Desorption ion source with post-desorption ionization in transmission geometry

Abstract

The invention relates to a device for generating ions from a sample material deposited on a substrate at least partially transparent to electromagnetic waves, comprising: - a support device having a holder for the substrate, - a desorption / ionization unit comprising a desorption device and an ionization device, the desorption device being arranged and designed to desorb the deposited sample material from a desorption site on the substrate using at least one energy burst, and the ionization device being arranged and designed to irradiate the desorbed sample material above the substrate using electromagnetic waves after at least one energy burst, wherein the electromagnetic waves pass through the substrate at a position corresponding to the desorption site before entering the desorbed sample material, and - an extraction device arranged and designed to extract ions from the desorbed sample material and transfer them to an analyzer. The invention also relates to a correspondingly arranged method.

Description

Technical Field

[0001] The invention relates to a device for generating ions from deposited sample material, in particular for use in analytical systems (eg mobility spectrometers, mass spectrometers and combined mobility-mass spectrometers), and also to the use of the generated ions for further investigation and analysis. Background Art

[0002] The prior art is described below in conjunction with specific aspects. However, this should not be construed as a limitation of the subsequent disclosure of the present invention. Useful further developments and improvements known from the prior art may also be applied above and beyond the relatively narrow scope of this introduction, and will be readily apparent to those skilled in the art after reading the disclosure following this introduction.

[0003] The combination of desorption of deposited samples and subsequent (post) ionization of the desorbed material has long been known in mass spectrometry. This post-desorption ionization improves the ionization efficiency and thus the measurement sensitivity, especially for molecules that are strongly diluted and / or difficult to ionize in the sample. An example is secondary ion mass spectrometry (SIMS), which is supplemented and extended by secondary neutral mass spectrometry (SNMS) using post-desorption ionization mode. A review article entitled "Use of Post-Ionisation Techniques to Complement SIMS Analysis, A Review With Practical Aspects" by HJ Mathieu et al. (High Temperature Materials and Processes, Vol. 17: No. 1-2, 1998, 29-44) relates to this topic.

[0004] SIMS, whether or not accompanied by SNMS, is mainly used for surface analysis, usually on untreated samples, where the atomic and molecular ions to be detected are directly generated by interaction with a high-energy primary ion beam in a high vacuum. On the other hand, matrix-assisted laser desorption / ionization (MALDI) uses a matrix material that forms crystals as its ionization medium. The crystalline matrix material in which the sample is embedded absorbs laser light (usually in the UV region of the spectrum) and responds to the laser bombardment by ablation, generation of a (excess) supply of charge carriers and transfer of charge carriers to the sample molecules that are simultaneously ablated.

[0005] MALDI mass spectrometry is also faced with the challenge of increasing the ionization yield of molecular ions, which has led to attempts in the field to adopt post-desorption ionization modes. One example of this can be found in the publication WO 2010 / 085720A1. It describes a method in which an ablation laser beam is directed from the front to a sample slide on which a sample has been deposited, and a beam of one or more post-desorption lasers ("POSTI lasers") is directed through a desorption cloud parallel to the sample slide and in a plane directly above it. A similar setup is described in the publication "Mass spectrometry imaging with laser-induced postionization" by Jens Soltwisch et al. (Science, 348 (6231), 211-215); in this case, it is referred to as MALDI-2.

[0006] As an alternative to ablation from the front, a post-desorption ionization mode of a MALDI ion source with a transmission geometry was also tested, in which the ablation laser beam passed through a suitably transparent sample slide so that the laser energy for ablation was introduced into the sample from behind. Examples of such arrangements can be found in the publications “Combining MALDI-2 and Transmission Geometry Laser Optics to Achieve High Sensitivity for Ultra-High Spatial Resolution Surface Analysis” by Eric C. Spivey et al. (Journal of Mass Spectrometry, Vol. 54, No. 4, April 2019, pp. 366-370), “Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution” by M. Niehaus et al. (Nature Methods, Vol. 16, pp. 925-931 (2019) and “Atmospheric Pressure MALDI Mass Spectrometry Imaging Using In-Line Plasma Induced Localization” by Efstathios A. Elia et al. Induced Postionization)" (Analytical Chemistry, 2020, 92, 23, 15285–15290), the latter involving plasma-induced post-ionization modes.

[0007] Extending this to the optical post-ionization modality, publication WO 2020 / 046892 A1 describes an apparatus and method for simultaneously illuminating a sample on a specimen slide from the front and back using a small diameter, laser-generated beam to image a mass spectrometer with subcellular spatial resolution. Some embodiments produce a beam of minimum diameter that substantially corresponds to the diffraction limit of twice the laser wavelength. It is believed that the high sensitivity of MALDI-TOF imaging with a single laser shot per laser for each pixel in the image can therefore be achieved.

[0008] In view of the above, there is a need to further significantly improve the sensitivity of LDI-MS (LDI = laser desorption / ionization), for example by MALDI-2, especially for imaging (mass spectrometry imaging - MSI). By reading the following disclosure, the skilled person will immediately understand the further objectives that can be achieved by the present invention. Summary of the invention

[0009] According to a first aspect, the present invention relates to a device for generating ions from a sample material deposited on a substrate which is at least partially transparent to electromagnetic waves, comprising: - a supporting device having a holder for the substrate, wherein the substrate can take the form of a glass plate which is transparent to electromagnetic waves in the ultraviolet, visible and / or infrared spectral regions, - a desorption / ionization unit comprising a desorption device and an ionization device, wherein the desorption device is arranged and designed to desorb the deposited sample material from a desorption site on the substrate using at least one energy burst, and wherein the ionization device is arranged and designed to irradiate the desorbed sample material above the substrate using electromagnetic waves after at least one energy burst, wherein the electromagnetic waves pass through the substrate at a position corresponding to the desorption site before entering the desorbed sample material, and - an extraction device arranged and designed to extract ions from the desorbed sample material and transfer them to an analyzer.

[0010] Guiding the transmitted electromagnetic waves through a substrate, wherein the sample material has already been (substantially to almost completely) desorbed by a previous energy burst or a short sequence of energy bursts at a desorption location which may comprise a portion of the total sample carrying area of ​​the substrate, allows the optical elements required for beam guidance, such as lenses or mirrors, to be kept away from the space where the ions are generated and guided. This makes the design of the ion source considerably easier, since these optical elements do not affect the potential in the ionization space for guiding the ions, in particular when the optical elements have a large aperture and are therefore close to the desorption location. Furthermore, the arrangement of the ionization device according to the invention is advantageous, since this eliminates the risk of contamination of the optical elements.

[0011] Furthermore, the electromagnetic wave beam used for post-desorption ionization penetrates the desorption cloud in a direction substantially consistent with the propagation direction of the desorption cloud (generally substantially parallel to the normal to the substrate surface). The interaction path between the electromagnetic wave and the desorbed sample material is thus prolonged, especially compared to prior art arrangements, such as MALDI-2, in which the post-desorption ionization pulse has an orientation substantially perpendicular to the propagation direction of the desorption cloud. Thus, the electromagnetic wave can excite the desorbed neutral molecules substantially over the entire range of the cloud, even if they are already far from the substrate surface, which results in a corresponding increase in the supply of charge carriers, rather than just in the relatively narrow focus of the transverse incident light beam located above the substrate as in the prior art. Furthermore, the electromagnetic wave irradiation used for post-desorption ionization can thus be prolonged for up to a period of several milliseconds before targeting the next desorption site on the substrate, which also increases the ionization probability and thus increases the ionization yield.

[0012] In various embodiments, the support device may include a chamber in which the holder for the substrate is located and the chamber is arranged and designed to create a conditioned environment for the substrate including the deposited sample material. For example, the chamber may be connected to a vacuum source to evacuate the environment of the deposited sample material, such as to a pump. The vacuum source may be arranged and designed to maintain a pressure significantly higher than a high vacuum (>10 -3 hPa) and less than about 10 2 Hectopascals (less than atmospheric pressure), for example a pressure of 1-10 hectopascals.

[0013] In various embodiments, the chamber can be connected to a gas supply device that is arranged and designed to feed an inert buffer gas, a reaction gas (e.g., methane), a humid gas (e.g., water vapor), and / or a dopant gas that is susceptible to absorbing electromagnetic waves into the chamber. For example, molecular nitrogen or helium are possible inert buffer gases. As described in the applicant's parallel application 102020120394.2, a dopant gas, such as a polar aprotic solvent such as acetone, a polar protic solvent such as isopropanol, or a non-polar solvent such as toluene, is preferably capable of absorbing electromagnetic waves, which react with the electromagnetic waves to excite and provide additional charge carriers, such as protons, which can be transferred directly to neutral, desorbed sample molecules in a chemical reaction through the excited molecules of the dopant gas or in a reaction cascade. The dopant gas is also preferably volatile and has a high vapor pressure to prevent excessive deposition on surfaces in the device, such as an ion source and downstream components of the analyzer.

[0014] In various embodiments, the desorption device is preferably arranged and designed to direct an energy beam onto the deposited sample material to trigger at least one energy burst. The energy beam may be a laser beam for ablating the deposited sample material. The laser beam may in particular be pulsed; furthermore, a plurality of identical or similar laser pulses generated in rapid succession may be used for desorption / ablation. Preferably, the energy beam passes through the substrate at a position corresponding to the desorption site before encountering the deposited sample material. Different types of energy bursts that may act on the deposited sample material for the purpose of desorption may be generated, for example, by locally focused acoustic waves with ultrashort pulse duration, for example in an acoustic transducer substrate that is partially transparent to electromagnetic waves.

[0015] In various embodiments, the ionization device may comprise a laser, a (broadband) discharge lamp or a light emitting diode (LED) for generating coherent electromagnetic waves. The wavelength of the laser light is preferably in the ultraviolet region of the spectrum, below 400 nm, for example 355 nm, 349 nm, 337 nm or 266 nm, which can be produced by many widely available solid-state and gas lasers. Lasers operating in pulsed operation or continuous wave operation in discontinuous mode may be used, or discharge lamps or LEDs with flash-like or continuous emission characteristics may be used. The discharge lamp may be an arc discharge lamp with high intensity broadband photon emission, for example a UV flash lamp such as a xenon flash lamp, or a hydrogen / deuterium discharge lamp or the like.

[0016] In various embodiments, the ionization device is preferably arranged and designed for irradiating the desorbed sample material by electromagnetic wave pulses that are temporally coordinated with at least one energy burst. In the case of a pulsed laser, the pulse duration can be a few nanoseconds. After at least one energy burst for desorption, before the support device moves the substrate to a different desorption position, the irradiation period of a continuous wave laser operating in a discontinuous mode can be a few microseconds. The irradiation time can sometimes last for tens of microseconds until the desorption cloud is greatly thinned or the density of the desorption cloud is significantly reduced. The time scale of such a process depends, among other things, on the ambient pressure at the desorption location, as well as on the setup and operation of the extraction device, such as gas dynamics and / or electromagnetic, such as continuous extraction of ions or pulsed extraction or a combination thereof, such as a continuous gas flow plus an extraction voltage that may be pulsed.

[0017] In various embodiments, the desorption device and the ionization device can use the same original beam of coherent electromagnetic waves, which can be adjusted to have different energies for desorption / stripping and ionization, respectively, as described, for example, in the applicant's application DE 102016 124 889 A1 (corresponding to GB 2 558 741 A, US 2018 / 0174815 A1 and CN 108206126 A). For example, on different multiplier paths, coherent light with an original wavelength of 1064 nanometers in the near infrared can be tripled to 355 nanometers for desorption / stripping, and quadrupled to 266 nanometers for post-desorption ionization. The necessary multiplier crystals can be located on parallel bifurcated branches in the optical path, after which the beam paths are merged again into a single path and then pass through optical imaging elements, such as lenses, support devices and the substrate itself.

[0018] In order to ensure that in each case only a light beam of a predetermined wavelength passes through the support device and the substrate, an electro-optical gate can be arranged where the light beam begins to diverge. Depending on the switching state, the electro-optical gate can guide the coherent electromagnetic wave of the first polarization to the first branch of the bifurcation, where the first multiplier path is located, for example, for energy tripling, while the coherent electromagnetic wave of the second polarization enters the second branch of the bifurcation, where the second multiplier path is located, for example, for energy quadrupling. For example, the polarization of the original light beam can be adjusted using the method described in the applicant's application DE 10 2015 115 416 A1 (corresponding to GB 2 542 500 A, US 2017 / 0076932 A1 and CN 106531607 A). Alternatively, a beam splitter may be positioned in front of the bifurcation, which redirects the original light beam onto two parallel paths simultaneously, and an electro-optical switch may be positioned for each path before the point where the two parallel paths merge again, the operation of the switches being coordinated so that at any one time only one switch is switched to allow passage, while the other blocks the beam path, for example by means of a switchable aperture.

[0019] In various embodiments, the extraction device may include at least one deflection electrode, which is arranged and designed so that the extracted ions change their direction of motion at least once. The deflection electrode can be provided with a voltage continuously, pulsed or discontinuously, which attracts or repels ions according to the electrode polarity, thereby causing a change in the direction of motion. In a further embodiment, the extraction device can use gas dynamics to extract ions from the desorbed sample material and pass them to the analyzer. For example, a transmission element such as a transmission capillary can be provided, which generates a gas flow to a downstream chamber or space at a lower pressure so as to entrain ions from the desorbed sample material. In a preferred embodiment, the extraction device combines gas dynamics, such as by generating a directional gas flow, and electrokinetic principles, such as by applying a voltage continuously or pulsed when necessary, so as to extract ions from the desorbed sample material and pass them to a downstream component, such as a mobility analyzer, a mass analyzer or a combined mobility-mass analyzer.

[0020] According to a second aspect, the present invention also relates to a method for generating ions from a sample material, comprising: - depositing the sample material on a substrate that is at least partially transparent to electromagnetic waves, - desorbing the deposited sample material from desorption sites on the substrate using at least one energy burst, - ionizing particles and / or molecules in the desorbed sample material above the substrate by irradiating them with electromagnetic waves, wherein the electromagnetic waves pass through the substrate at a position corresponding to the desorption site before they encounter the desorbed sample material, and - extracting ions from the desorbed sample material and transferring them to an analyzer.

[0021] In various embodiments, the sample material can include a tissue section, a homogenate, or a separate material deposited on a substrate. For example, the tissue section can be a thin slice of an animal organ, such as the liver, kidney, or brain of a laboratory mouse, which is studied and analyzed using a mass spectrometer and / or a mobility spectrometer with respect to the spatial distribution of the analyte molecules of interest. A particular embodiment can be that the material of the tissue section is desorbed by electromagnetic waves whose wavelengths are strongly absorbed (e.g., in the near infrared) by the water contained in the tissue section (especially over the entire layer thickness of the tissue in order to be able to work in transmission). For example, the material deposit can include a formulation produced dropwise (i.e., individually) on a sample support, such as a MALDI matrix formulation in a similar formulation array, such as in an AnchorChip TM The common feature of all these sample materials is that their substrates have a plurality of desorption sites which are spatially offset and / or spatially separated from one another and which are processed in a predetermined sequence using the above-described methods or devices.

[0022] Particularly preferred is a method as described above, carried out on a device as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention may be better understood by referring to the following drawings. The various elements in the drawings are not necessarily drawn to scale, but are mainly intended to illustrate the principles of the present invention (mainly schematic). The same reference numerals represent the same elements in various figures.

[0024] Figure 1A A schematic example embodiment of a device according to the invention is shown.

[0025] Figure 1B , Figure 1C and Figure 1D Schematically shows the Figure 1A There are three steps in the operation of the device.

[0026] Figure 2 Illustrative example embodiments are shown in which the desorption device and the ionization device use laser light from the same laser system to produce one energy burst or a series of energy bursts and post-desorption ionization.

[0027] Figure 3 A schematic example embodiment is shown, in which some components of the support device and the extraction device are located in a pumping chamber.

[0028] Figure 4A , Figure 4B and Figure 4C Three steps in the operation of another exemplary embodiment of the device according to the invention are schematically shown. DETAILED DESCRIPTION

[0029] While the invention has been described and illustrated with reference to several embodiments, those skilled in the art will recognize that changes may be made in form and details without departing from the scope of the technical teaching as defined in the following claims.

[0030] Figure 1AThe arrangement of a first exemplary embodiment is schematically shown. It shows a support device 2 with a holder 4 for carrying a substrate 6 of a sample material 8. The holder 4 can be a positioning device; for example, it can be designed in the style of an xy translation stage, which can be moved in two spatial directions in order to sequentially introduce several desorption sites on the substrate 6 into the desorption position. For example, alternative translation devices also allow movement in the z direction (perpendicular to the substrate surface) in order to adjust the optical focus and / or compensate for morphological differences between the sample material and the substrate material (e.g. glass). As an alternative to the illustrated embodiment in the form of a solid plate, the holder 4 can also take the form of a frame, which holds the substrate 6 essentially on the narrow sides, so that the large surfaces in front and behind are essentially unaffected and remain free. This arrangement is particularly advantageous for the passage of electromagnetic waves. The substrate 6 is preferably a sample support with standard dimensions, such as the dimensions of a microtiter plate often used for mass spectrometry and / or migration spectrometry measurements of desorbed molecules, and at least partially transmits electromagnetic waves. It can be a glass specimen slide commonly used in microscopes. As shown, the sample material 8 can be a single flat piece of sample material, such as a tissue section, which is scanned for mass spectrometry to generate a two-dimensional view ("map") of its molecular composition, for example, for biomolecules (such as proteins, peptides, lipids, glycans, etc.), drugs, metabolites, etc.

[0031] Figure 1A The view in shows the desorption / ionization unit 10 below the support device 2. The unit comprises a desorption device 12 and an ionization device 14. In this embodiment, the desorption device 12 comprises a laser system 16 for generating a high-energy laser beam, which is directed to the support device 2 by means of different optical elements (e.g. lenses and mirrors) so that it passes through the support device 2 and the substrate 6 at a position corresponding to the desorption site on the front side of the substrate 6. The energy beam can then interact with the sample material 8 at the desorption site on the front side of the substrate 6, and the applied energy causes the corresponding section of the sample material to be ablated into an expanding cloud above the substrate 6 at the desorption site. Directing the high-energy desorption beam to be transmitted through the substrate 6 allows the last imaging lens 18 to be positioned very close to the substrate 6, which makes it technically very easy to focus the beam into a very small focus at the desorption site on the surface of the substrate 6. The latter is a condition for scanning planar sample material 8, such as tissue sections for single cell analysis, at subcellular grid sizes (<1 micron).

[0032] In this embodiment, the ionization device 14 comprises a light source 20, which can be in the form of a laser system, for example. The ionization device 14 shown shares some of its optical elements with the desorption device 12. In the example shown, these shared optical elements are firstly a semi-transparent mirror 19, which is used to deflect the electromagnetic waves emitted by the light source 20 while transmitting the laser beam of the desorption device 12, and secondly an imaging lens 18, wherein the lens 18 can also be regarded as a placeholder structure for a more complex lens system. The lens 18 or the corresponding lens system preferably has a chromatic correction. The electromagnetic waves of the light source 20 are transmitted through the support device 4 and the substrate 6 in a manner coordinated in time with the desorption to the desorption cloud above the desorption site, where they interact directly or indirectly through secondary chemical reactions with the desorbed sample material in the gas phase and induce further ionization.

[0033] The holder 4 and the substrate 6 and finally the imaging lens 18 are preferably in such a form that they have substantially the same transmittance and imaging properties for electromagnetic waves of different wavelengths, if applicable, for example, 355 nanometers for the laser beam of the desorption device 12 and 266 nanometers for the electromagnetic beam of the ionization device 14. Suitable materials for these light-transmitting elements are, for example, quartz glass and calcium fluoride, preferably as a lens system in a chromatic compensation embodiment.

[0034] Figure 1A The view in shows an extraction device 22 above the support device 2. The extraction device 22 includes several electrodes to which voltage can be applied permanently or temporarily so as to extract ions from the desorption cloud and guide them to a mobility analyzer system, a mass analyzer system or a mobility-mass analyzer combination system (schematically represented as 24). In the first section, the extraction device 22 may include an electrode stack 26 in the form of an RF voltage funnel, i.e., a porous diaphragm arranged in sequence and having a central hole, the size of the central hole varying with the stack, in particular becoming smaller, so that the extracted ions have a stronger spatial focusing. Therefore, the porous diaphragm with the largest pore size faces the desorption site. There is a deflection electrode 28 behind the RF funnel 26 and on one side of the ion path, to which a voltage can be applied permanently or temporarily, the voltage repels ions of a specific polarity to deflect their path into a second RF funnel 30, which is located opposite the deflection electrode 28 and whose inner hole gradually becomes smaller in the direction away from the deflection electrode 28. The analyzer 24 can be located in an environment with a different pressure level, for example, at a lower pressure, and then receive and process the ions that have undergone a stronger spatial focusing. However, the neutral particles and molecules in the desorption cloud are not affected by the deflection electrodes 28 and are free to dissipate and thin out.

[0035] For the first step, Figure 1BAn energy burst for desorbing a sample material 8 is shown, wherein the laser system 16 of the desorption device emits a short high-energy laser pulse 32 which, after passing through the light-conducting element, the support means, the substrate 6, penetrates the sample material 8 from the rear at a position corresponding to the desorption site. The sample material 8 can be a flat tissue section which has been deposited on a glass plate and treated over the entire surface with a matrix material present in a crystalline state. The laser light can have a wavelength of about 355 nanometers and can be produced, for example, by frequency tripling the light of a Nd:YAG laser in the infrared region of the spectrum at 1064 nanometers. The prepared sample material 8 and the energy burst or a series of identical or similar energy bursts in rapid succession are coordinated in such a way that the sample material 8, including the matrix material, is almost completely ablated at the desorption site. This can be achieved very reliably by setting the number of pulses, the pulse length and the energy density of the laser beam. Flat sample material 8, for example a tissue section of about 10 micrometers thick treated with a matrix, can easily be completely desorbed on a local level.

[0036] For the second step, Figure 1C Depicted is how the stripped sample material expands and therefore becomes thinner in the gas phase away from the desorption site. In this example, the treatment with a MALDI matrix material on the substrate 6 ensures that the stripped matrix material provides charge carriers in the form of protons, which are transferred to the desorption cloud 34 while the stripped sample molecules and thereby complete the first ionization step. Unfortunately, the ionization efficiency of the simple MALDI process (i.e., MALDI-1) is relatively low, which means that a considerable portion of the stripped neutral sample molecules are not ionized by the charge carriers generated by applying one or more energy bursts. The ionization yield here may vary depending on the molecular species. For example, it is known that lipids ionize relatively poorly under standard MALDI conditions.

[0037] For the third step, Figure 1D As shown, in order to further increase the ionization yield of each energy burst or series of desorption energy bursts, a high-energy electromagnetic wave 36 is guided by the ionization device into the desorbed sample material above the desorption site, and it passes through the support device and the substrate 6 from back to front - just like the ablation radiation of the desorption device. In this way, it reaches the desorption cloud 34, where it interacts with the molecules to be ablated and in particular excites the matrix molecules, thereby providing a large number of charge carriers in the form of protons, which are transferred to the ablated neutral sample molecules. This is particularly beneficial for the ionization of highly diluted or difficult to ionize molecules in the sample material 8, such as lipids. The electromagnetic wave 36 can take the form of pulses generated and emitted in rapid succession, for example by a pulsed laser, or it can originate from an ionization device operating in a discontinuous mode, for example, time-limited operation of a continuous wave laser, wherein the time limit can be set in particular in the range of a few milliseconds, or originates from a time-limited emission period of a discharge lamp.

[0038] Figure 1B The desorption steps and Figure 1D The desorption and ionization steps between Figure 1C The states shown should be regarded as schematic. Figure 1C The duration of the state between desorption and post-desorption ionization shown in can be very short, such as a few microseconds or only a few nanoseconds. Figure 1C Embodiments are also conceivable in which the separate state of the desorption cloud expansion shown does not actually exist, i.e., one energy burst or a series of energy bursts for desorption and the electromagnetic wave pulse for post-desorption ionization occur in sequence with virtually no time delay, in which case the desorption device and the ionization device must cooperate with each other. A time delay that is small to almost non-existent can have the advantage that the desorbed sample material cloud is still very dense when the electromagnetic wave penetrates it, which increases the probability of interaction between photons and desorbed particles.

[0039] The ions formed can be extracted from the desorption cloud by means of permanent or temporary voltages on the electrodes of the extraction devices 26, 28, 30 and fed to the analyzer 24. Those electromagnetic waves 36 which have passed through the desorption cloud 34 without interacting with the molecules therein can be caught by a beam dump 38 located in the optical path after the extraction device; such a beam dump eliminates high-energy radiation from the device without reaching undesired or undesired places and without having any effect there.

[0040] From Figures 1A to 1D The ionization device 14 can be operated using high-energy laser radiation 32, for example with a wavelength of 266 nanometers, which can be generated by quadrupling a Nd-YAG laser originally having an infrared wavelength. If, contrary to expectations, the pulse or series of pulses of desorption ablation does not completely expose the substrate surface at the desorption site, the electromagnetic waves 36 used for post-desorption ionization can also ablate or desorb the residues of the sample material 8 located at the desorption site, in addition to the effect on the sample material that has already been desorbed or ablated. The electromagnetic waves 36 of the ionization device can also interact with fragments (fragments) of sample material formed during the desorption process and entrained in the desorption cloud, so as to transfer such fragments of sample material into the gas phase and ionize them. This additional post-ablation or post-desorption (residual) sample material can also further increase the yield of the generated ions and thus also contribute to achieving the goal.

[0041] After the ions resulting from desorption / stripping and subsequent post-desorption ionization have been extracted from the desorption cloud 34 and transferred to the analysis system 24, the holder 4 can move the substrate 6 to another desorption position (xy translation), possibly including adjustment of the focus (z translation), so that the still untouched part of the sample material 8 can be analyzed. If the sample material 8 is a tissue section, its surface can be scanned in a specific order to compile a molecular composition profile, for example with respect to lipids, proteins, peptides, glycans or similar biomolecules, as well as with respect to drugs and their breakdown products, (endogenous) metabolites, etc.

[0042] Figure 2 An improved arrangement of an embodiment is shown, in which both desorption / stripping and post-desorption ionization are affected by high energy electromagnetic waves 32, 36 transmitted through the holder 4 and the substrate 6, as shown in FIG. Figures 1A to 1D Here, the desorption device and the ionization device not only share several optical elements, but also share a laser system 40. For the sake of clarity, the extraction device is not shown in this example.

[0043] The difference between the desorption device and the ionization device here lies in particular in the assigned multiplier paths 42, 44, which can adjust the original beam or original pulse of the laser system 40 to different wavelengths. This can be achieved by a polarization-dependent beam splitter 46, which deflects the laser light of a specific polarization to the first multiplier path 42, which polarization can be applied in the laser resonator of the laser system 40, while the laser light with a different polarization is transmitted to the second multiplier path 44. Then, the beams or pulses of different conditions can be gathered again on one optical path before they pass through the holder 4 and the substrate 6. Alternatively, the beam splitter 46 can also pass the electromagnetic waves of the original laser beam or pulse to the two multiplier paths 42, 43 at the same time. Before the branch optical paths merge, the adjusted beam or pulse can be selected so that it is guided through the holder 4 and the substrate 6 through the switchable apertures 48A, 48B.

[0044] The intensity of the conditioned beam or pulse of each subpath can be set by a beam splitter (46). In particular, an attenuator can be inserted into at least one or each subpath in order to keep possible pulse-to-pulse variations small. Preferably, the switchable aperture can also blank the pulses by means of a fast electro-optical element. Other irrelevant wavelengths can also be filtered out by means of a Perlin-Broca prism and / or a dichroic filter.

[0045] As shown, the optical path lengths of the two subpaths are preferably the same to simplify the time coordination of the switching states of the apertures 48A, 48B. It should be understood that when the original wavelength of the laser system 40 is already suitable for one of the purposes, i.e., desorption / ablation or desorption followed by ionization, the subpaths do not require a multiplier path.

[0046] Figure 3 Shows Figures 1A to 1DA further modification of the setup is shown corresponding to Figure 1D The substrate 6 with the sample material 8 deposited thereon and one of the RF funnels 26 of the extraction device are located in a chamber 50 in which a regulated gas environment can be generated. A pump 52 is connected to the chamber 50 and maintains a predetermined pressure condition. For example, the pressure in the chamber 50 can be lower than atmospheric pressure (<10 2 hPa), preferably in a moderate vacuum (>10 -3 hPa), for example 1-10 hPa. The chamber 50 is simultaneously connected to a gas feeder 54, through which a buffer gas, a reaction gas (e.g., methane), a humid gas (e.g., water vapor) and / or a dopant gas can be fed into the chamber 50, so that the balance of gas inflow and outflow can be set and controlled. Inert gases such as nitrogen or helium are possible buffer gases. Possible dopant gases include many volatile solvents, which can act as additional ionizing media in the gas phase and interact with the electromagnetic waves 36 of the ionization device to provide charge carrier donors by optical or photoexcitation, which transfer charge carriers, such as protons, to neutral desorbed sample molecules. In this way, the optical or photoexcitation process in the gas phase is not limited to desorbed molecules (e.g., matrix molecules with MALDI ionization), and the supply of charge carriers can be expanded, for example, by proton donors in the gas phase above the desorption site.

[0047] Figures 4A to 4C Another embodiment is shown, in which the principle of the present disclosure is implemented. The support device and the ionization device here may correspond to Figures 1A to 1D The differences lie in particular in the desorption device and the extraction device, which will be explained in more detail below.

[0048] In this example, the desorption device includes a system that directs an energy beam 32* at a slightly oblique incident angle from the front side onto a substrate 6, which in this embodiment is coated with a separate sample deposit or sample preparation 8*. When the energy beam 32* is a laser beam that ablates a single sample 8*, and the sample material is prepared with a concentrated MALDI matrix substance, such as an organic acid, it can be called reflection mode ablation, which is similar to Figures 1A to 1D Ablation in transmission mode is shown to be the opposite.

[0049] In the example shown, the extraction device comprises an RF funnel 26 for receiving and spatially focusing ions from the desorption cloud, said funnel having local grooves across the stack of membrane electrodes for passing the energy beam 32* (not shown), and a plurality of deflection electrodes 56 arranged behind the RF funnel 26 opposite each other on one side of the propagation direction of the cloud, to which a voltage can be applied permanently or temporarily so that the direction of motion of the extracted ions changes twice, each time by approximately 90°. Further electrodes 58 of the ion guide can then guide the deflected ions to a connected analyzer (not shown), with a path substantially parallel to the original extraction direction of the ions through the RF funnel 26.

[0050] Figure 4A Depicted is the first step in the desorption of a single sample deposit 8* by a high-energy pulsed beam 32*. Figure 4B As shown, in a second step, a desorption cloud 34 generated by the material of the deposited and possibly prepared sample 8* expands above the desorption site on the substrate 6, in this case at a slightly inclined angle relative to the surface normal above the substrate 6 due to the correspondingly angled direction of incidence of the desorption pulse series 32*. In contrast, Figure 4C The triggering of the post-desorption ionization beam of a pulsed electromagnetic wave 36 is shown, which is transmitted through the support means and the substrate 6 from back to front at a position corresponding to the desorption site and completely penetrates the desorption cloud 34 in the propagation direction of the desorption cloud. The energy input into the cloud 34 is used to excite the desorbed neutral molecules, thereby increasing the supply of charge carriers, which increases the ionization probability and improves the detection sensitivity in a connected analyzer (not shown).

[0051] The generated ions substantially follow the path indicated by the arrows between the deflection electrodes 56, 58 of the extraction device, while the neutral components dissipate and thin out in the source region. On the other hand, the unused part of the electromagnetic wave 36 of the ionization device can be absorbed by the beam dump 38 in the optical path and neutralized.

[0052] After the ions formed during desorption and subsequent post-desorption ionization have been extracted from the desorption cloud 34 and transferred to the analysis system, the holder 4 can move the substrate 6 to a further desorption position (xy translation), including adjustment of the focus if necessary (z translation), so that a single sample 8* that is still unprocessed can be analyzed, which is typically used, for example, for AnchorChip TM Single MALDI spot preparation on a plate.

[0053] The present invention has been shown and described above with reference to a number of different embodiments thereof. However, it will be appreciated by those skilled in the art that various aspects or details of the present invention may be varied, if feasible, or various aspects or details of different embodiments may be arbitrarily combined without departing from the scope of the present invention. In general, the above description is for illustrative purposes only and not for the purpose of limiting the present invention, which is limited only by the appended claims, including any equivalent embodiments, as the case may be.

Claims

1. Apparatus for generating ions from a sample material deposited on a substrate at least partially transparent to electromagnetic waves, comprising: - a supporting device having a holder for the substrate, a desorption / ionization unit comprising a desorption device and an ionization device, the desorption device being arranged and designed to desorb the deposited sample material from a desorption site on the substrate using at least one energy burst, and the ionization device being arranged and designed to irradiate the desorbed sample material above the substrate using electromagnetic waves after the at least one energy burst, wherein the electromagnetic waves pass through the substrate at a position corresponding to the desorption site before entering the desorbed sample material, and - An extraction device arranged and designed to extract ions from the desorbed sample material and transfer them to the analyser.

2. Apparatus according to claim 1, wherein the support means comprises a chamber in which a holder for a substrate is located and which is arranged and designed to create a conditioned environment for the substrate comprising the deposited sample material.

3. The apparatus of claim 2, wherein the chamber is connected to a vacuum source to evacuate the environment of the deposited sample material.

4. The apparatus according to claim 2, wherein the chamber is connected to a gas supply device, which is arranged and designed to feed an inert buffer gas, a reaction gas, a humid gas and / or a dopant gas that easily absorbs electromagnetic waves into the chamber.

5. The apparatus of claim 1, wherein the desorption device is arranged and designed to direct an energy beam onto the deposited sample material to trigger at least one energy burst.

6. The apparatus of claim 5, wherein the energy beam is a laser beam for ablating deposited sample material.

7. The apparatus of claim 5, wherein the energy beam passes through the substrate at a location corresponding to a desorption site before encountering the deposited sample material.

8. The apparatus of claim 1, wherein the ionization device comprises a laser, a discharge lamp or a light emitting diode (LED) for generating coherent electromagnetic waves.

9. The apparatus of claim 8, wherein the laser is operated in pulsed mode or in continuous wave mode, or the discharge lamp or LED has flash-like or continuous emission characteristics.

10. The apparatus according to claim 1, wherein the ionization device is arranged and designed to irradiate the desorbed sample material by a pulse of electromagnetic waves coordinated in time with at least one energy burst.

11. The apparatus according to claim 5, wherein the desorption device and the ionization device use the same original beam of coherent electromagnetic waves, which are adjusted to have different energies for desorption / ablation and ionization, respectively.

12. The apparatus according to claim 1, wherein the extraction device comprises at least one deflection electrode, which is arranged and designed so that the extracted ions change their direction of movement at least once.

13. A method of generating ions from a sample material, comprising: - depositing the sample material on a substrate at least partially transparent to electromagnetic waves, - desorbing the deposited sample material from a desorption site on the substrate using at least one energy burst, - ionizing particles and / or molecules in the desorbed sample material above the substrate by irradiating them with electromagnetic waves which pass through the substrate at locations corresponding to desorption sites before they encounter the desorbed sample material, and - Extracts ions from the desorbed sample material and transfers them to the analyzer.

14. The method of claim 13, wherein the sample material comprises a tissue section, a homogenate, or a separate material deposited on a substrate.

15. The method of claim 13, wherein the method is performed on the device of claim 1.

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