Device and method for determining characteristics of a sample used in a charged particle microscope

Abstract

To determine whether a cold frozen and vitrified sample is suitable for observation with a charged particle microscope.SOLUTION: The invention relates to a device and a method for determining property of a sample 3 that is to be used in a charged particle microscope. The sample 3 comprises a specimen embedded within a matrix layer. The device comprises a light source 11 arranged for directing a beam of light towards the sample 3, and a detector 12 arranged for detecting light emitted from the sample 3 in response to the beam of light being incident on the sample 3. Finally, the device comprises a controller 13 that is connected to the detector 12 and arranged for determining property of the matrix layer based on signals received by the detector 12.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] Summary The present invention relates to a device and a method for determining the characteristics of a sample used in a charged particle microscope, the sample including a specimen embedded in a matrix layer such as an aqueous layer or an ice layer.

Background Art

[0002] Biology is a natural science that studies life and living organisms, including physical structures, chemical processes, molecular interactions, physiological mechanisms, expression, and evolution.

[0003] Cell biology is a field of biology that studies the structure and function of cells, that is, the basic units of life. Cell biology focuses on physiological characteristics, metabolic processes, signal transduction pathways, life cycles, chemical compositions, and the interactions between cells and their environments. In cell biology, molecular recognition between macromolecules governs all of the most sophisticated processes in cells. The most common macromolecules include biopolymers (nucleic acids, proteins, carbohydrates, and lipids), as well as large non-polymeric molecules (such as lipids and macrocyclic molecules).

[0004] Many researchers are interested in studying macromolecular complexes at high resolution in their natural environments in order to elucidate the structural mechanics and interactions of the macromolecular complexes. For this purpose, charged particle microscopes can be used. Charged particle microscopy, particularly in the form of electron microscopy (EM), is a well-known and increasingly important technique for imaging microscopic objects. Historically, the basic types of electron microscopes have evolved into numerous well-known devices such as transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning transmission electron microscopes (STEMs), and also, for example, so-called "dual beam" tools (such as FIB-SEMs) that additionally use, for example, a "machined" focused ion beam (FIB) to enable ion beam milling or ion beam induced deposition (IBID).

[0005] EM provides many methods for studying biological specimens. Conventional TEM is used to study the overall morphology of biological specimens, electron crystallography and single-particle analysis are utilized to study proteins and macromolecular complexes, and (cryo) electron tomography of vitrified sections (CEMOVIS) and cryo-EM aim at organelles and molecular structures. In cryo-EM and CEMOVIS, specimens are preserved by rapid freezing using vitrification techniques and observed by cryo-TEM. CEMOVIS further includes the step of sectioning the specimen at low temperature, which can be performed using cryo-FIB techniques.

[0006] To study a specimen, it is necessary to prepare a sample containing the specimen. This sample can include a carrier and a specimen embedded within a matrix layer, which matrix layer may be a liquid or solid such as water or ice and is provided on the carrier.

[0007] The preparation of samples for study by charged particle microscopes is often time-consuming and laborious. One of the drawbacks associated with these studies is that only after the sample has been prepared and examined by a charged particle microscope does the user know whether the sample preparation has been successful. This is especially true when preparing biological samples such as cryo-EM samples.

[0008] Preparing a cryo-EM sample includes, for example, the steps of taking an aqueous sample of biological material (i.e., the "specimen", usually a purified protein complex), applying it to a support structure (grid), reducing its dimensions to a layer as thin as possible (about 100 - 800 Å depending on the size of the biomolecule) by, for example, filter paper, and then freezing this layer at a speed sufficient to prevent the aqueous liquid from crystallizing.

[0009] Devices for preparing cryo-EM samples are known from WO02 / 077612A1 under the applicant's name. This device is commercially available under the trade name "Vitrobot". The device described in WO02 / 077612A1 comprises an environmental chamber, a holder for a sample or a carrier, at least one suction element which is a medium for absorbing a liquid or to which it can be attached, both being arranged within the environmental chamber, a suction element, and a cooling medium for cooling the sample. This suction element can move towards the sample or the carrier in a controlled manner.

[0010] Many aspects of this process for preparing biological samples are challenging. In this context, the step of reducing the layer dimensions by removing excess liquid - which can be done, for example, by a technique called "wicking" - is thought to be crucial for the quality and reproducibility of the liquid film.

[0011] Since the wicking step is difficult to establish in a reproducible way, US2017 / 350798A1 proposes methods and devices by which the need for excess fluid wicking can be minimized or eliminated by reducing the sample volume from the order of microliters to the order of picoliters.

[0012] US2010 / 181495A1 describes a method and a device for preparing a sample for a cryo-electron microscope. Here, a carrier is fixed to a holder, a liquid containing a specimen is applied to the carrier, and a suction device is used to remove excess liquid from the carrier. This suction device comprises filter paper in contact with the carrier. On opposite sides of the filter paper, a light source and a light sensor device are provided. The filter paper is illuminated with light, and a change in the optical properties of the filter paper due to the absorption of excess liquid by the filter paper is detected by the light sensor device. A control unit moves the blotting paper away from the carrier in response to that detected change in the optical properties.

[0013] Even when the blotting step presents challenges, many other aspects of the above process of preparing a biological sample are equally difficult and unpredictable. The purified complex may face microscopic surfaces, materials, and dynamics that alter the purified complex, or the sample may become contaminated during the process. Regarding vitrification techniques, it has also been observed that controlling the resulting ice thickness and the art of that ice is equally important.

[0014] When preparing cryo-EM samples using current preparation techniques, a very wide variety of qualities are still produced, and thus important areas of the EM grid cannot be used. In many cases, these variations can only be recognized after the sample on the grid has been tested by cryo-EM once. Therefore, valuable resources such as the scientist's preparation time and the use time of expensive EM may be wasted. This also applies to the preparation of other samples and is not limited to biological samples.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Summary of the Invention

[0016] From this perspective, an object of the present invention is to provide a device and method for determining the characteristics of a sample used in a charged particle microscope, and by using these, it is possible to prevent waste of valuable resources as described above.

[0017] For this purpose, the present invention provides a device for determining the characteristics of a sample used in a charged particle microscope, the sample including a specimen embedded in a matrix layer, and the device is defined according to claim 1. As defined herein, the device - A light source arranged to direct a light beam towards the sample, - A detector arranged to detect light emitted from the sample in response to the light beam incident on the sample, - A controller connected to the detector and arranged to determine the characteristics of the matrix layer based on the signal received by the detector.

[0018] Using the light source, directing it directly towards the sample and directly detecting the light emitted from the sample, it is possible to determine the characteristics of the matrix layer in a direct way. It should be noted that the matrix layer may be a liquid such as a liquid used to prepare the sample. The matrix layer may also be a solid such as a vitrified liquid used to prepare the sample, or any other (semi)transparent solid such as, for example, a resin. The light source can be selected in such a way that it is affected by the matrix layer. The matrix layer can, for example, reflect, diffract, transmit, and / or absorb light from the light source. By using a detector for detecting the reflected, diffracted, transmitted, and / or absorbed light, it is possible to distinguish between samples based on different characteristics of the matrix layer. Distinguishing or establishing those characteristics can be qualitative and / or quantitative.

[0019] In this device, it should be noted that the light source is directed directly at the sample, and the detector is used to measure the light directly reflected by the sample or transmitted through the sample. Therefore, this device provides a method for determining the characteristics of the matrix layer in a direct way. This enables the device to be used at various sample stages. For example, it is possible to use the device during the preparation of the sample, such as during the aforementioned absorption step where the matrix layer is an aqueous layer. In another embodiment, the device can be used after the sample has been vitrified, and the matrix layer is an ice layer. The matrix layer can in principle be any type of matrix layer that affects the emitted light by reflection, diffraction, transmission, and / or absorption. In a preferred embodiment, the matrix layer is an aqueous layer or an ice (vitreous) layer.

[0020] The light source can be directed at the surface of the sample, and the light source is arranged to emit light in a first direction. The detector can be arranged to receive the reflected light and / or transmitted light from the sample, and the normal of the detector is directed in a second direction different from the first direction.

[0021] In one embodiment, the first direction is not perpendicular to the normal of the sample.

[0022] In one embodiment, the second direction is not perpendicular to the normal of the sample.

[0023] Here, the preferred embodiments of the present invention, and their advantages, are described below.

[0024] In one embodiment, the device is arranged to collect spatially resolved data. Using this, it is possible to locally determine the characteristics of the matrix layer. For example, a 2D (x,y) coordinate system can be mapped to the sample surface, and the determined characteristics of the matrix layer can be assigned to one or more coordinates within that 2D coordinate system. In one embodiment, this can be used to establish a map of the sample surface with the corresponding characteristics of the matrix layer. This map provides the user with valuable insights into which areas can be used to further examine the sample and which areas can be most preferably avoided. Note that currently, in the case of cryo-EM samples, cryo-TEM is used in low magnification overview mode to evaluate ice quality. This activity shuts down the TEM for a significant amount of time. By using the device disclosed herein, the overview / map / lattice lines of the grid can be acquired in a rapid and efficient manner, thereby enabling the identification of areas that are potentially good for data collection.

[0025] In one embodiment, the controller is arranged to evaluate the sample based on the determined characteristics. This means that the controller can identify whether each part of the sample has been approved or rejected based on the determined characteristics of the matrix layer. For example, if it is determined that a (portion of) the matrix layer contains contamination or that the thickness of the matrix layer is not the desired thickness, the controller can indicate that the sample is not suitable for research. The controller can assign the evaluation to each 2D coordinate of the sample or assign an evaluation for the entire sample.

[0026] In one embodiment, the controller is arranged to determine at least one of a criterion for the thickness of the matrix layer, a criterion for the contamination of the matrix layer, and a criterion for the skill of the matrix layer. Accordingly, the characteristic can be a criterion for thickness and / or contamination. When the matrix layer is vitrified (frozen), the characteristic can be the skill (or state) of the vitrified matrix layer.

[0027] In one embodiment, the device comprises at least one optical element such as a lens. This optical element can be positioned in the optical path between the light source and the detector. This optical element can be arranged to focus and / or condition the light beam. In one embodiment, the optical element is arranged to focus the light beam onto the sample. In one embodiment, the optical element is arranged to focus the light beam onto the detector. A plurality of optical elements are provided and each is expected to have one or more characteristics as described above.

[0028] The at least one optical element can be a color selectivity filter. This color selectivity filter can be positioned in the optical path between the light source and the detector. This color selectivity filter is arranged to pass a limited band of the wavelength of light towards the detector. Note that the color selectivity filter can comprise a plurality of color selectivity filter elements, each arranged to pass a different limited band of the wavelength of light towards the detector. The plurality of color selectivity filter elements can be spatially arranged on the detector or can be an integral part of the detector. As an example, the detector can be selectively sensitive to different wavelength bands by design, such as when an RGB pixel camera is used where different pixels are sensitive to different colors. Additionally or alternatively, the plurality of color selectivity filter elements can be positioned sequentially, i.e., one by one in order, in the optical path.

[0029] In one embodiment, the detector is arranged to receive light transmitted through the sample. In one embodiment, the detector is arranged to receive light reflected from the sample. Note that this detector can comprise a plurality of detector units, and one of those detector units is arranged to receive light transmitted through the sample, and another one of those detector units is arranged to receive light reflected from the sample. Thereby, additional sample information is provided.

[0030] In one embodiment, the device comprises a scanning unit for applying a relative scanning motion of the light beam onto the sample. By using the relative scanning motion, it is possible to scan the sample at a relatively high resolution, for example, to establish a 2D characteristic map of the aforementioned sample.

[0031] This relative scanning motion is assumed to be established, for example, by using a sample stage to move the sample past the light source and / or to move the sample past the detector.

[0032] Determining the characteristics of the matrix layer using the light source and the detector can be achieved in a number of ways. Various embodiments will be described below. Note that these embodiments are not intended to be limiting, and combinations of these embodiments are also further envisioned.

[0033] In one embodiment, the light source includes a laser light source. The laser light source is relatively inexpensive. This laser light source is directed towards the surface of the sample, particularly the surface of the matrix layer. This laser light source can be arranged to project a line onto the sample. This line can be scanned across the sample by using the relative movement between the laser line and the sample. By preferably using the line light source in combination with a line sensor, it is possible to illuminate a relatively large portion of the sample at once while still retaining the spatial information of the sample. By using line scanning, the entire surface of the sample can be sorted in a quick and easy manner. This helps to establish spatially resolved data regarding the characteristics of the matrix layer. An additional advantage is that this equipment does not require the use of a relatively expensive 2D sensor.

[0034] Particularly in combination with the laser light source, it is assumed that the detector comprises a CMOS sensor. This CMOS sensor can be a CMOS line sensor, which can be used together with the laser line light source described above. Using the aforementioned optical elements, the transmitted light from the sample can be focused onto the CMOS detector. In one embodiment, the CMOS line sensor comprises a CMOS 16k line sensor and operates at a frame rate adapted to the relative movement between the sample and the light source and / or the detector. This frame rate can be higher than 250 fps, preferably higher than 500 fps, and more preferably higher than 1000 fps. Using this setting, it is possible to obtain a complete matrix layer characteristic map of a 3 mm grid within 15 seconds.

[0035] In one embodiment, the light source comprises an LED. The LED is relatively inexpensive. This light source can be arranged to direct a multi - color light beam towards the sample. This can be achieved when the device comprises multi - color LEDs. In an alternative embodiment, the device comprises an LED arranged to generate white LED illumination in combination with one or more filter elements. In one embodiment, one or more filter elements can be provided. In particular, a total of three color filter elements can be provided. These colors can be the primary colors red, green, and blue, so that the sample can be illuminated using at least three different colors.

[0036] When the sample is illuminated using at least three different colors, the thickness of the matrix layer can be obtained using the theory of multiple - beam interference from parallel films. This film interference occurs because light rays reflected from different layers have interference due to a phase difference. A light beam hitting the thin film at an angle θ with respect to the normal of the thin film has an optical path difference (OPD, or phase shift), and the optical path difference is equal to OPD = 2ndcos(θ). Currently, there are two very distinguishable cases, namely, constructive interference and destructive interference. Destructive interference occurs when the phase difference is 180°, and constructive interference occurs when the phase difference is 0° or 360°. Since the 180° phase difference depends on the wavelength λ, the destructive interference as a function of OPD is defined as 2ndcos(θ)=mλ i , where m is an integer. By introducing at least three different colors (different wavelengths), a system of simultaneous equations can be obtained and used to determine the thickness of the thin film. Similarly, the thickness of a multi - layer system (i.e., a grid including a matrix layer on top) can be determined. This method has been found to be particularly useful for determining the thickness of a vitrified layer. Furthermore, using three colors is advantageous for determining whether contamination has occurred within the sample.

[0037] In one embodiment, the detector comprises a color camera. This color camera can be arranged to receive reflected light, especially in combination with LEDs. This combination is relatively inexpensive.

[0038] In one embodiment, the light source can be arranged to generate a polarized analyzer beam. In this embodiment, the device is arranged to measure the properties of the matrix layer based on the polarization analysis method. Using this, the change in polarization during reflection or transmission of the light source on the sample can be measured and compared with a model. From the perspective of a (vitrified) biological sample, this is particularly useful for measuring the thickness and / or crystallinity of the matrix layer. In this embodiment, a polarization element provided between the light source and the sample can be used. Further, a second polarization element can be provided between the sample and the detector. Optionally, one or more compensator elements can be provided between the light source and the sample and between the sample and the detector. Appropriate polarization analysis settings are known to those skilled in the art.

[0039] It is envisaged that the light source comprises a three-color LED. In combination with the above polarized analyzer beam, it is possible to measure the thickness of the matrix layer (especially the thickness of the ice layer), as well as scattering and / or transmission (which are criteria for contamination and / or crystallinity). The polarization analysis equipment can be based on reflection, and as a result, the light source and the detector are arranged on the same side of the specimen. A combined scanning movement is also further envisaged. The spatial resolution can be obtained by deconvolution.

[0040] In one embodiment, the light source is arranged to generate light within the UV spectrum. Additionally or alternatively, the light source can be arranged to generate light within the IR spectrum. This embodiment is particularly advantageous for measuring ice artifacts. In the IR and UV, amorphous ice (which is typically considered the desirable type of ice for biological samples in cryo-EM) has different optical properties compared to crystalline ice (which is typically considered the undesirable type of ice). In particular, the IR absorption coefficient and refractive index differ between the two types of ice. Additionally, the UV absorption is different for amorphous ice compared to cubic ice. Thus, the use of UV and / or IR light can be advantageously used to determine the properties of the matrix layer and, in this embodiment, the quality of the matrix layer after vitrification.

[0041] According to one aspect, a sample preparation tool is provided that comprises the device defined herein. The sample preparation tool can be a cryo-EM sample preparation tool as described in WO02 / 077612A1, which is hereby incorporated by reference herein. The device is arranged near the absorption element to monitor the absorption step during sample preparation. The device can additionally or alternatively be arranged near the cooling medium to monitor the cooling (vitrification) of the sample.

[0042] According to one aspect, there is provided a charged particle microscope comprising a device as defined herein. This device can be provided near the sample holder of the charged particle microscope. In particular, the sample holder can be movable between a loading position and an inspection position. This device can be arranged such that the sample passes through the device during movement between the loading position and the inspection position, enabling inspection of the characteristics of the sample. For example, this enables the sample to be loaded and inspected by this device in a way that a 2D map of the sample having corresponding characteristics of the matrix layer can be established. The results can be shown to the user or stored to assist in further processing of the sample.

[0043] In one embodiment, the sample includes one or more pattern elements arranged to be detectable by a device as defined herein. Further, the one or more pattern elements can be arranged to be detectable by the charged particle microscope as well. When using the one or more pattern elements, it is possible to accurately register the position of the sample, and alignment in the charged particle microscope is similarly possible without having to return to a low magnification.

[0044] According to one aspect, there is provided a method for determining the characteristics of a sample used in a charged particle microscope as defined in claim 12. As defined herein, this method comprises - providing a sample comprising a specimen embedded in a matrix layer; - directing a light beam towards the sample; - detecting light emitted from the sample in response to the light beam incident on the sample; - using a controller to determine the characteristics of the matrix layer based on the detected light.

[0045] The advantages of this method, and further embodiments of this method, have already been made clear above. In summary, by directly illuminating a sample with a light beam and using a detector for detecting the light emitted from the sample, it is possible to determine characteristics of the matrix layer, such as the thickness or contamination of the matrix layer. The determination of the characteristics can be performed qualitatively or quantitatively, and the evaluation of the sample can additionally be performed.

[0046] The determined characteristics can be used as criteria for the thickness of the matrix layer and / or for the contamination of the matrix layer.

[0047] The methods and devices disclosed herein can be used advantageously during or after one or more of the following steps: - a preparation step of preparing a sample; - an application step of applying a matrix layer to the sample; - a removal step of removing an excessive amount of the matrix layer from the sample; - a vitrification step of vitrifying the sample having the matrix layer; - an imaging step of imaging the sample.

Brief Description of the Drawings

[0048] The present invention will now be described in more detail based on exemplary embodiments and the accompanying schematic diagrams.

Figure 1

Figure 2

Figure 3a

Figure 3b

Figure 4

Figure 5

Figure 6

Figure 7

[0049] Figures 1-4 show different stages of a method for preparing a biological sample 3 for use in a charged particle microscope, and a device 1 for determining the characteristics of the prepared sample 3 is used. Generally, the method for preparing a biological sample 3 comprises - providing a sample carrier 9 and providing a specimen 5 of interest embedded in a liquid matrix layer 7 on the sample carrier 9 for preparing the sample 3 (Figure 1); - a blotting step (Figure 2) using a blotting material 21 such as blotting paper to remove excess liquid from the matrix layer 7; - a vitrification step for vitrifying the liquid matrix layer containing the specimen 5 of interest, wherein a cryo-bath (Figure 3a) or a cryo-jet (Figure 3b) is used; - a storage step (Figure 4) for storing the vitrified sample 3 once vitrification has occurred.

[0050] These general steps for preparing cryo-EM samples are known to those skilled in the art and will not be described in further detail. The devices as disclosed herein can be advantageously used in sample preparation, particularly cryo-EM sample preparation as described above. The devices as disclosed herein can be advantageously used in each of the aforementioned sample preparation steps. It should be noted that in these Figures 1-4, the devices are shown schematically. For these reasons, some embodiments of the device will first be described with reference to Figures 5-7, and thereafter the use of the devices of Figures 1-4 will be described in more detail.

[0051] Here, turning to FIG. 5, a first embodiment of a device 1 for determining the characteristics of a sample 3 used in a charged particle microscope is shown. In each figure, similar or corresponding features are denoted using the same reference numerals. The illustrated device includes a light source 11 arranged to direct a light beam towards the sample 3, a detector 12 arranged to detect light emitted from the sample in response to the light beam incident on the sample 3, and a controller 13 connected to the detector 12 and arranged to determine the characteristics of the matrix layer 7 based on the signal received by the detector 12. In the illustrated embodiment, the light emitted by the light source 11 is directed towards the sample 3, and the detector 12 is arranged on the opposite side of the sample 3. In other words, the device 1 is arranged such that the sample can be placed between the light source 11 and the detector 12. The light hitting the sample 3 is, for example, scattered and / or absorbed, and the detector 12 detects a specific amount of light depending on the amount of scattering and / or absorption. Thus, the detector 12 is arranged to receive the light transmitted through the sample 3. Thereby, it is possible to determine characteristics of the matrix layer of the sample 3, such as, for example, the presence of the matrix layer and / or the thickness of the matrix layer.

[0052] In the embodiment shown in FIG. 5, the light source 11 can be a laser or an LED. The detector is arranged to detect the light emitted from the light source 11 and is thus arranged to receive laser light and / or LED light accordingly. The detector can be a CMOS sensor (especially in combination with a laser) or a color camera (especially in combination with an LED), as previously explained. Other light sources are further envisioned.

[0053] In the embodiment shown in FIG. 5, the light source 11 is directed towards the sample 3. The light beam can be directed to a single, relatively small spot on the sample 3. The light beam can be directed to a plurality of spots on the sample 3. Alternatively, the light beam can be directed to a single, relatively large spot on the sample 3. It is envisioned that the light beam is arranged to illuminate the sample 3 in a line pattern. This enables most of the sample 3 to be illuminated and simultaneously detected by a suitable detector. A scanning unit (20, not shown in FIG. 5) is provided that is arranged to establish relative movement between the sample 3 and the light source 11. Thus, the light beam, which can be a spot or a line, can be scanned across the sample 3, and as a result, spatially resolved data can be collected. This means that a 2D map of the sample, which is useful for further evaluation of the sample, and the respective properties of the matrix layer can be obtained.

[0054] Note that the controller 13 can be arranged to evaluate the sample 3 based on the determined properties. This evaluation can be performed based on the aforementioned spatially resolved data. The evaluation can also be performed for a plurality of properties in the same manner.

[0055] As an example, the determined characteristics of the matrix layer can be one or more of a reference for the thickness of the matrix layer and a reference for the contamination of the matrix layer. For example, the reference for the thickness of the matrix layer includes the determination of the overall thickness of sample 3 including matrix layer 7 and sample carrier 9. The reference for the thickness may be qualitative (e.g., pass or fail) or quantitative (e.g., 150 nm). In other words, it is assumed that the device is arranged to determine the thickness of the matrix layer over a predetermined range of values and with a predetermined accuracy (regardless of the presence or absence of sample carrier 9). In one embodiment, the device is arranged to detect and determine a thickness in the range of 0 to 1000 nm, more specifically in the range of 0 to 400 nm.

[0056] Figure 6 shows a second embodiment of device 1 disclosed herein. This device 1 includes a housing 10, in which a light source 11 and a detector 12 are provided. A translucent mirror 19 is positioned between the light source 11 and the detector 12 and is aligned with the optical axis O such that the transmitted light from the light source 11 is emitted along the optical axis O and the light reflected by the sample is emitted back along the optical axis O towards the detector 12. An optical element 15 is provided downstream of the light source 11. In the illustrated embodiment, the light source is a white LED and the optical element 15 is a lens element. Further, a final lens 18 is provided. The optical element 15 and the final lens 18 are arranged to focus the white LED light onto the specimen using a beam that is not substantially parallel (although almost parallel). In other words, the light beam emitted from the final lens 18 converges slightly onto the sample. By using a beam that is not slightly parallelized, the amount of reflected light to device 1 is increased, and in particular, when the sample surface is not perpendicular to the optical axis O, the signal received by the detector is increased. The optical element 15 can be, for example, a plano-convex lens having a focal length of 60 mm.

[0057] The device 1 shown in FIG. 6 comprises a further optical element 16 positioned between the detector 12 and the semi-transparent mirror 19. This further optical element 16 is an achromatic lens element.

[0058] Thus, the device 1 as shown in FIG. 6 is arranged such that a polarizer analyzer beam is generated. Polarizer analysis is an optical technique for examining the dielectric properties (birefringence or dielectric function) of thin films. Polarizer analysis measures the change in polarization of reflection or transmission and compares it with a model. Using polarizer analysis, the composition, roughness, thickness (depth), crystallinity, doping concentration (in semiconductor samples), electrical conductivity, and other material properties can be characterized. It is very sensitive to changes in the optical response of the incident radiation interacting with the material being examined. Thus, the polarizer analyzer beam provides an advantage for use in examining the properties of the matrix layer of a specimen that can be studied with a charged particle microscope when the sample is usually small.

[0059] In particular, the combination of creating spatially resolved data and achromatic polarization analysis enables the identification of special states of the sample that were previously undetectable. This special state is the so-called wetting of the back surface of the sample. It has been found that using a simple optical model based on interference and using three colors makes it possible to determine the absolute thickness of the sample (i.e., the sample holder 9 and the matrix layer 7). However, it is impossible to confirm the exact positions of some of the layers that cause the interference. For this purpose, in one embodiment, it is possible to use an optical model based on interference and diffraction, in which the diffraction order is caused by the propagation of light through a support structure having the characteristics of a repeating pattern such as regularly arranged array-like holes, whereby the absolute thickness of the entire sample, as well as the location of the matrix layer (i.e., the front side, the back side, and / or combinations thereof) can be determined. It should be noted that it is advantageous to illuminate the sample at an angle such that the diffraction order propagates to a detector perpendicular to the sample surface when detecting the first-order or higher-order diffraction beam. Note that for a particular combination of detected light, there are multiple solutions for the thickness of the sample. In that case, since the thickness of the matrix layer is in principle continuous over at least a part of the surface of the sample holder 9, it is possible to draw conclusions about the thickness using spatially resolved data.

[0060] Figure 7 shows another embodiment of the device 1. In this embodiment, the light source 11 is provided on one side of the sample 3, and the detector 12 is positioned on the side of the sample 3 opposite thereto. Thus, the sample is installed between the light source 11 and the detector 12. The light source 11 and the detector 12 are installed substantially along the line of the optical axis O. One or more optical elements 15, 16, such as lens elements and / or filter elements, can be provided between the light source 11 and the sample 3 and / or between the sample 3 and the detector 12. The detector 12 includes several different pixels 12a - 12c. Pixel 12a is sensitive to a first band of light wavelengths, pixel 12b is sensitive to a second band of light wavelengths, and pixel 12c is sensitive to a third band of light wavelengths. As shown in the figure, the detector 12 includes a number of different pixels 12a - 12c, and for the sake of brevity, only three pixels are shown using the reference symbols 12a - 12c. The detector 12 may be an RGB pixel camera. Pixel 12a is sensitive to red, pixel 12b is sensitive to green, and pixel 12c is sensitive to blue. It is conceivable to use multiple detectors to obtain the same or similar technical effects. The detector 12 (or multiple detectors) can be connected to the control unit 13, and based on the signal received by the detector 12, the control unit can determine the characteristics of the matrix layer of the sample 3. In this embodiment, the light source 11 is provided with scanning means 20 so that the light beam can move relative to the sample 3. This enables the generation of a 2D matrix layer characteristic map of the sample 3.

[0061] Here, returning to FIGS. 1 - 4, some usage examples of the device 1 are described. It should be noted that in principle, any one of the device embodiments as shown in FIGS. 5 - 7 can be used. In particular, the use of the embodiment shown in FIG. 6 is advantageous because it only requires access to one side of the sample.

[0062] In FIG. 1, device 1 is used during the preparation step of preparing a sample. In the case shown in the figure, device 1 is used during the application step of applying a matrix layer to sample 3. Here, a liquid matrix layer 7 containing the specimen 5 under study is applied to sample holder 9. Sample holder 9 may be a sample grid, which is known to those skilled in the art and may comprise a small (a few millimeters) copper disc with a fine mesh having a carbon foil on top. The liquid matrix layer 7 is applied to one side of the sample grid 9, and by using device 1, that is, by sensing an increase in the thickness of the object under study or a change in optical properties, the proper application of the liquid matrix layer can be checked.

[0063] In FIG. 2, device 1 is used in the removal step of removing an excessive amount of matrix layer 7 from the sample. Here, an absorbent filter 21 is used and gently pressed against sample 3 to allow the absorbent paper 21 to absorb the excess fluid from matrix layer 7. Thereby, a relatively thin layer 7 containing the specimen 5 under study can be retained on sample holder 9. Device 1 can be used during and / or after the absorption step to confirm that the absorption has been properly applied. If the absorption is not successful, further attempts can be made. If the absorption removes too much material, it can be determined whether to discard the current grid or reapply fresh liquid to the same grid.

[0064] In FIG. 3a, device 1 is positioned slightly above the liquid nitrogen bath 31. Sample 3 can be vitrified within this bath 31, and as soon as it is removed from the bath, the quality of the obtained sample 3 can be inspected using device 1. For example, the ice thickness, contamination, and / or crystallinity of the vitrified matrix layer can be investigated. In FIG. 3b, the device is used in a similar manner, but differs in that two opposing ethane jets are used in the vitrification process. It should be noted in this regard that the essence of the method for vitrifying a sample is not limited to the use of device 1 as disclosed herein. Device 1 can advantageously be used to investigate one or more properties of the matrix layer after the vitrification step.

[0065] In FIG. 4, it is schematically shown that sample 3 can be stored as soon as it is vitrified. Options for storage include storage box 41 or cassette, or bath storage 42 filled with, for example, liquid nitrogen. The quality of sample 3 can be inspected using device 1 before and after storage. However, generally, it is sufficient to check that the quality is appropriate after vitrification and evaluate the sample based on the results obtained.

[0066] Thus, from the above, as a matter of course, device 1 can advantageously be used at different positions within a sample preparation tool such as that disclosed in WO02 / 077612A1, which is hereby incorporated by reference herein.

[0067] Furthermore, a device as disclosed herein can be advantageously used in charged particle microscopes such as cryo-EM. In particular, the device can be positioned near the sample holder and / or sample loading device of cryo-EM. When inserting a sample of interest into a charged particle microscope, a device as disclosed herein can check - quantitatively or qualitatively - whether the sample is suitable for use in the charged particle microscope. Furthermore, it is possible to generate a 2D map of the relevant part of the sample. Thereby, the sample can be studied more quickly and more efficiently.

[0068] The desired protection is determined by the appended claims.

Claims

1. A device for determining the characteristics of a sample used in a charged particle microscope, wherein the sample includes a specimen embedded in a matrix layer, and the device includes: - A light source arranged to direct a light beam towards the sample, the light beam including a first beam of a first color, a second beam of a second color, and a third beam of a third color; - A detector arranged to detect light emitted from the sample in response to the light beam incident on the sample; - A controller connected to the detector and arranged to determine the characteristics of the matrix layer based on multi-beam interference between the first beam, the second beam, and the second beam indicated in the signal received by the detector.

2. The device according to claim 1, wherein the device is arranged to collect spatially resolved data.

3. The device according to claim 1 or 2, wherein the controller is arranged to evaluate the sample based on the determined characteristics.

4. The controller is arranged to: - Determine at least one of a reference for the thickness of the matrix layer; - A reference for contamination of the matrix layer; and - A reference for the skill of the matrix layer.

5. The device according to any one of claims 1 to 4, wherein the detector is arranged to receive light transmitted through the sample.

6. The device according to any one of claims 1 to 5, wherein the detector is arranged to receive light reflected from the sample.

7. The device according to any one of claims 1 to 6, further comprising a scanning unit for applying a relative scanning motion of the light beam on the sample.

8. The device according to any one of claims 1 to 7, wherein the light source includes a laser light source and the detector includes a CMOS line sensor.

9. The device according to any one of claims 1 to 7, wherein the light source includes an LED configured to emit the first beam of the first color, the second beam of the second color, and the third beam of the third color, and the detector includes a color camera.

10. The device according to any one of claims 1 to 7, wherein the light source is arranged to generate an analyzer beam. **Claim 11**: Further comprising one or more color filter elements configured to filter the light beam so as to include only the first beam of the first color, the second beam of the second color, and the third beam of the third color. The device according to any one of claims 1 to 7. **Claim 12** A sample preparation tool comprising the device according to any one of claims 1 to 11. **Claim 13** A charged particle microscope comprising the device according to any one of claims 1 to 11. **Claim 14** A method for determining characteristics of a sample used in a charged particle microscope, the method comprising: - providing a sample comprising a specimen embedded in a matrix layer; - directing a light beam towards the sample, the light beam including a first beam of a first color, a second beam of a second color, and a third beam of a third color; - detecting light emitted from the sample in response to the light beam incident on the sample; - using a controller to determine characteristics of the matrix layer based on multi-beam interference between the first beam, the second beam, and the second beam indicated in the detected light. **Claim 15** - a criterion for the thickness of the matrix layer, - a criterion for contamination of the matrix layer, and - a criterion for the skill of the matrix layer, the method according to claim 14 comprising determining at least one of. **Claim 16** The following steps: - a preparation step of preparing the sample, - an application step of applying a matrix layer to the sample, - a removal step of removing an excessive amount of the matrix layer from the sample, - a vitrification step of vitrifying the sample, and - an imaging step of imaging the sample, the method according to claim 14 or 15 comprising the step of rejecting and / or approving the sample before, during, or after one or more of.

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