Cohesive correlative light electron microscopy (CLEM), transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) microscope systems and methods

Abstract

An electron microscope system including a sample holder, a shared camera, a correlative light electron microscope (CLEM), a focused ion beam scanning electron microscope (FIB-SEM), and a transmission electron microscope (TEM). The CLEM includes a CLEM electron source to generate a first electron beam towards a sample and cause a first scattered beam for capture by the shared camera, and a light source configured to cause at least a portion of the sample to fluoresce for capture by the shared camera. The FIB-SEM includes a SEM electron source and a plasma source, the plasma source configured to generate an ion beam to mill the sample while supported by the sample holder. The TEM includes a TEM electron source to generate an electron beam towards the sample on the sample holder and cause a scattered beam for capture by the shared camera as a diffraction pattern.

Description

COHESIVE CORRELATIVE LIGHT ELECTRON MICROSCOPY (CLEM), TRANSMISSION ELECTRON MICROSCOPY (TEM), AND FOCUSED ION BEAM SCANNING ELECTRON MICROSCOPY (FIB-SEM) MICROSCOPE SYSTEMS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 684,629, filed August 19, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under HDTRA1 -21-1-0004 awarded by the Defense Threat Reduction Agency, and GM136508 awarded by the National Institutes of Health. The government has certain rights in the invention.SUMMARY OF THE DISCLOSURE

[0003] In one embodiment, a system is provided. The system includes an electron microscope system. The system includes: a sample holder configured to support a sample; a shared camera; a correlative light electron microscope (CLEM) comprising a CLEM electron source to generate a first electron beam towards the sample supported by the sample holder and cause a first scattered beam for capture by the shared camera as an electron microscope image, and a light source configured to cause at least a portion of the sample supported by the sample holder to fluoresce for capture by the shared camera as an optical image; a focused ion beam scanning electron microscope (FIB-SEM) comprising a SEM electron source and a plasma source, the plasma source configured to generate an ion beam to mill the sample while supported by the sample holder; and a transmission electron microscope (TEM) comprising a TEM electron source to generate an electron beam towards the sample on the sample holder and cause a scattered beam for capture by the shared camera as a diffraction pattern.

[0004] In another embodiment, a method for an electron microscope. The method includes: controlling a TEM electron source of an electron microscope to emit a first electron beam towards a sample supported by a sample holder of the electron microscope to generate a first atlas image of the sample with a shared camera; controlling a light source of a CLEM of the electron microscope to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera; identifying one or more targets in the first fluorescence overlay image; controlling a plasma source of a FIB-SEM to mill the sample based on identifying the one or more targets; controlling the TEM electron source to emit a second electron beam towards the sample while controlling the sample holder790482-00538 to rotate; capturing, by the shared camera, scatterings of the electron beam by the sample as diffraction images while the sample holder is rotated; and outputting microcrystal electron diffraction data based on the diffraction images.

[0005] The foregoing and other aspects and advantages of the present disclosure will appear from the following description. In the description, reference is made to the accompanying drawings that form a part hereof, and in which there is shown by way of illustration a preferred embodiment. This embodiment does not necessarily represent the full scope of the invention, however, and reference is therefore made to the claims and herein for interpreting the scope of the invention. Like reference numerals will be used to refer to like parts from Figure to Figure in the following description.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 illustrates a block diagram of a cohesive electron microscope (EM) system, in accordance with some embodiments.

[0007] FIG. 2 illustrates the cohesive electron microscope of FIG. 1 , in accordance with some embodiments.

[0008] FIG. 3 illustrates a cooling system for the electron microscope of FIG. 1, in accordance with some embodiments.

[0009] FIG. 4 illustrates a sample stage of the cohesive electron microscope of FIG. 1, in accordance with some embodiments.

[0010] FIG. 5 illustrates a control system and user input / output (I / O) devices of the cohesive electron microscope of FIG. 1, in accordance with some embodiments.

[0011] FIG. 6 illustrates a process for an electron microscope, in accordance with some embodiments.

[0012] FIG. 7A illustrates an example atlas of a grid with a sample thereon, in accordance with some embodiments.

[0013] FIG. 7B illustrates an example fluorescence overlay image of the grid with the sample thereon of FIG. 7 A, in accordance with some embodiments.

[0014] FIG. 8A illustrates a cross section of the sample of FIGS. 7A-7B, in accordance with some embodiments.

[0015] FIG. 8B illustrates a cross section of the sample of FIGS. 7A-7B after milling, in accordance with some embodiments.

[0016] FIG. 9 illustrates an example of the cohesive EM system of FIG. 1 incorporated within a housing, in accordance with some embodiments.790482-00538DETAILED DESCRIPTION

[0017] Microcrystal electron diffraction (MicroED) allows the collection of high- resolution electron diffraction data from extremely small (sub-micron-sized) protein microcrystals using an electron cryo-microscope (cryo-EM). To perform MicroED, a standard transmission electron microscopy (TEM) system may be modified to use a continuous rotationcontrolling device. However, identifying targets within a sample and preparing the targets for a MicroED is a nontrivial task that can be time consuming and difficult to successfully perform. In some examples, a target, as used herein, refers to a crystal (a crystal target), which is a two- or three-dimensional object with repeat structural motifs, regardless of size or identity of the repeat motif (which may be, e.g., a protein, small molecule, large molecule, material etc.). In some examples, a target may be a material, cell, or tissue that is not a crystal (a non-crystal target).

[0018] A cohesive electron microscope and methods implemented by a cohesive electron microscope are provided herein that address these and other issues. For example, by combining a transmission electron microscope (TEM), a correlative light electron microscope (CLEM), and a focused ion beam scanning electron microscope (FIB-SEM) system into a cohesive electron microscope, the cohesive electron microscope can: receive a sample, control the TEM to identify potential targets in the sample, control the CLEM to identity7targets that fluoresce from the potential targets, control the FIB-SEM to mill around the identified targets to prepare the identified targets for imaging, and then control the TEM to perform MicroED imaging of the prepared targets. Thus, the cohesive electron microscope can provide an automated and integrated workflow, increasing accuracy and throughput of a MicroED system.

[0019] FIG. 1 illustrates a block diagram of a cohesive electron microscope (EM) system 100, according to some embodiments. The cohesive EM system 100 includes a transmission electron microscope (TEM) system 105, correlative light electron microscope (CLEM) system 110, a focused ion beam scanning electron microscope (FIB-SEM) system 115, a sample stage 118, a shared camera 120, a vacuum system 125, a control system 130, and an user input / output (I / O) interface 135. One or more elements of the cohesive EM system 100 may be coupled to a communication bus 140 to enable communication among these elements. For example, in some embodiments, the control system 130 is configured to communicate with and control the other components of the cohesive EM system 100 to generate and process image data via the communication bus 134. As explained in further detail, the shared camera 120 may be able to capture TEM image data of a sample on the sample stage 118 generated by the TEM system 105, CLEM image data of the sample on the sample stage 118 generated by the CLEM790482-00538 system 110, and FIB-SEM image data of the sample on the sample stage 118 generated by the FIB-SEM system 115.

[0020] In some examples, the shared camera 120 includes a CMOS detector (or detector of another imaging technology) having a pixel array of at least 512 x 512 pixels (512 x 512 resolution), a physical pixel size of between 4-250 micrometers, radiation hardy, and a high dynamic range.

[0021] FIG. 2 illustrates a multi-column architecture of the cohesive EM system 100. More particularly, the cohesive EM system 100 includes a TEM central column 200, a CLEM angled column 205, and a FIB-SEM angled column 210. The TEM central column 200 includes an upper portion 211, an intermediate portion 212, and a lower portion 213. The upper portion houses a TEM electron source 215, one or more lenses (e.g.. condenser lenses Cl. C2. C3), and an objective 218. The intermediate portion includes an interface or connection point for the TEM central column 200, the CLEM angled column 205, and the FIB-SEM angled column 210, as well as the sample stage 118. The sample stage 118, illustrated in further detail in FIG. 4, may also be referred to as a piezo sample stage 118, and further includes a sample inlet 220. The sample stage 1 18 may support a sample to be imaged by the cohesive EM system 100. The lower portion of the TEM central column 200 includes one or more lenses, filters, and apertures (e.g., selected area aperture 225 and filter 230) and the shared camera 120. The TEM electron source 215 is configured to generate a first electron beam towards the sample.

[0022] The CLEM angled column 205 includes a CLEM electron source 235 configured to generate a second electron beam towards the sample and a CLEM light source 240 configured to generate a light beam towards the sample. The SIB-FEM angled column 210 includes a plasma source 245 configured to generate an ion beam towards the sample and the SEM electron source 250 is configured to generate a third electron beam towards the sample.

[0023] In FIG. 2, the CLEM, TEM, and FIB-SEM columns and sources therein are offset with respect to one another by 45 degrees (e.g., on a x-z plane, with the sample on an x- y plane). More particularly, the CLEM electron source 235 and the CLEM light source 240 are 45 degrees offset from the TEM electron source 215; the SEM electron source 250 and the plasma source 245 are 45 degrees offset from the TEM electron source 215; and the CLEM electron source 235 and the CLEM light source 240 are 90 degrees offset from the SEM electron source 250 and the plasma source 245. Stated another way, the second electron beam and the light beam from the CLEM electron source 235 and the CLEM light source 240 are 45 degrees offset from the first electron beam generated by the TEM electron source 215. The third electron beam and the ion beam from the SEM electron source 250 and the plasma source790482-00538245 are 45 degrees offset from the first electron beam generated by the TEM electron source 215. The second electron beam and the light beam from the CLEM electron source 235 and the CLEM light source 240 are 90 degrees offset from the third electron beam and the ion beam from the SEM electron source 250 and the plasma source 245. These offset angles of 45 and 90 degrees described herein may be 45 and 90 degrees within a tolerance range of, for example, + / - 1 degree. + / - 0.5 degrees, or + / - 0.1 degrees. In some examples, the offset angles of the columns and sources therein may be other values.

[0024] The shared camera 120 may be aligned with the other components of the TEM system 105 such that an optical axis of the TEM system 105, extending from the TEM electron source 215 to the shared camera 120, crosses through the center of a rotation axis 255 of the sample stage 118.

[0025] In some examples, the TEM central column 200, the CLEM angled column 205, and the FIB-SEM angled column 210 together define a chamber 260. The chamber 260 may be airtight after a sample is inserted in the system 100 onto the sample stage 118 and the sample inlet 220 is sealed. The chamber 260 may include the sample stage 118, the sources 215, 235, 240, 245, 250 (e.g., the emitting portions of the sources 215, 235, 240, 245, 250), the shared camera 120, the lenses Cl, C2, C3, the objective 218, and the filter 230. The vacuum system 125 may include a pump and may be coupled to the chamber 260. The control system 130 may control the vacuum system 125 (e g., the pump thereof) to generate a vacuum in the chamber 260 while the system 100 is operational (e.g., during imaging, milling, blocks 605-630 of FIG. 6, etc.).

[0026] In some examples, the system 100 is used for imaging and operation in cryogenic conditions. For example, FIG. 3 illustrates a cooling system 300 for use with the system 100. The cooling system 300 is coupled to a holder 305, which may be used to deliver a grid 310 having a sample thereon 315 to the sample stage 118. In some examples, the holder 305 may be rod-shaped. The cooling system 300 includes a cryogenic fluid supply 320, a cryogenic fluid supply line 325 (e.g., flexible tubing), support(s) 330, insulation 335 to insulate the cryogenic fluid supply line 325 within the chamber 260, and a decoupler 340. The cryogenic fluid may be a liquefied gas that can be kept in liquid state at temperatures below -150 degrees Celsius. In some examples, the cryogenic fluid is liquid nitrogen. The holder 305 may be coupled to the cryogenic fluid supply 320 via the cryogenic fluid supply line 325 to maintain the holder 305, grid 310, and / or sample 315 at cry ogenic temperatures during imaging and operation of the system 100. In some examples, the system 100 is used for imaging and operation at ambient conditions. In such examples, the holder may be not coupled to a790482-00538 cryogenic fluid supply or other components of the cooling system 300 that can maintain the holder and sample at cryogenic temperatures and, rather, the holder 305. the grid 310, and / or the sample 315 are at ambient temperature.

[0027] FIG. 4 illustrates an example of the sample stage 118 in further detail. More particularly, the sample stage 118 includes the sample inlet 220 with port 402 (also referred to as a holder port) enabling insertion and removal of a sample onto the sample stage 118. The sample stage 118 further includes the holder 305 that supports the grid 310 on which the sample 315 that is to be imaged may be provided. The grid 310 may be an electron microscopy grid. The grid 310 may have or be described as a mesh of windows formed by crossing x and y lines, where the area between each pair of adjacent x lines and crossing adjacent y lines is s window. The port 402 and the sample inlet 220 may receive the holder 305 having the grid 310 with the sample 315 thereon. The sample stage 118 further includes piezo actuators 420 configured to be controlled by the control system 130 to rotate up to 360 degrees. For example, by moving one piezo actuator up and the other piezo actuator down, the holder 305 (which may be generally cylindrical) may rotate. The piezo actuators 420 may be configured to rotate the holder 305 with linearity of rotation such that, at 0.1-10 degrees of rotation per second, the linearity of rotation of between 0. 1-10 degrees rotation / second greater than 99% linearity.

[0028] FIG. 4 further illustrates example beams that may be generated and transit through the various columns of the cohesive EM system 100. More particularly, FIG. 4 illustrates a first electron beam 430, a second electron beam 435, a first light beam 440, a third electron beam 445, a focused ion beam 450, and a fourth electron beam 455. The first electron beam 430 may be generated by the TEM electron source 215 and may travel through the TEM central column 200 towards the sample 315 (e.g., to generate an atlas, as described further below). The second electron beam 435 may be generated by the CLEM electron source 235 and may travel through the CLEM angled column 205 towards the sample 315. The first light beam 440 may be generated by the CLEM light source 240 and may travel through the CLEM angled column 205 towards the sample 315. The third electron beam 445 may be generated by the SEM electron source 250 and may travel through the FIB-SEM angled column 210 towards the sample 315. The focused ion beam 450 may be generated by the plasma source 245 and may travel through the FIB-SEM angled column 210 towards the sample 315. The fourth electron beam 455 may be generated by the TEM electron source 215 and may travel through the TEM central column 200 towards the sample 315 (e.g., to generate diffraction images for MicroED data, as described below). Because the sources of each of the TEM, CLEM, and FIB- SEM systems is positioned within the chamber 260, each of the beams 430, 435, 440, 445, 450,790482-00538 and 455 travel through the chamber 260, serving as a shared chamber, and reach the sample within the chamber 260.

[0029] In FIG. 4, a single line is labeled as both the beams 430 and 455 because both the beams 430 and 455 may travel along a generally similar path towards the sample 315. However, the beams 430 and 455 are separate beams generated at different points in time by the TEM electron source 215. The first electron beam 430 may also be referred to as an atlas TEM electron beam because it is generated by the TEM electron source 215 to generate an atlas of the sample 315, as described further below. The fourth electron beam 455 may also be referred to as an imaging TEM electron beam because it is generated by the TEM electron source 215 to generate imaging data of the sample 315 (e.g., to generate diffraction images for MicroED data, as described below). The first electron beam 430 and the fourth electron beam 455 may also be referred to as TEM electron beams because they are electron beams generated by the TEM electron source 215.

[0030] In FIG. 4, a single line is labeled as both the beams 435 and 440 because both the beams 435 and 440 may travel along a generally similar path towards the sample 315. However, the beams 435 and 440 are separate beams generated by different components (the CLEM electron source 235 and the CLEM light source 240). The second electron beam 435 may be referred to as a CLEM electron beam because it is generated by the CLEM electron source 235. The first light beam 440 may be referred to as a CLEM light beam because it is generated by the CLEM light source 240.

[0031] In FIG. 4, a single line is labeled as both the beams 445 and 450 because both the beams 445 and 450 may travel along a generally similar path towards the sample 315. However, the beams 445 and 450 are separate beams generated by different components (the plasma source 245 and the SEM electron source 250). The third electron beam 445 may also referred to as a SEM electron beam because it is generated by the SEM electron source 250.

[0032] FIG. 5 illustrates a block diagram of an example of the control system 130 and the user I / O devices 135. As illustrated, the control system 130 includes an electronic processor 500, a memory 505, and an input / output interface 510 coupled by a communication bus 512. The memory 505 includes one or more of a read only memory (ROM), random access memory (RAM), or other non-transitory computer-readable media. The electronic processor 500 is configured to, among other things, receive instructions and data from the memory 505 and execute the instructions to, for example, carry out the functionality of the control system 130 described herein, including the process 600 of FIG. 6. For example, the memory 505 includes one or more of control software 515 and image data 525. Generally, the electronic processor790482-00538500 may be configured to execute the control software 515 to generate, process, and store the image data 525, to control the TEM system 105, to control the CLEM system 110, to control the FIB-SEM system 115, to control the sample stage 118, to control the shared camera 120, to control the vacuum system 125, and to generate and output MicroED output. In some embodiments, instead of or in addition to executing software from the memory 505 to carryout the functionality of the control system 130 described herein, the electronic processor 500 includes one or more hardware circuit elements configured to perform some or all of this functionality.

[0033] The input / output interface 510 includes input and output interface elements that enable the electronic processor 500 to communicate with and control the other components of the system 100. including the user input / output devices 135. In some embodiments, the input / output interface 510 enables wireless and / or wired communication according to one or more known protocols (e.g., Wi-Fi, Bluetooth, USB, fiber, copper etc.). For example, the input / output interface 510 includes wired or wireless interface circuitry-, such as antennas, wired ports, and transceivers for transmitting and receiving signals using antennas and / or wired ports.

[0034] Although the control system 130 is illustrated as a single unit, in some embodiments, one or more components of the control system 130 is remote from the other components, is a distributed component, or a combination thereof. For example, in some embodiments, the memory 505 includes local memory co-located with the electronic processor 500 as well as remote memory located off-site and, for example, connected to the electronic processor 500 by one or more netw orks (e.g. , a local area netw ork or another wide area netw ork such as the Internet). Similarly, in some embodiments, the electronic processor 500 includes one or more local microprocessors and, in other embodiments, the electronic processor 500 is a distributed processing system including a combination of one or more local microprocessors and remote processors (e.g., cloud computing). The user input / output devices 135 include one or more devices enabling a user to interact with the system 100. As illustrated, the user input / output devices 135 includes a display 550 for displaying one or more images generated by the shared camera 120 and human input devices (HIDs) 555 (e.g.. one or more keyboards, switches, a mouse, touch pads, touch screens, etc.) for receiving input from an operator.

[0035] FIG. 6 illustrates a process 600 for an electron microscope, according to some examples. The process 600 is described as being carried out by the cohesive EM system 100. However, in some embodiments, the process 600 may be implemented by another electron microscope system. Additionally, although the blocks of the process 600 are illustrated in a790482-00538 particular order, in some embodiments, one or more of the blocks may be executed partially or entirely in parallel, may be executed in a different order than illustrated in FIG. 6, or may be bypassed.

[0036] In block 605, a control system controls a TEM electron source of an electron microscope to emit a first electron beam towards a sample supported by a sample holder of the electron microscope to generate a first atlas image of the sample with a shared camera. For example, with reference to FIGS. 1, 2, and 4, the control system 130 may control the TEM electron source 215 to generate a first electron beam 430 towards the sample 315 on the sample stage 118. The control system 130 may further control the shared camera 120 to capture an image or images to generate an atlas of the grid 310 and the sample 315 thereon. In some examples, to generate the atlas, the control system 130 controls the TEM electron source 215 and the shared camera 120 to image an entirety of the grid 310 using a low dose (e.g., a lower dose or at a power level than used for TEM imaging, such as in block 630, for example). In some examples, the control system 130 controls the TEM electron source 215 and the shared camera 120 to perform a raster scan of the 310. In both cases, the atlas generated may include image data for the grid 310 and the sample 315 thereon captured by the shared camera 120.

[0037] The control system 130 may analyze or process the image data to identify locations of crystals or other potential targets to be imaged. For example, the image data processing may identify windows in the grid 310 having shapes (e.g.. circles) or areas of contrast relative to the background or other portions of the grid 310. The identified windows having such contrasting elements may be considered target locations having potential targets to be imaged. The potential targets may be defined in terms of coordinates on the grid (e.g., x- y coordinates describing x and y location in millimeters, grid window location, and / or arbitrary units).

[0038] FIG. 7A illustrates an example atlas 700 of the grid 310 with the sample 315 thereon. The atlas 700, or atlas image, includes windows defined in terms of grid coordinates with letters on the x-axis and numbers on the y-axis. However, other coordinate systems are used in other examples. For example, values of actual measurements (e.g., in millimeters (mm)) may be used to define x-y coordinates (e.g., a window may be at position 300 mm, 600 mm, or at position 200 mm, 400 mm). As illustrated in FIG.7A, six windows are illustrated with target locations having potential targets. More specifically, windows Bl, C4, D2, E3, F4, and F5 are target locations with potential targets, two of which are labeled as potential target 705 and potential target 710. The potential targets are not necessarily drawn to scale on the atlas 700. In some examples, in block 605, the control system 130 may generate a list of the potential790482-00538 targets identified (e.g., in terms of coordinates on the grid 310).

[0039] In block 610, the control system controls a light source of a CLEM of the electron microscope to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera. For example, with reference to FIGS. 1, 2, and 4, the control system 130 may control the CLEM electron source 235 to generate a second electron beam 435 towards the target locations identified in block 605, and control the CLEM light source 240 to emit a first light beam 440 towards the sample 315 on the sample stage 118. The second electron beam 435 may cause targets of interest in the sample 315 to fluoresce, and the shared camera 120 may capture an optical image based on the first light beam 440 to capture those targets of interest that are fluorescing. In some examples, the second electron beam 435 is iteratively generated to individually target the target locations, one at a time, to attempt to cause each potential target to fluoresce. In some examples, between each generated second electron beam 435, the control system may control the CLEM light source 204 to generate the first light beam 440 and control the shared camera 120 to capture an image of the grid 310 and the sample 315. In some examples, after the second electron beam 435 has be generated to individually target each or a plurality of the target locations, one at a time, the control system 130 may control the CLEM light source 204 to generate the first light beam 440 and control the shared camera 120 to capture an image of the grid 310 and the sample 315. The resulting optical image(s) captured may be a fluorescence overlay image.

[0040] FIG. 7B illustrates an example fluorescence overlay image 712 of the grid 310 with the sample 315 thereon. In this example fluorescence overlay image 712, three potential targets fluoresce while three potential targets do not fluoresce. For example, the potential target 710 fluoresces and the potential target 715 does not fluoresce. The potential target 710 and other fluorescing potential targets may, accordingly, be referred to as a fluorescent target 720.

[0041] In block 615, the control system identifies one or more targets in the first fluorescence overlay image. For example, the control system 130 may process the fluorescence overlay image to identify a subset of the potential targets identified in block 605 as the targets. For example, the control system 130 may perform image processing configured to identify shapes or areas of a particular wavelength (e.g.. color) indicating a desired fluorescence level. Those shapes or areas may be identified as targets by the control system 130. For example, with reference to the example of FIGS. 7A and 7B, the control system 130 may identify as targets the three fluorescent targets in FIG. 7B (including the fluorescent target 720) of the original six potential targets identified in FIG. 7A. The targets identified in block 615 may again be defined in terms of coordinates of the grid 310. For example, in block 615, the control790482-00538 system 130 may generate a list of the targets identified (e.g., in terms of coordinates on the grid 310). In the example of FIG. 7B, the targets may be defined in terms of the window location of the target (e.g.. Bl, C4, and E3), and more precisely defined in terms of actual measurements (e.g., in mm) for each of the x and y coordinates.

[0042] In some examples, the control system 130 may further identify a height (z-axis) of the targets (e.g., crystals) within the sample 315. To do so, the control system 130 may adjust the focus of the CLEM electron source 235 and / or CLEM light source 240 up and down (e.g.. because the CLEM system 110 is a confocal system), and control the shared camera 120 to capture images of the sample 315 at the different heights at which the beam is focused (i.e., each captured image may correspond to a different height). The target in the resulting image captured by the shared camera 120 will generally be sharper when the beam is focused at the same height as the target. Thus, the shared camera 120 may infer, based on the sharpness of the fluorescing target within the captured images, the height of the targets within the sample 315. That is, the camera 120 may determine which captured image has the sharpest image of the target and assign the height of the beam focused for that captured image as the height of the target. Accordingly, in some examples, in block 615, the control system 130 may generate a list of the targets identified defined in terms of three-dimensional coordinates (e.g., x, y, and z coordinates) with respect to the grid 310. In the example of FIG. 7B, the targets may be defined in terms of the window location of the target (e.g. , B 1. C4, and E3), and more precisely defined in terms of actual measurements (e.g., in mm) for each of x, y. and z coordinates (where, for FIG. 7B, the z-axis extends in / out of the page).

[0043] In block 620, the control system controls a plasma source of a FIB-SEM to mill the sample based on identifying the one or more targets. For example, the control system 130 may control the plasma source 245 to mill the sample 315 where the targets were identified to a desired thickness. By milling the sample 315 where the targets were identified, excess portions of the sample that are not of interest for further imaging are removed. For example, excess portions of the sample may be removed from above, below, or both above and below the targets that are of interest for further imaging. The illustrations of FIGS. 8 A and 8B illustrate cross sectional diagrams for targets BL C4, and E3, labeled target 720, target 805. and target 810, respectively. More particularly, FIG. 8A illustrates a cross section 800 of the sample 315 (an x-z plane, with the y-axis extending in / out of the page) before milling, and FIG. 8B illustrates the cross section 800 of the sample 315 after milling. As show n in FIG. 8B. after milling, the sample 315. also referred to as a milled sample 815. is provided with portions of the sample having been milled aw ay. In the milled sample 815, portions of the sample above790482-00538 and below the targets have been milled away, leaving a portion of the sample 100-200 nanometers thick where the target(s) is / are located. The illustrations in FIGS. 8A-8B are not drawn to scale. For example, the milled sample 815 has a first milled-away portion 820 above the target 720 and a second milled away portion 825 below the target 720. The other targets 805 and 810 similarly have corresponding milled away portions of the milled sample 815 above and below the targets 805 and 810. The milled away portions may also be referred to as milled recesses.

[0044] To perform the milling, the control system 130 may also control the SEM electron source 250 to generate a third electron beam 445 (a SEM electron beam 445) towards the sample 315 and capture a resulting image at the shared camera 120 to accurately control the plasma source 245 to generate a focused ion beam 450 to mill the sample 315. For example, the SEM portion of the FIB-SEM may scan the surface of the sample 315 with the SEM electron beam 445 and generate an image or picture of the surface of the sample 315. This scanning can be done before, after, and during the milling process.

[0045] As discussed above and illustrated in FIGS. 8A-8B, the targets may be at different heights within the sample 315. To perform the milling accurately and without milling away the target(s), the control system 130 may use the height ofthe sample 315 estimated using the CLEM system 110, and mill above and below this height at the x-y location of the target. In some examples, after milling down to the desired thickness (e.g., 100-200 nanometers (nm), 100-300 nm. 200-300 nm, or 150-250 nm), the control system 130 may capture a further fluorescence overlay image using the CLEM system 110 and shared camera 120 and confirm that the target is still present at the x-y location (and, for example, has not been inadvertently milled away).

[0046] In block 625. the control system controls the TEM electron source to emit a second electron beam towards the sample while controlling the sample holder to rotate. For example, with reference to FIGS. 1, 2, and 4, the control system 130 controls the TEM electron source 215 to emit a fourth electron beam 455 towards the sample 315 while controlling the sample stage 118 to rotate about axis 255 (e.g., by controlling the piezo actuator(s) 420). More particularly, the control system 130 controls the TEM electronic source 215 to emit the fourth electron beam 455 at a first target of the one or more targets identified in block 615 and milled around in block 620 (e.g., at the x, y, z location of the first target). This emission of the electron beam occurs as the sample stage 118 is rotated at a linear rotation speed (e.g., 99% linear when rotating between 0. 1 to 10 degrees / second).

[0047] In block 630, the control system captures, by the shared camera, scatterings of790482-00538 the electron beam by the sample as diffraction images while the sample holder is rotated. For example, the control system 130 controls the shared camera 120 to capture a series of diffraction images capturing the scatterings of the fourth electron beam 455 by the sample 315 (e.g., after milling), each image corresponding to a different rotation angle of the sample stage 118 about the axis 255. The series of diffraction images may be captured at a particular capture rate (e.g., with a particular time period between each image capture) as the sample stage 118 rotates to generate a movie of a particular frame rate.

[0048] In block 635, the control system outputs microcrystal electron diffraction (MicroED) data based on the diffraction images. For example, the control system 130 may output MicroED data based on the diffraction images captured by the shared camera 120. In some examples, the control system 130 processes the diffraction images (diffraction image data) obtained in block 630, which includes the diffraction images captured at the various rotational angles of the sample stage 118. In some embodiments, to process the image data, the control system 130 processes each diffraction image to identify and index diffraction points. The control system 130 may identify diffraction points as a pixel (or group of pixels) within a diffraction image with an intensity above a threshold value, having a size within a certain range, and / or having a particular shape (e g., circular). The control system 130 may then index each diffraction point by determining the x-y position of the diffraction point within the diffraction image, and associating the diffraction point of one diffraction image with the diffraction points within other diffraction images caused by the same diffraction instance (e.g., by the same diffracted portion of the electron beam from the TEM electron source 215). By determining the x-y positions of the associated diffraction points in each diffraction image, and knowing the rotation angle of the sample stage 118 of each diffraction image (e.g., based on the characteristic data of the diffraction image, such as the time stamp, frame rate, and rotation rate), the associated diffraction points from the multiple diffraction images can be mapped to a point within a virtual three-dimensional space. In other w ords, the diffraction instance may be mapped to a point within the reciprocal three-dimensional space. The control system 130 may further perform this processing to map each diffraction instance to the reciprocal three- dimensional space, thereby generating a three-dimensional diffraction pattern. In some embodiments, other three-dimensional reconstruction techniques are used to generate a three- dimensional diffraction pattern from the captured diffraction images. In some examples, the control system 130 may further refine structures generated from processing the image data. In some embodiments, refining structures may include one or more of: refining lens aberrations, refining electron scattering, refining shape function, mosaicity refinement, ewald sphere790482-00538 correction, correcting for diffraction astigmatism, beam movement correction, beam center correction, z-axis correction, eucentric height correction, eucentric drift correction, anisotropic diffraction correction, dose weighting for beam damage, dark current correction, dynamical refinement, and completeness padding.

[0049] In block 635, outputting the MicroED data may include the electronic processor 500, storing the three-dimensional image on the memory 505 (e.g., as the image data 525), displaying the three-dimensional image on the display 550, and / or transmitting the three- dimensional image to another device viathe I / O interface 510 (e.g., for further storage, display, or dissemination). The MicroED data may include one or more of: 1) structure of a specimen, 2) a full identity of the specimen with or without prior knowledge, 3) information about sample components (e.g., if specimen is a mixture), 4) a percent (%) contamination of the specimen and identity of the contaminants, 5) purity information for the specimen, and 6) a three- dimensional graph of diffraction data generated from a combination of the obtained two- dimensional diffraction images. For example, the control system 130 may combine the obtained two-dimensional diffraction images to generate three-dimensional data for the sample (e.g., a three-dimensional graph of the diffraction data or a three-dimensional structure of a sub-micron protein). The three-dimensional data may be output as MicroED data and / or may be further analyzed and classified. For example, the three-dimensional data may serve as an input to a lookup table that links the input data to one or more outputs. For example, the three- dimensional data may be compared to a database of three-dimensional data sets to identify a closest match, where the database of three-dimensional data sets have associated therewith one or more of the atomic structure of a specimen, full identify of a specimen, information about sample components, percent contamination of the specimen and contaminant identities, and purify information.

[0050] In some examples, blocks 610, 615, and 620 are implemented iteratively to use CLEM to identify a target that fluoresces, estimate thickness (and / or z-axis location) of the identified target, and then mill around the identified target to prepare the identified targets for further imaging (e.g., MicroED). By iteratively performing these steps, multiple targets may be identified and prepared for further imaging. For example, the milling in block 620 may be iterative with intermediate checks to confirm continued fluorescence of the target and / or whether sample thickness has been reduced to a desired level. For example, the control system 130 may initially detemtine an estimated position of the target (e.g., in terms of x. y, and z coordinates) as described with respect to block 615. Then, in block 620. the control system 130 may mill the sample 315 above and / or below the target based on the estimated position (e.g.,790482-00538 at the x, y coordinates but above and / or below the z coordinate of the target). The control system 130 may control the plasma source 245 to mill the sample 315 above and / or below the target a predetermined amount or an amount based on the z-position of the target within the sample 315. Then, the control system 130 may control the CLEM light source 240 to emit a further light beam towards the sample 315 to generate a further fluorescence overlay image, as described with respect to block 610. The control system 130 may analyze the fluorescence overlay image to determine whether the target is still present and fluorescing (e.g., at the same x, y coordinates of the sample 315), as described with respect to identifying fluorescent targets in block 615. When the target is no longer fluorescing, the control system 130 may remove the target from the list of targets generated in block 615 and move on to mill the next target (if present).

[0051] When the target is confirmed to be fluorescing, the control system 130 may estimate thickness of the sample 315 at the estimated x-y position of the target. For example, to estimate thickness, the control system 130 may iteratively capture images at an angle before and after milling and calculate the thickness based on these real-space images.

[0052] When the estimated thickness is within a desired range (e.g., within 100-200 nanometers (nm), 100-300 nm, 200-300 nm, or 150-250 nm), the control system 130 may determine that milling for the target is complete. For example, the control system 130 may determine whether the estimated thickness of the sample at the location of the target is greater than a thickness threshold (and milling should continue) or below the thickness threshold (and milling should cease). In some examples, in response to the control system 130 determining that the estimated thickness of the sample at the location of the target is greater than the thickness threshold, the control system 130 may repeat the process by further milling the sample (above and / or below the target), generating a further fluorescence overlay image, to confirm that the target is still present and fluorescing, and checking the thickness of the sample at the location of the target, until the thickness of the sample at the location of the target is below the thickness threshold. The control system 130 may then move to the next target, if present, or move to block 625 (e.g., when no further targets remain to be milled).

[0053] Thus, in some examples, the control system 130 may iteratively mill the sample above, below, or both above and below a location of a target; determine that a thickness of the sample at the location of the target has not reached a desired thickness; and generate a further fluorescence overlay image for the sample with the shared camera to confirm that the first target fluoresces. The control system 130 may cease these iterations upon determining that the thickness of the sample at the location of the target has reached the desired thickness (e.g., is790482-00538 below a thickness threshold (e.g., of 200 nm, 250 nm, 300 nm, or another value).

[0054] Additionally, in some examples, blocks 625. 630, and 635 may repeat for each target identified and milled around in blocks 615 and 620. Thus, MicroED data may be generated for each prepared target of multiple prepared targets in the sample.

[0055] In some examples, before executing block 635, the control system 130 includes a microcrystal electron diffraction (MicroED) setup process. For example, in a first MicroED setup process, the control system 130 may capture setup diffraction images at at least three different holder positions to determine a height (z-position) of the target to be imaged. For example, the control system 130 may (i) control the TEM electron source 215 to emit an electron beam (a first setup beam) towards the sample when the holder 305 is positioned at a first rotational position (e.g., -20 degrees) and control the shared camera to capture resulting diffraction in a first setup image, (ii) control the TEM electron source 215 to emit another electron beam (a second setup beam) towards the sample when the holder 305 is positioned at a second rotational position (e.g., +20 degrees) and may control the shared camera to capture resulting diffraction in a second setup image; and (iii) control the TEM electron source 215 to emit another electron beam (a third setup beam) towards the sample when the holder 305 is positioned at a third rotational position (e.g., 0 degrees) and may control the shared camera to capture resulting diffraction in a third setup image. The control system 130 may then autocorrelate the first, second, and third setup images (e g., by correlating diffraction points within each of the images). Based on the correlation, the known rotational positions of the holder 305 when the three setup images were captured, and known geometry of the cohesive EM system 100, the control system 130 may determine the z-position of the target that would cause such diffraction points within each of the setup images. With the z-position of the target determined, the control system 130 may proceed to block 625 to emit the second electron beam towards the sample 315 and, more particularly, focused on the x, y, z-position of the target of the sample 315.

[0056] In a second MicroED setup process, the control system 130 may capture setup diffraction images at different focus positions for the TEM electron source 215. The control system 130 may analyze the setup diffraction images to determine which image (and. thus, focus setting) results in a minimum spot size and / or reflection size within the setup diffraction images. The control system 130 determines the focus setting that was applicable when the determined image was captured. The control system 130 may then determine the height (z- position) of the target based on the focus setting and known geometry of the cohesive EM system 100. For example, the focus setting may be a numerical value within a range, where790482-00538 each potential numerical value within the range maps to a height (z-position). In some examples of the second MicroED setup process, the shared camera 120 captures the diffraction images and changes focus in real time until a spot size and / or reflection size within the diffraction images is minimized, thereby indicating that the system is in-focus (e.g., focused on the target). Based on the focus settings and know n geometry of the cohesive EM system 100, the control system 130 may determine the z-position of the target determined, the control system 130 may proceed to block 625 to emit the second electron beam towards the sample 315 and, more particularly, focused on the x, y, z-position of the target of the sample 315.

[0057] While several techniques described herein are described with respect to the Cartesian (x, y, and z) coordinate system, in some examples, other three-dimensional coordinate systems (e.g., spherical, cylindrical, etc.) are used or implemented in place of Cartesian coordinates but with otherwise similar approaches to the techniques.

[0058] FIG. 9 illustrates an example of the cohesive EM system 100 incorporated within a housing 900 including an first housing portion 905 and a second housing portion 910. The first housing portion 905 includes the TEM central column 200, the CLEM angled column 205, and the FIB-SEM angled column 210. The first housing portion 905 may house the components of the cohesive EM system 100 illustrated in FIG. 2, including the chamber 260, the TEM electron source 215, the CLEM electron source 235, the CLEM light source 240, the plasma source 245, the SEM electron source 250, the sample stage 118, the piezo actuators 420, among other components.

[0059] The second housing portion 910 may include the control system 130 and, for example, other electronics of the cohesive EM system 100 described herein but not illustrated in FIG. 2 (e.g., the vacuum system 125, the user I / O devices 135, etc.). Thus, for example, the second housing portion may include a vacuum pump of the vacuum system 125 that is controlled to provide a vacuum within the chamber 260 of the first housing portion 905. Further, the user I / O devices 135 may include a touch screen display, a keypad, and other components (e.g., as described herein) to enable a user to interact with and control the cohesive EM system 100, as well as view results, outputs, and data collected by the cohesive EM system 100 (e.g., as generated via execution of the process 600, including MicroED data and intermediate data).

[0060] By integrating the TEM, CLEM, and FIB-SEM systems together in the cohesive EM system 100 (e.g., and into a housing such as the housing 900), the overall volume occupied by the cohesive EM system 100 is reduced relative to separate TEM, CLEM, and FIB-SEM systems or combinations of only two of the three of these systems. The housing 900 has a790482-00538 height 915 and a width 920. For example, in some examples, the height 915 is approximately four feet or less, the width 920 is approximately four feet or less, and a depth of the housing 900 is approximately four feet or less. Thus, in some examples, the cohesive EM system 100 is a compact system.

[0061] The present disclosure has described one or more embodiments as non-limiting examples, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the application. Features of the disclosed non-limiting examples can be combined and rearranged in various ways. Furthermore, the non-limiting examples of the disclosure provided herein are not limited in application to the details of construction and the arrangement of components set forth in the description or illustrated in the drawings. The embodiments are capable of being practiced or of being carried out in various ways.

[0062] Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

[0063] Also, the use the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "right", "left", "front", "back", "upper", "lower", "above", "below", "top”, or "bottom" and variations thereof herein is for the purpose of description and should not be regarded as limiting. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

[0064] Unless otherwise specified or limited, phrases similar to “at least one of A, B. and C,” “one or more of A, B, and C,” etc., are meant to indicate A, or B, or C, or any combination of A, B, and / or C, including combinations with multiple or single instances of A, B, and / or C.

[0065] In some non-limiting examples, aspects of the present disclosure, including computerized implementations of methods, can be implemented as a system, method,790482-00538 apparatus, or article of manufacture using standard programming or engineering techniques to produce software, firmware, hardware, or any combination thereof to control a processor device, a computer (e.g., a processor device operatively coupled to a memory), or another electronically operated controller to implement aspects detailed herein. Accordingly, for example, non-limiting examples of disclosed embodiments can be implemented as a set of instructions, tangibly embodied on a non-transitory computer-readable media, such that a processor device can implement the instructions based upon reading the instructions from the computer-readable media. Some non-limiting examples of the invention can include (or utilize) a device such as an automation device, a special purpose or programmable computer including various computer hardware, software, firmware, and so on, consistent with the discussion below.

[0066] The term “article of manufacture” as used herein is intended to encompass a computer program accessible from any computer-readable device, carrier (e.g., non-transitory signals), or media (e.g., non-transitory media). For example, computer-readable media can include but are not limited to magnetic storage devices (e.g., hard disk, floppy disk, magnetic strips, tapes and so on), optical disks (e.g., compact disk (CD), digital versatile disk (DVD), and so on), smart cards, and flash memory devices (e g., card, stick, and so on). Additionally, it should be appreciated that a carrier wave can be employed to carry computer-readable electronic data such as those used in transmitting and receiving electronic mail or in accessing a network such as the Internet or a local area network (LAN). Those skilled in the art will recognize many modifications may be made to these configurations without departing from the scope or spirit of the claimed subject matter.

[0067] Certain operations of disclosed methods, or of systems executing those methods, may be represented schematically in the figures or otherwise discussed herein. Unless otherwise specified or limited, representation in the figures of particular operations in particular spatial order may not necessarily require those operations to be executed in a particular sequence corresponding to the particular spatial order. Correspondingly, certain operations represented in the figures, or otherwise disclosed herein, can be executed in different orders than are expressly illustrated or described, as appropriate for particular non-limiting examples of the invention. Further, in some non-limiting examples, certain operations can be executed in parallel, including by dedicated parallel processing devices, or separate computing devices configured to interoperate as part of a large system.

[0068] As used herein in the context of computer implementation, unless otherwise specified or limited, the terms “component,” “system,” “module,” etc. are intended to790482-00538 encompass part or all of computer-related systems that include hardware, software, a combination of hardware and software, or software in execution. For example, a component may be, but is not limited to being, a processor device, a process being executed (or executable) by a processor device, an object, an executable, a thread of execution, a computer program, or a computer. By way of illustration, both an application running on a computer and the computer can be a component. One or more components (or system, module, and so on) may reside within a process or thread of execution, may be localized on one computer, may be distributed between two or more computers or other processor devices, or may be included within another component (or system, module, and so on).

[0069] As used herein, the term, “controller” and “processor” and “computer” include any device capable of executing a computer program, or any device that includes logic gates configured to execute the described functionality. For example, this may include a processor, a microcontroller, a field-programmable gate array, a programmable logic controller, etc. As another example, these terms may include one or more processors and memories and / or one or more programmable hardware elements, such as any of types of processors, CPUs, microcontrollers, digital signal processors, or other devices capable of executing software instructions.

[0070] FURTHER EXAMPLES

[0071] Example 1: A method, apparatus, and / or non-transitory computer-readable medium storing processor-executable instructions for execution to perform actions, comprising: a sample holder configured to support a sample; a shared camera; a correlative light electron microscope (CLEM) comprising a CLEM electron source to generate a CLEM electron beam towards the sample supported by the sample holder and cause a first scattered beam for capture by the shared camera as an electron microscope image, and a light source configured to cause at least a portion of the sample supported by the sample holder to fluoresce for capture by the shared camera as an optical image; wherein a focused ion beam scanning electron microscope (FIB-SEM) comprising a SEM electron source and a plasma source, the plasma source configured to generate an ion beam to mill the sample while supported by the sample holder; and a transmission electron microscope (TEM) comprising a TEM electron source to generate a TEM electron beam towards the sample on the sample holder and cause a scattered beam for capture by the shared camera as a diffraction pattern.

[0072] Example 2: The method, apparatus, and / or non-transitory computer readable medium of Example 1. further comprising: a chamber comprising: a central column comprising the TEM electron source and the sample holder, a CLEM angled column comprising the CLEM790482-00538 electron source and the light source, and a FIB-SEM angled column comprising the plasma source.

[0073] Example 3: The method, apparatus, and / or non-transitory computer readable medium of Example 2, wherein the chamber is a vacuum chamber configured to provide a vacuum for the CLEM, the FIB-SEM, the TEM, the sample, and the shared camera.

[0074] Example 4: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 1 to 3, wherein the CLEM electron source and the light source in the CLEM angled column are offset 45 degrees with respect to the TEM electron source in the central column, wherein the plasma source in the FIB-SEM angled column is offset 45 degrees with respect to the TEM electron source in the central column, and wherein the CLEM electron source and the light source in the CLEM angled column are offset 90 degrees with respect to the plasma source in the FIB-SEM angled column.

[0075] Example 5: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 1 to 4, further comprising: a control system including an electronic processor and a memory, wherein the control system is in communication with and configured to control the CLEM, the FIB-SEM, and the TEM.

[0076] Example 6: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 1 to 5, further comprising: a control system including an electronic processor and a memory, the control system configured to: control the TEM electron source to emit an atlas TEM electron beam towards the sample to generate a first atlas image of the sample with the shared camera; control the light source of the CLEM to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera; identify one or more targets in the first fluorescence overlay image; control the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets; control the TEM electron source to emit the TEM electron beam towards the sample while controlling the sample holder to rotate; capture, by the shared camera, scatterings of the electron beam by the sample as diffraction images while the sample holder is rotated; and output microcrystal electron diffraction data based on the diffraction images.

[0077] Example 7: The method, apparatus, and / or non-transitory computer readable medium of Example 6, wherein the output microcrystal electron diffraction data includes one or more of the following: an atomic structure of the sample, an identify of the sample, an identification of components of the sample, an amount of contamination of the sample, an identification of contaminants of the sample, and a three-dimensional graph of diffraction data generated from a combination of the diffraction images.790482-00538

[0078] Example 8: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 1 to 7. further comprising: a control system including at an electronic processor and a memory, the control system configured to perform at least one from a group of: control the shared camera to capture an image of the sample; identify a polymorph in the sample; identify racemization in the sample; control the light source of the CLEM to emit a first light beam towards the sample to generate a first fluorescence image of the sample with the shared camera; control the TEM electron source to emit an atlas TEM electron beam towards the sample to generate a first TEM image of the sample with the shared camera; control the SEM electron source to emit a SEM electron beam towards the sample to generate a first SEM image of the sample with the shared camera; control the plasma source of the FIB-SEM to mill the sample; and control the TEM electron source to emit the TEM electron beam towards the sample while controlling the sample holder to rotate to generate diffraction images with the shared camera, and output microcrystal electron diffraction data based on the diffraction images.

[0079] Example 9: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 1 to 8, further comprising a cryogenic fluid supply line configured to receive a cryogenic fluid to maintain the sample at cryogenic temperature during operation of one or more of the CLEM, FIB-SEM, and TEM.

[0080] Example 10: A method, apparatus, and / or non-transitory computer-readable medium storing processor-executable instructions for execution to perform actions, comprising: controlling a TEM electron source of an electron microscope to emit an atlas TEM electron beam tow ards a sample supported by a sample holder of the electron microscope to generate a first atlas image of the sample with a shared camera: controlling a light source of a CLEM of the electron microscope to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera; identifying one or more targets in the first fluorescence overlay image; controlling a plasma source of a FIB-SEM to mill the sample based on identifying the one or more targets; controlling the TEM electron source to emit a TEM electron beam towards the sample while controlling the sample holder to rotate; capturing, by the shared camera, scatterings of the electron beam by the sample as diffraction images while the sample holder is rotated; and outputting microcrystal electron diffraction data based on the diffraction images.

[0081] Example 11 : The method, apparatus, and / or non-transitory computer readable medium of Example 10. wherein the electron microscope comprises a chamber comprising a central column comprising the TEM electron source and the sample holder, a CLEM angled790482-00538 column comprising a CLEM electron source and the light source of the CLEM, and a FIB- SEM angled column comprising the plasma source: wherein controlling the plasma source of the FIB-SEM to mill the sample includes directing an ion beam from the FIB-SEM through the FIB-SEM angled column; wherein controlling the TEM electron source to emit the TEM electron beam towards the sample includes emitting the TEM electron beam through the central column; and wherein controlling the light source of the CLEM to emit the first light beam towards the sample includes emitting the first light beam through the CLEM angled column.

[0082] Example 12: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 11, wherein the CLEM electron source and the light source in the CLEM angled column are offset 45 degrees with respect to the TEM electron source in the central column, wherein the plasma source in the FIB-SEM angled column is offset 45 degrees with respect to the TEM electron source in the central column, and wherein the CLEM electron source and the light source in the CLEM angled column are offset 90 degrees with respect to the plasma source in the FIB-SEM angled column.

[0083] Example 13: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 12, further comprising: providing, by a vacuum system, a vacuum in the chamber for the light source of the CLEM, the plasma source of the FIB-SEM, the TEM electron source, the sample, and the shared camera.

[0084] Example 14: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 13, further comprising: controlling, by a control system including an electronic processor and a memory, the TEM electron source to emit the atlas TEM electron beam; controlling, by the control system, the light source of the CLEM to emit the first light beam; identifying, by the control system, the one or more targets in the first fluorescence overlay image; controlling, by the control system, the plasma source of the FIB- SEM to mill the sample based on identifying the one or more targets; controlling, by the control system, the TEM electron source to emit the TEM electron beam; controlling, by the control system, the shared camera to capture the scatterings of the electron beam by the sample as diffraction images while the sample holder is rotated; and outputting, by the control system, the microcrystal electron diffraction data based on the diffraction images.

[0085] Example 15: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 14, wherein the microcrystal electron diffraction data that is output is automatically generated by the electron microscope and includes one or more of the following: an atomic structure of the sample, an identity of the sample, an identification of components of the sample, an amount of contamination of the sample, an identification of790482-00538 contaminants of the sample, and a three-dimensional graph of diffraction data generated from a combination of the diffraction images.

[0086] Example 16: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 15, further comprising: maintaining, using a cryogenic fluid, the sample at cryogenic temperature during operation of one or more of the CLEM, FIB-SEM, and TEM.

[0087] Example 17: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 16, wherein controlling the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets comprises: determining x, y, z coordinates of a first target of the one or more targets; milling at the x. y coordinates of the first target at an upper position above the z-coordinate of the first target; and milling at the x, y coordinates of the first target at a lower position below the z-coordinate of the first target.

[0088] Example 18: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 17, further comprising: wherein controlling the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets comprises: determining a location of a first target of the one or more targets in the sample, the location defined as a position in a three dimensional coordinate system; milling the sample above, below, or both above and below the location of the first target; after the milling, determining that a thickness of the sample at the location is greater than a thickness threshold; further milling the sample above, below, or both above and below the location of the first target in response to determining that the thickness of the sample at the location is greater than a thickness threshold; after the further milling, determining that the thickness of the sample at the location is less than a thickness threshold; and ceasing milling for the first target in response to determining that the thickness of the sample at the location is less than a thickness threshold.

[0089] Example 19: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 18, further comprising: after the milling, the further milling, or both: controlling the light source of the CLEM of the electron microscope to emit another light beam towards the sample to generate a further fluorescence overlay image for the sample with the shared camera; and confirming that the first target fluoresces based on the fluorescence overlay image.

[0090] Example 20: The method, apparatus, and / or non-transitory computer readable medium of any of Examples 10 to 19, further comprising: iteratively: (i) milling the sample above, below-, or both above and below a location of the first target of the one or more targets;790482-00538(ii) determining that a thickness of the sample at the location of the first target has not reached a desired thickness; and (iii) generating a further fluorescence overlay image for the sample with the shared camera to confirm that the first target fluoresces.

Claims

790482-00538CLAIMSWhat is claimed is:

1. An electron microscope system comprising: a sample holder configured to support a sample; a shared camera; a correlative light electron microscope (CLEM) comprising a CLEM electron source to generate a CLEM electron beam towards the sample supported by the sample holder and cause a first scattered beam for capture by the shared camera as an electron microscope image, and a light source configured to cause at least a portion of the sample supported by the sample holder to fluoresce for capture by the shared camera as an optical image; a focused ion beam scanning electron microscope (FIB-SEM) comprising a SEM electron source and a plasma source, the plasma source configured to generate an ion beam to mill the sample while supported by the sample holder; and a transmission electron microscope (TEM) comprising a TEM electron source to generate a TEM electron beam towards the sample on the sample holder and cause a scattered beam for capture by the shared camera as a diffraction pattern.

2. The electron microscope system of claim 1, further comprising: a chamber comprising: a central column comprising the TEM electron source and the sample holder, a CLEM angled column comprising the CLEM electron source and the light source, and a FIB-SEM angled column comprising the plasma source.

3. The electron microscope system of claim 2, wherein the chamber is a vacuum chamber configured to provide a vacuum for the CLEM, the FIB-SEM, the TEM, the sample, and the shared camera.

4. The electron microscope system of claim 2,790482-00538 wherein the CLEM electron source and the light source in the CLEM angled column are offset 45 degrees with respect to the TEM electron source in the central column, wherein the plasma source in the FIB-SEM angled column is offset 45 degrees with respect to the TEM electron source in the central column, and wherein the CLEM electron source and the light source in the CLEM angled column are offset 90 degrees with respect to the plasma source in the FIB-SEM angled column.

5. The electron microscope system of claim 1 , further comprising: a control system including an electronic processor and a memory. wherein the control system is in communication with and configured to control the CLEM, the FIB-SEM, and the TEM.

6. The electron microscope system of claim 1 , further comprising: a control system including an electronic processor and a memory, the control system configured to: control the TEM electron source to emit an atlas TEM electron beam towards the sample to generate a first atlas image of the sample with the shared camera; control the light source of the CLEM to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera; identify one or more targets in the first fluorescence overlay image; control the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets; control the TEM electron source to emit the TEM electron beam tow ards the sample while controlling the sample holder to rotate; capture, by the shared camera, scatterings of the TEM electron beam by the sample as diffraction images while the sample holder is rotated; and output microcrystal electron diffraction data based on the diffraction images.

7. The electron microscope system of claim 6, wherein the output microcrystal electron diffraction data includes one or more of the following:790482-00538 an atomic structure of the sample, an identity of the sample. an identification of components of the sample, an amount of contamination of the sample, an identification of contaminants of the sample, and a three-dimensional graph of diffraction data generated from a combination of the diffraction images.

8. The electron microscope system of claim 1 , further comprising: a control system including at an electronic processor and a memory, the control system configured to perform at least one from a group of: control the shared camera to capture an image of the sample; identify a polymorph in the sample; identify racemization in the sample; control the light source of the CLEM to emit a first light beam towards the sample to generate a first fluorescence image of the sample with the shared camera; control the TEM electron source to emit an atlas TEM electron beam towards the sample to generate a first TEM image of the sample with the shared camera; control the SEM electron source to emit a SEM electron beam towards the sample to generate a first SEM image of the sample with the shared camera; control the plasma source of the FIB-SEM to mill the sample; and control the TEM electron source to emit the TEM electron beam towards the sample while controlling the sample holder to rotate to generate diffraction images with the shared camera, and output microcrystal electron diffraction data based on the diffraction images.

9. The electron microscope system of claim 1, further comprising a cry ogenic fluid supply line configured to receive a cryogenic fluid to maintain the sample at cryogenic temperature during operation of one or more of the CLEM, FIB-SEM, and TEM.

10. A method for an electron microscope, the method comprising: controlling a TEM electron source of an electron microscope to emit an atlas TEM electron beam towards a sample supported by a sample holder of the electron microscope to generate a first atlas image of the sample with a shared camera;790482-00538 controlling a light source of a CLEM of the electron microscope to emit a first light beam towards the sample to generate a first fluorescence overlay image for the sample with the shared camera; identifying one or more targets in the first fluorescence overlay image; controlling a plasma source of a FIB-SEM to mill the sample based on identifying the one or more targets; controlling the TEM electron source to emit a TEM electron beam towards the sample while controlling the sample holder to rotate; capturing, by the shared camera, scatterings of the TEM electron beam by the sample as diffraction images while the sample holder is rotated; and outputting microcrystal electron diffraction data based on the diffraction images.

11. The method of claim 10, wherein the electron microscope comprises a chamber comprising a central column comprising the TEM electron source and the sample holder, a CLEM angled column comprising a CLEM electron source and the light source of the CLEM, and a FIB-SEM angled column comprising the plasma source; wherein controlling the plasma source of the FIB-SEM to mill the sample includes directing an ion beam from the FIB-SEM through the FIB-SEM angled column; wherein controlling the TEM electron source to emit the TEM electron beam towards the sample includes emitting the TEM electron beam through the central column; and wherein controlling the light source of the CLEM to emit the first light beam towards the sample includes emitting the first light beam through the CLEM angled column.

12. The method of claim 11, wherein the CLEM electron source and the light source in the CLEM angled column are offset 45 degrees with respect to the TEM electron source in the central column, wherein the plasma source in the FIB-SEM angled column is offset 45 degrees with respect to the TEM electron source in the central column, and790482-00538 wherein the CLEM electron source and the light source in the CLEM angled column are offset 90 degrees with respect to the plasma source in the FIB-SEM angled column.

13. The method of claim 11, further comprising: providing, by a vacuum system, a vacuum in the chamber for the light source of the CLEM, the plasma source of the FIB-SEM, the TEM electron source, the sample, and the shared camera.

14. The method of claim 10, further comprising: controlling, by a control system including an electronic processor and a memory, the TEM electron source to emit the atlas TEM electron beam; controlling, by the control system, the light source of the CLEM to emit the first light beam; identifying, by the control system, the one or more targets in the first fluorescence overlay image; controlling, by the control system, the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets; controlling, by the control system, the TEM electron source to emit the TEM electron beam; controlling, by the control system, the shared camera to capture the scatterings of the TEM electron beam by the sample as diffraction images while the sample holder is rotated; and outputting, by the control system, the microcrystal electron diffraction data based on the diffraction images.

15. The method of claim 10, wherein the microcrystal electron diffraction data that is output is automatically generated by the electron microscope and includes one or more of the following: an atomic structure of the sample, an identity of the sample, an identification of components of the sample, an amount of contamination of the sample,790482-00538 an identification of contaminants of the sample, and a three-dimensional graph of diffraction data generated from a combination of the diffraction images.

16. The method of claim 10, further comprising: maintaining, using a cryogenic fluid, the sample at cryogenic temperature during operation of one or more of the CLEM, FIB-SEM, and TEM.

17. The method of claim 10, wherein controlling the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets comprises: determining x, y , z coordinates of a first target of the one or more targets; milling at the x, y coordinates of the first target at an upper position above the z-coordinate of the first target; and milling at the x, y coordinates of the first target at a lower position below the z-coordinate of the first target.

18. The method of claim 10, further comprising: wherein controlling the plasma source of the FIB-SEM to mill the sample based on identifying the one or more targets comprises: determining a location of a first target of the one or more targets in the sample, the location defined as a position in a three dimensional coordinate system; milling the sample above, below, or both above and below the location of the first target; after the milling, determining that a thickness of the sample at the location is greater than a thickness threshold; further milling the sample above, below, or both above and below the location of the first target in response to determining that the thickness of the sample at the location is greater than a thickness threshold; after the further milling, determining that the thickness of the sample at the location is less than a thickness threshold; and ceasing milling for the first target in response to determining that the thickness of the sample at the location is less than a thickness threshold.

19. The method of claim 18, further comprising:790482-00538 after the milling, the further milling, or both: controlling the light source of the CLEM of the electron microscope to emit another light beam towards the sample to generate a further fluorescence overlay image for the sample with the shared camera; and confirming that the first target fluoresces based on the further fluorescence overlay image.

20. The method of claim 10, further comprising: iteratively, milling the sample above, below, or both above and below a location of the first target of the one or more targets; determining that a thickness of the sample at the location of the first target has not reached a desired thickness; and generating a further fluorescence overlay image for the sample with the shared camera to confirm that the first target fluoresces.

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