can optionally be stored. Specifically, the information about the sample grid and / or the sample thereon is stored in a location in the database associated with the first mode GUID, the second mode GUID, and / or the sample grid. The information can include the sample image on the sample grid generated by cryo-EM, information about the sample, information about cryo-EM, etc. The information can be generated by cryo-EM (or an associated computing device), or can be input by a user.

[0064] Figures 5 - 10 An example cryo-EM compatible GUID is illustrated, which can be imaged with cryo-EM after exposure to the vitrification process. The Cryo-EM compatible GUID according to the present invention is not limited to Figures 5 - 10 those shown. Additionally, Figures 5 - 10 the cryo-EM compatible GUIDs shown can be used in combination with each other, or the cryo-EM compatible GUIDs according to the present disclosure can include combinations of different Figures 5 - 10 features depicted therein.

[0065] Figure 5 A portion of a cryo-EM compatible sample grid 500 including a cryo-EM compatible GUID is illustrated, the cryo-EM compatible GUID including a non-linear arrangement of holes. Specifically, Figure 5 A portion of a cryo-EM compatible sample grid 500 including a plurality of internal support structures 106 is illustrated, the internal support structures defining a plurality of apertures 108 for holding a sample. Although not depicted in Figure 5 it, the apertures 108 can include a carbon foil configured to hold the sample while allowing the sample to be inspected by a cryo-EM system. The non-linear arrangement of the holes 502 is shown as an arrangement of substantially circular holes. In some embodiments, a portion of the non-linear arrangement of the holes can encode a global identifier, and different portions of the non-linear arrangement of the holes can encode an identifier and / or the orientation of the sample grid.

[0066] Figure 6 Illustrated is a portion of a cryo-EM compatible sample grid 600 including a cryo-EM compatible GUID 602, the cryo-EM compatible GUID including respective holes located at intersections of internal support structures. Figure 7 Illustrated is a portion of a cryo-EM compatible sample grid 700 including a cryo-EM compatible GUID 702, the cryo-EM compatible GUID including a linear arrangement of holes along internal support structures. Although Figures 5 - 7 each of the holes of GUIDs 502, 602, and 702 is depicted as having a sample size and shape, in other embodiments, the respective holes may have different sizes and / or shapes.

[0067] Figure 8 Illustrated is a portion of a cryo-EM compatible sample grid 800 including a cryo-EM compatible GUID 802, the cryo-EM compatible GUID including a plurality of teeth protruding into aperture 108. Similarly, Figure 9 Illustrated is a portion of a cryo-EM compatible sample grid 900 including a cryo-EM compatible GUID 902, the cryo-EM compatible GUID including a notch protruding into support structure 106. In some embodiments, although Figure 8 and 9 each is depicted as having a uniform size and shape in, the size and shape of the notch and / or teeth may vary within the GUID.

[0068] Figure 10 Illustrated is a portion of a cryo-EM compatible sample grid 1000 including a cryo-EM compatible GUID 1002, the cryo-EM compatible GUID including a non-linear arrangement of holes on an identification surface 1004. The identification surface 1004 is located within aperture 108 and is thermally isolated from a plurality of internal support structures 106 by one or more connecting bridges 1006. Each individual connecting bridge 10006 is configured to thermally isolate the identification surface 1004 from one or more internal support structures 106.

[0069] Figure 11 Illustrated is a cross-sectional view 1100 of a sample grid having a multimodal cryo-EM compatible GUID, the multimodal cryo-EM compatible GUID illustrating the performance of a second mode cryo-EM GUID after vitrification of the sample grid. Specifically, Figure 11Illustrated is a cross-sectional view of a sample grid 1102 bisected along the length of an internal support structure 1104. The sample grid 1102 is shown as having a top side 1106 and a bottom side 1108, with the top side facing the electron source when placed in a microscope for evaluation and the bottom side opposite the top side 1106. Figure 11 Also depicted is a foil 1110 (e.g., a carbon foil) positioned on the top side 1106 of the sample grid 1102. Also depicted is a cryo-EM compatible GUID 1112 that includes a plurality of holes 1114 drilled in the internal support structure 1104.

[0070] Additionally, Figure 11 An exploded view 1120 of a region 1118 of the sample grid 1102 is included, which illustrates the operation of the cryo-EM compatible GUID 1112 after vitrification of the sample grid 1102. Specifically, the exploded view 1120 illustrates the detectability of the cryo-EM compatible GUID 1112 when one or more of the plurality of holes 1114 are partially or fully obscured by dirt or ice 1122. An electron incidence path 1124 is depicted along an electron axis 1126, through which electrons from an electron source are incident onto the sample grid 1102, and an electron scattering path 1128 is depicted, which illustrates the path of the electrons after being scattered by dirt or ice 1122 in the hole 1114. The exploded view 1116 also shows a first pupil diameter 1130 of a projector lens when the microscope is in the LM mode (objective lens off), and a second pupil diameter 1132 of the projector lens when the microscope is in the SA mode (objective lens on). When operating in the LM mode, such a typical projector lens has a half-angle corresponding to 13 mrad, while when operating in the SA mode, such a projector lens typically has a half-angle corresponding to 130 mrad.

[0071] As described above, the individual holes 1114 are visible in an electron microscope because electrons can pass through a single hole 1114 and into a lens that guides the electrons to a detection system. This means that for an individual hole 1114 to be visible in an electron microscope, the electron scattering path 1128 must pass through the pupil diameter of the projector lens. For this reason, the cryo-EM compatible GUID 1112 is more visible in an electron microscope when the microscope is operating in the SA mode. Additional improvement in the detection of electrons passing through the plurality of holes 1114 can be achieved by defocusing the electron beam to the bottom side 1108 of the internal support structure 1104 and / or the exit plane of the holes in the plurality of holes 1114. For example, in some embodiments, reading the cryo-EM compatible GUID 1112 may include focusing the electron beam of an electron microscope onto the bottom side 1108 of the internal support structure 1104 of the sample grid.

[0072] Figure 12and 13 is a graphical representation of electron simulation results for different heights / thicknesses through the model material GlassyCarbon. The density and atomic mass of GlassyCarbon are relatively close to the expected mixture of the vitrified sample solution, which may fill the pores of the cryo-EM compatible GUID. Figure 12 is a graphical representation of the 300 kV scattering angles of the material within the pores of the cryo-EM compatible GUID 1200. Specifically, Figure 12 depicts the number (out of 100,000) of simulated electrons exhibiting each scattering angle (measured in mrad) at different heights / thicknesses of the model material GlassyCarbon. For the simulation, the spread of the outgoing trajectories of the transmitted electrons is recorded at the bottom plane of the GlassyCarbon slab.

[0073] Figure 13 is a graphical representation of the fraction of incident electrons transmitted through the material within the pores of the cryo-EM compatible GUID at 300 kV 1300. Specifically, Figure 13 depicts the fraction of electrons at each height / thickness of the model material GlassyCarbon transmitted through the objective lens of the projection lens system of the electron microscope at different objective lens semi-angles (measured in mrad). In graphical representation 1200, it can be seen that using a small semi-angle, such as <13 mrad when the objective lens is disabled (i.e., the TEM is operating in the LM mode), even a thin layer of a few microns of material filling the pores will move the electron trajectories outside the acceptance angle of the projection lens system. However, graphical representation 1200 also shows that by enabling the objective lens in the projection lens system (i.e., the TEM is operating in the SA mode), for which the acceptance angle is significantly larger (typically in the range of 130 mrad or greater), a larger number of scattered electrons are captured. As a result, when the TEM is operating in the SA mode, even when the entire pores of the cryo-EM compatible GUID are filled with 30 microns of material, a large enough detectable signal can be obtained.

[0074] Examples of the inventive subject matter according to the present disclosure are described in the paragraphs listed below.

[0075] A1. A cryo-compatible sample grid, the grid comprising:

[0076] An external support structure that defines an area of the grid for holding one or more samples;

[0077] A plurality of internal support structures that define a plurality of apertures, each individual aperture being configured to hold a sample;

[0078] A first identifier located on the external support structure; and

[0079] A second identifier, which is located within a region of the grid for holding one or more samples, wherein the second identifier is readable by an electron microscope.

[0080] A1.1. A cryogenic-compatible sample grid according to paragraph A1, wherein the first identifier is readable by an optical detector.

[0081] A1.1.1. A cryogenic-compatible sample grid according to paragraph A1, wherein the first identifier is readable by a visible light detector implemented in one or more of a sample storage device, a sample transfer device, a sample modification device, a vitrification device, or an electron microscope.

[0082] A1.2. A cryogenic-compatible sample grid according to any one of paragraphs A1 - A1.1, wherein the second identifier is readable by an electron detector.

[0083] A2. A cryogenic-compatible sample grid according to any one of paragraphs A1 - A1.2, wherein the cryogenic-compatible sample grid is configured to:

[0084] Receive one or more samples;

[0085] Perform a vitrification process, wherein one or more samples are vitrified; and

[0086] Facilitate examination of the vitrified samples by an electron microscope after vitrification.

[0087] A3. A cryogenic-compatible sample grid according to any one of paragraphs A1 and A2, wherein each of the first identifier and the second identifier is vitrification-compatible.

[0088] A3.1. A cryogenic-compatible sample grid according to paragraph A3, wherein each of the first identifier and the second identifier does not add thermal mass to the cryogenic-compatible sample grid.

[0089] A4. A cryogenic-compatible sample grid according to any one of paragraphs A1 - A3.1, wherein the first identifier is readable before the (said) vitrification process.

[0090] A4.1. A cryogenic-compatible sample grid according to paragraph A4, wherein the first identifier is readable after the vitrification process.

[0091] A4.2. A cryogenic-compatible sample grid according to any one of A4 - A4.1, wherein the first identifier is generated on the cryogenic-compatible sample grid by at least one of the following:

[0092] Surface roughening of the surface of an external support structure by a laser or an ion beam;

[0093] Surface structuring with a laser or an ion beam;

[0094] Mechanical stamping on the surface of the external support structure;

[0095] Mechanical scratching to deform the surface of the external support structure;

[0096] Attaching a mark on the external support structure;

[0097] Chemical coloring of the surface of the external support structure;

[0098] Chemical etching to remove the surface material of the external support structure;

[0099] Electrical discharge machining (EDM) to remove the surface material of the external support structure;

[0100] Drilling the external support structure; and

[0101] Coloring the surface of the external support structure using a photoresist process.

[0102] A4.3. A cryogenic-compatible sample grid according to any one of paragraphs A4 - A4.2, wherein the first identifier is one of a barcode, a QR code, a data matrix, and an alphanumeric code.

[0103] A4.4. A cryogenic-compatible sample grid according to any one of paragraphs A4 - A4.3, wherein the first identifier is a globally unique identifier.

[0104] A4.5. A cryogenic-compatible sample grid according to any one of paragraphs A4 - A4.4, wherein the first identifier comprises a first part encoding a globally unique identifier and a second part encoding a specific attribute of the sample grid.

[0105] A5. A cryogenic-compatible sample grid according to any one of paragraphs A2 - A4.4, wherein the second identifier is readable after vitrification.

[0106] A5.1. A cryogenic-compatible sample grid according to paragraph A5, wherein the second identifier is repeated at multiple positions within the area of the grid for holding one or more samples.

[0107] A5.2. A cryogenic-compatible sample grid according to any one of paragraphs A5 - A5.1, wherein the second identifier comprises one or more teeth that project outward from a specific internal support structure of a plurality of internal support structures and into corresponding apertures.

[0108] A5.2.1. A cryogenic-compatible sample grid according to paragraph A5.2, wherein each of the one or more teeth is separated from each other by a distance within the range of 1 mu to 10 mu.

[0109] A5.3. Based on the cryogenic-compatible sample grid of any one of paragraphs A5 - A5.2.1, wherein the second identifier includes a plurality of holes.

[0110] A5.3.1. Based on the cryogenic-compatible sample grid of paragraph A5.3, wherein at least one of the plurality of holes has a different size from a different hole among the plurality of holes.

[0111] A5.3.2. Based on the cryogenic-compatible sample grid of any one of paragraphs A5.3 - A5.3.1, wherein each of the plurality of holes is substantially circular.

[0112] A5.3.2.1. Based on the cryogenic-compatible sample grid of paragraph A5.3.2, wherein each of the plurality of holes is located at the intersection of the internal support structures.

[0113] A5.3.3. Based on the cryogenic-compatible sample grid of any one of paragraphs A5.3 - A5.4.3.1, wherein the plurality of holes are arranged in a linear arrangement.

[0114] A5.3.3.1. Based on the cryogenic-compatible sample grid of paragraph A5.3.3, wherein the linear arrangement is positioned along the internal support structures of the plurality of internal support structures.

[0115] A5.3.3.1.1. Based on the cryogenic-compatible sample grid of paragraph A5.3.3.1, wherein the linear arrangement is positioned along the edge of the internal support structures of the plurality of internal support structures, such that a small piece with an open window of carbon is visible.

[0116] A5.3.3.2. Based on the cryogenic-compatible sample grid of paragraph A5.3.3, wherein the second identifier is repeated at multiple positions, and the linear arrangement or each instance of the second identifier is positioned along different internal support structures of the plurality of internal support structures.

[0117] A5.3.4. Based on the cryogenic-compatible sample grid of any one of paragraphs A5.3 - A5.3.2, wherein the plurality of holes are arranged in a non-linear arrangement.

[0118] A5.3.4.1. Based on the cryogenic-compatible sample grid of paragraph A5.3.4, wherein the non-linear arrangement is not a rectangular or orthogonal pattern.

[0119] A5.3.5. Based on the cryogenic-compatible sample grid of any one of paragraphs A5.3 - A5.3.2, wherein the second identifier is readable when one or more teeth and / or holes containing the second identifier are filled with ice from the vitrification process.

[0120] A5.3.6. Based on the cryogenic-compatible sample grid of any one of paragraphs A5.3 - A5.5, wherein each of the plurality of holes has a diameter in the range of 1 mu to 10 mu.

[0121] A5.4. A cryogenic-compatible sample grid according to any one of paragraphs A5.3 - A5.3.2, 5.3.4.1, and 5.3.6, wherein the area of the grid for holding one or more samples includes an identification surface, and the identification surface includes a second identifier.

[0122] A5.4.1. A cryogenic-compatible sample grid according to paragraph A5.4, wherein the identification surface is thermally isolated from a plurality of internal support structures.

[0123] A5.4.2. A cryogenic-compatible sample grid according to any one of paragraphs A5.4 - A5.4.1, wherein the identification surface is connected to one or more internal support structures by one or more connecting bridges.

[0124] A5.4.2.1. A cryogenic-compatible sample grid according to paragraph A5.4.2, wherein each connecting bridge is configured to thermally isolate the identification surface from one or more internal support structures.

[0125] A5.5. A cryogenic-compatible sample grid according to any one of paragraphs A5 - A5.5.1, wherein the second identifier is a globally unique identifier.

[0126] A5.5.1. A cryogenic-compatible sample grid according to paragraph A5.5, wherein the second identifier is associated with the first identifier.

[0127] A5.5.2. A cryogenic-compatible sample grid according to paragraph A5.5, wherein the second identifier is the same as the first identifier.

[0128] A5.5.3. A cryogenic-compatible sample grid according to any one of paragraphs A5.5 - A5.5.2, wherein the second identifier also identifies the orientation of the sample grid.

[0129] A5.6. A cryogenic-compatible sample grid according to any one of paragraphs A5 - A5.5.3, wherein the individual components of the second identifier are positioned at a closed density such that they can be imaged without montage and / or stage movement.

[0130] A5.7. A cryogenic-compatible sample grid according to any one of paragraphs A5 - A5.6, wherein the second identifier is located near a marker, and the marker is visible at low magnification.

[0131] A6. A cryogenic-compatible sample grid according to any one of paragraphs A1 - A5.8, wherein each sample of one or more samples contains a solution containing one or more specimens.

[0132] A6.1. A cryogenic-compatible sample grid according to paragraph A6, wherein during the vitrification process the solution is frozen such that the solution is vitrified, thereby facilitating examination of one or more specimens with an electron microscope.

[0133] B1. A method for identifying a cryogenic-compatible sample grid, the method comprising:

[0134] generating an electron microscope image of the cryogenic-compatible sample grid, wherein the cryogenic-compatible sample grid has undergone a vitrification process;

[0135] identifying regions of the electron microscope image that include a vitrification-compatible identifier; and

[0136] determining the identity of the cryogenic-compatible sample grid based on the vitrification-compatible identifier.

[0137] B1.1. The method according to paragraph B1, further comprising determining the identity of a sample on the sample grid based on the vitrification-compatible identifier.

[0138] B1.2. The method according to any one of paragraphs B1 - B1.1, wherein the electron microscope is a transmission electron microscope with an enabled objective lens.

[0139] B1.3. The method according to any one of paragraphs B1 - B1.2, wherein generating the electron microscope image comprises setting the defocus of the electron microscope towards the bottom of the internal support structure of the compatible sample grid and away from the foil supporting the sample.

[0140] The method according to paragraph B1.3, wherein setting the defocus of the electron microscope towards the bottom of the internal support structure of the compatible sample grid comprises focusing the electron beam at the plane of the bottom surface of the sample grid.

[0141] The method according to paragraph B1.3, wherein setting the defocus of the electron microscope towards the bottom of the internal support structure of the compatible sample grid comprises focusing the electron beam at the plane of the bottom surface of the internal support structure.

[0142] The method according to paragraph B1.3, wherein setting the defocus of the electron microscope towards the bottom of the internal support structure of the compatible sample grid comprises focusing the electron beam at the exit plane of a hole, tooth or notch of the vitrification-compatible identifier.

[0143] B2. The method according to any one of paragraphs B1 - B1.3, wherein the vitrification-compatible identifier is a second identifier according to any one of paragraphs A1 - A6.1, and the cryogenic-compatible sample grid further comprises a first identifier according to any one of paragraphs A1 - A6.1.

[0144] B2.1. According to the method of any one of paragraphs B1 and B2, wherein the cryogenic-compatible sample grid is the cryogenic-compatible sample grid according to any one of paragraphs A1 - A6.1.

[0145] B3. According to the method of any one of paragraphs B2 and B2.1, which further comprises:

[0146] Scanning a first identifier; and

[0147] Storing information about the cryogenic-compatible sample grid and / or the sample on the cryogenic-compatible sample grid in a database associated with the first identifier.

[0148] B3.1. According to the method of paragraph B3, which further comprises accessing the database based on the first identifier.

[0149] B3.1.1. According to the method of paragraph B3.1, which further comprises:

[0150] Retrieving information from the database based on the first identifier; and

[0151] Taking actions in the vitrification device according to this information.

[0152] B3.2. According to the method of any one of paragraphs B3 and B3.1, which further comprises storing information about the cryogenic-compatible sample grid in the database.

[0153] B4. According to the method of any one of paragraphs B1 - B3.2, which further comprises accessing the (said) database based on a second identifier.

[0154] B4.1. According to the method of paragraph B4, which further comprises storing information about the cryogenic-compatible sample grid in the database.

[0155] B4.1.1. According to the method of paragraph B4.1, wherein the information includes an electron microscope image.

[0156] B4.2. According to the method of any one of paragraphs B4 - B4.1.1, which further comprises causing the electron microscope to perform actions based on the second identifier and the information in the database.

[0157] B4.2.1. According to the method of paragraph B4.2, wherein causing the electron microscope to perform actions comprises:

[0158] Accessing the object settings associated with the second identifier; and

[0159] Causing the electron microscope to operate according to the object settings associated with the second identifier.

[0160] B4.2.2. According to the method of paragraph B4.2, wherein causing the electron microscope to perform actions comprises:

[0161] Access the object settings associated with the second identifier; and

[0162] Move the sample grid to the milling position and initiate milling by a focused ion beam.

[0163] B4.2.3. The method according to paragraph B4.2, wherein causing the electron microscope to perform an action comprises:

[0164] Access the object settings associated with the second identifier; and

[0165] Cause the electron microscope to capture an image of the sample grid.

[0166] B4.3. The method according to any one of paragraphs B4 - B4.2.3, further comprising invoking a protocol based on the second identifier.

[0167] B4.3.1. The method according to paragraph B4.3, wherein the protocol includes providing a graphical user interface that presents options for imaging one or more samples in a cryo - compatible sample grid.

[0168] B4.3.2. The method according to any one of paragraphs B4.3 - B4.3.1, wherein the protocol includes providing a graphical user interface that presents options for imaging one or more samples in a cryo - compatible sample grid.

[0169] B5. The method according to any one of paragraphs B2 - B4.3.2, wherein generating an electron microscope image of a cryo - compatible sample grid with an electron microscope comprises:

[0170] Generating a first image of a first part of the second identifier with the electron microscope; and

[0171] Generating a second image of a second part of the second identifier, the second image being at least partially different from the first part of the identifier.

[0172] B5.1. The method according to paragraph B5, further comprising identifying the cryo - compatible sample grid based on the first image and the second image. C1. Use a cryo - compatible sample grid according to any one of paragraphs A1 - A6.1.

[0173] C1. Use a cryo - compatible sample grid according to any one of paragraphs A1 - A6.1.

[0174] D1. Use a cryo - compatible sample grid according to any one of paragraphs A1 - A6.1 to perform any one of the methods according to paragraphs B1 - B5.1.

Claims

1. A cryogenic-compatible sample grid, comprising: An external support structure that defines an area of the cryogenic-compatible sample grid for holding one or more samples; A plurality of internal support structures that define a plurality of apertures, each individual aperture being configured to hold a sample; A first identifier located on the external support structure; And A second identifier located within the area of the grid for holding the one or more samples, wherein the second identifier can be read by an electron microscope and encodes a global identifier associated with the cryogenic-compatible sample grid.

2. The cryogenic-compatible sample grid according to claim 1, wherein the first identifier can be read by a visible light detector implemented in one or more of a sample storage device, a sample transfer device, a sample modification device, a vitrification device, or the electron microscope.

3. The cryogenic-compatible sample grid according to claim 1, wherein the cryogenic-compatible sample grid is configured to: Receive the one or more samples; Perform a vitrification process, wherein the one or more samples are vitrified; and Facilitate examination of the vitrified samples with the electron microscope after vitrification.

4. The cryogenic-compatible sample grid according to any one of claims 1-3, wherein the first identifier is generated on the cryogenic-compatible sample grid via at least one of the following: Surface roughening of the surface of the external support structure by a laser or an ion beam; Surface structuring with a laser or an ion beam; Mechanical imprinting on the surface of the external support structure; Mechanical scratching to deform the surface of the external support structure; Chemical etching to remove surface material of the external support structure; Electrical discharge machining (EDM) to remove surface material of the external support structure; Attaching a marker on the external support structure; Chemically coloring the surface of the external support structure; Drilling holes in the external support structure; And Coloring the surface of the external support structure using a photoresist process.

5. The cryogenic-compatible sample grid according to claim 1, wherein the first identifier comprises a first part encoding a globally unique identifier and a second part encoding an attribute of the cryogenic-compatible sample grid.

6. The cryogenic-compatible sample grid according to any one of claims 1-3 and 5, wherein the second identifier is readable after the cryogenic-compatible sample grid has undergone a vitrification process.

7. The cryogenic-compatible sample grid according to claim 6, wherein the second identifier comprises one or more teeth that project outward from an internal support structure of the plurality of internal support structures and into a corresponding aperture, and each of the one or more teeth is separated from each other by a distance in the range of 1 mu to 10 mu.

8. The cryogenic-compatible sample grid according to claim 6, wherein the second identifier comprises a plurality of holes, each of the plurality of holes having a diameter in the range of 1 mu to 10 mu.

9. The cryogenic-compatible sample grid according to claim 8, wherein at least one of the plurality of holes has a different size from the different holes among the plurality of holes.

10. The cryogenic-compatible sample grid according to claim 8, wherein the plurality of holes are arranged in a linear arrangement that is positioned along the internal support structures of the plurality of internal support structures.

11. The cryogenic-compatible sample grid according to claim 6, wherein the second identifier is readable when one or more teeth or holes containing the second identifier are filled with ice from the vitrification process.

12. The cryogenic-compatible sample grid according to claim 6, wherein the region of the grid for holding the one or more samples includes an identification surface that includes the second identifier, and wherein the identification surface is thermally isolated from the plurality of internal support structures by one or more connecting bridges that connect the identification surface to the internal support structures.

13. The cryogenic-compatible sample grid according to any one of claims 1-3 and 5, wherein the second identifier also identifies the orientation of the cryogenic-compatible sample grid.

14. The cryogenic-compatible sample grid according to any one of claims 1-3 and 5, wherein the second identifier is located near a marker, and wherein the marker is visible at low magnification.

15. A method for identifying a cryogenic-compatible sample grid according to claim 1, the method comprising: generating an electron microscope image of the cryogenic-compatible sample grid with an electron microscope, wherein the cryogenic-compatible sample grid has undergone a vitrification process; identifying a region of the electron microscope image that includes a vitrification-compatible identifier, the identifier comprising a plurality of holes, incisions, or teeth in the sample grid, wherein the vitrification-compatible identifier encodes a global identifier associated with the cryogenic-compatible sample grid; and determining the identity of the cryogenic-compatible sample grid based on the vitrification-compatible identifier.

Images