DE102005006125B4 - Laser system for the ionization of a sample through matrix-assisted laser desorption in mass spectrometric analysis - Google Patents

Abstract

Laser system (300) for the ionization of a sample (301) by matrix-assisted laser desorption in mass spectrometric analysis, characterized in that the laser system (300) contains an adjustable device with which the laser radiation is spatially modulated, and the intensity distribution of the laser radiation on the sample (301) in one setting of the device consists of a single intensity peak and in another setting consists of a plurality of intensity peaks, the adjustable device a) one behind the other in the beam path of the laser system (300) a lens array (306), a variable optical system (307) , a first focusing optic (309) and a second focusing optic (304), and wherebyib) the variable optical system (307) can be adjusted so that in different settings there is one of several optical planes behind the lens array (306) into the front focal plane of the first focusing optics (309) images, and wherebyic) the sample (301) is in the rear focal plane of the second focusing optics (304).

Description

The invention relates to a laser system for ionizing a sample by means of matrix-assisted laser desorption in mass spectrometric analysis.

The invention consists in providing an adjustable laser system which in one setting generates a single intensity peak and in another setting a plurality of intensity peaks on the sample, the single intensity peak and / or the intensity peaks with respect to the half-width, the intensity, the spatial arrangement and / or the spatial degree of modulation are adjustable.

State of the art

In mass spectrometric analysis, two methods for soft ionization of biological macromolecules have become established in the last 10 to 15 years: matrix-assisted laser desorption / ionization (MALDI, abbreviation for "Matrix Assisted Laser Desorption Ionization") and electrospraying (ESI, abbreviation for " Electro Spray Ionization "). The biological macromolecules to be analyzed are referred to below as analyte molecules. In the MALDI method, the analyte molecules are usually prepared on the surface of a sample carrier in a solid matrix, while in the ESI method they are dissolved in a liquid. Both methods have a major influence on the mass spectrometric analysis of biological macromolecules in genomics, proteomics and metabolomics; its inventors were awarded the Nobel Prize in Chemistry in 2002.

In a prepared MALDI sample, the matrix molecules are present in a 10 ³ to 10 ^5- fold excess over the analyte molecules and form a polycrystalline matrix in which the analyte molecules are incorporated individually inside the crystals or at their grain boundaries. The prepared MALDI sample is briefly irradiated with a laser pulse that is strongly absorbed by the matrix molecules. The pulsed irradiation causes the matrix to explode from the solid state of aggregation into the gas phase of an evaporation cloud (desorption). The ionization of the analyte molecules usually takes place through their protonation or deprotonation in reactions with matrix molecules or matrix ions, the analyte ions being predominantly simply charged after leaving the vaporization cloud. The degree of ionization of the analyte molecules is only about 10 ^-4 . One speaks of soft ionization when an analyte molecule is isolated and transferred into the gas phase and ionized without breaking the bond.

The matrix-assisted laser desorption / ionization is, despite the linear absorption by the matrix, a non-linear process which, for pulsed laser radiation with a duration of a few nanoseconds, only starts from an intensity threshold of around 10 ⁶ watts per square centimeter. For soft ionization, the maximum intensity is at an upper limit of approximately 10 ⁷ watts per square centimeter. With a typical duration of the laser pulses of around ten nanoseconds, the aforementioned intensity limits result in a fluence of between 10 and 100 millijoules per square centimeter.

The MALDI process is complex and is influenced by many factors, some of which are interdependent. Since the MALDI method was first published in 1988, many parameters have been investigated and varied. Nevertheless, the processes in the matrix and in the vaporization cloud that lead to the ionization of the analyte molecules are not yet fully understood and are still being intensively researched ( K. Dreisewerd, Chem Rev. 103 (2003), 395-425: "The Desorption Process in MALDI" ).

The chemical parameters of the MALDI process, such as the matrix substances themselves, the concentration ratio between matrix and analyte molecules and the preparation conditions, have been examined in detail. For analyte molecules of different chemical substance classes, such as proteins or nucleic acids, over one hundred different chemical matrix substances are known, such as sinapic acid, DHB (abbreviation for "2,5-dihydroxy-bencoic acid"), CHCA (abbreviation for "α-cyano- 4-hydroxy cinnamic acid ", cinnamic acid) or HPA (abbreviation for" 3-hydroxypicolinic acid "). The matrix substances show strong absorption in the wavelength range between 330 and 360 nanometers. A MALDI sample can be prepared in different ways, for example with the "Dried Droplet" preparation or the "Thin Layer" preparation. In the "Dried Droplet" preparation, the matrix substance is dissolved together with the analyte molecules in a solvent, applied to a sample carrier and then slowly dried in air. In thin-layer preparation, on the other hand, the matrix substance without analyte molecules is dissolved in a volatile solvent, such as acetone or acetonitrile, and applied to the sample carrier. Compared to the "Dried Droplet" preparation, the volatile solvent evaporates very quickly and enables the creation of a thin, homogeneous matrix layer. A solution with analyte molecules is then applied to the thin matrix layer, which partially redissolves it and the analyte molecules are incorporated into the matrix during the subsequent drying process. While a homogeneous MALDI- Sample with microcrystals is created, larger crystals form in the "Dried Droplet" preparation and the surface of the MALDI sample shows a pronounced morphology with different sample thicknesses.

In terms of the physical parameters of the MALDI process, the duration of the laser pulses, the intensity in the laser focus and the wavelength of the pulsed laser radiation have so far been considered.

For commercially available mass spectrometers with MALDI, pulsed laser systems in the ultraviolet spectral range (UV) are predominantly used nowadays. There are different laser types and wavelengths to choose from: nitrogen laser (λ = 337nm), excimer laser (λ = 193nm, 248nm, 308nm), Nd: YLF laser (λ = 349nm) and Nd: YAG laser (λ = 266nm, 355nm), where For the MALDI process, only the nitrogen laser and the Nd: YAG laser at a wavelength of 355 nanometers are commercially important and the nitrogen laser is used most frequently by a large margin. The laser medium of the nitrogen laser is a gas, while the Nd: YAG laser is a YAG crystal (yttrium aluminum garnet: Y ₃ Al ₅ O ₁₂ ) doped with neodymium ions. With the Nd: YAG laser, the strongest laser line, which is at a wavelength of 1064 nanometers, is converted into the specified wavelengths in non-linear optical crystals. The duration of the laser pulses used in the MALDI method is typically between 1 and 20 nanoseconds in the UV, but pulse durations in the picosecond range have also been used in academia.

For research purposes, laser systems that emit in the infrared spectral range (IR) are occasionally used for the MALDI process: Er: YAG (λ = 2.94 µm) and CO2 (λ = 10.6 µm). While in the UV-MALDI process energy is supplied to the matrix molecules via excited electronic states, in the IR-MALDI process molecular vibrations of the matrix molecules are excited. The pulse duration of the IR laser systems in the IR-MALDI process is between 6 and 200 nanoseconds. In contrast to the UV-MALDI process, the IR-MALDI process uses not only solid matrices but also liquid matrices, such as glycerine.

Laser systems used in the MALDI process differ not only in terms of their wavelength, but also in terms of their spatial beam profile. In solid-state lasers, such as the Nd: YAG LAser or the Er: YAG laser, the laser medium is a crystal doped with ions. The laser medium is located in an optical resonator, which ensures that the spatial beam profile consists of a basic transverse mode or a few transverse beam modes. The radial intensity distribution of the transverse fundamental mode corresponds to a Gaussian function and is rotationally symmetrical to the direction of propagation of the laser radiation. Such a laser beam can be focused on a minimal diameter limited only by the diffraction.

The nitrogen laser with a wavelength of 337 nanometers is by far the most frequently used laser type in the MALDI process, and this wavelength is the most intense laser line of the nitrogen laser. Gaseous nitrogen, which is excited by an electrical discharge between two electrodes, is used as the laser medium. Since the most intense laser line has a high gain, a laser pulse can already reduce the population inversion of the energy states if it only passes through the electrodes once. Even when using resonator mirrors, many transverse beam modes are superimposed in the beam profile of the nitrogen laser, so that the minimum diameter of a laser focus of commercial nitrogen lasers at a wavelength of 337 nanometers is only about three micrometers. The typical diameter of the area irradiated in MALDI applications is around 20 to 200 micrometers. The beam profile of the nitrogen laser has an almost rectangular plateau on the electrodes (in English "flat top"), with the width and height of the beam profile being determined by the distance or height of the discharge electrodes. The repetition frequency of the laser pulses in the nitrogen laser is limited to around 100 Hertz if a rapid gas exchange is not ensured. For MALDI applications, nitrogen lasers with a typical repetition frequency of 50 Hertz are used.

In practice, the electrical gas discharge in the nitrogen laser is not the same at every point between the electrodes and creates a spatially inhomogeneous amplification profile that does not even out during the short duration of the laser activity, but transfers to the beam profile of the nitrogen laser. The nitrogen laser thus has a spatially modulated plateau-shaped beam profile with intensity maxima and minima, which is imaged or focused on the sample. These inhomogeneities of the nitrogen laser that are inherent in the beam profile mean that the intensity distribution of the laser radiation on the sample is spatially modulated and always has a large number of intensity peaks.

The pulsed solid-state lasers used to date in the MALDI process mostly have a beam profile that comes very close to a single Gaussian beam mode. If a pulsed laser beam is focused or imaged on the sample, a Gaussian intensity distribution with a single intensity peak results at the location of the sample. The The width of an intensity peak is usually indicated by the so-called half-width. In the area of the half-width, the intensity is greater than half the maximum intensity of the intensity peak. The half-width for solid-state lasers in the UV can theoretically be less than one micrometer, but in MALDI applications it is typically between 20 and 200 micrometers. Even if, in principle, repetition frequencies of the laser pulses of a few hundred kilohertz are possible with solid-state lasers, most MALDI applications currently work with a repetition frequency of up to 200 Hertz. The energy fluctuations from laser pulse to laser pulse are typically smaller with solid-state lasers than with nitrogen lasers.

According to the prior art, a spatially homogeneous intensity distribution is often sought on the sample in order to average out the inhomogeneities of the prepared MALDI sample, such as, for example, in the case of uneven storage of the analyte molecules in the matrix. In order to obtain a homogeneous intensity distribution on the sample with a Gaussian beam profile of a solid-state laser, the beam profile can be spatially homogenized by the propagation in a fiber and then mapped onto the sample. For this purpose, the laser beam is coupled into a fiber in which a large number of transverse fiber modes with different radial intensity distributions can propagate (multimode fiber). As the coupled laser beam propagates in the multimode fiber, energy is transferred from the Gaussian beam mode into many transverse fiber modes that are superimposed at the fiber exit. If the temporal coherence of the laser radiation used is sufficiently small or the multimode fiber is sufficiently long, the intensity distribution at the fiber output results from the sum of the intensity distributions of the individual transverse fiber modes. The large number of transverse fiber modes with different radial intensity profiles thus results in a homogeneous intensity distribution at the fiber exit. If the output of the multimode fiber is now mapped, a plateau-shaped intensity distribution is also obtained on the sample. This method of homogenizing the beam profile is also used with nitrogen lasers in order to minimize the inherent inhomogeneities in the beam profile.

The quality of a mass spectrometric analysis is generally determined by the following parameters: the mass accuracy, the mass resolution, the detection capability, the quantitative reproducibility and the signal-to-noise ratio. The quality of a mass spectrometric analysis increases if at least one parameter is improved and the other parameters are not impaired as a result. The mass accuracy includes both a systematic deviation of the measured mean ion mass from the true ion mass (mass accuracy or, better, mass inaccuracy) as well as the statistical spread of the individual measured values around the mean value of the ion mass (mass precision). The mass resolution shows which ion masses can still be differentiated in the mass spectrometric analysis. In practice, however, not only the quality but also the robustness of the mass spectrometric analysis is important. A mass spectrometric analysis is robust if its quality changes little when the measurement parameters vary, such as the energy of the laser pulses or the preparation conditions of the MALDI sample.

The ion signal of a mass spectrometer with MALDI is proportional to the ionization efficiency, the desorbed sample volume and the concentration of the analyte molecules in the sample. The ionization efficiency results from the number of analyte ions that can be evaluated divided by the number of analyte molecules in the desorbed sample volume, i.e. what percentage of the analyte molecules from the sample volume removed by the laser irradiation are available as ions for a mass spectrometric analysis. In the event that analyte molecules are already ionized in the matrix before the desorption process, the number of analyte molecules increases by the number of analyte ions already ionized. Since the desorbed sample volume can be increased relatively easily by the irradiated sample area and by the fluence, the ionization efficiency is an important parameter for optimizing the MALDI process low sample consumption). With a typical degree of ionization of only 10 ^{-4 there} is the possibility of considerably improving the MALDI process. The definition of the ionization efficiency of the MALDI process also takes into account the losses that occur due to the fragmentation of analyte molecules during transfer into the gas phase and thus reduce the number of analyte ions that can be evaluated.

In principle, classical sector field mass spectrometers and quadrupole mass spectrometers as well as quadrupole ion trap mass spectrometers and ion cyclotron resonance mass spectrometers can be used for the mass spectrometric analysis of the analyte ions generated in the MALDI process. However, time-of-flight mass spectrometers with axial insertion are particularly suitable, since they require a pulsed ion current to measure the time of flight (TOF, abbreviation for "Time Of Flight"). The time for the start of the time-of-flight measurement is specified by the ionizing laser pulse. The MALDI process is originally for has been developed for use in a vacuum. In more recent developments, matrix-assisted laser desorption / ionization is also used at atmospheric pressure (AP MALDI, abbreviation for "Atmospheric Pressure Matrix Assisted Laser Desorption Ionization"). Here the ions are generated with a repetition frequency of up to 2 kilohertz and with the help of an ion guide system, a time-of-flight mass spectrometer with orthogonal injection (OTOF, abbreviation for "Orthogonal Time Of Flight"), a quadrupole ion trap mass spectrometer or an ion cyclotron resonance Mass spectrometer supplied. In an OTOF mass spectrometer, the ions generated in the MALDI process can be fragmented and stored before the measurement of the flight time is started with an electronic pulse.

With special analysis methods, the intensity on the sample is increased to such an extent that the generated ions have enough internal energy to dissociate. Depending on the length of time between the generation of the ions and their dissociation, one speaks of a decay within the ion source (ISD, abbreviation for "In Source Decay") or outside the ion source (PSD, abbreviation for "Post Source Decay").

There are also imaging mass spectrometric analysis methods (IMS, abbreviation for "Imaging Mass Spectrometry") in which the MALDI process is used to generate the ions. In the IMS, a thin tissue section, which is obtained from a human organ with a microtome, for example, is prepared with a matrix substance and examined spatially resolved by mass spectrometry. The spatial resolution of the mass spectrometric examination can be achieved either by scanning individual small points of the tissue section or by stigmatic imaging of the generated ions. In the scanning process, the pulsed laser beam is focused on a small diameter on the sample, with a mass spectrum being measured for each individual scanning point. A one- or two-dimensional frequency distribution for individual proteins is created from the large number of individual, spatially resolved mass spectra. With stigmatic imaging, an area of up to 200 by 200 micrometers is homogeneously irradiated with a laser pulse. The ions generated in the process are imaged point by point on a spatially resolving detector. So far, only the frequency distribution of an ion mass can be recorded with a single laser pulse, since there are no sufficiently fast spatially resolving ion detectors. However, the measured ion mass can be varied from laser pulse to laser pulse.

The publication of the patent application US 2003/0025074 A1 describes a high throughput laser desorption / ionization mass spectrometer (LDI) that uses an ion source with a plurality of lasers that fire in tandem at one or more samples to increase the rate at which ion packets are generated in the ion source .

The international application WO 2005/079360 A2 discloses an arrangement of optical devices for the rapid structuring of laser profiles which are used for desorption and / or ionization sources in analytical mass spectrometry.

Registration DE 10 2004 044 196 A1 the applicant describes laser systems for MALDI ionization that generate laser beams with specific beam profiles for desorption.

Object of the invention

It is the object of the present invention to provide a laser system for the ionization of a sample by matrix-assisted laser desorption in mass spectrometric analysis, which ensures a high quality and robustness of the mass spectrometric analysis with different chemical parameters, analysis methods and mass spectrometers, in particular with different sample preparations and imaging mass spectrometric analysis methods.

Solution of the task

The object is achieved by a laser system according to the attached patent claim 1. Advantageous designs of the laser system can be found in the attached subclaims 2, 3 and 4.

The present invention is based on the far-reaching knowledge that the quality and robustness of the mass spectrometric analysis of ions generated with the MALDI method, with different chemical parameters (e.g. sample preparations), analysis methods and mass spectrometers, largely depend on the intensity distribution on the MALDI -Sample to be determined. A laser system according to the invention can therefore be adjusted in such a way that the intensity distribution of the laser radiation on the MALDI sample consists of a single intensity peak in one setting and a plurality of intensity peaks in another setting. Furthermore, with a laser system according to the invention, the half-width or the intensity of the individual intensity peaks or both can be set. There are at least two intensity peaks among a large number of intensity peaks understand which can be adjusted with a laser system according to the invention all or in part with respect to the half-width, the intensity, the spatial arrangement and / or the spatial degree of modulation. The number of intensity peaks can also be changed with a laser system according to the invention.

In complete contrast to the previous state of the art, it has been shown that an intensity distribution with a large number of fine intensity peaks is advantageous for the quality and robustness of the mass spectrometric analysis when the MALDI samples are produced with the thin-layer preparation. If the analyte ions are analyzed in particular in a time-of-flight mass spectrometer with an axial injection, the ionization efficiency, the mass resolution and the signal-to-noise ratio can be significantly increased compared to a spatially homogeneous intensity distribution and an intensity distribution with only a single, coarse intensity peak. In the case of sample preparation according to the third-droplet method, on the other hand, an intensity distribution with a single, relatively coarse intensity peak can surprisingly have advantages over an intensity distribution with many fine intensity peaks.

In particular, with the two imaging mass spectrometric analysis methods, different intensity distributions are advantageous. In order to achieve a high spatial resolution in the mass spectroscopic analysis using the scanning method, the intensity distribution on the sample should consist of a single fine intensity peak with a small half-width. A large number of fine intensity peaks can in turn favor stigmatic imaging, since in thin-layer preparation the number of analyte ions generated on the irradiated surface is increased through optimized ionization efficiency. If the positions of the intensity peaks on the sample are also known, the spatially resolved detector signal can easily be assigned to a location on the sample.

A laser system according to the invention generally consists of a laser medium, an energy supply to excite the laser activity, an optical resonator and optical and electro-optical components for spatial and temporal modulation of the laser radiation. In the following, a laser system is understood to mean the entire structure of optical, electrical and electro-optical components that are necessary for generating and modulating the laser radiation from the laser medium to the location of the MALDI sample. The components for spatial modulation of the laser radiation can be located both inside the optical resonator close to the laser medium and outside the optical resonator. Examples of such components are lenses, lens arrays consisting of a large number of lenses, mirrors, active Q-switches for pulse generation, diffractive optical elements (e.g. grids) and non-linear optical crystals. It is clear to the person skilled in the art that not all components mentioned have to be used in a laser system according to the invention and that further components can be added.

In contrast to the previous state of the art, a laser system according to the invention can generate very different intensity distributions on the MALDI sample, which enable the quality and robustness of the mass spectrometric analysis to be optimized for the respective chemical parameters, analysis methods and mass spectrometers. The intensity distribution is adjusted either manually or automatically by a control software to the respective conditions. For the person skilled in the art it is of course evident that the implementation of a laser system according to the invention is possible in diverse embodiments.

Brief description of the images

The shows a laser system in which a lens array and a spherical lens can be moved perpendicular to the optical axis of the laser system.

The to show a laser system in which a spherical lens can be moved along the optical axis and, as a result, different optical planes behind a lens array are mapped onto the sample in a reduced size.

The shows a laser system in which a large number of parallel bundles of rays are generated, all of which have different directions to the optical axis and are focused on the sample by a spherical lens.

Preferred Embodiments

The shows a first preferred embodiment of a laser system according to the invention ( 100 ). The laser unit ( 103 ) is a Nd: YLF laser that generates a time-pulsed laser beam at a frequency tripled wavelength of 349 nanometers. The active laser medium is a (LiY _1.0-x Nd _x F ₄ ) crystal doped with neodymium ions. The laser pulses of the Q-switched laser unit ( 203 ) have a pulse duration of around 10 nanoseconds. The spatial beam profile corresponds to a good approximation of a single Gaussian beam mode. The energy of the laser pulses can be measured with one in the laser unit ( 103 ) integrated attenuator. The type of laser medium and that of the laser unit ( 103 ) generated wavelength are for all embodiments of the present invention are not essential; all wavelengths suitable for the MALDI process can be used equally.

The Lens ( 106 ) and the lens array ( 107 ) can one after the other through a mechanism in the beam path of the laser system ( 100 ) so that the rear focal planes of the lens ( 106 ) and the lens array ( 107 ) in the plane of the diaphragm ( 108 ) lie. A kind of turret mechanism is available, which is known from microscopy for various objectives. The lens array ( 107 ) and the lens ( 106 ) generate in the plane of the diaphragm ( 108 ) a spatial intensity distribution that consists of a large number of intensity peaks or a single intensity peak. The aperture ( 108 ) is through the lens ( 109 ) into the intermediate image level ( 110 ), which in turn through the lens ( 104 ) and the deflection mirror ( 105 ) reduced to the sample ( 101 ) is mapped. The image scale is typically around 1: 6. The use of the intermediate image plane ( 110 ) is structurally advantageous, since the mechanical and optical elements that are necessary to generate the various intensity distributions can be arranged at a distance from the sample in the beam path.

The sample ( 101 ) is with other samples not shown on the sample carrier ( 102 ) and contains the analyte molecules built into a solid matrix. If the swell intensity for the MALDI process on the sample ( 101 ) is exceeded, the explosive evaporation of the matrix begins. The analyte molecules are transferred into the gas phase with the matrix and are present in the vaporization cloud to a certain extent as analyte ions. Through the deflection mirror ( 105 ) the laser system ( 100 ) spatially decoupled from the mass spectrometer, not shown, and thereby facilitates the transfer of the ions generated in the MALDI process into the mass spectrometer.

The lens array ( 107 ) has a base area of 25 square millimeters, on which spherical lenses are typically arranged at a distance of 120 micrometers in a square grid. Each individual lens of the lens array ( 107 ) has a focal length of around 10 millimeters. On the sample, the intensity peaks are 20 micrometers apart and have a half width of 10 micrometers. The single lens ( 106 ) has a focal length of 25 millimeters and generates a single intensity peak on the sample with a half-width of about one micrometer.

Between the intensity peaks, the sample ( 101 ) may not be ionized evenly at all points. To the sample ( 101 ) to be used as completely as possible with a series of laser pulses, it may therefore be necessary to determine the position of the intensity peaks relative to the sample ( 101 ) to change. This can be achieved, for example, by turning the deflecting mirror ( 105 ) is tilted or the sample carrier ( 102 ) is moved. A shift in the imaging lens ( 109 ) perpendicular to the optical axis is possible.

Is used in the laser system ( 100 ) instead of the lens ( 109 ) a zoom lens is used, the image scale between the diaphragm planes ( 108 ) and ( 110 ) must be set so that the distance between the panels ( 108 ) and ( 110 ) preserved. For example, the distance between the intensity peaks and the half-width of the individual intensity peaks can be set by means of a variable imaging scale. The individual intensity peak can be changed continuously from a fine intensity peak with a half-width less than 10 micrometers to a coarse intensity peak with a half-width greater than 100 micrometers.

Furthermore, the lens array ( 107 ) also consist of a large number of cylindrical lenses that generate a large number of line foci in the rear focal plane. The line foci can also be understood as intensity peaks, which, however, have two different half-widths along and across the line focus. Of course, in addition to the lens ( 106 ) and the lens array ( 107 ) swivel further lenses or lens arrays into the beam path using the same mechanism so that in the diaphragm plane ( 108 ) and thus on the test ( 101 ) generate more than two different intensity distributions.

The to show a second preferred embodiment of a laser system according to the invention ( 200 ). The laser unit ( 203 ) consists of a Nd: YAG laser that generates a time-pulsed laser beam at a frequency tripled wavelength of 355 nanometers. The laser pulses of the Q-switched laser unit ( 203 ) have a pulse duration of about 7 nanoseconds. The spatial beam profile corresponds almost to a Gaussian beam mode. The energy of the laser pulses can be measured with one in the laser unit ( 203 ) integrated attenuator.

In the generates the lens array ( 206 ) a multitude of intensity peaks in the rear focal plane. The lens array ( 206 ) consists, as in the first embodiment, of a multiplicity of spherical lenses and has similar geometric parameters. The entire lens array ( 206 ) is made entirely of quartz glass. The Lens ( 207 ) forms the rear focal plane of the lens array ( 206 ) in a 1: 1 mapping into the intermediate image plane ( 208 ) which in turn through the lens ( 204 ) eight times reduced to the sample ( 201 ) is mapped. The individual foci of the lens array ( 206 ) are thus mapped onto the sample in a reduced size; a multitude of intensity peaks arise there. In contrast to the first embodiment, the lens array remains ( 206 ) always in the same optical plane, while the lens ( 207 ) in the to is moved along the optical axis.

In the is the lens ( 207 ) so in the direction of the intermediate image plane ( 208 ) moved so that the plane directly behind the lens array ( 206 ) reduced to the intermediate image level ( 208 ) is mapped. Since the laser beam is directly behind the lens array ( 206 ) is imaged and the lens array ( 206 ) has a small thickness and is transparent, arises in the intermediate image plane ( 208 ) a reduced image of the laser beam. The laser beam has in front of the lens array ( 206 ) a diameter of about one millimeter. Through the two lenses ( 204 ) and ( 207 ) a single intensity peak with a half width of about 80 micrometers is generated on the sample. Will the lens ( 207 ) in the direction of the lens array ( 206 ) shifted and this enlarged to the intermediate image level ( 208 ) mapped, arises on the sample ( 201 ) a single intensity peak with a half width of about 200 micrometers.

In the is the lens ( 207 ) at a single focal length in front of the intermediate image plane ( 208 ) and focuses the laser beam. The intensity distribution in the intermediate image plane ( 208 ) has, in addition to a dominating main maximum, further secondary maxima. The secondary maxima arise due to the diffraction of the laser beam at the lens array ( 206 ). The main maximum corresponds to the zeroth diffraction order. Since the lens array ( 206 ) has relatively coarse structures in the range of 100 micrometers, the intensities of the secondary maxima are orders of magnitude lower than the intensity of the main maximum. The half width of the main maximum in the intermediate image plane ( 208 ) is about 5 microns. Due to imaging errors and the limited resolution, the main maximum of the intensity distribution on the sample ( 201 ) only a half width of 3 micrometers. The intensities of the secondary maxima are on the sample ( 201 ) so low that the threshold for the MALDI process is not reached there and thus the MALDI process only takes place in the area of a single fine intensity peak.

To the sample ( 201 ) to be used evenly between the intensity peaks, the lens array ( 206 ) rotated so that the positions of the intensity peaks on the sample ( 201 ) to be changed. The sample ( 201 ) and further samples on the sample carrier ( 202 ) can be spatially scanned one after the other with a single intensity peak by placing the sample carrier ( 202 ) is moved. The fine intensity peak that is generated with the laser system ( 200 ) in is very well suited to achieve an imaging mass spectrometric analysis with high spatial resolution using the raster method.

A very advantageous extension of the second embodiment is that so-called fractal Talbot planes also follow the rear focal plane of the lens array ( 206 ) into the intermediate image level ( 208 ) can be mapped. The Talbot effect occurs in all periodic structures and thus also in the lens array ( 206 ) ( K. Besold et al., Pure Appl. Opt. 6 (1997), 691-698: "Practical limitations of Talbot imaging with microlens arrays" ). The distance z _{T of} the Talbot plane from the rear focal plane of the lens array ( 206 ) results from the distance p between the periodically arranged lenses of the lens array ( 206 ) and the wavelength λ: z _T = 2 · p ² / λ. Intensity peaks occur in the Talbot plane, which like the lens foci in the rear focal plane of the lens array ( 206 ) are arranged. Interestingly, there are also fractal Talbot planes between the rear focal plane and the Talbot plane, in which the number of intensity peaks is multiplied and the half width is reduced. By mapping suitable fractal Talbot planes, even the distance between the intensity peaks on the sample ( 201 ) can be set. In particular, the degree of spatial modulation of the intensity peaks can also be set in that the fractal Talbot planes are not sharply imaged, as a result of which the intensity between the intensity peaks does not completely disappear.

The shows a third preferred embodiment of a laser system according to the invention ( 300 ). The laser unit ( 303 ) here is again a Nd: YAG laser that generates a time-pulsed laser beam at a frequency tripled wavelength of 355 nanometers. The spatial beam profile corresponds almost to a Gaussian basic mode. The energy of the laser pulses can be measured with one in the laser unit ( 203 ) integrated attenuator.

The lens array ( 306 ) generates a multitude of intensity peaks in the rear focal plane, which can be detected by a zoom lens ( 307 ) into the front focal plane ( 308 ) the lens ( 307 ) can be mapped. The lens array ( 306 ) has similar geometrical and optical parameters as in the first two embodiments. The zoom lens ( 307 ) consists of two spherical lenses that can be moved independently of each other. From each intensity peak in the focal plane ( 308 ) creates the lens ( 307 ) a parallel bundle of rays, each bundle of rays having a different angle to the optical axis. In the For the sake of clarity, only the bundle of rays is shown parallel to the optical axis. The sample ( 301 ) is located in the rear focal plane of the lens ( 304 ) so that the different parallel beams hit the sample ( 301 ) be focused. Since the parallel bundles of rays hit the lens ( 304 ), each bundle of rays results in a single intensity peak which, depending on the direction and angle of the bundle of rays, has a certain position on the sample ( 301 ) Has. In this way, on the sample ( 301 ) generates a multitude of intensity peaks. Due to the focal lengths of the lenses ( 304 ) and ( 309 ) the distance between the intensity peaks on the sample ( 301 ) at a given distance between the intensity peaks in the focal plane ( 308 ).

A major advantage of this embodiment is that the distance between the two lenses ( 304 ) and ( 309 ) is not determined by a figure, but is in principle arbitrary. Furthermore, the zoom lens ( 307 ) the image scale between the rear focal plane of the lens array ( 306 ) and the focal plane ( 308 ) can be continuously adjusted. This makes it possible in a very advantageous way to determine the distances between the intensity peaks on the sample ( 301 ). As shown in the second embodiment, it is of course also possible to use fractal Talbot planes or other optical planes in which the intensity peaks have a higher periodicity or have a low spatial degree of modulation.

With the zoom lens ( 307 ) the plane directly behind the lens array ( 306 ) into the focal plane ( 308 ) can be mapped. This results in the focus plane ( 308 ) generates a single intensity peak that is passed through the two lenses ( 304 ) and ( 309 ) to the test ( 301 ) is transferred. With the zoom lens, the image scale can be continuously changed and the half-width of the individual intensity peaks on the sample ( 301 ) can be set.

As in the first two embodiments, the sample ( 301 ) can be used up evenly by adjusting the position of the intensity peaks on the sample ( 301 ) is changed during a sequence of laser pulses. This can be achieved, for example, by the deflection mirror ( 305 ) is tilted or the sample carrier ( 302 ) is moved or preferably the lens array ( 306 ) is rotated.

Instead of the lens array ( 206 ) a combination of a Dammann grating and a single spherical lens can also be used to produce a variety of intensity peaks. A Dammann grating is a diffractive optical element (DOE) that diffracts the laser beam into different orders like a normal grating, but distributes the laser beam evenly over a large number of orders. The different diffraction orders of the Dammann grating, which can be viewed as parallel bundles of rays with different directions to the optical axis, are focused by the single spherical lens in its rear focal plane and result in a large number of intensity peaks. These intensity peaks can, as described in the third embodiment, through the zoom lens ( 307 ) into the focal plane ( 308 ) can be mapped. Possibly the generation of the parallel bundles of rays passing through the lens ( 304 ) to the test ( 301 ) are focused entirely through a single Damman grating that is variably adjustable. Such a variable Damman grating can in principle consist of a programmable chip which is used in liquid crystal screens or projectors.

With the knowledge of the invention, it is possible for the person skilled in the art to design further configurations of laser systems according to the invention.

Claims (4)

Laser system (300) for the ionization of a sample (301) by matrix-assisted laser desorption in mass spectrometric analysis, characterized in that the laser system (300) contains an adjustable device with which the laser radiation is spatially modulated, and the intensity distribution of the laser radiation on the sample (301) in one setting of the device consists of a single intensity peak and in another setting of a plurality of intensity peaks, the adjustable device a) one behind the other in the beam path of the laser system (300) a lens array (306), a variable optical system (307 ), a first focusing optic (309) and a second focusing optic (304), and wherein b) the variable optical system (307) can be set in such a way that in different settings there is one of several optical planes behind the lens array ( 306) into the front focal plane of the first focusing optics (30 9), and where c) the sample (301) is located in the rear focal plane of the second focusing optics (304). Laser system according to Claim 1 , characterized in that the variable optical system images the plane directly behind the lens array (306) or the rear focal plane of the lens array (306) or both in the front focal plane of the first focusing optics (309). Laser system according to Claim 1 or 2 , characterized in that the scale of the image is adjustable. Laser system according to one of the Claims 1 to 3 , characterized in that the lens array (306) is moved after each laser pulse or a few laser pulses.

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