DEVICE AND METHOD FOR THE SPECTROMETRIC ANALYSIS OF SAMPLE MATERIAL

Abstract

The disclosure relates to devices and methods for the spectrometric analysis of sample material located on a sample support, and in particular on a flat sample support plate, using axial time-of-flight analysis. One operating mode of the devices and methods comprises an adjustment of the pulse focal position for the abrupt ablation and/or abrupt desorption of sample material in a z-direction that is perpendicular to a tangential plane at the location of ablation and/or desorption at the sample support, and the selection of a suitable setting for an acceleration with time lag of the ablated and/or desorbed and ionized sample material onto a flight path. It can be particularly advantageous to use these devices and methods in mass spectrometry imaging (MSI). The devices and methods can, in particular, be used with laser desorption/ionization (LDI) and specifically matrix-assisted laser desorption/ionization (MALDI).

Claims

1. A device for the spectrometric analysis of sample material located on a sample support, comprising: an axial time-of-flight analyzer with a flight path emanating from the sample support, an ionization device that is arranged and designed to locally impact sample material on the sample support using ablation and/or desorption pulses, to ionize locally ablated and/or desorbed sample material and to adapt a pulse focal position along a z-direction that is substantially perpendicular to a tangential plane on an impingement point of an ablation and/or desorption pulse at the sample support, as a function of a sample material location in the z-direction, an extraction device that is arranged and designed to accelerate ionized sample material onto the flight path with a time lag, where the acceleration with time lag is coordinated with an ablation and/or desorption pulse and is performed using a setting that can be selected from a plurality of different settings that are designed for a plurality of predetermined sample material locations in the z-direction, a probing device that is arranged and designed to determine a sample material location in the z-direction for an impingement point of an upcoming ablation and/or desorption pulse, and a control and/or guidance system that communicates with the axial time-of-flight analyzer, the ionization device, the extraction device and the probing device and that is arranged and designed to control the extraction device in such a way that the determined sample material location in the z-direction is used to select a setting for the acceleration with time lag that follows the upcoming ablation and/or desorption pulse.

2. The device according to claim 1, wherein the plurality of settings comprises a corresponding plurality of time lags for the acceleration with time lag.

3. The device according to claim 2, wherein the plurality of time lags is allocated to discrete sample material locations in the z-direction in a reference table.

4. The device according to claim 2, wherein the plurality of time lags is recorded in a nanosecond grid containing intervals which are selected from among a group including: eight nanoseconds, six nanoseconds, four nanoseconds, two nanoseconds, one nanosecond.

5. The device according to claim 2, wherein the plurality of settings is parameterized in an equation as a function of the determined sample material location in the z-direction.

6. The device according to claim 5, wherein the equation is parameterized linearly, in accordance with: Time lag (height h)=a*h+b, where h is a relative reference sample material location in the z-direction at the ablation and/or desorption location, and a and b are constants of a regression from calibration data.

7. The device according to claim 1, wherein the control and/or guidance system is arranged and designed to convert times of flight to masses m or mass-related values, e.g. m/z, using time-of-flight correction values that can be selected from a plurality of time-of-flight correction values designed for a plurality of predetermined sample material locations in the z-direction.

8. The device according to claim 7, wherein a time-of-flight correction value is selected for the conversion using the determined sample material location in the z-direction.

9. The device according to claim 1, wherein the time-of-flight analyzer is arranged and designed with a rectilinear flight path or curved flight path, e.g. using at least one reflector.

10. The device according to claim 1, wherein the ionization device is arranged and designed to impact the sample material in transmission mode through the sample support, or in reflection mode with ablation and/or desorption pulses.

11. A method for the spectrometric analysis of sample material located on a sample support, executed using a device according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The invention can be better understood by referring to the following illustrations. The elements in the illustrations are not necessarily to scale, but are primarily intended to illustrate the principles of the invention (mostly schematically). In the illustrations, the same reference numbers designate corresponding elements in the different views.

[0038] FIG. 1 is a schematic representation of an arrangement of a device for the spectrometric analysis of sample material located on a sample support, in accordance with principles of the present disclosure (transmission mode arrangement).

[0039] FIG. 2 illustrates time-of-flight correction values as a function of the measured time of flight for different sample material locations in the z-direction.

[0040] FIG. 3 is a schematic representation of an additional arrangement of a device for the spectrometric analysis of sample material located on a sample support, in accordance with principles of the present disclosure (reflection mode arrangement).

[0041] FIG. 4 illustrates a position and angle adaptation of a pulse focal position with a non-perpendicular incidence of a pulsed beam onto a sample support and the sample material (z z), in accordance with principles of the present disclosure.

[0042] FIG. 5A shows a diagram in which an optimal time lag for acceleration with time lag is plotted against a sample material location in the z-direction.

[0043] FIG. 5B shows a diagram in which the mass resolving power is plotted against a sample material location in the z-direction, both with application of the principles of the present disclosure and (for reference) with a static setting.

DETAILED DESCRIPTION

[0044] While the invention has been illustrated and explained with reference to a number of embodiments, those skilled in the art will recognize that various modifications in form and detail can be made without departing from the scope of the technical teaching, as defined in the attached claims.

[0045] FIG. 1 shows a first arrangement of a device for the spectrometric analysis of sample material located on a sample support (2), and depicts, in particular, how a sample material location in a z-direction can be detected and how a pulse focal position can be adapted to the detected sample material location in the z-direction in order to bring about optimal ablation and/or desorption.

[0046] It shows an axial time-of-flight analyzer with an ionization device from which ionized sample material is pulsed, with a time lag, onto a flight path (4) that is deflected by a reflector (6) and therefore exhibits a non-rectilinear course before ending at a detector (8). The ion formation area is located between a sample support (2), on which the sample material has been deposited, and an extraction electrode (10) with a central aperture, which can, by means of a control and/or guidance system, be pulsed onto an electrical potential that attracts the ablated and/or desorbed and ionized sample material. The single extraction electrode (10) may also take the form of a more complex extraction electrode system. For illustrative purposes, the sample support (2) is depicted as having a stepped design, which means that there are always different sample material locations in a z-direction that corresponds to an ion extraction direction and that is preferably parallel to a surface normal of the sample support at the ablation or desorption location, even if other influencing variables, such as the sample material thickness or the fixation of the sample support (2) in a translation stage (not shown), are ideal. This type of stepped sample support (2) can, for example, be used for calibrating the settings of both the acceleration with time lag and the time-of-flight correction. Alternatively, a flat sample support held in a translation stage with a means of adjustment in the z-direction may be used for this type of calibration. The sample support (2) is at least partially conductive and is connected to a potential supply, e.g. a voltage source (12), in order to receive an electrical reference potential.

[0047] The translation stage (not shown) to which the sample support (2) is coupled can be moved in an xy-plane in steps that go down to a few micrometers. A means of adjustment in the z-direction is optional, but not necessary within the context of the present disclosure. The dashed outline of the sample support (2) indicates that it is transparent for electromagnetic waves in order to allow the application of ablation and/or desorption pulses (14) in transmission mode. The sample support may, for example be depicted as an ITO glass specimen slide. The pulses (14) can be delivered using a laser (16), e.g. a nitrogen gas laser or Nd:YAG laser with frequency multiplication to the ultraviolet spectral range. An imaging lens (18) or a lens system in the beam path can be moved along the z-direction using a suitable mechanism, thereby adjusting the object distance and the image plane so that a pulse focus can be set to different points along the z-axis. A control and/or guidance system can be organized centrally in a single computer or locally using different circuits or processors coupled together. The control and/or guidance system in the present disclosure comprises a trigger generator (20) for the temporally coordinated release of laser pulses, a delay generator (22) and an electronic circuit (24) for generating an electrical extraction pulse, which can also take the form of a temporally varying potential profile, for example in order to take account of the acceleration behavior of ionic species with different masses.

[0048] Along the flight path (4) beyond the extraction electrode (10), there is a lateral probing device (26) in the form of a combined image generation and capture system that can project predetermined light patterns onto the sample support in incident light at a predetermined angle, and depict the surface of the sample support carrying the sample material along the same optical axis (28). Although not shown in the illustration, it is also possible for the axis of the projected light pattern and the observation axis to be located opposite each other, on different sides of the extraction axis, so that the angle of incidence of the light corresponds to the angle of reflection of the observation axis. The light pattern may comprise, for example, a light spot or a row of light bars that are imaged onto the sample support (2) at the location of an upcoming ablation and/or desorption, which is preferably located on the extraction axis. The image capture system may have a video camera with upstream imaging optical system which can be used to localize the light pattern on the sample material or sample support (2) and whose lateral displacement from a reference position in an xy-plane, arising from the oblique incidence of the light pattern projection, makes it possible to determine the sample material location in the z-direction. This type of method allows location differences along the z-direction to be detected in the range of a few micrometers, e.g. between around 5 and 10 micrometers.

[0049] A possible operational sequence can look like this: the translation stage (not shown) moves the sample support (2) to a certain position in the xy-plane so that a certain part of the sample material lies on the extraction axis. In the present example, it may be, as shown, the reference position z.sub.1, which reflects a central position between the sample support surface and the extraction electrode (10), compared to the reference positions z.sub.0 and z.sub.2. The imaging system throws a light pattern onto the location where the sample material should be if it is correctly located in the z-direction. If this is the case, e.g. because the image capture system does not detect any lateral displacement of the projected light pattern, the pulse focal position does not need to be adapted, and the laser pulse (14) for ablation and/or desorption (including ionization where applicable, e.g. with MALDI) and the delayed downstream electrical extraction pulse can be triggered by the trigger generator (20) or the delay generator (22) without any further changes. If, however, a lateral displacement of the light pattern is detected, as is for example the case on the stepped sample support (2) with spacings z.sub.0 and z.sub.2 compared to z.sub.1, an adaptation command is generated and transmitted to the movement mechanism of the imaging lens (18) in the beam path of the laser, so that the lens (18), which may be part of a more complex lens system, is moved to a position and the pulse focal position adapted such that the area of the lowest lateral spread of the pulse (14) in the z-direction is preferably at the location of the determined sample material location in the z-direction.

[0050] At the same time, the sample material location in the z-direction is instantaneously transmitted to the delay generator (22), which then selects a setting for acceleration with time lag that best corresponds to the determined sample material location. This can be done by selecting the most closely matching time lag value from a reference table in the control and/guidance system's memory. Alternatively, a time lag can also be calculated in real time from the reference points set out in a reference table, e.g. by interpolation, extrapolation, or regression. Alternatively, the dependency of the time lags on the sample material location in the z-direction can be stored as an equation in a control and/or guidance system processor and used to derive the required time lag after applying or entering the determined sample material location in the z-direction. When the adjustment of the pulse focal position is complete and the corresponding setting for the acceleration with time lag has been set up in the circuit (24) for applying the extraction potential, the laser pulse (14) and the electrical extraction pulse that follows with a time lag can be triggered. As explained above, the extraction pulse may be temporally variable, for example in order to take account of the acceleration behavior of ionic species with different masses.

[0051] In order to take into account the varying sample material location in the z-direction also when converting times of flight to mass units or mass-related units such as m/z, a plurality of corresponding time-of-flight correction values for various sample material locations in the z-direction may be saved for retrieval in the control and/or guidance system. When the sample material location in the z-direction has been determined, the control and/or guidance system may process the spectral data delivered from the detector (8) accordingly, by converting the measured times of flight to corrected times of flight and then converting these values to masses or charge-related masses m/z in accordance with a predetermined mass calibration. An example of the time-of-flight correction values is shown in FIG. 2 with the plotting of the time difference t in nanoseconds as a function of the measured time of flight for different sample material locations in the z-direction (in this case 25, 50, 75 and 100 micrometers of deviation from a zero position t.sub.0 m). The points on the y-axis for the zero value indicate the fact that no time-of-flight correction value is needed for an optimal pulse focal position because the correction values are zero. The more the actual sample material location in the z-direction deviates from this zero position, the larger the time-of-flight correction values will be, e.g. a 6 nanosecond correction for a z-direction position of 50 micrometers at a measured time of flight of around 68 000 nanoseconds (=68 microseconds) or a 12 nanosecond correction for a z-direction position of 75 micrometers at a measured time of flight of around 94 000 nanoseconds (=94 microseconds). The parameters for the best-fit lines in the measured reference points, which were detected using a Bruker rapifleX, are specified.

[0052] It is also possible to add reference molecules to the sample material, e.g. as an admixture of the matrix substance for the MALDI method in order to use the ionic species resulting from these reference molecules for a real-time calibration of the recorded spectral data. This procedure is also referred to as the lock-mass method. It is possible to use ionic species of the matrix substance as reference molecules, e.g. with the inclusion of dimers, trimers or even higher polymers of the matrix substance.

[0053] FIG. 3 shows a further arrangement of a device for the spectrometric analysis of sample material located on a sample support (2), and also depicts how a sample material location in a z-direction can be detected and how a pulse focal position can be adapted to the detected sample material location in the z-direction in order to bring about optimal ablation and/or desorption.

[0054] It shows an axial time-of-flight analyzer with an ionization device from which ionized sample material is pulsed, with a time lag, onto a flight path (4) that is deflected by a reflector (6) and therefore exhibits a non-rectilinear course before ending at a detector (8). A rectilinear variant of the flight path (4) is shown in dashed contour, where the detector (8) is directly opposite the ion-receiving side of an extraction electrode (10) and the sample support surface carrying the sample material. This type of time-of-flight analyzer design shortens the flight path (4) and reduces the time of flight accordingly, but can increase the ion detection sensitivity. As before, the ion formation area is located between the sample support (2), on which the sample material has been deposited, and the extraction electrode (10) with a central aperture, which can, by means of a control and/or guidance system, be pulsed onto an electrical potential that attracts the ablated and/or desorbed and ionized sample material. The extraction electrode (10) may also take the form of a more complex extraction electrode system. The sample support (2), which is shown stepped here too, is at least partially conductive and is connected to a voltage source (12) as a potential supply in order to receive an electrical reference potential.

[0055] A translation stage (not shown) to which the sample support (2) is coupled can be moved in an xy-plane in steps that go down to a few micrometers. A means of adjustment in the z-direction is optional, but not necessary within the context of the present disclosure. The dashed outline of the sample support (2) indicates that it is transparent for electromagnetic waves in order to allow the optical determination of the sample material location in the z-direction.

[0056] In the embodiment shown, the ionization device comprises a laser (16), which can irradiate the pulses onto the sample support (2) in incident light at an angle to a surface normal. The laser (16) may be, for example, a nitrogen gas laser or Nd:YAG laser with frequency multiplication to the ultraviolet spectral range. An imaging lens (18) or a lens system in the beam path (14) can be moved along the direction of the pulse incidence onto the sample support (2) using a suitable mechanism, thereby adjusting the object distance and the image plane so that a pulse focus can be set to different points along the z-axis. It is important to take into account that a non-perpendicular incidence direction of the beam pulse (14) will mean that merely moving the lens to change the pulse focal position will also bring about a lateral displacement (x-y) of the pulse focus on the sample support (2), i.e. in the xy-plane. This displacement can, however, be derived from basic geometric principles, and can be easily corrected, for example by means of an angular deflection () using a pair of fast-actuating micromirrors (not shown) through which the laser pulse passes on its beam path (14), as shown concisely in FIG. 4.

[0057] As before, a control and/or guidance system can be organized centrally in a single computer or locally using different circuits or processors coupled together. The control and/or guidance system in the present disclosure comprises a trigger generator (20) for the temporally coordinated release of laser pulses, a delay generator (22) and an electronic circuit (24) for generating an electrical extraction pulse, which can also take the form of a temporally varying potential profile, for example in order to take account of the acceleration behavior of ionic species with different masses.

[0058] In an area behind the sample support (2), away from the ion-optical setups such as the extraction device and flight tube, in which the flight path (4, 4) is located, a probing device in the form of an optical microscope (26) is positioned. The optical microscope (26) contains a light source that can illuminate the back of the sample support in incident light. The observation axis (28) of the optical microscope (26) runs perpendicular to the back of the sample support, and is therefore optically insensitive to purely lateral movements of the sample support (2) along or parallel to the xy-plane, as may be performed by the translation stage (not shown). An image capture system of the optical microscope (26) is arranged and designed for determining the position of maximum contrast along the z-direction or the extraction axis. For this purpose, a focus stacking method can be used, in which different object distances in the z-direction are set, e.g. by adjusting an objective lens in the optical microscope (26), and the setting with the greatest image contrast can be determined as a function of the position in the z-direction.

[0059] A possible operational sequence can look like this: the translation stage (not shown) moves the sample support (2) to a certain position in the xy-plane so that a certain part of the sample material lies on the extraction axis. In the present example, it may be the reference position z.sub.1, which has a central distance to the extraction electrode (10), compared to the positions z.sub.0 and z.sub.2. The light source of the optical microscope (26) illuminates the sample material and the sample support (2) from the back, and applies a focus stacking method in order to determine the maximum contrast in this position. If the determined maximum contrast corresponds to the standard position in the z-direction, for which the acceleration with time lag setting is optimal, and for which there is no need for any correction of the times of flight, the pulse focal position does not need to be adjusted and the laser pulse at the time of ablation and/or desorption and the delayed downstream electrical extraction pulse can be triggered by the trigger generator (20) or the delay generator (22). If, however, a deviation from the standard position in the z-direction is detected, as is for example the case on the stepped sample support (2) with distances z.sub.0 and z.sub.2 in relation to z.sub.1, adaptation commands are generated and transmitted to the movement mechanism for the imaging lens (18) and the micromirrors (not shown) in the beam path (14) of the laser (16) so that the lens (18) is moved into a position and the pulse focal position adapted such that the area of the lowest lateral spread of the pulse in the z-direction is at the location of the determined sample material location in the z-direction, taking into account the lateral correction in the xy-plane by the corresponding optical system e.g. micromirror, see FIG. 4.

[0060] At the same time, the current sample material location in the z-direction is transmitted to the delay generator (22), which then selects a setting for acceleration with time lag that best corresponds to the determined sample material location. This can be done by selecting the most closely matching time lag value from a reference table. Alternatively, a time lag can also be calculated in real time from the reference points set out in a reference table, e.g. by interpolation, extrapolation, or regression. Alternatively, the dependency of the time lag on the sample material location in the z-direction can be stored as an equation in a control and/or guidance system processor and used to derive the time lag to be applied after applying or entering the determined sample material location in the z-direction. When the adjustment of the pulse focal position is complete and the corresponding setting for the acceleration with time lag has been set up in the circuit (24) for applying the extraction potential, the laser pulse and the electrical extraction pulse that follows with a time lag can be triggered.

[0061] FIG. 5A shows an experimentally determined dependency of the time lag of the delayed acceleration on a determined sample material location in the z-direction, on the basis that the mass resolution is to stay largely constant despite a changed sample material location. The investigated range for the sample material location in the z-direction was 0-100 micrometers, where 0 micrometers represents the largest possible distance from an extraction electrode, as shown as z.sub.0 in FIG. 1, for example, and where 100 micrometers represents a significantly smaller distance from the extraction electrode, as shown as z.sub.2 in FIG. 1. The observed mass is m/z 3147. The time lag changes from 160 nanoseconds to 200 nanoseconds between the two extreme positions, and behaves in a stable linear fashion, which allows parameterization with a best-fit line, whose parameters are given in the figure.

[0062] FIG. 5B shows the impact on the adjustment of the time lag on the sample material location in the z-direction and on the mass resolution compared to a scenario in which a static time lag is used for the delayed acceleration. The adjustment allows a stable high mass resolution of 50,000, whereas the static setting by means of a stroke setting in the z-direction leads to a reduction to around half the mass resolution (25,000). These results, which were obtained from the measured data from a Bruker rapifleX, which schematically corresponds to the set-up shown in FIG. 3, confirm the benefit brought about by the principles of the present invention for axial time-of-flight analysis.

[0063] The invention has been described above with reference to different, specific example embodiments. It is to be understood, however, that various aspects or details of the embodiments described can be modified without deviating from the scope of the invention. Furthermore, the features and measures disclosed in connection with different embodiments can be combined as desired if this appears practicable to a person skilled in the art. Moreover, the above description serves only as an illustration of the invention and not as a limitation of the scope of protection, which is exclusively defined by the appended Claims, taking into account any equivalents which may exist.