Apparatus and method for determining properties of a sample to be used in a charged particle microscope

Abstract

The invention relates to an apparatus and a method for determining a property of a sample to be used in a charged particle microscope. The sample comprises a specimen embedded in a matrix layer. The apparatus comprises a light source arranged for directing a light beam towards the sample and a detector arranged for detecting light emitted from the sample in response to the light beam being incident on the sample. Finally, the apparatus comprises a controller connected to the detector and arranged for determining a property of the matrix layer based on a signal received by the detector.

Description

[0001] The present invention relates to an apparatus and method for determining properties of a sample to be used in a charged particle microscope, the sample comprising a test specimen embedded in a matrix layer, e.g. a water-containing layer or an ice layer.

[0002] Biology is the natural science of life and living organisms, including their physical structure, chemical processes, molecular interactions, physiology, developmental and evolutionary biology.

[0003] Cell biology is a branch of biology that studies the structure and function of the cell, the basic unit of life. Cell biology involves the physiological properties, metabolic processes, signaling pathways, life cycle, biochemical composition, and interactions of the cell with its environment. In cell biology, molecular recognition between macromolecules controls all the most complex processes in cells. The most common macromolecules include biological polymers (nucleic acids, proteins, carbohydrates, and lipids) and large non-polymeric molecules (e.g. lipids and macrocycles).

[0004] Many researchers are interested in studying macromolecular complexes in their natural environment at high resolution in order to reveal the structural dynamics and interactions of the complexes. For this purpose, a charged particle microscope can be used. Charged particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, in particular in the form of electron microscopy (EM). Historically, the basic categories of electron microscopy have evolved into many well-known device categories, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM), and into various subcategories, such as the so-called “dual-beam” tools (e.g. FIB-SEM), which additionally employ a “machining” focused ion beam (FIB), e.g. allowing supportive activities like ion beam milling or ion beam induced deposition (IBID).

[0005] EM offers a variety of methods for studying biological test specimens: conventional TEM for studying the overall morphology of biological test specimens; electron crystallography and single-particle analysis for studying proteins and macromolecular complexes; and (cryo-) electron tomography of vitreous sections and cryo-EM (CEMOVIS) for cellular organelles and molecular architecture. In cryo-EM and CEMOVIS, the test specimen is preserved by rapid freezing using vitrification techniques and observed by low-temperature TEM. CEMOVIS additionally comprises a low-temperature sectioning step of the test specimen, which can be performed using low-temperature FIB techniques.

[0006] For studying a test specimen, a sample comprising the test specimen is needed. The sample can comprise a carrier and the test specimen is provided on the carrier embedded in a matrix layer, which can be liquid or solid, e.g. water or ice.

[0007] Samples prepared for investigation in a charged particle microscope are often time-consuming and laborious. One of the disadvantages associated with these investigations is that the user only knows whether the preparation of the biological sample was successful after the sample has been prepared and investigated in the charged particle microscope. This is especially true for the preparation of biological samples, such as cryo-EM samples.

[0008] The preparation of a cryo-EM sample, for example, comprises the following steps: taking an aqueous sample of biological material (i.e. a "test sample", often a purified protein complex), applying it to a support structure (grid), reducing its size to a layer as thin as possible (~ 100-800 A, depending on the size of the biological molecules) by means of a filter paper, and then rapidly freezing this layer to prevent the aqueous liquid from crystallizing.

[0009] A device for preparing a cryo-EM sample is known from WO 02 / 077612 A1 in the name of the applicant. This device is commercially available under the trade name "Vitrobot". The device described in WO 02 / 077612 A1 comprises an environmental chamber, a holder for a sample or carrier, and at least one blotting element, a medium for absorbing liquid being disposed in or attachable to the at least one blotting element, both being disposed in the environmental chamber, and a cooling medium for cooling the sample. The blotting element can be moved towards the sample or carrier in a controlled manner.

[0010] Many aspects of this process of preparing a biological sample are challenging. In this context, the step of reducing the layer size by removing excess liquid (e.g. can be done by a technique called "blotting") is considered to play a decisive role for the quality and reproducibility of the liquid film.

[0011] As it is difficult to establish the blotting step in a reproducible manner, US 2017 / 350798 A1 proposes a method and device in which the requirement to blot off the excess liquid can be minimized or eliminated by reducing the volume of the sample from the microliter scale to the picoliter scale.

[0012] In US 2010 / 181495 A1, a method and a device for preparing a sample for a cryo-electron microscope are described. Here, a carrier is fixed to a holder, a liquid containing a test sample is applied to the carrier, and an excess of liquid is removed from the carrier using a blotting device. The blotting device comprises a filter paper in contact with the carrier. On the opposite side of the filter paper, a light source and an optical 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 filter paper absorbing the excess liquid is detected by the optical sensor device. A control unit moves the blotting paper away from the carrier depending on the detected change in the optical properties.

[0013] Even though the blotting step poses challenges, many other aspects of the above process of preparing a biological sample are fraught with challenges and unpredictability. The purified complex can encounter changes in the micro-surface, material and dynamics of the purified complex, or the sample can become contaminated during the process. With respect to vitrification techniques, it can be observed that it is also necessary to obtain control over the resulting ice thickness and crystallization of the ice.

[0014] The preparation of cryo-EM samples using current preparation techniques still results in a wide variety of quality, so that large areas of the EM grid cannot be used. Often, only upon examination of the sample on the grid in cryo-EM, the variations are visible. Thus, valuable resources, including scientist preparation time and expensive EM usage time, can be wasted. The same is true for the preparation of other samples, and is not limited to biological samples.

[0015] In view of this, it is an object of the present invention to provide an apparatus and a method for determining a property of a sample to be used in a charged particle microscope, whereby wasting valuable resources as described above can be prevented.

[0016] To this end, the present invention provides an apparatus for determining a property of a sample to be used in a charged particle microscope, the sample comprising a specimen embedded in a matrix layer, wherein the apparatus is defined according to claim 1. As defined herein, the apparatus comprises:

[0017] - a light source arranged for directing a light beam towards the sample;

[0018] - a detector arranged for detecting light emitted from the sample in response to the light beam being incident on the sample; and

[0019] - a controller connected to the detector and arranged for determining a property of the matrix layer based on a signal received by the detector.

[0020] By using a light source and directing it directly towards the sample, and directly detecting light emitted from the sample, it is possible to directly determine a property of the matrix layer. It is noted that the matrix layer can be a liquid, e.g. a liquid used for preparing the sample. The matrix layer can also be a solid, e.g. a vitrified liquid used for preparing the sample, or any other (semi-)transparent solid, e.g. a resin. The light source can be chosen such that it is affected by the matrix layer. The matrix layer can e.g. reflect, diffract, transmit and / or absorb light from the light source. By using a detector to detect the reflected, diffracted, transmitted and / or absorbed light, it is possible to distinguish between samples based on different properties of the matrix layer. The distinguishing or determining of the property can be qualitative and / or quantitative.

[0021] It is noted that in the present device, the light source is directed directly at the sample, and the detector is used to measure light that is directly reflected by or directly transmitted through the sample. Hence, the present device provides a method for directly determining the properties of the matrix layer. This allows the use of this device at various stages of the sample. For example, it is possible to use the device during sample preparation, for example during the blotting step as previously described, wherein the matrix layer is an aqueous layer. In another embodiment, it is possible to use the device after vitrification of the sample, wherein the matrix layer is an ice layer. The matrix layer can in principle be any type of matrix layer that influences 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.

[0022] The light source can be directed at a surface of the sample, wherein the light source is arranged to emit light in a first direction. The detector can be arranged to receive reflected and / or transmitted light from the sample, wherein a normal of the detector is directed in a second direction different from the first direction.

[0023] In an embodiment, the first direction is not perpendicular to a normal of the sample.

[0024] In an embodiment, the second direction is not perpendicular to the normal of the sample.

[0025] Preferred embodiments of the present application and advantages thereof will now be explained in the following.

[0026] In an embodiment, the device is arranged to collect spatially resolved data. Thereby, it is possible to determine the properties of the matrix layer locally. For example, it is possible to map a 2D (x,y) coordinate system to the sample surface, and to assign a determined property of the matrix layer to one or more coordinates in the 2D coordinate system. In an embodiment, this can be used to establish a map of the sample surface with corresponding properties of the matrix layer. This map provides the user with a useful insight into which parts can be used for further investigation of the sample, and which parts are best avoided. It is noted at this point that for cryo-EM samples, cryo-TEM is used in low magnification overview mode to assess ice quality. This activity blocks the TEM for a considerable amount of time. By using the device as disclosed herein, an overview / map / profile of the grid can be obtained in a quick and efficient manner, and this allows to identify areas that are potentially beneficial for data collection.

[0027] In an embodiment, the controller is arranged to evaluate the sample based on the determined properties. This means that the controller can identify based on the determined properties of the matrix layer whether certain parts of the sample are approved or rejected. For example, when it is determined that the matrix layer (part of) contains contaminants, or that the thickness of the matrix layer does not meet the requirements, the controller can indicate that the sample is not suitable for investigation. The controller can assign an evaluation to a respective 2D coordinate of the sample, or can assign an evaluation for the entire sample.

[0028] In embodiments, the controller is arranged for determining at least one of a degree of thickness of the substrate layer, a degree of contamination of the substrate layer, a degree of crystallization of the substrate layer. Thus, the property can be the thickness and / or the degree of contamination. In case the substrate layer is vitrified (frozen), then the property can be the crystallization (or state) of the vitrified substrate layer.

[0029] In embodiments, the apparatus comprises at least one optical element, e.g. a lens. The optical element can be located within the optical path between the light source and the detector. The optical element can be arranged for focusing and / or conditioning the light beam. In embodiments, the optical element is arranged for focusing the light beam onto the sample. In embodiments, the optical element is arranged for focusing the light beam onto the detector. It is conceivable to provide multiple optical elements, each having one or more features as described above.

[0030] The at least one optical element can be a color selection filter. The color selection filter can be located within the optical path between the light source and the detector. The color selection filter is arranged to pass a restricted waveband of light towards the detector. It is noted that the color selection filter can comprise multiple color selection filter elements, each color selection filter element being arranged for passing a different restricted waveband of light towards the detector. Multiple color selection filter elements can be spatially organized over the detector, or can be an integral part of the detector. As an example, the detector can be designed to selectively sense different wavebands, e.g. in case of an RGB pixel camera, wherein different pixels are sensitive to different colors. Additionally or alternatively, multiple color selection filter elements can be positioned in sequence, i.e. one after the other, within the optical path.

[0031] In embodiments, the detector is arranged for receiving light transmitted through the sample. In embodiments, the detector is arranged for receiving light reflected from the sample. It is noted that the detector can comprise multiple detector units, wherein one of the detector units is arranged for receiving light transmitted through the sample, and another one of the detector units is arranged for receiving light reflected from the sample. This provides additional sample information.

[0032] In embodiments, the apparatus comprises a scanning unit for imparting a relative scanning motion of the light beam onto the sample. By using a relative scanning motion, it is possible to scan the sample with a relatively high resolution, e.g. to establish a previously described 2D property map of the sample.

[0033] It is conceivable that the relative scanning motion is established by moving the sample past the light source and / or moving the sample past the detector, e.g. by using a sample stage.

[0034] Determining the properties of the matrix layer by means of a light source and a detector can be realized in various ways. Various embodiments will be explained hereinafter. It is noted that these embodiments are not intended to be limiting and that combinations of these embodiments are also conceivable.

[0035] In an embodiment, the light source comprises a laser light source. Laser light sources are relatively cheap. The laser light source is directed towards the sample surface, in particular towards the surface of the matrix layer. The laser light source can be arranged for projecting a line onto the sample. The line can be scanned over the entire sample by using a relative motion between the laser line and the sample. By using a line source, preferably in combination with a line sensor, it is possible to illuminate a relatively large portion of the sample at once, while still maintaining the spatial information of the sample. By using line scanning, the entire sample surface can be screened quickly and easily. This contributes to establishing spatially resolved data on the properties of the matrix layer. An additional advantage is that this setup does not require the use of relatively expensive 2D sensors.

[0036] It is conceivable, in particular in combination with a laser light source, that the detector comprises a CMOS sensor. The CMOS sensor can be a CMOS line sensor, which can be used with the laser line source described above. The aforementioned optical elements can be used to focus the transmitted light from the sample onto the CMOS detector. In an embodiment, the CMOS line sensor comprises a CMOS 16k line sensor operating at a frame rate suitable for the relative motion between the sample and the light source and / or the detector. The frame rate can be higher than 250 fps, preferably higher than 500 fps, more preferably higher than 1000 fps. With this setup, it is possible to obtain a complete matrix layer property map of a 3 mm grid within 15 seconds.

[0037] In an embodiment, the light source comprises an LED. LEDs are relatively cheap. The light source can be arranged for directing a polychromatic light beam towards the sample; this can be realized in case the device comprises a polychromatic LED. In an alternative embodiment, the device comprises an LED which is arranged for combining with one or more filter elements to create a white LED illumination. In an embodiment, one or more filter elements can be provided. In particular, color filter elements for a total of three colors can be provided. These colors can be the primary colors red, green and blue, so that the sample can be illuminated with at least three different colors.

[0038] When the sample is illuminated with at least three different colors, the multi-beam interference theory from parallel films can be used to obtain the thickness of the matrix layer. Interference of the light rays is created because of the interference due to the phase difference of the light rays reflected at different layers. A light beam that is incident on a thin film (with thickness d and refractive index n) at an angle θ with respect to the normal of the film has an optical path difference (OPD or phase shift) of OPD = 2 n d cos(θ). Now two cases are well distinguished: constructive interference and destructive interference. Destructive interference occurs when the phase difference is 180 º ° and constructive interference occurs when the phase difference is 0° or 360°. Since the 180° phase difference is dependent on the wavelength λ, the destructive interference as a function of OPD then is limited to 2 n d cos(θ) = m λ i , where m is an integer. By introducing at least three different colors (different wavelengths), a system of equations can be obtained with which the thickness of the thin film can be determined. Similarly, the thickness of a multi-layer system (i.e. a grid of matrix layers on top of each other) can be determined. This method is found to be particularly useful for determining the thickness of vitrified layers. In addition, using three colors is advantageous for determining whether contamination is created in the sample.

[0039] In an embodiment, the detector comprises a color camera. The color camera can be arranged for receiving the reflected light, in particular in combination with the LED. This combination is relatively cheap.

[0040] In an embodiment, the light source is arranged for generating an ellipsometer beam. In this embodiment, the apparatus is arranged for measuring the properties of the matrix layer based on ellipsometry. Thereby, the change in polarization upon reflection or transmission of the light source on the sample can be measured and compared to a model. This is particularly useful for measuring the thickness and / or crystalline properties of the matrix layer, taking into account the (vitrified) biological sample. In this embodiment, a polarizing element can be used that is arranged between the light source and the sample. Furthermore, a second polarizing element can be arranged between the sample and the detector. Optionally, one or more compensator elements can be arranged between the light source and the sample and between the sample and the detector. Suitable ellipsometry setups are known to the person skilled in the art.

[0041] It is conceivable that the light source comprises a 3-color LED. In combination with the above ellipsometer beam, it is possible to measure the matrix layer thickness (in particular ice layer thickness) as well as the scattering and / or transmission (which is a measure for the degree of contamination and / or crystalline properties). The ellipsometry setup can be based on reflection, such that the light source and the detector are arranged on the same side of the sample. Also a combined scanning motion is conceivable. Spatial resolution can be obtained by deconvolution.

[0042] In embodiments, the light source is arranged for generating light in the UV spectrum. Additionally or alternatively, the light source can be arranged for generating light in the IR spectrum. This embodiment is particularly advantageous for measuring the crystallization of ice. In IR and UV, amorphous ice (typically considered to be the type of ice wanted for biological samples in cryo-EM) has different optical properties compared to crystalline ice forms (typically considered to be the type of ice not wanted). In particular, the IR absorption coefficient and the refractive index are different between the two types of ice. Furthermore, the UV absorption of amorphous ice is different compared to cubic ice. Thus, the use of UV and / or IR light can advantageously be used to determine the properties of the matrix layer, and in this embodiment the quality of the matrix layer after vitrification.

[0043] According to an aspect, a sample preparation tool is provided comprising a device as defined herein. The sample preparation tool can be a cryo-EM sample preparation tool as described in WO 02 / 077612 Al, which is incorporated herein by reference. The device can be arranged in the vicinity of the blotting element to monitor the blotting step during sample preparation. The device can additionally or alternatively be arranged in the vicinity of the cooling medium to monitor the cooling (vitrification) of the sample.

[0044] According to an aspect, a charged particle microscope is provided comprising a device as defined herein. The device can be provided in the vicinity of a sample holder of the charged particle microscope. In particular, the sample holder can be movable between a loading position and an inspection position. The device can be arranged such that a sample moves through the device during movement between the loading position and the inspection position and the properties of the sample can be inspected. This allows for example that a sample is loaded and inspected by the device such that a 2D map of samples with corresponding properties of the matrix layer can be established. The results can be shown to a user or otherwise stored to aid further processing of the sample.

[0045] In embodiments, the sample comprises one or more pattern elements which are arranged to be detectable by the device defined herein. In addition, the one or more pattern elements can be arranged such that they are also detectable by the charged particle microscope. When using one or more pattern elements, it is possible to accurately record the position of the sample and it is also possible to align in the charged particle microscope without the need to go back to a low magnification.

[0046] According to an aspect, a method of determining properties of a sample to be used in a charged particle microscope is provided as defined in claim 12. The method comprises the following steps as defined herein:

[0047] - providing a sample comprising a test embedded within a matrix layer;

[0048] - directing a light beam at the sample;

[0049] - detecting light emitted from the sample in response to the light beam being incident on the sample; and

[0050] - determining, using a controller, a property of the matrix layer based on the detected light.

[0051] The advantages of this method and further embodiments thereof have been elucidated above. In summary, by directly illuminating a sample with a light beam, and using a detector to detect light emitted from the sample, it is possible to determine a property of a matrix layer, such as the thickness or contamination of the matrix layer. The property can be determined qualitatively or quantitatively, and an assessment of the sample can additionally be made.

[0052] The determined property can be a degree of thickness of the matrix layer and / or a degree of contamination of the matrix layer.

[0053] The method and apparatus disclosed herein can be used in an advantageous manner during or after one or more of the following phases:

[0054] - a preparation step of preparing the sample;

[0055] - an application step of applying a matrix layer to the sample;

[0056] - a removal step of removing excess matrix layer from the sample;

[0057] - a vitrification step of vitrifying the sample with the matrix layer; and

[0058] - an imaging step of imaging the sample.

[0059] The application will now be elucidated in more detail based on exemplary embodiments and the attached schematic drawings, in which:

[0060] Figure 1 - shows an apparatus for determining a property of a sample during a first use example;

[0061] Figure 2 - shows an apparatus for determining a property of a sample during a second use example;

[0062] Figure 3a and b - shows an apparatus for determining a property of a sample during a third use example;

[0063] Figure 4 - shows an apparatus for determining a property of a sample during a fourth use example;

[0064] Figure 5 - shows a first embodiment of an apparatus for determining a property of a sample;

[0065] Figure 6 - shows a second embodiment of an apparatus for determining a property of a sample;

[0066] Figure 7 - A third embodiment of a device for determining sample properties is shown;

[0067] Figures 1 to 4 The diagram illustrates different stages of a method for preparing a biological sample 3 for use in a charged particle microscope, wherein a device 1 for determining the properties of the prepared sample 3 is used. Generally, the method for preparing the biological sample 3 includes the following steps:

[0068] - Provide a sample carrier 9 and provide the sample 5 of interest, embedded in the liquid matrix layer 7, onto the sample carrier 9 to prepare sample 3 ( Figure 1 );

[0069] - An ink-absorbing step that removes excess liquid from the matrix layer 7 using an ink-absorbing material 21, such as blotting paper. Figure 2 );

[0070] - A vitrification step for vitrifying the liquid matrix layer containing the sample 5 of interest, wherein a low-temperature bath solution was used ( Figure 3a ) or low-temperature nozzle ( Figure 3b );as well as

[0071] -Storage steps for storing vitrified sample 3 once vitrification occurs ( Figure 4 ).

[0072] Those skilled in the art are familiar with the general procedures for preparing low-temperature EM samples, and therefore will not explain them in detail. The apparatus disclosed herein can be advantageously used in sample preparation, particularly in the preparation of low-temperature EM samples as described above. The apparatus disclosed herein can be advantageously used in each of the aforementioned sample preparation steps. It should be noted that in these... Figures 1 to 4 The device is schematically indicated in the text. For these reasons, reference will be made first. Figures 5 to 7 Several embodiments of the device are explained below, and will be explained in more detail later. Figures 1 to 4 The use of the device.

[0073] Turn now Figure 5, a first embodiment of an apparatus 1 for determining properties of a sample 3 to be used in a charged particle microscope is shown. In the figures, like or corresponding features are denoted using the same reference signs. The shown apparatus comprises a light source 11 arranged for directing a light beam towards the sample 3, a detector 12 arranged for detecting light emitted from the sample in response to the light beam being incident on the sample 3, and a controller 13 connected to the detector 12 and arranged for determining properties of the matrix layer 7 based on signals received by the detector 12. In the shown embodiment, 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 apparatus 1 is arranged such that the sample can be located between the light source 11 and the detector 12. For example, light falling on the sample 3 is scattered and / or absorbed and the detector 12 detects an amount of light depending on the amount of scattering and / or absorption. Thus, the detector 12 is arranged to receive light transmitted through the sample 3. From this, properties of the matrix layer of the sample 3 can be determined, for example the presence of the matrix layer and / or the thickness of the matrix layer.

[0074] In Figure 5 the shown embodiment, the light source 11 can be a laser or an LED. The detector is arranged for detecting light emitted from the light source 11 and is thus correspondingly arranged for receiving laser and / or LED light. As mentioned before, the detector can be a CMOS sensor (in particular in combination with a laser) or a color camera (in particular in combination with an LED). Other light sources are also conceivable.

[0075] In Figure 5 the shown embodiment, the light source 11 is directed towards the sample 3. The light beam can be directed towards a single relatively small spot on the sample 3. The light beam can be directed towards multiple spots on the sample 3. Alternatively, the light beam can be directed towards a single relatively large spot on the sample 3. It is conceivable that the light beam is arranged to illuminate the sample 3 in a line pattern. This allows a large portion of the sample 3 to be illuminated and at the same time be detected by a suitable detector. It is conceivable that a scanning unit (20, not shown in Figure 5 ) is provided which is arranged for establishing a relative movement between the sample 3 and the light source 11. Thus, the light beam, which can be a point spot or a line, can be scanned over the sample 3 in order to collect spatially resolved data. This means that a 2D map of the sample and the respective properties of the matrix layer can be obtained which is useful for further evaluation of the sample.

[0076] It is noted that the controller 13 can be arranged for evaluating the sample 3 based on the determined properties. The evaluation can be based on the spatially resolved data as described above. Also multiple properties can be evaluated.

[0077] As an example, the determined property of the matrix layer can be one or more of a degree of thickness of the matrix layer and a degree of contamination of the matrix layer. For example, the degree of thickness of the matrix layer includes determining the total thickness of the sample 3, including the matrix layer 7 and the sample carrier 9. The degree of thickness can be qualitative (e.g. pass or fail) or quantitative (e.g. 150 nm). In other words, it is conceivable that the device is arranged for determining the thickness of the matrix layer (with or without the sample carrier 9) within a predetermined range of values and with a predetermined accuracy. In an embodiment, the device is arranged for detecting and determining a thickness in the range of 0 to 1000 nm, more specifically in the range of 0 to 400 nm.

[0078] Figure 6 A second embodiment of the device 1 disclosed herein is shown. The device 1 comprises a housing 10 in which a light source 11 and a detector 12 are provided. A semi-transparent mirror 19 is positioned between the light source 11 and the detector 12 and is aligned with the optical axis O such that light transmitted from the light source 11 is emitted along the optical axis O and 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 shown 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 for focusing the white LED light onto the specimen in a substantially non-parallel (but almost parallel) beam. In other words, the light beam emitted from the final lens 18 is slightly converging onto the sample. The use of a slightly non-parallel beam increases the amount of reflected light towards the device 1 and increases the signal received by the detector, in particular when the sample surface is not perpendicular to the optical axis O. The optical element 15 can for example be a plano-convex lens with a focal length of 60 mm.

[0079] Figure 6 The shown device 1 comprises a further optical element 16 which is positioned between the detector 12 and the semi-transparent mirror 19. The further optical element 16 is an achromatic lens element.

[0080] Thus, as Figure 6 The shown device 1 is arranged such that an ellipsometer beam is generated. Ellipsometry is an optical technique used to study the dielectric properties (complex refractive index or dielectric function) of thin films. Ellipsometry measures the change in polarization upon reflection or transmission and compares the polarization change to a model. Ellipsometry can be used to characterize composition, roughness, thickness (depth), crystalline properties, doping concentration (in semiconductor samples), electrical conductivity, and other material properties. It is very sensitive to changes in the optical response of the incident radiation that interacts with the material under study. Thus, the ellipsometer beam provides the advantage of studying the matrix layer properties of a specimen to be studied in a charged particle microscope in which the sample is typically very small.

[0081] In particular, the combination of spatially resolved data and achromatic ellipsometry makes it possible to distinguish a special condition of the sample, which was not detectable before. This special condition is the so-called backside wetting of the sample. It has been found that using a simple optical model based on interference and using three colors, it is possible to determine the absolute thickness of the sample, i.e. the sample holder 9 and the matrix layer 7. However, it is not possible to see the exact position of the several layers that cause the interference. For this reason, in an embodiment, it is possible to use an optical model based on interference and diffraction, wherein the diffraction orders are caused by the light propagation through a support structure having a repeating pattern of features, similar to a regular array of holes, and it is thus possible to determine the absolute thickness of the total sample and the position of the matrix layer, i.e. the front side, the backside and / or a combination thereof. It should be noted that when detecting the first or higher order diffraction beams, it is advantageous to illuminate the sample at an angle such that the diffraction orders propagate perpendicular to the sample plane to the detector. It should be noted that for certain combinations of detected light, there are multiple solutions for the sample thickness. In that case, it is possible to use the spatially resolved data to conclude on the thickness, since the thickness of the matrix layer is in principle continuous over at least part of the surface of the sample holder 9.

[0082] In Figure 7 , another embodiment of the apparatus 1 is shown. In this embodiment, the light source 11 is arranged at one side of the sample 3 and the detector 12 is positioned at the opposite side of the sample 3. Thus, the sample is placed between the light source 11 and the detector 12. The light source 11 and the detector 12 are placed substantially in line with the optical axis O. One or more optical elements 15, 16, such as lens elements and / or filter elements, can be arranged between the light source 11 and the sample 3 and / or between the sample 3 and the detector 12. The detector 12 comprises a plurality of different pixels 12a to 12c, wherein the pixels 12a are sensitive to a first waveband of light, the pixels 12b are sensitive to a second waveband of light and the pixels 12c are sensitive to a third waveband of light. As shown, the detector 12 comprises a multitude of different pixels 12a to 12c and for the sake of brevity only three pixels are indicated using reference numerals 12a to 12c. The detector 12 can be an RGB pixel camera, wherein the pixels 12a are sensitive to red, the pixels 12b are sensitive to green and the pixels 12c are sensitive to blue. It is conceivable to use multiple detectors to obtain the same or a similar technical effect. The detector 12 (or multiple detectors) can be connected to the control unit 13 and based on the signals received by the detector 12, the control unit is able to determine the properties of the matrix layer of the sample 3. In this embodiment, the light source 11 is provided with scanning means 20 such that the light beam can be moved relative to the sample 3. This allows generating a 2D matrix layer property map of the sample 3.

[0083] Now returning to Figures 1 to 4 , several use cases of the apparatus 1 will be explained. It should be noted that in principle it is possible to use asFigures 5 to 7 Any of the embodiments of the device shown. In particular, the use Figure 6 The embodiment shown is advantageous in that it only requires access to one side of the sample.

[0084] In Figure 1 , the device 1 is used during a preparation step of preparing the sample. In the case shown, the device 1 is used during an application step of applying a matrix layer to the sample 3. Here, a liquid matrix layer 7 containing a specimen 5 to be studied is applied to a sample holder 9. The sample holder 9 can be a sample grid known to the person skilled in the art and can comprise small (few millimeter) copper discs comprising a fine mesh with a carbon foil on top. The liquid matrix layer 7 is applied to one side of the sample grid 9 and the device 1 can be used to check the successful application of the liquid matrix layer, i.e. by sensing an increase in thickness or a change in optical properties of the object under study.

[0085] In Figure 2 , the device 1 is used in a removal step of removing excess matrix layer 7 from the sample. Here, a blotting filter 21 is used and pressed lightly against the sample 3 to allow excess liquid from the matrix layer 7 to be absorbed by the blotting paper 21. This allows a relatively small layer 7 containing a specimen 5 to be studied to remain on the sample holder 9. The device 1 can be used during and / or after the blotting step to observe that blotting was successful. If blotting was not successful, further attempts can be made. If blotting removed too much material, it can be decided to discard the current grid or to reapply fresh liquid to the same grid.

[0086] In Figure 3a , the device 1 is positioned slightly above a liquid nitrogen bath 31. The sample 3 can be vitrified in this bath 31 and, once removed from the bath, the device 1 can be used to check the quality of the obtained sample 3. For example, the ice thickness, the contamination and / or the crystalline properties of the vitrified matrix layer can be studied. In Figure 3b , the device is used in a similar way, with the difference that two opposing ethane nozzles are used in the vitrification process. In this sense, it should be noted that the nature of the method of vitrifying a sample is not limited to the use of the device 1 as disclosed herein. The device 1 can advantageously be used to study one or more properties of the matrix layer after the vitrification step.

[0087] In Figure 4 , it is schematically shown that the sample 3, once vitrified, can be stored. Options for storage include a storage box 41 or a box, or a bath storage device 42 filled with liquid nitrogen or the like. The device 1 can be used to check the quality of the sample 3 before or after storage. However, in general, it is sufficient to check the quality immediately after vitrification and to assess the sample according to the obtained results.

[0088] From the above it follows that the device 1 can thus advantageously be used at different positions in a sample preparation tool, such as the one disclosed in WO 02 / 077612 Al, which document is included herein by reference.

[0089] Further, the device disclosed herein can advantageously be used in charged particle microscopes, such as cryo-EM. In particular, the device can be positioned in the vicinity of a sample holder and / or a sample loader of a cryo-EM. When a sample to be investigated is inserted into the charged particle microscope, the device disclosed herein can quantitatively or qualitatively check whether the sample is suitable for use in the charged particle microscope. Further, it is possible to generate a 2D map of the relevant part of the sample. This way the sample can be investigated faster and more efficiently.

[0090] The desired protection is determined by the claims appended hereto.

Claims

1. An apparatus (1) for determining the properties of a sample (3) to be used in a charged particle microscope, the sample (3) comprising a specimen (5) embedded within a matrix layer (7), wherein the apparatus (1) comprises: - A light source (11) is arranged to direct a light beam to the sample, and the light beam includes a first beam of a first color, a second beam of a second color, and a third beam of a third color; - Detector (12), which is arranged to detect light emitted from the sample in response to the light beam being incident on the sample; as well as - A controller (13), which is connected to the detector and is arranged to determine the characteristics of the matrix layer based on a multibeam interference pattern between the first, second and third beams shown in the signal received by the detector.

2. The apparatus according to claim 1, wherein the apparatus (1) is arranged for collecting spatially resolved data.

3. The apparatus according to claim 1 or 2, wherein the controller (13) is arranged to evaluate the sample (3) based on the determined characteristics.

4. The apparatus according to claim 1 or 2, wherein the controller (13) is arranged to determine at least one of the following: - The degree of thickness of the matrix layer; - The degree of contamination of the matrix layer; and - The degree of crystallization of the matrix layer.

5. The apparatus according to claim 1 or 2, wherein the detector (12) is arranged to receive light transmitted through the sample (3).

6. The apparatus according to claim 1 or 2, wherein the detector (12) is arranged to receive light reflected from the sample (3).

7. The apparatus according to claim 1 or 2, wherein the apparatus (1) includes a scanning unit (20) for applying a relative scanning motion of the light beam to the sample (3).

8. The apparatus according to claim 1 or 2, wherein the light source (11) comprises a laser light source.

9. The apparatus according to claim 1 or 2, wherein the light source (11) comprises an LED.

10. The apparatus according to claim 1 or 2, wherein the light source (11) is arranged to generate an elliptic polarimeter beam.

11. The apparatus of claim 8, wherein the detector (12) comprises a CMOS line sensor.

12. The apparatus of claim 9, wherein the detector (12) comprises a color camera.

13. A sample preparation tool comprising the apparatus according to any one of claims 1 to 12.

14. A charged particle microscope comprising the apparatus according to any one of claims 1 to 12.

15. A method for determining the properties of a sample to be used in a charged particle microscope, the method comprising the steps of: - Provide samples including specimens embedded within the matrix layer; - Point the light beam at the sample, and the light beam includes 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; as well as The properties of the matrix layer are determined using a controller based on the multibeam interference pattern between the first, second, and third beams shown in the detected light.

16. The method of claim 15, further comprising the step of determining at least one of the following: - The degree of thickness of the matrix layer; - The degree of contamination of the matrix layer; and - The degree of crystallization of the matrix layer.

17. The method of claim 15 or 16, further comprising the steps of rejecting and / or approving the sample before, during, or after one or more of the following stages: - The preparation steps for preparing the sample; - An application step of applying the matrix layer onto the sample; -A removal step to remove excess matrix layer from the sample; - A vitrification step that vitrifies the sample; - Imaging steps to image the sample.

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