Method and apparatus for preparing samples for cryo-electron microscopy - Patents.com

Abstract

To provide various methods and devices for searching the optimum sample condition of a sample for cryogenic electron microscopy.SOLUTION: A sample inspection device includes: a first chamber 231 formed between a top electron transparent layer 217 and a bottom electron transparent layer 218 for holding a first sample; a plurality of pillars 212 each of which extends from the top electron transparent layer 217 to the bottom electron transparent layer 218, within the first chamber 231; and a window 201 formed with a portion of at least one of the top electron transparent layer 217 and the bottom electron transparent layer 218 for inspecting the first sample in the first chamber 231, the window covering at least one of the plurality of pillars 212.SELECTED DRAWING: Figure 2B

Description

[Technical field]

[0001] This description relates generally to methods and apparatus for preparing samples for single particle analysis, and more particularly to optimizing sample quality for cryo-electron microscopy imaging based single particle analysis. [Background technology]

[0002] Single particle analysis based on cryogenic electron microscopy (cryo-EM) images can resolve the structures of biological macromolecules at near atomic resolution. Sample preparation is a critical step to obtain high-quality cryo-EM images of macromolecules. As an example, to resolve protein structures, protein samples are prepared by mixing the protein with one or more buffer solutions. The sample composition, as well as the buffer conditions, including PH levels, salt concentrations, surfactant concentrations, and detergent concentrations, need to be optimized to solubilize and stabilize the protein for optimal cryo-EM results. However, preparing and imaging vitrified protein samples with different sample conditions requires extensive trial-and-error testing and can be very time-consuming. Summary of the Invention

[0003] In one embodiment, a sample inspection device is used to screen sample conditions. The sample inspection device includes one or more chambers formed between an upper electron-transparent layer and a lower electron-transparent layer for holding a first sample, a plurality of pillars in the first chamber, each pillar of the plurality of pillars extending from the upper electron-transparent layer to the lower electron-transparent layer, and a window formed in a portion of at least one of the upper and lower electron-transparent layers for inspecting the first sample in the first chamber, the window covering at least one of the plurality of pillars. In this way, samples with multiple sample conditions can be vitrified and imaged in the same sample inspection device.

[0004] In another embodiment, a method for inspecting a sample using a sample inspection device includes flowing a first sample through a first chamber of the sample inspection device, the first chamber being formed between an upper electron transparent layer and a lower electron transparent layer, and a plurality of pillars being arranged in the first chamber, each pillar of the plurality of pillars extending from the upper electron transparent layer to the lower electron transparent layer; directing a charged particle beam toward a window of the sample inspection device, the window being formed in a portion of at least one of the upper electron transparent layer and the lower electron transparent layer, the window covering at least one of the plurality of pillars; and forming a first image of the first sample based on the charged particles transmitted through the sample inspection device.

[0005] It should be understood that the above Summary is provided to introduce in a simplified form a selection of concepts that are further described in the Detailed Description. It is not intended to identify key or essential features of the claimed subject matter, the scope of which is uniquely defined by the claims that follow the Detailed Description. Moreover, the claimed subject matter is not limited to implementations that solve any disadvantages noted above or in any part of this disclosure. [Brief description of the drawings]

[0006] [Figure 1] 1 shows a cryogenic electron microscope system. [Figure 2A] 1 shows a sample inspection device with one chamber. [Figure 2B] 1 shows a sample inspection device with one chamber. [Figure 2C] 1 shows a sample inspection device with one chamber. [Figure 3A] 1 shows the sample inspection device with the two chambers disconnected. [Figure 3B] 1 shows the sample inspection device with the two chambers disconnected. [Figure 3C] 1 shows the sample inspection device with the two chambers disconnected. [Figure 4]1 shows an exemplary sample inspection device with four chambers cut out. [Figure 5A] 1 shows an exemplary sample inspection device with two connected chambers. [Figure 5B] 5B shows various sample state distributions in the sample inspection device of FIG. 5A. [Figure 6] 1 illustrates a method for manufacturing a sample inspection device. [Figure 7] 7 shows a procedure for manufacturing a sample inspection device according to the method of FIG. 6. [Figure 8] 1 illustrates an exemplary method for screening specimen conditions using a disconnected chamber sample inspection device. [Figure 9] 1 illustrates an exemplary method for screening sample conditions using an exemplary sample inspection device with an associated chamber. [Figure 10] An exemplary method for screening sample conditions using both a disconnected chamber sample inspection device and a connected chamber sample inspection device is shown.

[0007] Like reference numerals refer to corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The following description relates to a system and method for screening samples for cryogenic electron microscopy (cryo-EM) based single particle analysis. Cryo-EM imaging can be performed using the imaging system shown in FIG.

[0009] To perform single particle analysis, samples undergo multiple sample preparation steps to achieve optimal sample conditions before being imaged with a cryo-EM imaging system. Sample conditions may include buffer type and PH level, salt and detergent type and concentration, and surfactant concentration. Sample conditions may also include nanodisc size, lipid to protein ratio, and critical micelle concentration.

[0010] The sample preparation steps may include purifying the sample, mixing the purified sample with a buffer, and vitrifying the mixture. One of the sample preparation steps is mixing a sample, such as a protein, with one or more buffers to solubilize and stabilize the sample. For example, proteins may be stabilized by adding constructs such as nanodiscs, crosslinkers, and / or scaffolds. By mixing the sample with one or more buffers, the buffer system of the sample is exchanged from the buffer system used during the purification process and / or sample storage to a buffer system suitable for cryo-EM imaging.

[0011] Sample quality for single particle analysis may be determined based on a cryo-EM image of the sample. Sample quality may include particle quality, sample grid quality, and cryo-EM image quality. Particle quality may include one or more of tertiary and quaternary structure, aggregation state, stability, particle binding, particle size, and particle similarity. Sample grid quality may include one or more of ice formation, and particle distribution and particle orientation on the grid. Image quality may include one or more of contrast and resolution.

[0012] Although multiple techniques are available for screening samples for protein expression and purification without cryo-EM imaging, sample quality for single particle analysis can only be accurately and reliably determined based on cryo-EM images of vitrified samples. As a result, sample conditions can only be accurately and reliably assessed based on cryo-EM images. Because it is difficult to predict sample conditions for a new sample based on known optimal sample conditions for other samples, multiple repeated runs of sample preparation and cryo-EM imaging are required to search for optimal sample conditions for a new sample.

[0013] To address this issue, various examples of sample inspection devices and methods for screening sample conditions using the sample inspection device are presented to accelerate the search for optimal sample conditions. The sample inspection device may have one chamber (FIGS. 2A-2C), multiple fluidically disconnected chambers (FIGS. 3A-3C and FIG. 4), or multiple fluidly connected chambers (FIG. 5A) formed between an upper electron-transparent layer and a lower electron-transparent layer. Samples with different sample conditions may be loaded into one or more chambers and inspected by imaging through a window formed by at least a portion of the electron-transparent layer. A plurality of pillars are arranged in the chamber. A window covers at least one of the pillars. Each pillar has one end bonded to the upper electron-transparent layer and the other end bonded to the lower electron-transparent layer. Samples may flow around the pillars. The pillars provide mechanical strength and robustness to the window. More importantly, bonding the pillars to the electron-transparent layer may prevent bulging and vitrification of the device during sample loading.

[0014] In the case of a sample inspection device with disconnected chambers, individual sample conditions may be imaged and analyzed. In the case of a sample inspection device with fluidically connected chambers, a gradient of sample conditions may be formed in the mixing region, and a continuous variety of sample conditions may be imaged and analyzed. FIG. 5B shows an exemplary sample condition in the mixing region of the device of FIG. 5A. By providing windows covering multiple samples or samples with different sample conditions, multiple locations of the sample and / or multiple samples may be imaged with minimal translation of the imaging beam or sample. Additionally, a large window area may provide a large area with uniform thickness and heat capacity, facilitating the building of high quality ice during vitrification.

[0015] The inner surface of the sample inspection device may be pre-treated to facilitate entry of the sample into the chamber. The inner surface may be pre-treated during manufacture of the sample inspection device. Alternatively or additionally, the inner surface may be pre-treated prior to loading the sample into the device.

[0016] 2A-2C, 3A-3C, 4 and 5A show example configurations of a sample inspection device with the relative placement of various components. These figures are not drawn to scale. When elements are shown to be in direct contact with or directly connected to one another, then such elements may be referred to as being in direct contact with or directly connected to one another, respectively. Similarly, elements shown consecutively or adjacent to one another may be consecutively or adjacent to one another, respectively. As another example, elements that are positioned apart from one another with only space between them and no other components may, in at least one example, be so referred to. Additionally, as shown in the figures, the topmost element or point of an element on the z-axis may be referred to as the "top" of the component, and the bottommost element or point of an element on the z-axis may, in at least one example, be referred to as the "bottom" of the component. As used herein, top / bottom, up / down, above / below may be relative to the z-axis and may be used to describe the placement of the elements of the figures relative to one another.

[0017] FIG. 6 shows an exemplary method for manufacturing a sample inspection device. FIG. 7 shows a procedure for manufacturing a sample inspection device according to the method of FIG. 6. The sample inspection device can be made from two silicon wafers, each of which is deposited with a layer of electron-transparent material. The two deposited wafers are bonded together by a patterned layer. The patterned layer forms a plurality of pillars and a bonding layer.

[0018] Multiple disconnected chamber sample inspection devices may be used individually to screen individual sample conditions, as shown in FIG. 8. Multiple connected chamber sample inspection devices may be used to screen a series of different sample conditions, as shown in FIG. 9. Connected chamber sample inspection devices may be used together with disconnected chamber sample inspection devices to quickly identify optimal sample conditions, as shown in FIG. 10. Connected chamber devices may be used to first identify an optimal range of sample conditions. Disconnected chamber devices may then be used to identify optimal sample conditions within the optimal range.

[0019] Referring to FIG. 1, a cryo-EM imaging system according to an embodiment of the present disclosure is shown. The cryo-EM system may include a transmission electron microscope (TEM) system 100 for imaging a vitrified sample. The TEM system 100 includes an electron source 10 that emits charged particles along an emission axis 110, such as an electron beam 11, toward a column 12. The electron source 10 may generate high-energy electrons, i.e., electrons having a typical energy of about 10 keV to 1,000 keV. In some embodiments, the column 12 may include one or more condenser lenses, such as condenser lenses 120 and 121, and one or more apertures, such as aperture 122. The column 12 collimates the electrons generated from the electron source 10 and directs the electron beam onto a sample inspection device 14. The sample inspection device 14 may be held by a sample holder 13. The sample inspection device may be any of the sample inspection devices described herein. The sample holder 13 may adjust the sample position by tilting and / or translating the sample. The sample holder may be kept at a temperature significantly below zero for cryogenic imaging. A condenser aperture 15 may optionally be inserted in the beam path to allow the electron beam to irradiate only selected regions of the sample.

[0020] The TEM system 100 may be used to obtain both a diffraction pattern and an EM image of a sample. In a diffraction imaging mode, the scattered electrons may be transmitted through the sample inspection device 14, sequentially through the objective lens 123, the intermediate lens 126, and the projection lens 127, as shown by solid lines, before being collected by the detector 25. The unscattered beam may be blocked by the beam stopper 17. The collected scattered electrons may be used to form an electron diffraction pattern, such as a selected area electron diffraction (SAED) pattern. In an EM imaging mode, the beam stopper 17 is removed from the beam path. The electrons transmitted from the sample inspection device 14 sequentially through the objective lens 123, the intermediate lens 126, and the projection lens 127, as shown by dashed lines, to form a TEM image of the sample on the detector 25.

[0021] The detector 25 may detect the received electrons and send a signal to the image processor 24 to form an image. The detector 25 may include an amplifier to amplify the signal before sending it to the image processor 24. In one example, the detector 25 may be a CCD camera or a CMOS camera. In some embodiments, different detectors may be used to detect signals generated by the interaction of the electrons with the sample. For example, the detector may detect x-rays and / or photons.

[0022] The controller 30 may control the operation of the TEM system 100 either manually in response to an operator's command or automatically according to computer readable instructions stored in a non-transitory memory (or computer readable medium) 32 of the controller 30. The controller 30 may include a processor 24 and be configured to execute computer readable instructions stored in the memory 32 and control various components of the TEM system 100 to implement any of the methods described herein. For example, the controller may adjust the imaging mode to acquire a diffraction pattern or an EM image by adjusting the position of the selected area aperture 15 and the objective aperture 124. The controller 30 may adjust the dose of the electron beam directed toward the sample by adjusting the current density. For example, the current density may be adjusted by adjusting the condenser optics (such as the condenser lenses 120 and 121). The controller 30 may adjust the profile of the incident beam by adjusting one or more apertures and / or lenses of the column 12. The controller 30 may adjust the position and / or orientation of the sample relative to the incident beam by adjusting the sample holder 13. The controller 30 may be configured to automatically acquire images at one or multiple locations of the sample inspection apparatus 14. The controller 30 may be further coupled to a display 31 for displaying notifications and / or images of the sample. The controller 30 may receive user input from a user input device 33. The user input device 33 may include a keyboard, a mouse, or a touch screen.

[0023] Although a TEM system is described as an example, it should be understood that the diffraction pattern and EM image may be acquired with other microscope systems. As an example, the diffraction pattern may be acquired by an optical-based microscope system. As an example, the diffraction pattern may be an X-ray diffraction pattern acquired from an X-ray diffraction system. As another example, the EM image may be acquired from a scanning transmission electron microscope (STEM) system. This discussion of a TEM system is provided merely as an example of one suitable imaging modality.

[0024] 2A-2C show an example of a sample inspection device 200 having a single chamber. An inlet and an outlet are fluidly connected to the chamber. Sample can flow into the chamber through the inlet and be inspected through a window covering at least a portion of the chamber.

[0025] 2A is a top view of the sample inspection device 200. The sample inspection device 200 may be rectangular. For example, the sample inspection device may be 10×3.3 mm 2The window 201, the inlet 203, and the outlet 204 are formed in the top layer 211 of the sample inspection device. The window 201, the inlet 203, and the outlet 204 may be formed by creating tapered recesses 202, 205, and 206, respectively, in the top layer 211. The openings of the recesses (202, 205, and 206) in the xy plane decrease as the openings approach the chamber 231. The recesses (202, 205, and 206) have the same depth as the thickness of the top layer 211. In one example, the recesses (202, 205, and 206) are created by etching a silicon wafer. The thickness of the top layer 211 may be 300 μm. The minimum dimension of the window 201 in the xy plane may be greater than 100 μm. The large window area provides a large area with uniform thickness and heat capacity, which may facilitate building high quality ice during vitrification. In one example, the window 201 is a square with a perimeter of more than 100 μm. In another example, the window 201 is a square with a perimeter of about 200 μm. In other examples, the window 201 can be other shapes, such as a rectangle or a circle. In one example, the inlet 203 and the outlet 204 are square with a perimeter of 80-180 μm. In other examples, the inlet 203 and the outlet 204 can be other shapes, such as a rectangle or a circle. The window 201 covers at least a portion of the chamber 231. The inlet 203 and the outlet 204 are in fluid communication with the chamber 231. The sample can flow from the inlet 203 to the chamber 231 via the channel 209 and from the chamber 231 to the outlet 204 via the channel 210. The sample in the chamber can be inspected through the window 201.

[0026] FIG. 2B is a cross-sectional view of the sample inspection device 200 in the xz plane along A-A' in FIG. 2A. Two electron-transparent layers 217 and 218 are disposed between the upper layer 211 and the lower layer 215. The upper electron-transparent layer 217 is in direct contact with the upper layer 211, and the lower electron-transparent layer 218 is in direct contact with the lower layer 215. The upper electron-transparent layer 217 and the lower electron-transparent layer 218 are bonded by a bonding layer 232 and one or more pillars 212. The thickness of the electron-transparent layers 217 and 218 may be 20 nm. A tapered recess 242 is formed in the lower layer 215 and aligned with the recess 202 so that charged particles such as electrons can transmit through the window 201 to the sample inspection device. Similar to the recess 202, the opening of the recess 242 in the xy plane decreases as the opening approaches the chamber 231. The height of the recess 242 is the same as the thickness 252 of the lower layer 215. The thickness 251 of the upper layer 211 may be the same as the thickness 252 of the lower layer 215.

[0027] The window 201 includes at least a portion of one of the two electron-transparent layers 217 and 218. The portion of the electron-transparent layer 217 that forms the window 202 is of continuous material. The chamber 231 is formed between the electron-transparent layers 217 and 218. The distance between the two electron-transparent layers (or the height of the chamber) can be 50-100 nm. A plurality of pillars 212 are arranged between the two electron-transparent layers 217 and 218 to allow the sample to flow around the pillars. Each of the pillars 212 extends from the upper electron-transparent layer 217 to the lower electron-transparent layer 218. Each of the pillars 212 has one end bonded to the upper electron-transparent layer 217 and the other end bonded to the lower electron-transparent layer 218. By bonding each end of the pillars to one of the two electron-transparent layers, the sample inspection device can retain a shape during the processes of sample loading, sample vitrification, and sample imaging. In one example, the shape of the pillars in the xy cross section can be circular, elliptical, or rectangular. The window 201 covers at least one of the pillars 212. The cross-sectional area of ​​each pillar in the xy plane is 3 μm 2 ~350μm 2The pillars (212, 213) and the bonding layer 232 may be fabricated from the same material in the same process step. Charged particles may pass through the window 201 between the gaps of the pillars 212, thereby imaging the sample surrounding the pillars. In other words, the window 201 has at least an area that allows the charged particles to pass through and at least an area where the charged particles are blocked by the pillars. The area of ​​the window may be increased by arranging the pillars in a chamber covered with a window. The increased window area allows for a larger imaging area and a shorter stage travel distance while inspecting the sample with a charged particle imaging system. Furthermore, the large window area facilitates homogenous cooling of the sample during vitrification. Furthermore, the pillars provide mechanical support for the window, increasing the robustness of the sample inspection device.

[0028] Channels 209 and 210 are formed in the bottom layer 215 and connect the inlet 203 and outlet 204, respectively, to the chamber 231. The channels may be created by etching into the bottom layer 215. The height 255 of the channels 209 and 210 may be 0.1-10 μm. In some embodiments, a plurality of pillars 213 may be optionally arranged in the chamber, with one end bonded to the top electron-transparent layer 217. In one embodiment, the chamber 231 is evacuated. The inlet 203 and outlet 204 are sealed by a thin film. In one example, the inlet 203 and outlet 204 are sealed by the top electron-transparent layer 217, as shown in FIG. 2B. In another embodiment, the sample inspection device has only an inlet and no outlet. The inlet may be sealed with a thin film. The sample may flow into the chamber by poking and breaking the thin film.

[0029] Buffer chambers 207 and 208 are arranged between inlet 203 and chamber 231, and between chamber 231 and outlet 204, respectively. Sample flows from the inlet through buffer chamber 207 to the area of ​​chamber 231 covered by window 201. In some embodiments, the buffer chambers can be part of chamber 231, as shown in Figures 2B-2C. Buffer chambers 207 and 208 are between two electron-transparent layers 217 and 218. Pillars are not arranged in the buffer chambers. In other embodiments, the buffer chambers can be part of channels 209 and 210. Buffer chambers without pillars can avoid backflow of sample from chamber 231 towards inlet 203.

[0030] FIG. 2C is a cross-sectional view of the sample inspection device 200 in the xy plane along B-B' of FIG. 2B. A part of the chamber 231 is covered by the window 201. The edge of the window is depicted by the dashed line 230. The buffer chambers 207 and 208 fluidly connect the chamber area covered by the window with the inlet 203 and the outlet 204, respectively. The window 201 covers a plurality of pillars 212. In other words, the window 201 is supported by a plurality of pillars 212. The sample can flow sequentially from the inlet 203, through the channel 209 and the buffer chamber 207, to the area of ​​the chamber 231 covered by the window 201. If the sample inspection device includes an outlet 204, the sample can be discharged from the area of ​​the chamber 231 covered by the window 201 to the outlet through the buffer chamber 208 and the channel 210.

[0031] In some embodiments, the sample inspection device may be circular, for example, the sample inspection device may be circular with a diameter of 3 mm.

[0032] 3A-3C show an example of a sample inspection device 300 with two disconnected chambers. Each chamber has an inlet fluidly connected to the chamber. In this example, the sample inspection device 300 does not include an outlet. Samples in both chambers may be inspected through a window. In some examples, one or more of the chambers may be connected to one or more outlets.

[0033] 3A is a top view of the sample inspection device 300. The window 301, the first inlet 303, and the second inlet 304 are formed by creating tapered recesses 302, 305, and 306, respectively, in the top layer 311. The window 301 covers both a portion of the first chamber 331 and a portion of the second chamber 332. The first chamber 331 and the second chamber 332 are not fluidically connected. In the area covered by the window 301, the first chamber 331 and the second chamber 332 are separated by a wall 360. The first inlet 303 is fluidically connected to the first chamber 331 by a channel 309. The second inlet 304 is fluidically connected to the second chamber 332 by a channel 310.

[0034] The thickness of the top layer 311 may be 300 μm. The smallest dimension of the window 301 in the xy plane may be greater than 100 μm. In one example, the window 301 is a square with a perimeter of greater than 100 μm. In another example, the window 301 is a square with a perimeter of about 200 μm. In other examples, the window 301 may be other shapes, such as rectangular or circular. In one example, the inlet 303 and the outlet 304 are square with a perimeter of 80-180 μm. In other examples, the inlet 303 and the outlet 304 may be other shapes, such as rectangular or circular.

[0035] FIG. 3B is a cross-sectional view of the sample inspection device 300 in the xz plane along C-C' of FIG. 3A. Two electron-transmitting layers 317 and 318 are disposed between the upper layer 311 and the lower layer 315. The upper electron-transmitting layer 317 is in direct contact with the upper layer 311, and the lower electron-transmitting layer 318 is in direct contact with the lower layer 315. The upper electron-transmitting layer 317 and the lower electron-transmitting layer 318 are coupled by the coupling layer 311 and the pillar 312. The pillars (312, 313, and 373) and the coupling layer 311 can be fabricated from the same material in the same process step. The thickness of the electron-transmitting layers 317 and 318 can be 20 nm. The openings of the tapered recesses 305, 302, and 306 decrease, and the openings approach the upper electron-transmitting layer 317. The depth of the tapered recess is the same as the thickness 351 of the upper layer 311.

[0036] A tapered recess 342 is formed in the lower layer 315 and aligned with the recess 302 so that charged particles, such as electrons, can transmit through the window 301 to the sample inspection device. The opening of the recess 342 in the xy plane decreases as the opening approaches the lower electron-transparent layer 318. The height of the recess 342 is the same as the thickness 352 of the lower layer 315. The thickness 351 of the upper layer 211 can be the same as the thickness 352 of the lower layer 315.

[0037] Channels 309 and 310 are formed in bottom layer 315 to connect first inlet 303 to first chamber 331 and second inlet 304 to second chamber 332, respectively. The channels may be created by etching into bottom layer 315. Height 355 of channel 309 and height 356 of channel 310 may be 0.1-10 μm. Pillars 313 and 373 may be optionally disposed in channel 309 and / or channel 310. Pillars 313 and 373 may be bonded to top electron-transparent layer 317. In some embodiments, first inlet 303 and second inlet 304 are sealed by a thin film from first chamber 331 and second chamber 332, respectively. In one example, the thin film is formed by top electron-transparent layer 317, as shown in FIG. 3B. First chamber 331 and second chamber 332 may be evacuated. The liquid sample can flow into the chamber by poking or breaking the membrane.

[0038] FIG. 3C is a cross-sectional view of the sample inspection device 300 in the xy plane along D-D' of FIG. 3B. The window 301 covers both a part of the first chamber 331 and a part of the second chamber 332. The edge of the window is depicted by a dashed line 330. The buffer chamber 307 fluidly connects the area of ​​the first chamber covered by the window 301 with the inlet 303. The buffer chamber 308 fluidly connects the area of ​​the second chamber covered by the window 301 with the inlet 304. The window 301 covers a plurality of pillars 312. In other words, the window 301 is supported by a plurality of pillars 312. The first sample can flow sequentially from the first inlet 303 through the channel 309 and the buffer chamber 307 to the area of ​​the chamber 331 covered by the window 301. A second sample may flow sequentially from the second inlet 304 , through the channel 310 and the buffer chamber 308 , to the region of the chamber 332 covered by the window 301 .

[0039] FIG. 4 is a cross-sectional view of a portion of a sample inspection device 400 with four chambers 451, 452, 453, and 454 formed between two electron-transparent layers bonded by a bonding layer 402 cut through. A window 401 covers at least a portion of each chamber. The window edge 470 is depicted with dashed lines. The area covered by the window 401 in the xy plane is indicated by a diagonal line in the window edge 470. The window 401 comprises at least a portion of a continuous upper electron-transparent layer. The smallest dimension of the window 401 in the xy plane may be greater than 100 μm. Similar to the sample inspection device of FIGS. 3A-3C, the sample inspection device may not have an outlet, and the inlet is sealed by a thin membrane. Four different samples may be transferred to different chambers of the sample inspection device and imaged through the window 401.

[0040] The first chamber 411 and the third chamber 431 are separated from the second chamber 421 and the fourth chamber 441 by a wall 403. The first chamber 411 and the second chamber 421 are separated from the third chamber 431 and the fourth chamber 441 by a wall 404. Each chamber may include an area in which a plurality of pillars are arranged and an area without pillars. The area without pillars is also referred to as a buffer chamber herein. For example, the first chamber 411 includes an area in which a plurality of pillars 412 and a pillar-free buffer chamber 410 are arranged. The second chamber 421 includes an area in which a plurality of pillars 422 and a pillar-free buffer chamber 420 are arranged. The third chamber 431 includes an area in which a plurality of pillars 432 and a pillar-free buffer chamber 430 are arranged. The fourth chamber 441 includes an area in which a plurality of pillars 442 and a pillar-free buffer chamber 440 are arranged. The window 401 covers at least one pillar in each of the chambers. In other words, at least one entire pillar in the xy plane is covered by the window 401 in the xy plane. Herein, the pillars are shown in a stadium shape in the xy plane. The cross section of the pillars can be other shapes, such as rectangular or circular. In some embodiments, the pillars can be different shapes in one sample inspection device.

[0041] Each of the chambers is fluidly connected to a different inlet (not shown) via a channel (451, 452, 453, or 454). A number of pillars (461, 462, 463, and 464) may optionally be arranged within the channel.

[0042] In one embodiment, the buffer chamber may be included in the channel instead of the chamber. In some embodiments, the number of chambers in the sample inspection device may be any integer, such as 16, 32, or 64.

[0043] FIG. 5A is a cross-sectional view of a portion of an exemplary sample inspection device 500. The sample inspection device 500 includes connected chambers formed between two electron-transparent layers bonded by a bonding layer 502. A first chamber 521 and a second chamber 522 receive a first sample and a second sample from different inlets (not shown), as indicated by arrows 551 and 522, respectively. The first chamber 521 and the second chamber 522 are fluidly separated upstream of the device (i.e., in the direction opposite to the x-axis arrow direction) and fluidly connected downstream of the device by a wall 506. The first sample and the second sample are mixed downstream (i.e., in the direction of the x-axis arrow) in a mixing region 591 of the sample inspection device. The flow directions of the portions of the first sample and the second sample are as indicated by arrows 553 and 554, respectively.

[0044] The first sample, the second sample, and the mixture of samples can be inspected through a window formed by at least a portion of the two electron-transparent layers. The window covers an area 501 in the xy plane. The area 501 is covered by a continuous portion of an electron-transparent layer (such as an upper electron-transparent layer). A dashed line 510 depicts an edge of the window-covered area 501. The window-covered area 501 includes a mixing area 591 where the samples are mixed. The window may also cover a portion of the wall 506. The window-covered area 501 includes at least one pillar of a plurality of pillars 511 arranged between the two electron-transparent layers. Herein, the pillar 511 is circular in the xy plane. In other examples, the cross section of the pillar 511 may be other shapes, such as a stadium or a rectangle.

[0045] Each chamber (521 and 522) of the sample inspection device 500 may include a pillar-free buffer chamber (531 and 532) upstream of the region of the window-covered region 501. In some embodiments, the sample inspection device may include three or more connected chambers for mixing three or more samples. A window may cover the region where the samples are mixed.

[0046] The first sample and the second sample may be transferred to the sample inspection device at the same time. A laminar steady flow may be established for each sample from the moment of transferring the samples to the sample inspection device. Sample mixing occurs at the boundary of these two parallel flows in the mixing region. A gradient of sample conditions may be formed in the mixing region. In one example, if a steady sample flow remains from the transfer of the samples to the sample inspection device until the time of vitrification, the sample condition is a function of the sample position in the mixing region. In another example, the sample condition may be a function of the sample position as well as the waiting period from the transfer of the samples to the time of vitrification. Different sample conditions, such as sample ratios, may be observed at different locations in the mixing region at different times. The spatial distribution of the sample ratios in the mixing region 591 changes over time.

[0047] FIG. 5B shows the ratio between sample 2 and sample 1 along a line 590 that crosses the mixing region 591 of FIG. 5A. In one example, if both samples maintain laminar and steady flow from the transfer of the samples to the sample inspection apparatus until the time of vitrification, the sample ratio 581 does not change with time. The sample ratio 581 decreases as the x-axis distance from the end of wall 506 increases. In another example, after the samples are transferred to the sample inspection apparatus at T1, the sample ratio 581 changes with time. For example, at a time T2, which is later than time T1, the sample ratio 582 increases from the sample ratio 581 at T1.

[0048] The sample inspection device shown in Figures 2A-2C, 3A-3C, 4 and 5A includes one or more chambers covered by a single window. The single window comprises a portion of a continuous electron transparent layer. The window is covered and supported by at least one pillar bonded between two electron transparent layers. The pillar may have a cross section of a single shape or multiple shapes. In some examples, all or a portion of the inner surfaces of the sample inspection device (i.e., the surfaces that directly contact the sample during sample loading and imaging) may be pretreated to facilitate sample loading. Pretreatment may be performed at room temperature.

[0049] Surface pretreatment can be performed via different experimental approaches. In one example, the surface can be pretreated by a surface activation method at room temperature. The inner surface of the sample inspection device can be treated with a chemical solution to allow for proper surface wetting, facilitating sample ingress into the device and facilitating sample adhesion to the chamber. The surface of the sample inspection device can be treated by incubating the solution on the surface or by flowing the solution through the surface. For example, a buffer containing an optimal concentration of detergent or a hydrophilic antifouling compound, e.g., PEG that does not interfere with the structure and function of the target protein, can be passed through the sample inspection device before transferring or loading the sample into the device to modify the inner surface of the device. In some examples, the chemicals can be covalently bonded to the inner surface of the device.

[0050] In another example, the interior surfaces of the sample inspection device may be treated by plasma cleaning, for example where charged air is applied at high voltage to increase the hydrophilicity of the surface via radical reactions.

[0051] In yet another example, the interior surface of the sample inspection device may be pretreated by irradiating the interior surface with radiation, such as ultraviolet radiation (UV). The interior surface is exposed to UV radiation to alter the surface tension and surface free energy, which enhances the wettability of the surface.

[0052] Figure 6 is an exemplary method 600 for manufacturing a sample inspection device. Figure 7 illustrates steps of the method 600 for manufacturing the sample inspection device of Figures 2A-2C.

[0053] At 602, two silicon wafers are prepared. In one example, the diameter of each wafer in the xy plane may be 4 inches. The thickness of the wafers may be 300 μm.

[0054] At 604, a layer of silicon nitride is deposited on one surface of each wafer. The thickness of the silicon nitride layer may be 20 nm. In one example, the silicon nitride layer is deposited by low pressure chemical vapor deposition (LPCVD). Plot 701 in FIG. 7 shows an xz cross section of a first wafer 711 on which an electron-transparent silicon nitride layer 712 has been deposited.

[0055] At 606, a patterned layer is deposited on the silicon nitride layer of the first wafer. The thickness of the patterned layer may be 50-100 nm. The patterned layer may be borophosphosilicate glass (BPSG). In one example, the patterned layer is deposited by plasma enhanced vapor deposition (PECVD). Plot 702 of FIG. 7 shows a patterned layer 724 deposited on the silicon nitride layer 712. The patterned layer 724 may form one or more of pillars 721 in a chamber (such as chamber 231 in FIGS. 2A-2C), pillars 722 in a channel (such as channel 209 in FIGS. 2A-2C), and a bonding layer (such as bonding layer 232 in FIGS. 2A-2C).

[0056] At 608, channels are etched into the second wafer. The channels may be formed by etching a silicon nitride layer deposited on the second wafer. The depth of the channels may be between 0.1 and 10 μm. Plot 703 shows channels 733 etched into a second wafer 731 having a silicon nitride layer 732 deposited thereon. The depth 735 of the channels 733 may be between 0.1 and 10 μm.

[0057] At 610, the first wafer from step 606 and the second wafer from step 608 may be aligned and bonded in a vacuum or low pressure inert gas by direct or fusion bonding. Plot 704 shows the processed first wafer of plot 702 and the processed second wafer of plot 703 aligned and bonded to one another.

[0058] At 612, the bonded wafers are etched. The bonded wafers may be etched to expose the silicon nitride layers. Plot 705 of FIG. 7 shows recesses 751, 752, 753, and 754 etched into the bonded wafers to expose silicon nitride layers 712 and 732. Thus, one or more of windows, inlets, and outlets may be formed on the bonded wafers.

[0059] At 614, the etched wafer may be diced into a plurality of individual devices.

[0060] The inner surface of the sample inspection device may be pretreated. In one example, the inner surface of the sample inspection device may be pretreated in step 610 prior to aligning and bonding the wafer. The inner surface may be irradiated with radiation, plasma cleaning, or heating. The inner surface may be pretreated by sequentially immersing some or all of the inner surface of the device in one or more chemical solutions. The inner surface may be pretreated by sequentially flowing the solutions over the inner surface. The solutions may be buffers containing optimal concentrations of detergents or anti-fouling chemicals such as PEG. In another example, the inner surface of the sample inspection device may be pretreated by heating the device after etching the boundary wafer in step 612. In yet another example, the inner surface may be pretreated by flowing solutions through the device after aligning and bonding the wafer in step 610.

[0061] Figure 8 shows a method 800 for screening sample conditions of multiple samples using a disconnected chamber sample inspection device, such as the sample inspection device of Figures 3A-3C and 4. Samples with different sample conditions can be transferred to different chambers of the sample inspection device and imaged. Optimal sample conditions can be selected based on cryo-EM images of the samples.

[0062] At 802, a plurality of samples with different sample conditions are prepared. For example, a purified protein solution may be mixed with a plurality of buffer solutions to obtain samples with different sample conditions. The samples may have different protein to buffer ratios, different protein concentrations, or different buffer concentrations.

[0063] In 803, the sample inspection device may be optionally pretreated before transferring or loading the sample into the device. The sample inspection device may be pretreated by plasma cleaning or by flowing a solution through the device. For example, a buffer solution containing an optimal concentration of detergent or an anti-fouling chemical such as PEG may be sequentially flowed through the inspection device before transferring the sample to the device to modify the interior surface. In some examples, if the inlet and / or outlet of the sample inspection device are sealed. Before pretreating the sample inspection device, first break the seal.

[0064] At 804, each of the prepared samples is transferred to one chamber of the sample inspection device. The samples may be transferred to the sample inspection device simultaneously or sequentially. For example, a small amount of sample may be placed in an inlet recess of the sample inspection device. A membrane sealing the inlet is then poked and broken to allow the small amount of sample to flow and fill the portion of the chamber covered by the window.

[0065] At 806, the samples transferred to the sample inspection device are vitrified. For example, a sample inspection device loaded with multiple samples may be vitrified by plunge freezing.

[0066] At 808, a sample image is obtained from each chamber of the sample inspection apparatus, for example, using the cryo-EM imaging system of FIG. 1. To image the sample in the chamber, a charged particle beam may be directed toward a location on the window and the sample surrounding one of the pillars covered by the window. An image of the sample may be formed based on the charged particles transmitted through the sample inspection apparatus. Multiple images of the sample at different locations of the chamber may be taken, where the sample at the different locations has the same sample conditions.

[0067] At 810, optimal sample conditions that provide the best sample quality may be determined based on the sample images acquired from each chamber. The sample quality of each sample may be evaluated based on the images of each sample acquired at 808. The optimal sample conditions correspond to the best sample quality and may be identified and used for sample preparation in single particle analysis.

[0068] In this way, samples with different sample conditions can be vitrified and imaged with a single device. Individual sample conditions can be evaluated. In some examples, instead of loading one type of protein with different sample conditions into the device chamber, multiple samples with various structures can be loaded into multiple chambers of one sample inspection device to determine the optimal stabilization method for the protein. In other examples, different protein complexes of a target protein with various ligands or compounds can be loaded and examined using one sample inspection device. In some examples, multiple versions of different proteins or protein complexes in different states (such as open, closed, or rotating) can be loaded and examined using one sample inspection device.

[0069] 9 illustrates a method 900 for screening sample conditions of multiple samples using a sample inspection device with an attached chamber, such as the sample inspection device of FIG. 5. The samples are transferred to the sample inspection device and mixed in the device. Images may be taken at different locations of the mixing area covered by a window of the sample inspection device. An optimal range of sample conditions may be determined based on the images and the corresponding imaging locations.

[0070] At 902, multiple samples are prepared, for example, a first sample is a purified protein solution and a second sample is a buffer solution.

[0071] At 903, the sample inspection device may optionally be pretreated before transferring or loading the sample into the device. The sample inspection device may be treated by plasma cleaning or by flowing a solution through the device. For example, a buffer solution containing an optimal concentration of a detergent or an anti-fouling chemical such as PEG may be sequentially flowed through the inspection device before transferring the sample into the device to modify the interior surface.

[0072] At 904, the samples are mixed in a sample inspection device with connected chambers. Mixing the samples includes transferring the samples to the sample inspection device by flowing each sample into one inlet of the device. For example, a small amount of sample may be placed in a recess in the inlet of the sample inspection device. A membrane sealing the inlet is then broken so that the small amount of sample may flow into an area of ​​the chamber covered by a window. The samples may flow simultaneously into the device and be mixed downstream of the device in a mixing area covered by a window.

[0073] At 906, the sample in the sample inspection device is vitrified after a waiting period after flowing the sample through the sample inspection device. The waiting period may be predetermined. The spatial distribution of the sample conditions in the mixing region may change with the waiting time. The sample in the sample inspection device may be vitrified by plunge freezing.

[0074] At 908, sample images are obtained from multiple locations in the mixing region of the sample inspection apparatus, for example using the cryo-EM imaging system of FIG. Each location in the mixing region may have different sample conditions. In one example, images may be taken at predetermined locations.

[0075] At 910, the sample quality of the acquired sample images may be evaluated to identify locations where the images have good sample quality.

[0076] At 912, an optimal range of sample conditions is determined. Determining the optimal range of sample conditions may include determining the sample conditions at the identified locations from 910. In some examples, the sensitivity of the sample conditions may be determined by identifying differences between the multiple locations based on the images from 908. Based on the sample conditions at the identified locations, an optimal range of sample conditions may be determined.

[0077] In one example, at 914, the sample condition at each of the identified locations may be determined experimentally. A mixed sample at each identified location may be extracted and analyzed to obtain the sample condition. For example, a mixed sample may be extracted by breaking out a sample inspection device using a focused ion beam system.

[0078] In another example, at 916, the sample conditions may be determined based on the identified locations and a calibrated sample condition distribution. The calibrated sample condition distribution may be theoretically generated by simulation based on parameters including one or more of the sample characteristics, wait time, temperature, and flow rate of the sample when transporting the sample to the sample inspection device. Alternatively, the calibrated sample condition distribution may be experimentally generated by measuring the sample conditions of a calibration sample with the sample inspection device.

[0079] In this way, samples can be mixed, vitrified, and imaged in a single device. A continuous range of sample conditions can be obtained and evaluated for mixed samples.

[0080] 10 shows a method 1000 for screening sample conditions using both a connected chamber sample inspection device and a disconnected chamber sample inspection device. The connected chamber sample inspection device may be used to first determine an optimal range of sample conditions by screening a large variation in sample conditions. A number of samples having sample conditions within the optimal range may then be individually screened to determine the optimal sample conditions using the disconnected chamber sample inspection device.

[0081] In 1002, an optimal range of sample conditions is determined using the method 900 of FIG. 9. For example, a protein solution and one or more buffers may be transferred to a sample inspection device with a connected chamber. Based on the cryo-EM images, a sample condition that may result in optimal sample quality is determined. Because a wide range of sample conditions may be obtained using a sample inspection device with a connected chamber, buffer screening using the method 900 provides a global search for an optimal range. However, because the spatial distribution of sample conditions in the mixing region varies continuously, the sample conditions in each sample image may vary. Furthermore, a limited number of mixed samples may be imaged at a particular sample condition. Thus, the method 900 may provide an optimal range of sample conditions that includes the optimal sample condition, rather than a specific optimal sample condition.

[0082] At 1004, a plurality of samples are prepared having sample conditions within the optimal range determined at 1002. The samples may be screened using method 800 of FIG. 8 to determine optimal sample conditions within the optimal range. Multiple images of the samples having particular sample conditions may be acquired, each image may be taken at a different location in the chamber. Thus, sample quality may be accurately and reliably assessed.

[0083] In some embodiments, each of steps 1002 and 1004 may be repeated to screen different buffers and / or to pinpoint optimal sample conditions.

[0084] In this way, a global sample condition screening is first performed using the chamber-connected sample inspection device to identify the optimal range. Localized sample condition screening is then performed within the optimal range to identify the optimal conditions. The multi-scale screening strategy can significantly reduce the sample condition screening time and facilitate the identification of global optimal sample conditions for single particle analysis.

[0085] The technical effect of providing a window covering at least one of the multiple pillars for sample inspection is to provide mechanical strength of the window-covered area of ​​the sample inspection device. The technical effect of bonding the pillar to two electron-transparent layers is to prevent bulging during sample loading and vitrification. The technical effect of imaging multiple samples in a chamber is that sample quality may be better controlled for vitrification and imaging. The technical effect of mixing multiple samples in the sample inspection device is that a continuous distribution of sample conditions may be obtained. The technical effect of controlling the waiting time between flowing the sample into the sample inspection device and vitrification of the sample is that the distribution of sample conditions in the mixing area may be adjusted.

[0086] In one embodiment, a sample inspection apparatus includes a first chamber formed between an upper electron-transparent layer and a lower electron-transparent layer for holding a first sample, a plurality of pillars in the first chamber, each pillar of the plurality of pillars extending from the upper electron-transparent layer to the lower electron-transparent layer, and a window formed in a portion of at least one of the upper and lower electron-transparent layers for inspecting a first sample in the first chamber, the window covering at least one of the plurality of pillars. In a first example of the apparatus, the window allows inspection of a first sample surrounding at least one of the plurality of pillars covered by the window. A second example of the method optionally includes the first example and further includes a second chamber formed between the upper and lower electron-transparent layers for holding a second sample, the window covering a portion of the second chamber for inspecting a second sample in the second chamber. A third example of the method optionally includes one or more of the first to second examples, further including a plurality of pillars arranged between the upper electron-transparent layer and the lower electron-transparent layer of the second chamber. A fourth example of the method optionally includes one or more of the first to third examples, further including the first chamber and the second chamber being fluidly connected in an area covered by the window. A fifth example of the method optionally includes one or more of the first to fourth examples, further including one end of each pillar of the plurality of pillars being coupled to the upper electron-transparent layer and the other end of the pillar being coupled to the lower electron-transparent layer. A sixth example of the method optionally includes one or more of the first to fifth examples, further including an inlet and a buffer chamber, the inlet being fluidly connected to the first chamber via the buffer chamber, and the pillars not arranged in the buffer chamber. A seventh example of the method optionally includes one or more of the first through sixth examples, further including at least a portion of the upper electron transparent layer and / or the lower electron transparent layer in contact with the first sample being pretreated to facilitate transfer of the first sample to the first chamber. An eighth example of the method optionally includes one or more of the first through seventh examples, further including an area of ​​the window that does not cover at least one of the plurality of pillars allowing the charged particle beam to transmit through the sample inspection device.A ninth example of the method optionally includes one or more of the first through eighth examples, further including a distance between the two electron transmitting layers being less than 100 nm.

[0087] In another embodiment, a method of inspecting a sample using a sample inspection device includes flowing a first sample into a first chamber of the sample inspection device, the first chamber being formed between an upper electron-transparent layer and a lower electron-transparent layer, a plurality of pillars being arranged in the first chamber, each pillar of the plurality of pillars extending from the upper electron-transparent layer to the lower electron-transparent layer, directing a charged particle beam toward a window of the sample inspection device, the window being formed of a portion of at least one of the upper electron-transparent layer and the lower electron-transparent layer, the window covering at least one of the plurality of pillars, and forming a first image of the first sample based on the charged particles transmitted through the sample inspection device. In a first example of the method, the method further includes treating an inner surface of the inspection device before flowing the first sample into the first chamber. A second example of the method optionally includes the first example and further includes that directing the charged particle beam toward the window includes directing the beam to a location of the window that does not cover at least one of the plurality of pillars. A third example of the method optionally includes one or more of the first through second examples, further including vitrifying the first sample in the sample inspection device after flowing the first sample into the first chamber and before directing the charged particle beam toward the window of the sample inspection device.A fourth example of the method optionally includes one or more of the first through third examples, further including directing the charged particle beam toward the window of the sample inspection device to form a first image of the first sample based on the charged particles transmitting through the sample inspection device includes directing the charged particle beam at a first location in the first chamber, and forming a first image of the first sample based on the charged particles transmitted through the sample inspection device.A fifth example of the method optionally includes one or more of the first through fourth examples, and further includes flowing a second sample into a second chamber of the sample inspection device, the second chamber being formed between an upper electron transparent layer and a lower electron transparent layer, the second chamber being fluidically isolated from the first chamber, and a window covering at least a portion of the second chamber, directing the charged particle beam toward a second location in the second chamber, and forming a second image of the second sample based on the charged particles transmitted through the sample inspection device. A sixth example of the method optionally includes one or more of the first through fifth examples, and further includes the first sample and the second sample having different sample conditions, and the method further includes comparing the first image and the second image to determine optimal sample conditions. A seventh example of the method optionally includes one or more of the first to sixth examples, further including: while flowing the first sample into the first chamber, flowing the second sample into a second chamber formed between the upper electron transparent layer and the lower electron transparent layer, and mixing the first sample and the second sample in a mixing region of the sample inspection device covered by a window, where directing the charged particle beam toward the window includes directing the charged particle beam at a plurality of locations in the mixing region, where a ratio between the first sample and the second sample at the plurality of locations is different. An eighth example of the method optionally includes one or more of the first to seventh examples, further including: forming an image based on the charged particles transmitted through the sample inspection device includes forming an image of each of the plurality of locations based on the charged particles transmitted through the sample inspection device, where the method further includes comparing the plurality of images to determine optimal sample conditions. A ninth example of the method optionally includes one or more of the first through eighth examples, and further includes vitrifying the first sample, the second sample, and a mixture of the first sample and the second sample in a sample inspection device prior to directing the charged particle beam toward the window, flowing the first sample and the second sample into the sample inspection device, and determining a ratio between the first sample and the second sample at the multiple locations based on the duration between vitrification.

Claims

1. 1. A method of testing a sample using a sample testing device, comprising: flowing a first sample through a first chamber of the sample inspection device, the first chamber being formed between an upper electron transparent layer and a lower electron transparent layer, a plurality of pillars being arranged within the first chamber, each pillar of the plurality of pillars extending from the upper electron transparent layer to the lower electron transparent layer; flowing a second sample into a second chamber formed between the upper electron transparent layer and the lower electron transparent layer; directing a charged particle beam toward a window of the sample inspection device, the window being formed of a portion of at least one of the upper electron transparent layer and the lower electron transparent layer, the window covering at least one of the plurality of pillars and a portion of the second chamber; forming a first image of the first sample based on the charged particles transmitted through the sample inspection device.

2. 10. The method of claim 1, further comprising treating an interior surface of the sample inspection device prior to flowing the first sample into the first chamber.

3. 2. The method of claim 1, wherein directing the charged particle beam toward the window comprises directing the charged particle beam to a location of the window that does not cover the at least one of the plurality of pillars.

4. 4. The method of claim 1, further comprising vitrifying the first sample and the second sample in the sample inspection device after flowing the first sample and the second sample into the sample inspection device and before directing the charged particle beam towards the window of the sample inspection device.

5. 5. The method of claim 4, further comprising: directing the charged particle beam towards the window of the sample inspection device to form the first image of the first sample based on the charged particles passing through the sample inspection device; and forming the first image of the first sample based on the charged particles passing through the sample inspection device.

6. directing the charged particle beam toward a second location in the second chamber; 6. The method of claim 5, further comprising forming a second image of the second sample based on the charged particles transmitted through the sample inspection device.

7. 7. The method of claim 6, wherein the first sample and the second sample have different sample conditions, the method further comprising determining an optimal sample condition by comparing the first image and the second image.

8. 5. The method of claim 4, further comprising: flowing the second sample into the second chamber while flowing the first sample into the first chamber; mixing the first sample and the second sample in a mixing area of ​​the sample inspection device covered by the window; and acquiring sample images from multiple locations within the mixing area, wherein a ratio between the first sample and the second sample at the multiple locations is different.

9. 9. The method of claim 8, wherein forming an image based on the charged particles transmitted through the sample inspection device comprises forming an image of each of a plurality of locations based on the charged particles transmitted through the sample inspection device, the method further comprising comparing a plurality of the images to determine optimal sample conditions.

10. 10. The method of claim 8, further comprising determining a ratio between the first sample and the second sample at a plurality of locations based on a duration between flowing the first sample and the second sample through the sample inspection device and the vitrification.

Images