Methods and systems for sample preparation

Abstract

The sample is held by a sample stage in the microscope system and milled to expose a region of interest (ROI) within the sample. The sample is milled based on the ROI location determined by sample images acquired with light beams irradiated from different axes. Sample images are acquired while the sample is held using the same sample stage for milling.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 471,208, filed on June 5, 2023. The entire contents of the above - mentioned application are incorporated herein by reference.

[0002] (Field of the Invention) This specification generally relates to methods and systems for analyzing samples, and more particularly to preparing samples for inspection under a charged - particle microscope using optical imaging.

Summary of the Invention

[0003] In one embodiment, a method for preparing a sample includes irradiating a sample held by a sample stage with an optical beam along a first axis, detecting photons emitted from the sample along the first axis to obtain at least a first sample image; irradiating the sample with an optical beam along a second axis, detecting photons emitted from the sample along the second axis to obtain at least a second sample image; determining the position of a region of interest (ROI) within the sample based on the first sample image and the second sample image; and milling the sample held by the sample stage based on the position of the ROI.

[0004] In another embodiment, the microscope system comprises a sample chamber, a sample stage positioned within the sample chamber, a light source for generating a light beam, an objective lens, a detector for detecting photons emitted from the sample passing through the objective lens, a milling source for generating a milling beam, and a controller, the controller comprising a processor and non-temporary memory for storing computer-readable instructions, wherein the processor executes computer-readable instructions, thereby configuring the microscope system to irradiate the sample held by the sample stage with a light beam along a first axis via the objective lens and acquire at least a first sample image via the detector, irradiate the sample with a light beam along a second axis via the objective lens and acquire at least a second sample image via the detector, determine the location of a region of interest (ROI) in the sample based on the first and second sample images, and mill the sample held by the sample stage with the milling beam based on the location of the ROI.

[0005] It should be understood that the above summary is provided in a simplified form to introduce a selection of concepts that will be further explained in the detailed description. It is not intended to identify any important or essential features of the claimed subject matter, the scope of which is uniquely defined by the claims that follow the detailed description. Furthermore, the subject matter of the claims is not limited to embodiments that resolve any defects described above or in any part of this disclosure. [Brief explanation of the drawing]

[0006] [Figure 1] An example of a microscope system is shown. [Figure 2] This image shows a sample stage placed in the sample chamber of a microscope system. [Figure 3] This is a flowchart of the method for preparing the sample. [Figure 4A] Here is an example of how to obtain a sample image. [Figure 4B] Here is an example of how to obtain a sample image. [Figure 5] Here's another example of obtaining a sample image. [Figure 6] This 2D diagram demonstrates how acquiring images from two different directions improves the accuracy of localizing a region of interest (ROI) by reducing uncertainty.

[0007] The same reference number refers to the corresponding part across several figures in the drawing. [Modes for carrying out the invention]

[0008] The following description concerns systems and methods for sample preparation, particularly for preparing frozen biological samples to be imaged by transmission electron microscopy (TEM). TEM imaging requires thin samples, such as thin sections, to allow electrons to pass through. Thin sections can be prepared by milling or micromachining bulk samples using laser and / or charged particle beams. Regions of interest (ROIs) embedded in the bulk sample need to be accurately identified and localized to ensure that the prepared thin section contains the ROIs. One method for identifying and localizing ROIs is to use fluorescence optical microscopy (FM). Fluorescence data may include multiple 2D scans, which can consist of lateral (tile set) or axial (stack) series. However, accurately localizing ROIs in a 3D bulk sample based on fluorescence data can be difficult because the lateral resolution of the fluorescence imaging system is higher than its axial resolution. The ROI position in the axial direction of the incident beam can be estimated based on the intensity of the fluorescence signal or by optical sectioning (acquiring multiple 2D fluorescence images with various imaging depth stacks), but the accuracy of axial positioning is intrinsically limited by the low axial resolution of the imaging system. Furthermore, FM may have limited imaging depth for mapping the entire ROI. Samples need to be prepared accurately and quickly for examination under a charged particle microscope. For example, samples need to be prepared and thinned (by milling, etc.) so that the ROI can be accurately and quickly localized and further analyzed under a charged particle microscope (TEM, etc.).

[0009] The above problem can be addressed by determining the ROI position based on optical images of the sample acquired from different angles of light incidence (i.e., different views / axes) relative to the sample surface. For example, a first sample image is acquired by illuminating the sample along a first axis, and a second sample image is acquired by illuminating the sample along a second, different axis. In other words, the first sample image is acquired by illuminating the sample at a first angle of incidence, and the second sample image is acquired by illuminating the sample at a second angle of incidence. The sample image is acquired by collecting / detecting photons emitted from the sample along the same optical axis of the corresponding illumination beam. In one example, the sample image is acquired in reflection mode, using the same objective lens to focus the illumination beam onto the sample and collect photons emitted from the sample in response to the illumination. The ROI of the sample is imaged in both the first and second images. The position of the ROI can be determined based on the first and second sample images. The sample can then be milled or micromachined based on the position of the ROI. By localizing the ROI based on sample images acquired from different incidence angles, the ROI can be localized with higher spatial resolution in any direction compared to the axial resolution of the microscope system. The sample is imaged and milled while held on the same sample stage in a vacuum chamber. Imaging and milling in a vacuum chamber allows the sample to be processed / milled without breaking the vacuum. Milling may be performed by a milling beam generated from a milling source. The milling beam may be a laser beam or a charged particle beam. The milling source may be a laser source, an electron source, or an ion source. By using the same sample stage for both imaging and milling of the sample, the ROI position and the milling position can be represented using the same coordinate system. In some examples, the coordinate system may be the sample stage coordinate system. Thus, conversion between different coordinate systems can be avoided.In some examples, an ROI may include one or more targets and criteria, and the target location may be expressed based on a coordinate system defined by the criteria, such as the position of the target relative to the criteria.

[0010] An ROI may be one or more regions within a sample. An ROI may include one or more targets to be examined or analyzed, as well as one or more internal and / or external references. For example, the references may be fluorescent beads embedded in the sample. The targets may be parts of a cell, such as mitochondria or cell membranes. In other examples, the references may be located outside the sample volume. For example, the sample is placed on a TEM grid. One or more fluorescent beads may be placed on or next to the surface of a cell on a supporting membrane. In another example, the references may be external stains of a cell. The sample image may be a fluorescence image including wide-field, reflective, or confocal fluorescence images. Fluorescence signals from the ROI may be generated due to endogenous or exogenous fluorescence. The location of the ROI may be presented by one or more 3D coordinates. Determining the location of the ROI involves mapping the ROI within the sample. In one example, the contour or boundary of the target may be depicted based on 3D coordinates. In another example, the geometric center of the target may be determined based on 3D coordinates.

[0011] Determining the location of an ROI based on first and second sample images may include determining the first coordinates of the ROI in the first sample image and the second coordinates of the ROI in the second sample image. The first and second coordinates are 3D coordinates (i.e., including X, Y, and Z components). The first and second coordinates may also be determined based on signal intensity. For example, the first and second coordinates may include the location of the ROI in the first and second sample planes, respectively. The first and second sample planes are orthogonal to the first and second axes, respectively. The locations of the first and second sample planes may be determined based on the intensity of the fluorescence signal. The first and second coordinates may be relative coordinates, such as coordinates with respect to a reference. Relative coordinates may be transformed into sample-stage coordinates through a correlation step. For example, the correlation step may perform the coordinate transformation using common features of the first image corresponding to the relative coordinates and the second image corresponding to the sample-stage coordinates.

[0012] Determining the ROI's position based on first and second sample images may include determining the ROI's position based on the orientation of the first and second axes relative to the sample stage. For example, the coordinates of the ROI in a plane perpendicular to the first axis are determined based on the first image, and the coordinates of the ROI in a plane perpendicular to the second axis are determined based on the second image. In this way, the ROI is located using the higher lateral resolution of the imaging system. The ROI is located with a spatial resolution higher than the axial resolution of the optical imaging system.

[0013] Determining the ROI location based on first and second sample images may involve merging the first and second sample images and determining the ROI location based on the merged sample image. The first and second sample images may be 2D images, and the merged image may be a 3D sample image. The first and second sample images may be merged based on their own coordinates (such as stage coordinates), endogenous / exogenous markers or sample features, or other types of images acquired together with the first and second sample images. Merging sample images is computationally intensive and may increase the overall sample preparation duration compared to determining the first and second coordinates of the ROI within the first and second images.

[0014] In one example, the sample is illuminated along first and second axes by tilting the sample stage by operating it. The objective lens of the optical microscope can maintain a fixed position relative to the sample chamber. Thus, the acquisition of first and second images is made possible by the sample stage. The first and second directions may be at the same angle with respect to the normal (Z direction) of the sample stage. Based on the first and second sample images, the position of the ROI in a third direction (such as the Z direction of the sample stage) can be determined with better resolution compared to the axial resolution of the beam alone.

[0015] In some examples, multiple first sample images and multiple second sample images are acquired at different imaging depths along the first and second axes, respectively. Based on the stack of sample images, the locations of multiple ROIs or large-scale ROIs can be determined. Furthermore, the ROI locations along the first and second axes can be determined more precisely, for example, based on signal intensity.

[0016] The ROI location determined from the sample image is represented using the coordinate system of the sample stage. The sample can then be milled using a light beam or a charged particle beam. Milling can be performed while the sample is held by the same sample stage for optical imaging. The sample stage is located in a sample chamber under vacuum. Optical imaging and charged particle imaging / milling can be performed without moving the sample outside the sample chamber. Milling can be performed according to a milling box positioned based on the determined ROI location. The sample can be milled from one or more directions to remove material adjacent to or around the ROI. The sample can be milled to form a thin section.

[0017] In some examples, one or more third sample images may be obtained by illuminating the sample from a third direction different from the first and second directions. The ROI position may be determined or updated based on the third image.

[0018] In some examples, after removing material from a sample by milling, an optical sample image can be obtained from the milled sample. By removing material, the ROI signal in the optical sample image may become stronger, and therefore the ROI location can be determined more accurately. The milling and optical imaging processes may be performed iteratively. In some examples, a sample image (such as a FIB image) can be obtained during milling. In some examples, the sample may be imaged using a charged particle beam, such as an electron beam, after the sample has been milled. In some examples, in addition to optical images, charged particle images, such as SEM / FIB images of the sample surface, can be acquired to further confirm, monitor, and determine the ROI location within the sample. For example, SEM / FIB images may be used in addition to a set of 2D or 3D optical images to monitor or determine the ROI location within the sample. For example, a unidirectional optical stack (e.g., an optical stack along the depth direction) can be combined with a FIB view to calculate the ROI and / or reference position. In some examples, the stack of sample images may be acquired at different imaging depths (or sample depths). Stacked images can be obtained by adjusting the depth of field and / or during the process of removing material from a sample by milling. Image stacks can include optical images such as SEM or FIB images, or charged particle images. Images in the stack can be aligned using one or more artificial marks (criteria such as beads or ion beam milled structures). Artificial marks can also be used to cross-correlated optical images and charged particle images (such as SEM / FIB images) to locate ROIs. In some examples, a 3D image of the sample can be reconstructed from the image stack.

[0019] Looking at Figure 1, which is a very schematic depiction of an embodiment of a charged particle microscope (CPM) system in which the present invention can be implemented. More specifically, one embodiment of a FIB-SEM is shown. System coordinates are shown as 110. The microscope 100 comprises an electron-optical column 1 that produces a beam 3 (in this case, an electron beam) of charged particles propagating along the electron optical axis 101. The electron optical axis 101 may be aligned with the Z axis of the system. The column 1 is mounted in a vacuum chamber (or sample chamber) 5 which comprises a sample stage 7 and associated actuator 8 for holding / positioning a sample 6. A micromanipulator 49 may be actuated by actuator 23 to manipulate a sample / test piece, such as a thin section extracted from the sample 6. The vacuum chamber 5 is evacuated using a vacuum pump (not shown). Also shown are vacuum ports 9 which may be opened for the introduction / extraction of supplies (parts, samples) into / out of the vacuum chamber 5. The microscope 100 may have multiple such ports 9 as needed.

[0020] Column 1 comprises an electron source 10 and an illuminator 2. The illuminator 2 comprises lenses 11 and 13 for focusing the electron beam 3 onto the sample 6, and a deflection unit 15 (for performing beam steering / scanning of the beam 3). The microscope 100 further comprises, among other things, a controller / computer processing unit 26 for controlling the deflection unit 15, lenses 11 and 13, a micromanipulator 49, and detectors 19 and 21, and for displaying information collected from the detectors 19 and 21 on a display unit 27.

[0021] In addition to the electron column 1 described above, the microscope 100 also includes an ion optical column 31. This includes an ion source 39 and an illuminator 32, which generate / direct an ion beam 33 along an ion optical axis 34. The ion axis 34 is inclined with respect to the electron axis 101 so as to provide easy access to the sample. As described above, such a focused ion beam (FIB) column 31 can perform processing / machining operations such as cutting, milling, etching, deposition, etc. on the sample 6 using, for example, the ion column 31. Further, an image of the sample 6 can be generated using the ion column 31. Note that the ion column 31 may be capable of arbitrarily generating various different ion species. Thus, a reference to the ion beam 33 is not necessarily to be regarded as specifying a particular species within the beam at any given time - in other words, the beam 33 may include ion species A for operation A (such as milling) and ion species B for operation B (such as implantation), where species A and B can be selected from various possible options. The ion source 39 may be a liquid metal ion source or a plasma ion source.

[0022] Similarly, shown is a gas injection system (GIS) 43, using which local injection of a precursor gas can be performed for the purpose of performing gas-assisted etching or deposition. Such a gas can be stored / buffered in a reservoir 41 and administered through a fine nozzle 42, resulting in its appearance, for example, near the intersection of the axes 101 and 34. 同様に、図示されているのは、気体圧入システム(Gas

[0023] The microscope 100 can include a laser source (not shown) for directing a laser beam at the sample. A portion of the sample can be milled or removed using the laser beam. The laser beam can also be used as a light source for exciting a fluorescence signal that is captured for an image from the view.

[0024] The sample may be milled by one or more of the FIB and the laser beam according to a milling box virtually placed by a user or a controller. The milling box defines a 3D area to be milled with respect to the sample. While placing the milling box, the incident angle of the FIB or the laser beam can also be determined. The 3D area can be defined using a sample stage coordinate system that can correlate the beam (FIB and / or laser beam).

[0025] An optical microscope 50 is disposed in the vacuum chamber 5. The microscope may be a fluorescence microscope, a reflection microscope, a transmitted light microscope, or a confocal microscope. The microscope is configured to image a sample 6 held by a sample stage 7. The sample position can be adjusted by operating the sample stage to achieve different incident angles of the light beam and image different areas of the sample. The imaging depth may be adjusted by adjusting one or more optical elements of the microscope. In one example, all optical elements of the microscope are disposed in the vacuum chamber. In another example, one or more optical elements or components 51 of the microscope are located outside the vacuum chamber. The optical microscope can be controlled by a controller 26 to acquire an image of the sample 6. The optical microscope can include a camera (a pixelated detector in the case of a wide-field scenario or a scanning source with an amplified detector in the case of a point-scanning method) capable of detecting an optical signal from the sample. An excitation source for optical imaging may be incorporated into the optical microscope or may be a separate component from the optical microscope.

[0026] The detectors 19 and 21 are selected from various potential detector types that can be used to examine different types of "induced" radiation emitted from the sample 6 in response to irradiation by the (acting) beam 3 and / or the beam 33. The detector 19 is, for example, a silicon drift detector (SDD) or silicon lithium (Silicon The detectors may be X-ray detectors such as lithium, Si(Li) detectors. Detector 21 may be an electron detector in the form of, for example, a solid-state photomultiplier tube (SSPM) or a vacuum photomultiplier tube (PMT). This may be used to detect backscattered and / or secondary electrons emitted from the sample. Those skilled in the art will understand that many different types of detectors can be selected in the illustrated setup, including, for example, annular / segmented detectors. By scanning beam 3 or beam 33 over sample 6, stimulated emission, including, for example, X-rays, infrared / visible / ultraviolet rays, secondary ions, secondary electrons (SE) and / or backscattered electrons (BSE), is emitted from the sample. Since such stimulated emission is position-sensitive (due to the scanning motion), the information obtained from detectors 19 and 21 will also be position-dependent.

[0027] Signals from detectors 19, 21 and the optical microscope 50 pass along control lines (buses) 25, are processed by the controller 26, and are displayed on the display unit 27. Such processing may include operations such as coupling, integration, subtraction, false coloring, edge enhancement, and other processes known to those skilled in the art. Furthermore, an automated recognition process may be included in such processing. The controller includes non-temporary memory 29 for storing computer-readable instructions and a processor 28. The methods disclosed herein may be carried out by executing computer-readable instructions within the processor. For example, the controller may control the microscope to image and mill a sample, collect data, and process the collected data. The controller may display an image on the display. The controller may adjust the ion beam energy by adjusting one or more lenses and / or ion sources. The controller may adjust the sample position via actuators coupled to the sample stage.

[0028] Figure 2 is a photograph of the inside of the vacuum chamber of the charged particle microscope system. A sample placed on the sample stage 204 can be imaged or processed by one or more of the following: a FIB formed by the ion optical column 202 or an electron beam formed by the electron optical column 203. Fluorescence images can be acquired by an optical microscope including an objective lens 201. Since the sample is imaged and processed in the same sample chamber while still on the sample stage, the same coordinate system can be used for imaging and processing.

[0029] In some embodiments, the optical microscope may be configured to irradiate the sample from an incident angle different from the incident angle of the charged particle beam. In one example, the optical microscope is positioned on the same side of the charged particle column relative to the sample stage. Figures 1 and 2 show the optical microscope positioned above the sample stage. In other words, the light beam and the charged particle beam are directed toward the sample from the same side (e.g., the top side) of the sample. In another example, the optical microscope may be positioned below the sample stage, and the charged particle column is positioned above the sample stage. Thus, the light beam irradiates the sample from below the sample stage.

[0030] Figure 3 shows a method 300 for preparing a sample by milling the sample and exposing the ROI. Method 300 can be performed using the microscope 100 shown in Figure 1.

[0031] In 302, the sample is loaded into the sample chamber. The sample may be loaded into the vacuum sample chamber via a load lock. Loading the sample includes positioning the sample on the sample stage within the sample chamber. The sample may be positioned based on images from a navigation camera.

[0032] In 304, the sample position is adjusted so that the light beam is directed towards the sample along a first optical axis. In response to the illumination of the light beam in 304, one or more first sample images are acquired in 306. The sample position may be adjusted by operating an actuator coupled to the sample stage. The sample may be first translated to an imaging position below the optical microscope and then tilted toward the objective lens of the optical microscope, allowing the light beam to image the ROI along the first axis. In one example, the portion of the sample to be imaged may be determined based on known knowledge of the potential location of the ROI. In another example, a series of optical or charged particle images may be acquired to determine the potential location of the ROI before acquiring the first and second sample images. The first sample image to be acquired may be a single image or multiple images acquired at various imaging depths.

[0033] In 308, the sample position is adjusted so that the light beam is directed toward the sample along the second axis. In response to the illumination of the light beam in 308, one or more second sample images are acquired in 310. The ROI is imaged in both the first and second images. The sample stage may be adjusted so that the Z axis of the sample stage is at the same angle from the first and second axes.

[0034] In 312, the position of at least one ROI, imaged in both the first and second images, is determined. In some examples, the ROI may include a sample target and / or reference. In one example, the first coordinates (x1, y1, z1) of the ROI are determined in the first image, and the second coordinates (x2, y2, z2) of the same ROI are determined in the second image. A third, more precise coordinate (x3, y3, z3) of the ROI may be determined based on the first and second coordinates. Relative measurements between the ROI and the reference may also be required to maintain scale and relative movement.

[0035] Therefore, the position of the ROI is determined based on the orientation of the first and second axes. In one example, the position of the ROI in a first plane perpendicular to the first axis is determined based on a first image, and the position of the ROI in a second plane perpendicular to the second axis is determined based on a second image.

[0036] Figures 4A and 4B show examples of light beam directions relative to sample 406 for acquiring the first and second images. The first image is acquired when the light beam 402 from the objective lens 401 is directed onto sample 406 along the Z-axis of the sample stage, as shown in Figure 4A. Next, the sample is rotated 90 degrees and the light beam 402 is directed onto the sample along the X-axis of the sample stage to acquire the second image. Thus, the ROI 405 can be mapped with a higher lateral resolution 403 compared to the lower axial resolution 404.

[0037] Figure 5 shows another example of the direction of the light beam relative to the sample stage. The sample stage 508 is adjusted so that the light beam 509 is directed from the objective lens 501 towards the sample 506 along the first axis 502, and a first image is acquired. Then, the sample stage 508 is adjusted (e.g., tilted) so that the light beam 510 is directed towards the sample along the second axis 503, and a second image is acquired. The first angle 511 between the first axis 502 and the normal (Z-axis) 504 of the sample stage is the same as the second angle 512 between the second axis 503 and the normal 504 of the sample stage. Compared to imaging along the Z-axis only, the ROI localization accuracy along the Z-axis in the overlapping region of beams 509 and 510 can be higher independently of the axial resolution of beams 509 and 510. Therefore, combining the two views allows for more accurate localization of the ROI.

[0038] Figure 6 further illustrates in 2D that the accuracy of ROI localization is improved by reducing uncertainty by acquiring images from two different directions. The sample is imaged from two directions 601 and 602. Each of the first and second directions has the same tilt angle α from the Z axis. The X and Z axes are coordinates relative to the sample stage. Region 603 represents the uncertainty of ROI placement when the sample is imaged along the Z axis. The size and shape of region 603 are determined by the resolution of the imaging system. By imaging along directions 601 and 602, the uncertainty of placement along view 1 604 and view 2 605 is ±δ. By imaging from directions 601 and 602, the uncertainty of placement along the X and Z axes is as follows:

[0039]

number

[0040] In another example, the first and second images are combined or merged into a 3D image stack, and the ROI location is determined in the merged 3D image.

[0041] In some examples, multiple first and second images are acquired from different imaging depths. The location of the ROI along the imaging axis (or beam axis) can be determined based on signal intensity. For example, the ROI may be captured in multiple sample images acquired along different imaging depths along the first axis. The ROI location along the first axis can be determined by interpolating the signal intensity of the ROI in the multiple sample images.

[0042] In some examples, the ROI includes at least a target and at least a reference. Both the target and the reference are imaged in the first and second images. The coordinates of the target in the sample may be determined based on its relative position from the reference, which is evaluated from the first and second images. In some examples, images of one or more charged particles of the sample can be obtained, and the reference is also imaged within the charged particle images. The coordinates of the target may be further determined based on the charged particle images. For example, a stack of the first and second sample images may be correlated with the charged particle images based on the reference to determine the target position.

[0043] In 314, a milling box is positioned based on the ROI location determined in 312, and then the sample is milled based on the milling box. The sample is milled to expose the ROI. Because the ROI can be accurately mapped by optical imaging, it can be exposed by milling the ROI with high precision.

[0044] In 316, the milled sample is imaged. Images may be acquired during or after milling. In one example, the sample is milled with a FIB, and FIB images can be acquired during the milling process. In another example, the exposed sample surface may be imaged with an optical beam and / or a charged particle beam. For example, the milled sample may be imaged with an optical microscope and / or an electron beam. Images of the milled sample may be displayed to the user. In some examples, the optical image stack and the charged particle image may be aligned or correlated based on references acquired within the optical image and the charged particle image.

[0045] In step 318, based on the image of the milled sample obtained in step 316, method 300 determines whether the ROI has been successfully exposed. If the ROI has been successfully exposed and no further milling is required, method 300 terminates and the sample preparation process is complete. If, otherwise, further milling is required, method 300 proceeds to step 320.

[0046] In step 320, method 300 determines whether the ROI location needs to be further mapped. In one example, the ROI can be remapped with greater accuracy because the signal intensity of the ROI increases after material adjacent to the ROI is removed by milling. In another example, the optical image may be retaken to determine the location of a different region of the ROI or a different ROI within the sample. If the ROI location needs to be determined or further mapped, method 300 proceeds to 304 to perform optical imaging from a different direction. If the ROI location does not need to be remapped, method 300 proceeds to 314 to further mill the sample based on the existing milling box or to position another milling box based on the image of the milled sample acquired in 316.

[0047] The technical benefit of irradiating a sample from different directions is that the ROI is mapped in 3D with a resolution higher than the axial resolution of the optical microscope system. The technical benefit of acquiring a sample image and milling the sample while the sample is positioned on the same sample stage is that the same coordinate system can be used for imaging and milling. Furthermore, the imaging and milling processes can be performed iteratively within the same system for better ROI mapping and monitoring of the milling process.

Claims

1. A method for preparing a sample, A light beam is shone onto the sample held by the sample stage along a first axis, and photons emitted from the sample along the first axis are detected to obtain at least a first sample image. The method involves irradiating the sample with the light beam along the second axis and detecting the photons emitted from the sample along the second axis to obtain at least a second sample image, Based on the first and second sample images, the location of the region of interest (ROI) within the sample is determined, Based on the position of the ROI, the sample held by the sample stage is milled, Methods that include...

2. Determining the position of the ROI based on the first sample image and the second sample image is: Determining the first coordinates of the ROI in the first sample image, Determining the second coordinates of the ROI in the second sample image, The method according to claim 1, comprising determining the position of the ROI based on the first coordinate and the second coordinate.

3. The method according to claim 2, wherein the first coordinate and the second coordinate are coordinates corresponding to the sample stage.

4. The method according to claim 1, wherein the first sample image and the second sample image are fluorescence images.

5. The method according to claim 4, further comprising determining the position of the ROI based on the intensity of the fluorescence image.

6. The method according to claim 1, further comprising: irradiating the sample with the light beam along a third axis; obtaining at least a third sample image by detecting photons emitted from the sample along the third axis; and determining the position of the ROI based on the third sample image.

7. The method according to claim 1, wherein determining the position of the ROI based on the first sample image and the second sample image includes determining the position of the ROI based on the orientation of the first axis and the second axis with respect to the sample stage.

8. The method according to claim 1, further comprising obtaining an image of charged particles of at least the sample, and determining the position of the ROI based on the image of the charged particles.

9. The method according to claim 8, wherein determining the position of the ROI in the sample based on the first sample image and the second sample image includes determining the relative position of the ROI with respect to a reference based on one or more of the first sample image, the second sample image, and the charged particle image, and determining the position of the ROI based on the relative position.

10. The method according to claim 1, wherein milling the sample held by the sample stage based on the position of the ROI includes determining a milling angle based on the position of the ROI.

11. The method according to claim 1, wherein the sample is milled with a first charged particle beam or laser beam.

12. The method according to claim 1, further comprising tilting the sample by operating the sample stage between irradiating the sample along the first axis and irradiating the sample along the second axis.

13. The method according to claim 12, wherein the first angle between the first axis and the normal to the sample stage and the second angle between the second axis and the normal to the sample stage are the same.

14. The method according to claim 1, wherein determining the position of the ROI within the sample based on the first sample image and the second sample image comprises merging the first sample image and the second sample image and determining the position of the ROI within the merged image.

15. The method according to claim 1, wherein the sample is a frozen biological sample, and the milled sample is a thin section analyzed by a transmission electron microscope.

16. The method according to claim 1, wherein acquiring at least the first sample image includes acquiring a plurality of sample images at different imaging depths using the light beam irradiated along the first axis, acquiring at least the second sample image includes acquiring a plurality of sample images at different imaging depths using the light beam irradiated along the second axis, and determining the position of the ROI based on the first and second sample images includes determining the position of the ROI based on the plurality of sample images acquired using the light beam irradiated along the first axis and the plurality of sample images acquired using the light beam irradiated along the second axis.

17. A microscope system, The sample room and A sample stage is placed inside the sample chamber for placing the sample, A light source for generating a light beam, The objective lens, A detector for detecting photons emitted from the sample passing through the objective lens, A milling source for generating a milling beam, The microscope system comprises a controller, the controller including a processor and non-temporary memory for storing computer-readable instructions, and the microscope system executes the computer-readable instructions in the processor. The light beam is irradiated onto the sample held by the sample stage along a first axis via the objective lens, and at least a first sample image is acquired via the detector. The light beam is irradiated onto the sample along the second axis through the objective lens, and at least a second sample image is acquired via the detector. Based on the first and second sample images, the location of the region of interest (ROI) within the sample is determined. A microscope system configured to mill the sample held by the sample stage using the milling beam based on the position of the ROI.

18. The microscope system according to claim 17, wherein the milling source is a first charged particle source, the microscope system further comprises a second particle source for generating a second charged particle beam, and the microscope system is further configured to mill the sample with the first charged particle beam and then acquire an image of the ROI with the second charged particle beam.

19. The microscope system according to claim 17, further configured to acquire a third sample image by irradiating the milled sample with the light beam, update the position of the ROI based on the third sample image, and mill the sample using the milling beam based on the updated position of the ROI using a charged particle beam.

20. The microscope system according to claim 17, further comprising an actuator for operating the sample stage, wherein the microscope system is further configured to tilt the sample so that it is irradiated along a second axis after irradiating the sample along a first axis.

Images