Patent Application
20240177986
The invention relates to methods and devices for desorbing and ionizing of sample material which is deposited on a sample support. Principles of the disclosure may be used in imaging ion spectrometry, for example, particularly in imaging ion spectrometry with ion generation using matrix-assisted laser desorption and ionization (MALDI).
The Prior Art is explained below with reference to a specific aspect. This is not to be understood as a limitation, however. Useful developments and modifications to the invention may also be applicable beyond the comparatively narrow scope of this introduction, and will become readily apparent to practitioners skilled in the art after reading the disclosure of the invention which follows this introduction.
The MALDI method has been used for ion spectrometry analyses for a long time. In the case of ultraviolet vacuum MALDI, soluble analyte molecules are embedded in a light-absorbing, crystallizing matrix substance and are then irradiated with coherent ultraviolet light pulses. The UV light is absorbed by the crystallized matrix substance, which is then desorbed into a material cloud, and the embedded analyte molecules are desorbed along with the substance. The characteristics of the desorption process and the material cloud cause charge carriers to be formed and transferred to the analyte molecules, with the result that charged analyte molecules or analyte ions are generated. These analyte ions can then be guided and analyzed using electromagnetic fields, e.g., in the process of a mobility and/or mass analysis that sorts and detects charged molecules or ions according to their collision-cross-section-to-charge ratio or their mass-to-charge ratio.
Benefits of this established MALDI method lie in the fact that the analyte molecules are ionized very gently, with practically no fragmentation, and that the generated analyte ions have a largely uniform charge state, usually z=1. In complex samples in particular, however, it has been found that different molecule classes respond differently to the MALDI method, in particular, depending on the matrix substance used. For example, certain biomolecules are ionized such that they are sufficiently detectable, whereas other biomolecules in the ion currents that are acquired from a mixture are quantitatively underrepresented by comparison. This differing responsiveness is particularly evident in the investigation of tissue sections in imaging mass spectrometry, for example, and can limit the informational value of the acquired measurement data. It has been observed, for example, that lipids in the spectra of tissue sections may be overrepresented compared to proteins and peptides.
Some time ago, a post-desorption ionization method was therefore proposed that can increase the conversion rate of lower-concentrated molecules. The principle essentially consists in laterally transmitting an additional coherent ultraviolet light pulse into the material cloud of the MALDI desorption. This method is referred to as MALDI-2. The interaction of the light pulse with the particles in the material cloud brings about an expansion of the supply of charge carriers, which improves the ionization yield, particularly for lower-concentrated analyte molecules. But higher-concentrated biomolecules can also benefit from a post-ionization modality. Phosphatidylethanolamines (PEs), for example, are barely detected in MALDI measurements compared to phosphatidylcholines (PCs), despite the fact that both are comparably abundant in tissue. With MALDI-2, PEs are heavily ionized and are then reliably detectable in the acquired spectra.
An important publication on the MALDI-2 method is the study by Jens Soltwisch et al. (Science, 10 Apr. 2015⋅Vol 348, Issue 6231, 211-215), in which this name was coined. This study uses a wavelength-tunable post-ionization laser to trigger secondary MALDI-like ionization processes in the gas phase. An increase in the ion yield by up to two orders of magnitude is reported for numerous lipid classes, fat-soluble vitamins and saccharides that were imaged in animal and plant tissue with a 5-micrometer-wide laser spot. The pressure of the cooling gas in the ion source, the laser wavelength, the pulse energy and the time lag between the two laser pulses are described as decisive parameters for the triggering of the secondary ionization processes.
A number of prior art publications that may be relevant for the present disclosure are briefly acknowledged in the following list:
The monograph by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425) deals with, among other things, post-ionization experiments on the characterization of the MALDI method, in particular, in Section V. Plume Dynamics.
The patent publication WO 2010/085720 A1 discloses a method and a device for the efficient measurement of an ionized MALDI desorption cloud when post-ionization methods (POSTI) are combined with a medium vacuum MALDI ion mobility orthogonal time-of-flight mass spectrometer (MALDI-IM-oTOF-MS). A related work is the article by Amina S. Woods et al. (J Proteome Res. 2013 Apr. 5; 12(4): 1668-1677).
The article by M. Niehaus et al. (Nature Methods Vol. 16, 925-931 (2019)) deals with MALDI-2 mass spectrometry in transmission mode for the imaging of cells and tissues with sub-cellular resolution.
MALDI measurements from very extensive sample material are becoming increasingly important; consider tissue sections in imaging mass spectrometry with areas in the order of magnitude of a few square centimeters or fields of very densely packed individual preparations in a high-throughput analysis, e.g., 1536 single preparations on a MALDI sample support. Measurements from sample supports loaded in this way can take a very long time; several hours or even days in the case of large tissue sections. To reduce the spectral data recording time, a dynamic operation of the MALDI desorption laser beam was proposed for the measurement procedure, combining a large number of rapid orientation changes of the desorption laser beam for the sampling of a predetermined finite area on the sample material with a few rather time-consuming adjustments of the MALDI sample support in order to move to different areas. This means that the entire surface of a sample support can be probed quicker than by simply adjusting the heavy, and therefore rather slow, translation stage that carries the sample support. An example is provided in the patent publication DE 10 2018 112 538 B3 (corresponds to US 2019/0362958 A1 and GB 2 574 709 A), in particular, with reference to
Scanning of the sample support surface that exclusively uses the laser beam is subject to technical limits in that, on the one hand, the desorption laser beam must not hit the sample material at too great an angle and, on the other hand, ablated and ionized sample material must be transferred into other components of a connected analytical system via interfaces that are usually stationary. This limits the beam deflection to a distance of a few hundred micrometers between the two furthest impingement points on a predetermined finite area. A standard MALDI sample support, on the other hand, has the dimensions of a microtitration plate (127.76 millimeters×85.48 millimeters×14.35 millimeters), meaning that the available surface—even with incomplete sample loading—cannot be completely covered solely by the desorption laser beam without spatial adjustment of the sample support. Rather, a plurality of predetermined finite areas on the sample support can usually be defined on a sample support.
In view of the above discussion, there is a need for an improvement in the methods and devices for desorbing and ionizing of sample material, particularly in relation to sensitivity to weakly ionizing molecular substrates. Further objectives that can be achieved by the invention will be immediately apparent to the person skilled in the art from reading the disclosure below.
According to a first aspect, the present disclosure relates to a method for desorbing and ionizing of sample material deposited on a sample support, comprising: —repeatedly locally impacting sample material on the sample support using a first energetic radiation and triggering of local desorption of sample material into the gas phase above the sample support, while varying a position of the first energetic radiation relative to the sample support and aiming at a plurality of impingement points on the sample material on the sample support; —pulsed impacting the locally desorbed sample material using a second energetic radiation, which is aimed into the locally desorbed sample material, and triggering of ionization and/or increasing a degree of ionization of the locally desorbed sample material, with a direction of propagation of the second energetic radiation being in a plane that is substantially perpendicular to a surface normal of the sample support and positioned above the sample support, and with a focus position and/or beam waist position of the second energetic radiation being re-aligned such that it is positioned substantially opposite a current impingement point at the sample material on the sample support; and —transferring of ionized sample material, originating from the locally desorbed sample material which has been impacted with the second energetic radiation, into an ion-processing device.
The height of the plane above the sample support may be in the range of 300-1000 micrometers, and particularly 500 micrometers. The position of the plane above the sample material and sample support is preferably substantially constant, e.g., with rather small deflections, by a few degrees, of the alignment of the first energetic radiation. In certain embodiments, the height of the plane above the sample material and sample support may be changed, e.g., increased or decreased, temporarily and for a short period of time. With desorption and ionization in a vacuum, the environment where the sample support with sample material is kept may be maintained at a pressure in the range of 0.5-10 hectopascal, e.g., by means of suitably connected pumps. A timespan or time lag between the triggering of the first energetic radiation and the second energetic radiation is preferably within the range of 0.5-1000 microseconds. The directions of propagation of the first energetic radiation and the second energetic radiation can be substantially perpendicular to each other, in particular, at an angle of between 45 and 135 degrees between each other, for example. The first energetic radiation can be delivered in incident light, i.e., from a side of the sample support on which the sample material is deposited, or in transmitted light, i.e., from a side of the sample support that faces away from the side on which the sample material is deposited.
In various embodiments, a direction of incidence of the first energetic radiation can be changed relative to a surface normal of the sample support, and a plurality of impingement points aimed at. This method of operation accelerates the rastering of a sample support with sample material deposited over its surface, since changes in the beam orientation, e.g., using reflective optical elements such as galvanometric micromirrors, can be carried out a lot quicker and easier than by moving the very heavy translation stage on which the sample support is deposited and/or prepared together with the sample material. Movements of the translation stage are preferably carried out when the range of motion of the first energetic radiation over a predetermined finite area on the sample support is exhausted.
In various embodiments, the sample material can be prepared with a light-absorbent matrix substance. For the desorption, a MALDI method may be possible in incident light (in reflection mode) or in transmitted light (in transmission mode), depending on the requirements. The MALDI method requires a certain sample preparation with a light-absorbent matrix substance, e.g., sinapic acid, 2.5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid or 2.5-dihydroxyacetophenone, all of which absorb strongly in the ultraviolet spectral range. A laser light from a nitrogen laser with a wavelength of around 337 nanometers is suitable for the first energetic radiation, for example, as is a laser light from a frequency-tripled solid-state Nd: YAG laser at around 355 nanometers. The second energetic radiation can comprise a laser light pulse with a wavelength of 266 nanometers, for example. For the second energetic radiation, all wavelengths below the two-photon limit can generally be used for the ionization of the matrix substance used, i.e., mostly wavelengths that are shorter than or equal to 290 nanometers for matrix substances with ionization energies of around eight electronvolts. The energy of the first energetic radiation is preferably in the range of 0.1-50 microjoules; the lower limit can be applied particularly in the case of small laser foci on the sample material, as can be set with transmission MALDI, for example. The energy of the second energetic radiation can be in the range of 100-600 microjoules, for example, with particular preference given to 300-500 microjoules.
In various embodiments, the first energetic radiation can be delivered using a transmitted light optical system that is positioned and designed so that the first energetic radiation is applied to the sample support from a backward direction after passing through the sample support. The embodiment as a transmitted light optical system makes it possible to keep the front desorption and ion formation area free from beam-guiding elements that could interfere with the ion extraction. The ion extraction from the ion formation area can be performed substantially linearly parallel to a surface normal of the sample support or can also comprise changes of direction, e.g., deflections by 90°, which may be triggered by suitably arranged deflection electrodes. Moreover, a transmitted light optical system allows greater focusing of the first energetic radiation for a spatially very limited ablation of the sample material, meaning that considerably higher spatial-lateral resolutions can be achieved compared to when using incident light optical systems, such as reflection MALDI. A laser beam can be used to achieve ablation surfaces, and therefore pixel surfaces, with diameters in the single-digit micrometer range and—with particularly careful fine adjustment—even in the submicrometer range, e.g., with a diameter of 0.5-5 micrometers.
In various embodiments, the sample material can have a plurality of spot preparations or a two-dimensional or flat tissue section. In particular, a microtomized tissue section can be used as sample material. Examples of this are brain tissue and retina tissue, e.g., from rodents. The sample material can, in particular, be taken from a frozen piece of tissue or formalin-fixed paraffin-embedded (FFPE) tissue, which may require additional processing steps prior to the analysis, e.g., “deparaffinization” and “de-crosslinking”, also known as antigen retrieval. The thickness of a tissue section for analysis can be 2-20 micrometers, or in particular, 2-15 micrometers for MALDI applications in transmitted light. For reflection MALDI, the sections may also be thicker, e.g., 2-40 micrometers. The analysis of tissue sections is becoming increasingly important, particularly in the area of clinical applications aimed at determining the pathological states of a tissue and differentiating them from non-pathological states, or the cell response to the administration of pharmaceutical substances. A plurality of spot preparations may, for example, comprise a dense field of 1536 or more individual preparations on the sample support, which have been produced using a dried-droplet method. Active substance candidate detection for pharmacological investigations, for example, is worth mentioning as an area of application.
In various embodiments, the sample support can comprise a glass plate, metal plate or ceramic plate. The surface of the sample support that carries the sample material is preferably designed to be electrically conductive so that it can form an electrical reference potential and allow and/or simplify the handling of the desorbed and ionized sample material. This design has a positive impact particularly in the case of axial extraction of the ionized sample material from the ion formation area, i.e., extraction that is substantially conducted parallel to a surface normal of the sample support. Suitable options include, for example, polished steel plates or plates with lyophilic anchor sites in a lyophobic environment such as the AnchorChips™ from Bruker. For the use of the first energetic radiation in transmitted light, glass specimen slides coated with indium tin oxide (ITO) can be used in particular.
In various embodiments, the first energetic radiation and/or the second energetic radiation can be delivered by a pulse laser or pulsed laser. In particular, the first energetic radiation can be applied to the sample in pulses. The clock-pulse rate of a pulse sequence can be in the range of a few hertz, e.g., 1-20 pulses per second, up to 103 or 104 Hz. A clock-pulse rate of the second energetic radiation can be matched to the clock-pulse rate of the first energetic radiation, and each individual desorption cloud can be irradiated with a suitable delay of a few microseconds, which enhances the formation of a desorption cloud, starting from the application of the first energetic radiation. The delay can be, for example, 0.5-100 microseconds, depending on the height of the direction of propagation plane above the sample support and on the pressure level, and can preferably be 5-20 microseconds, particularly under medium vacuum pressures of a few hectopascals and a height of the direction of propagation plane above the sample support of around 500 micrometers.
In various embodiments, the position of the sample support relative to the first energetic radiation and/or the direction of propagation of the second energetic radiation can be changed or re-aligned using one or more mirrors and/or one or more lenses. For the first energetic radiation, an optical arrangement with a Kepler telescope can be used, for example, as described in the patent publication DE 10 2011 112 649 A1 (corresponds to GB 2 495 805 A and US 2013/0056628 A1). For the second energetic radiation, the use of galvanometric micromirror pairs is preferred, with each micromirror able to be rotated around an axis of rotation in order to change the direction of emergence of a reflected beam. The use of at least one pair of flexible rotating mirrors makes it possible to change the beam orientation in a way that allows an almost parallel offset compared to a preset standard beam alignment, while simultaneously keeping the direction of propagation at one height above the sample material and sample support. This means that the alignment of the second energetic radiation can be quickly and reliably corrected to match the varying impingement points at the sample material on the sample support, so that optimal irradiation of a propagating desorption cloud is always achieved. By providing an additional pair of flexible rotating galvanometric mirrors with axes of rotation aligned perpendicularly to those of the other micromirrors, it is also possible to temporarily change, e.g., increase or decrease, the height of the direction of propagation of the second energetic radiation for a short period.
In various embodiments, the ion-processing device can be designed as an analyzer, and, in particular, as a mobility analyzer, mass analyzer, or coupled or hybrid mobility-mass analyzer. Ion-guiding intermediate stages, e.g., radio-frequency voltage ion guides such as rod multipoles or RF funnel arrangements. can be arranged upstream from the actual analyzer, or multiple analyzers in series, and also in different sections between such analyzers in series. Various analyzers and intermediate stages can also be operated at different vacuum levels.
An ion mobility analyzer separates charged molecules or molecular ions according to their collision cross-section to charge ratio, sometimes designated by σ/z or Ω/z. The basis for this is the interaction between the ionic species and an electric field that couples with the charge of the ions, and the simultaneous effect of a buffer gas that influences the average cross-sectional area of the ion. Already known are, particularly, drift-tube mobility separators with static electric field gradients, which drive ions through an essentially stationary gas. Here, the drift velocity of an ionic species is given by the propulsive force of the electric field and the decelerating force of the collisions with the gas particles. Equally common are trapping ion mobility separators (TIMS) with a continuous laminar gas flow driving the ions forward, said gas flow being counteracted by a gradually changed electric field gradient with correspondingly variable deceleration force. Traveling-wave mobility separators are also worthy of mention.
A mass analyzer, on the other hand, separates charged molecules or molecular ions according to their mass-to-charge ratio, usually designated as m/z. Time-of-flight analyzers can be used, for which both linear and reflector setups and/or setups with orthogonal acceleration into the flight region can be chosen. Other types of mass-dispersive separators can also be used, e.g., quadrupole mass filters (“single quads”), triple quadrupole analyzers (“triple quads”), ion cyclotron resonance cells (ICR), Kingdon-type analyzers such as the Orbitrap® (Thermo Fisher Scientific), and others. It is evident that analyzers and separators of the previously mentioned types can be coupled to enable ionic species to be separated multi-dimensionally, i.e., according to more than one physical-chemical property, such as m/z and σ/z or Ω/z.
In various embodiments, the focus position and/or beam waist position of the second energetic radiation can be re-aligned (i) perpendicularly to and/or (ii) along the direction of propagation of the second energetic radiation. The beam-waist position or focus position is preferably realized along the direction of propagation of the second energetic radiation using a lens system in the beam path that contains at least one movable optical lens that can be used to adjust the focal length settings of the entire optical system for the second energetic radiation. Regarding re-alignment perpendicularly to the direction of propagation of the second energetic radiation, pairs of galvanometric micromirrors are preferably used, which can each be rotated around a separate axis of rotation and which change a direction of emergence of the reflected second energetic radiation.
It may be expedient to change the height of the beam-waist position or focus position above the sample material and sample support if a very pronounced deflection of the first energetic radiation has been set in relation to a standard impingement point. As a result of the divergence of the second energetic radiation, a very pronounced deflection of the impingement point may bring about a risk of areas on the sample material and sample support coming into contact with peripheral areas of the second energetic radiation, thereby generating interfering background in the spectral data. Setting a temporarily and briefly greater height above the sample material and sample support for such extreme deflections allows a compromise to be reached between reducing this risk of forming ionic or chemical background in the spectral data and maintaining a favorable beam path of the second energetic radiation. It may also be advisable to temporarily set a lower height above the sample material and sample support for a short period, e.g., in order to place the interaction of the second energetic radiation and the desorbed sample material in an area of the desorption cloud where the particle density is so high that it significantly enhances charge carrier formation and charge carrier transfer to uncharged molecules in the cloud, which can bring about an increase in the yield of ionized desorbed sample material. Such an embodiment is also attributable to the invention.
According to an additional aspect, the present disclosure relates to a device for desorbing and ionizing of sample material deposited on a sample support, comprising: —a desorption device for generating and guiding the first energetic radiation; —an ionization device for generating and guiding the second energetic radiation; —a first adjustment device for setting and changing the position of the first energetic radiation relative to the sample support; —a second adjustment device for setting and re-aligning the focus position and/or beam waist position of the second energetic radiation; and a guidance system that communicates with the desorption device, the ionization device, the first adjustment device and the second adjustment device, and that is designed and programmed to coordinate and perform a method as described herein above.
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 numerals designate corresponding elements in the different views.
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.
The ions then enter the RF quadrupole rod system (18), which can be operated both as a simple ion guide and as a mass filter to select a species of precursor ions to be fragmented. The unselected or selected ions are then fed into the RF quadrupole ion storage device (19) and can be fragmented by high-energy collisions according to their acceleration. The ion storage device (19) has a gastight casing and is charged with collision gas, such as nitrogen or argon, through the gas feeder (20) in order to focus the ions by means of collisions and to collect them in the axis.
At specified times, ions are extracted from the ion storage device (19) by a switchable extraction lens (21), which shapes the ions into a fine primary ion beam (22) and sends them to the ion pulser (23). The ion pulser (23) pulses out a section of the primary ion beam (22) orthogonally into the drift region, which is at a high electrical potential, thus generating the new ion beam (24). The ion beam (24) is reflected in the reflector (25) with velocity focusing and measured in the detector (26). The mass spectrometer is evacuated by connected pumps (27), (28) and (29).
A first laser system (111) is, in this example, positioned at an oblique angle above the translation stage (132) and is designed to direct a laser beam (112) onto predetermined locations within a predetermined finite area on the sample support (115) in different orientations (solid, dotted and dashed contours) without any movement of the translation stage (132), e.g., a design as reflection MALDI; the latter designation is derived from the fact that, in the broadest sense, the desorbed and ionized sample material leaves the sample support (115) against the direction of incidence of the first laser beam (112). The finite area can have a diameter or an edge length of 100-1000 micrometers, for example. A guiding element positioned above the sample support (115), such as the indicated RF ion funnel (117), is able to collect the desorbed and electrically charged sample material and to transfer it to a connected analyzer (not shown), via intermediate stages if necessary and/or using axial extraction or extraction with changes of direction. To this end, extracting electrical potentials can be either permanently or intermittently applied to the RF ion funnel (117), coordinated with the desorption pulses of the first energetic radiation (112).
Also shown is a post-ionization modality in the form of a second laser system (111*) positioned and designed so that a second laser beam (112*) can be laterally focused into a desorption cloud of sample material, e.g., according to the MALDI-2 method, see
The alignment and focusing of the second laser beam (112*) is usually rigid and so cannot be changed or adapted without complex human intervention, meaning that there is just one optimal position for a desorption cloud in order for it to be optimally impacted by the second beam (112*). The aim here is always ionization, or at least an increase in the degree of ionization if the desorption process itself already involves ionization, as with a MALDI preparation. For this reason, the ablation or desorption location would always need to be moved to the fixed focus position or beam waist position of the second laser, which can only be achieved by moving the translation stage (132), which is slow and time-consuming, as indicated above. If a change is made to the alignment of the first laser beam (112) relative to the sample support (115), or rather to a surface normal (134) of the sample support (115), see
The situation is more serious if a change in alignment of the first energetic radiation (112) deflects the impingement point on the sample support (115) along a direction that is substantially perpendicular to the direction of propagation of the second beam (112*). In such a situation, the problem of complete spatial divergence of the second beam (112*) and the desorption cloud emerges a lot more quickly than does slipping out of focus in the case of deflection along the direction of propagation of the second beam (112*). This case is schematically represented by way of example in
A guidance system (242) communicates with the adjustment device (not shown) of the sample support (215), the second laser system (211*), the adjustment device of the first beam (not shown), the pairs of mirrors (238, 238′) and the lens system (240), and coordinates their operation so that the impingement and ablation point on the sample support (215) and the waist position or focus position of the second beam (212*) are always located substantially opposite one another. Communication is indicated by the double-chain-dotted lines (244).
Principles of the invention make it possible to enlarge, in particular, the areas on the sample material that can be impacted by simply adjusting the first energetic radiation, without having to move the heavy and slow translation stage on which the sample support is located. This allows the setting of larger deflections in the beam guide of the first energetic radiation. This can help to accelerate the spatially resolved processing of a sample support on which sample material is deposited, compared to methods known from the Prior Art, since the total number and frequency of the sample support movements required for rastering can be reduced still further.
The invention has been described above with reference to different, specific example embodiments. It is to be understood, however, that various aspects or particulars 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102022131740 | Nov 2022 | DE | national |
