METHOD FOR MASS SPECTROMETRIC ANALYSIS AND THREE-DIMENSIONAL IMAGING OF THE SURFACE OF SAMPLES

Description

[0001] The invention describes a method for measuring and analyzing the chemical composition and three-dimensional structure of sample surfaces. Description and introduction of the general field of the invention

[0002] The goal of spatially resolved (imaging) microscale surface analysis is to qualitatively and / or quantitatively depict the chemical composition of a sample (e.g., a biological tissue or object) with high lateral resolution. In this context, microscale means that the size of a measurement point is between 0.5 µm and 10 µm.

[0003] A sample is the entirety of the material to be examined. This material can be a chemical substance or mixture of substances, whether liquid (such as drinking water samples or urine samples) or solid (such as a rock sample), or a mixture of different aggregate states (e.g., a moist soil sample). In spatially resolved (imaging) microscale surface analysis, samples are examined in the solid aggregate state.

[0004] Unlike an optical microscope image, such an analytical image reveals the quantitative distribution of a specific substance within the sample. Common methods allow the determination of the chemical composition on flat samples and visualization of the chemical composition in two-dimensional representations, as well as the generation of three-dimensional chemical distribution images from a multitude of two-dimensional images (measurements). These methods include laser-assisted mass spectrometry techniques, such as laser desorption / ionization mass spectrometry (LDI-MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS), and laser ablation post-ionization mass spectrometry.

[0005] Desorption refers to the process by which (predominantly) uncharged atoms or molecules leave the surface of a solid.

[0006] Ionization is any process in which one or more charge carriers are removed from or added to an atom or molecule, leaving the atom or molecule as a positively or negatively charged ion. State of the art

[0007] A common MALDI-MS method for two-dimensional mapping of concentrations of specific molecules is described, for example, in US Pat. No. 5,808,300 A1. In this method, the sample surface is divided into individual measurement points, and a focused laser beam is applied to each defined point on the sample surface, detecting the released ions. This process is then repeated for every other point on the sample surface. It is not possible to simultaneously obtain information about the topography of the sample and its surface composition. There is no connection between the two-dimensional spatial distribution of a substance and the topography of a sample. Topography here refers to the height or thickness of a sample at a specific location.

[0008] In contrast, confocal laser scanning microscopy provides a three-dimensional image but no direct information about the chemical composition of the sample.

[0009] EP 1 739 719 A2 discloses a method for generating a sharp, high-resolution image of an area on a sample plate for an ion source, for example, to identify analytes to be examined and extracted with an ionizing laser. For this purpose, the area of ​​the sample plate is positioned in the field of view of an imaging device. Subsequently, a plurality of images with different depths of field are created, which are combined to form a two- or three-dimensional high-resolution overall image.

[0010] WO 2016 090 356 A1 describes a mass cytometry system in the form of an LA-ICP-MS for imaging a biological sample, using a solid-state laser to generate a laser beam for ablating the biological sample in an ablation chamber. The laser beam is focused onto the sample using an optical arrangement, with the sample arranged on a movable sample carrier. A plasma inductively coupled to the ablation chamber collects the ablated portions of the biological sample, which are then fed to a mass spectrometer.

[0011] The publication "Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis" by Spengler et al., in the American Society for Mass Spectrometry (2002), describes a device and method for laterally resolved mass spectrometric surface analysis. The device enables the imaging of the distribution of selected sample components of natural and artificial surfaces. The samples are analyzed either by time-of-flight laser desorption ionization (LDI) mass spectrometry or, after preparation with a suitable matrix, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry.

[0012] US 2008 149 822 A1 combines modified MALDI mass spectrometers and SNOM devices in a system and method for analyzing and imaging a sample. The system comprises an ionization region, a sample holder, a laser, and a mass spectrometer with a vacuum chamber. An atmospheric pressure interface connects the ionization region to the mass spectrometer. An optical near-field microscopy (SNOM) instrument scans the sample with a near-field probe. A platform with the sample holder can be moved via a controller to a distance within the near-field distance of the near-field probe, with a recording device detecting the distance between the probe and the sample.

[0013] US 7,180,058 B1 discloses a MALDI / LDI source comprising an ion optical device and beam focusing optics arranged on opposite sides of a locally transparent sample carrier. A laser located near the back of the sample carrier emits a beam that is focused by beam focusing optics and traverses the transparent area of ​​the sample carrier to impinge on the sample. The ions generated by irradiating the sample are collected by an ion optical device located near the front surface of the sample carrier. Task

[0014] The object of the invention is to provide a method for enabling simultaneous analysis of the two-dimensional, spatial chemical composition and topography of a sample. Solution to the task

[0015] This object is achieved according to the invention by a method having the features of claim 1. Advantageous embodiments and further developments of the present invention emerge from the subclaims.

[0016] A device 100, 200, which is not the subject of the invention, for carrying out the method according to the invention is intended for use with various mass spectrometers. It can be used with all mass spectrometers known to those skilled in the art that have an atmospheric pressure inlet system or operate at reduced or elevated pressure in the sample chamber. These include, among others: quadrupole, ion trap, ion cyclotron, and orbital trap mass spectrometers, as well as time-of-flight (TOF) mass spectrometers. The device is suitable for all methods of desorption and / or ionization of surfaces (desorption methods), for example, LDI (laser desorption ionization), MALDI (matrix-assisted laser desorption ionization), and laser ablation with post-ionization.The device 100, 200 for mass spectrometric analysis and three-dimensional imaging of the surface of objects comprises at least one energy source 20, 10 for desorption or desorption and ionization of atoms of the surface of a sample by means of a desorption / ionization beam 2 and their transmission to a mass spectrometer 7.

[0017] Furthermore, the device 100, 200 comprises a displacement table 1, which is designed such that a sample to be examined can be applied to it and wherein the beam path of the energy source 20, 10 and the displacement table 1 are designed to be movable relative to one another.

[0018] Furthermore, the device 100, 200 comprises an interface 50 for exchanging data with a mass spectrometer 7. The interface 50 is designed such that measurement data about the chemical composition of the sample surface at a measuring point 60 can be transmitted from a mass spectrometer via the interface 50 to the evaluation unit 40, so that the chemical composition for the measuring point 60 can be determined by the evaluation unit 40. The interface 50 can be, for example, an ISA, PCI, USB, or EIA-232 interface. An interface for wireless data transmission such as WLAN, Wi-Fi, IEEE 802.11, or Bluetooth is also possible.

[0019] Furthermore, the device 100, 200 comprises a light source 8, 10, at least one first optical system 3 and at least one light detector 4 and an evaluation unit 40. These are designed such that a measuring point 60 on the sample is illuminated by the light source 8, 10 and light reflected or scattered from the measuring point 60 is detected by the light detector 4, so that the surface of a sample can be detected three-dimensionally and the topography of this surface can be determined by the evaluation unit 40.

[0020] The light detector 4 preferably consists of a component with which radiation can be detected in a position-sensitive manner (e.g. a quadrant detector, a position-sensitive detector, a CCD sensor or a video camera chip).

[0021] The first optical system 3 preferably comprises one or more of the following components: lenses, apertures, mirrors, filters, telecentric lenses, and light guides. These are arranged such that the light from the light source 8, 10 can be shaped or guided on its path from the sample surface to the light detector 4 in such a way that the latter can optimally process the reflected and / or scattered light.

[0022] Furthermore, the device 100, 200 comprises a control unit 30 for controlling the translation stage 1, the energy source 20, 10 and the light source 8, 10, which is designed such that the surface of a sample can be irradiated point by point by the energy source 20 and the light source 8, 10.

[0023] In a second embodiment of the device 100, 200, the energy source 10, 20 is embodied as a desorption or ionization laser. The preferred wavelength lies in the ultraviolet, visible, or infrared range. Typical lasers used are KrF excimer lasers (λ = 248 nm) and frequency-quadrupled Nd:YAG lasers (λ = 266 nm). In contrast to other ionization methods (e.g., electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI)), lasers as energy sources 10, 20 allow the generation of ions independent of electric fields, since ions can only be formed in the laser beam. However, it is also possible to use a different energy source (20), e.g., an electrospray emitter or a primary ion or neutral particle source.

[0024] In a third embodiment of the device 100, 200, the light source 8, 10 is embodied as a laser. The preferred wavelength lies in the visible optical range, between 320 nm and 750 nm. This wavelength range represents a compromise between the highest possible resolution and the least possible impact on the surface.

[0025] In a fourth embodiment of the device 100, 200, it further comprises an ion guide channel 6 for guiding ions released from the surface of a sample by the energy source 20, 10. This channel is designed so that ions can be guided in a targeted manner to a connected mass spectrometer 7.

[0026] In a fifth embodiment of the device 100, 200, it further comprises a focusing system 11 for focusing the radiation from the energy source 20, 10 onto the surface of a sample. The focusing system 11 can comprise one or more of the following components: mirrors, lenses, apertures, filters, light guides, a lens with a central bore 5, and telecentric lenses.

[0027] In a sixth embodiment, the device 100, 200 further comprises a second optical system 12, namely a focusing system 12 for focusing the radiation from the light source 8 onto the surface of a sample. This allows only the microscale measuring point 60 to be illuminated, preventing scattered signals from adjacent areas. This is particularly important if the light source does not include internal optics. The second focusing system can comprise one or more of the following components: mirrors, lenses, apertures, filters, light guides, lenses with a central bore 5, and telecentric lenses.

[0028] The focusing system 12 for the light source 8 and the focusing system 11 for the energy source 20 can be used individually or in combination. They are controlled via the control unit 30.

[0029] In a seventh embodiment of the device 200, the energy source 10 for desorption / ionization and the light source 10 are formed by the same laser. ( Fig.4 This allows for a simpler and more cost-effective design of the device. A uniform focusing system is preferably used for this light source / energy source 10 in order to precisely irradiate a measuring point 60. The focusing system can be constructed from mirrors, lenses, apertures, filters, light guides, lenses with a central bore 5, and telecentric lenses.

[0030] Preferably, the light detector 4 of the device 100, 200 is designed such that it is positioned at an angle α of more than 0° (preferably more than 45°) relative to the sample surface, whereby it generates a measurement image of the sample surface and / or the measurement point 60.

[0031] The method according to the invention for mass spectrometric analysis and imaging of the surface of a sample provides a) that a sample to be examined is placed on the translation stage, with the sample being located on a sample carrier on the translation stage, b) that the sample to be examined is examined point-wise in scanning mode by moving the translation stage or the energy source, step by step or in continuous movement, c) wherein a working plane of the object is defined for this microscale scanning examination, d) wherein a microscale measuring point is generated by the light source and imaged on the sample by the second optical system, e) wherein the reflected / scattered measuring beam of the light source is imaged on the light detector by the first optical system, f) wherein the position of the image of the measuring point on the light detector is evaluated by the evaluation unit,and g) wherein the relative position change of the measuring point is calculated and the deviation of the z-position from the defined working plane of the object is calculated from this information, h) that with the help of this deviation, the current z-position of the object at the measuring point is moved into the specified working plane, i) wherein the displacement value is stored as height information for the measuring point, j) that chemical information (chemical composition) of the measuring point is recorded on the surface of the object using a detection method, k) wherein the desorption beam of the energy source - after approaching the working plane for the measuring point - is used to desorb and ionize analyte material from the microscale measuring point or to ionize it in a subsequent step, and l) wherein the ions move to an ion guide channel of the mass spectrometer, wherein the ion guide channel is designed to guide ions,which were released from the surface of a sample by the energy source, and m) that after scanning the sample surface by moving the translation stage in the x and y directions and by regulating the z coordinate for each measuring point, the topography of the sample surface is imaged by evaluating the z coordinate, whereby the mass spectrometric analysis of the ions from each microscale measuring point provides the chemical information about the composition of the sample.

[0032] In the inventive method for mass spectrometric analysis and imaging of the surface of a sample using a device 100, 200 according to one of the aforementioned embodiments, the sample to be examined is examined point by point in scanning mode by moving the translation stage or the energy source 20. Scanning mode refers to the systematic, regular scanning or measuring of the surface, either in a step-by-point sequence or in a temporally synchronized, continuous sequence of movement of the translation stage or the energy source and the resulting construction of a raster image. The main idea is to obtain an overall image of the surface with relatively limited measuring instruments through a large number of individual measurements. For this purpose, the x and y coordinates of each measurement point are always determined.The x and y coordinates describe the position of a point on a plane that is parallel to the surface of the sample.

[0033] In a second embodiment of the method, the z-coordinate or z-position of individual points on the sample surface is additionally determined. The z-coordinate is assumed to be the spatial direction perpendicular to the sample surface.

[0034] In a third embodiment of the method, the output signals of the light detector of the device are used to control the scanning operation, whereby a rasterization (movement in the xy plane) and a determination of the topography are carried out by evaluating the z coordinate.

[0035] In a fourth embodiment of the method, the position of the translation table 1 is specifically changed by the control unit 30 to realize the scanning operation.

[0036] In a fifth embodiment of the method, in order to realize the scanning operation, the position of the imaging system for focusing the energy source 20, 10 is specifically changed by the control unit 30.

[0037] In a sixth embodiment of the method, raw mass spectrometric data are obtained from the sample to be analyzed. The raw mass spectrometric data for a measurement point 60 are understood to include the mass-to-charge ratios, the ion signal intensities, and the coordinates (x, y, z).

[0038] In a seventh embodiment of the method, a multidimensional data matrix is ​​generated by assigning the calculated mass-to-charge number centroids, intensities, and Cartesian coordinates to each detected mass spectrometric signal.

[0039] In an eighth embodiment of the method, height and distribution information is translated into grayscale or color coding and pictorial representations are generated. Evaluation procedure:

[0040] To carry out the evaluation procedure, a sample to be examined is first placed on a translation table 1. It is preferably located on a sample carrier on the translation table 1. In the figures Fig.1 , 2 , 3 and 4shows how a microscale measuring point 60 is generated by a light source 8, 9, 10 and imaged onto the sample by a second optical system 12. The reflected / scattered measuring beam from the light source 8, 10 is imaged onto the light detector 4 by a first optical system 3. The position of the image of the measuring point on the light detector 4 is evaluated by the evaluation unit 40. The relative position change of the measuring point is calculated, and from this information, the deviation of the z-position from the working plane of the object previously defined for the microscale scanning examination is calculated. Using this value, the current z-position of the object at the measuring point is moved into the specified working plane. The displacement value is saved as height information for the measuring point.The chemical information (chemical composition) of the measurement point on the object's surface is recorded using a suitable detection method, such as laser desorption / ionization mass spectrometry (LDI-MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS), or desorption electrospray ionization mass spectrometry (DESI-MS). Laser-based spatially resolved methods have a high numerical aperture and thus a shallow depth of field at high spatial resolution (i.e., for small measurement points). Therefore, for a mass spectrometric investigation of the measurement point, the working plane for the respective measurement point must be approached with high precision.Other desorption / ionization methods sometimes have different geometric constraints for successful analysis of a measurement point, which also require moving to the working plane. For DESI-MS, for example, the angles and distances between the DESI beam, sample measurement point, and the inlet port of the mass spectrometer must be maintained.

[0041] In the illustrations Fig.1 , 2 , 3 , 4 The sample holder can also be positioned at an angle different from the angles shown. Image analysis methods

[0042] In an alternative embodiment of the method according to the invention, a light detector 4 with a first optical system 3 is positioned at an angle α of more than 0° relative to the sample surface. The sample surface is illuminated by the light source 8, 9, 10. By evaluating the information of the illuminated, imaged sample surface, the optimal focal plane of the desorption / ionization beam 2 of the energy source 20 is determined. With correct arrangement, for example, the highest contrast value or the maximum standard deviation of a 5x5 pixel image area corresponds to the focal plane of the desorption / ionization beam 2. The contrast value C is given, for example, by C = (I MAX -IA ) / IA , where I MAX is the maximum intensity of the image detected from the sample and IA is the average intensity of the image detected from the sample. By measuring the position, for exampleThe z-position can be corrected relative to the measurement point, e.g., the highest contrast value or the largest standard deviation, in order to move the sample at the measurement point into the focal plane of the ionization laser. The height information is derived from the travel path of the translation stage in the z-coordinate. Furthermore, it is possible to determine the position of the highest contrast or the largest standard deviation through systematic iteration and focus variation. The z-position for the given measurement point can also be derived from this information. In the embodiment of the device as shown in . Fig.4 Furthermore, as shown, the measuring point 60 on the sample generated by the desorption / ionization beam of the energy source 10 can be used directly to determine the height information.

[0043] After the working plane for the measurement point has been approached using one of the described methods, the selected desorption beam is used to desorb and ionize analyte material from the microscale measurement point, or to ionize it in a subsequent step. The ions move to the ion guide channel 6 of one of the described mass spectrometers 7. The mass spectrometric analysis provides direct information about the chemical composition of the microscale measurement point.

[0044] For each measurement point, the z-position of the measurement point is first moved into the working plane, thus determining the sample height at the measurement point. Subsequently, mass spectrometric analysis is performed to determine the chemical composition, before the next measurement point is approached, which in turn is analyzed for height and chemical information. After scanning the sample surface by moving in the x and y directions and adjusting the z-coordinates for each measurement point, the topography of the sample surface is mapped by evaluating the z-coordinates. The mass spectrometric analysis of the ions from each microscale measurement point provides the chemical information about the composition.

[0045] The obtained multidimensional information (topography, location-dependent chemical distribution and chemical composition) is evaluated with the developed software and visualized in multidimensional representations ( Fig. 5 .).

[0046] In a further embodiment, it is intended to use the device and the method in time-of-flight mass spectrometry.

[0047] Alternatively, the device is intended to be used in quadrupole, ion trap, ion cyclotron resonance or orbital trap mass spectrometry. Figure legends and list of reference symbols

[0048] Fig. 1 shows a device 100 for carrying out the method according to the invention. Fig. 2 shows the device 100 in a further embodiment using a focusing system 12 for a light source 8 and a focusing system 11 for an energy source 20. Fig. 3 shows the device 100 in the embodiment using a pierced lens 5. Fig. 4 In the embodiment, the device 200 shows that the desorption / ionization laser is also used as a light source for height determination. Fig. 5represents the data evaluation process. The spatial information (x, y, and z coordinates) generated by the device 100, 200 is read into the evaluation unit. The mass spectrometric raw data generated by the mass spectrometer 7 is also read into the evaluation unit via the interface. The evaluation unit generates a topographic image and a chemical distribution image of the sample. In a further step, the topographic image and the chemical distribution image are converted into a three-dimensional chemical distribution image and displayed. List of reference symbols

[0049] 1Translation stage 2Desorption / ionization beam of the energy source 3First optical system 4Light detector 5Lens with central bore 6Ion guide channel 7Mass spectrometer 8, 10Light source 11, 12Second optical system / focusing system 100, 200Device 20, 10Energy source 30Control unit 40Evaluation unit 50Interface 60Measuring point

Claims

1. Method for mass spectrometrically analysing and imaging the surface of a sample with a device (100, 200) comprising ▪ at least one energy source (20, 10) for desorption or desorption and ionisation of atoms or molecules of the surface of a sample for onward transmission to a mass spectrometer (7), ▪ a translation stage (1) which is designed in such a way that a sample to be analysed can be mounted on it and wherein the beam path of the energy source (20, 10) and the translation stage (1) are designed to be movable relative to each other, ▪ a light source (8, 10), ▪ a control unit (30) for controlling the translation stage (1), the energy source (20, 10) and the light source (8, 10), wherein the latter is designed such that the surface of a sample can be irradiated in a point-like manner at a measuring point (60) by the energy source (20, 10) and the light source (8, 10), ▪ an interface (50) for exchanging data with a mass spectrometer, wherein the interface (50) is designed such that measurement data on the chemical composition of the surface of the sample at a measurement point (60) can be transmitted from a mass spectrometer to the evaluation unit via the interface (50), so that the chemical composition of the measurement point (60) can be determined by the evaluation unit (40), ▪ wherein the device (100, 200) further comprises a first optical system (3), a light detector (4) and an evaluation unit (40), and ▪ wherein the light source (8, 10), the first optical system (3) and the light detector (4) are designed such that a measuring point (60) on a sample can be illuminated by the light source (8, 10) via a second optical system (12) and light reflected or scattered from the measuring point (60) can be detected by the light detector (4) with upstream first optical system (3), and a) that a sample to be analysed is placed on the translation stage (1), the sample being located on a sample carrier on the translation stage (1), b) that the sample to be examined is examined by moving the translation stage (1) or the energy source (20, 10) in a point-like manner in scanning mode in steps or in continuous movement, c) wherein a working plane of the object is defined for this microscale scanning examination, d) wherein a microscale measuring point (60) is generated by the light source (8, 10) and imaged on the sample by the second optical system (12), e) wherein the reflected / scattered measuring beam of the light source (8, 10) is imaged on the light detector (4) by the first optical system (3), f) wherein the position of the image of the measuring point on the light detector (4) is analysed by the evaluation unit (40), and g) wherein the relative change in position of the measuring point is calculated and the deviation of the z-position from the defined working plane of the object is calculated from this information, and working plane of the object is calculated, h) this deviation is used to move the current z-position of the object in the measuring point (60) into the predetermined working plane, i) whereby the displacement value is stored as height information for the measuring point (60), j) wherein a chemical information chemical composition of the measuring point (60) on the surface of the object is recorded by a detection method, k) wherein the desorption beam of the energy source (20, 10) - after moving to the working plane for the measuring point (60) - is used to desorb and ionise analyte material from the microscale measuring point (60) or to ionise it in a subsequent step, and l) wherein the ions move to an ion guide channel (6) of the mass spectrometer (7), the ion guide channel (6) being designed to guide ions released from the surface of a sample by the energy source (20, 10), and m) after scanning the sample surface by moving the translation stage (1) in the x and y directions and by regulating the z-coordinate for each measuring point (60), the topography of the sample surface is mapped by evaluating the z-coordinate, the mass spectrometric analysis of the ions from each microscale measuring point (60) providing the chemical information on the composition of the sample.

2. Method for analysis according to claim 1, characterised in that the multidimensional information obtained, namely topography, location-dependent chemical distribution and chemical composition, is evaluated using software and visualised in multidimensional representations.

3. Method for analysis according to claim 1 or 2, characterised in that output signals of the light detector or sensor control the device in scanning mode, whereby a rasterisation and a determination of the topography is carried out by evaluating the z-coordinate.

4. Method for analysis according to one of claims 1 to 3, characterised in that the position of the sliding table is specifically changed by the control unit (30) in order to implement the scanning operation.

5. Method for analysis according to one of claims 1 to 4, characterised in that the position of the focusing system (11) for focusing the radiation of the energy source (20, 10) is specifically changed by the control unit (30) in order to implement the scanning operation.

6. Method for analysis according to one of claims 1 to 5, characterised in that raw mass spectrometric data are obtained from the substances to be examined.

7. Method according to one of claims 1 to 6, characterised in that a multidimensional data matrix is generated by assigning the calculated mass-to-charge number centroids, intensities and Cartesian coordinates to each detected mass spectrometric signal.

8. Method for analysis according to one of claims 1 to 7, characterised in that the height and distribution information is translated into greyscale or colour codes and pictorial representations are generated.