Systems and methods for sub-micron laser extraction for spatially-resolved mass spectrometry

Abstract

Systems and methods are provided that facilitate laser-based spatially-resolved ablation and ionization of a material, with low sample volume and little or no fragmentation. Sub-nanosecond pulses are directed onto a sample with a delay of less than 1 ns. Within an initial 50 fs after the onset of sample irradiation, one of the pulses generates an initial free electron density, and after the initial 50 fs, the other pulse increases the free electron density such that ablation occurs, resulting in an ablation volume having a sub-diffraction-limited lateral area that is smaller than a diffraction-limited 1 / e2 spot size of at least one of the first laser pulse and the second laser pulse, and a depth of less than 1 micron. The pulse intensities are provided such that ablation occurs without substantial analyte fragmentation, facilitating, for example, analysis and identification via mass spectrometry.

Description

SYSTEMS AND METHODS FOR SUB-MICRON LASER EXTRACTION FOR SPATIALLY- RESOLVED MASS SPECTROMETRYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 738,103, titled “SYSTEMSAND METHODS FOR SUB-MICRON LASER EXTRACTION FOR SPATIALLY-RESOLVED MASS SPECTROMETRY” and filed on December 23, 2024, the entire contents of which is incorporated herein by reference.BACKGROUND OF THE DISCLOSURE

[0002] The present disclosure relates to mass spectrometry and imaging mass spectrometry.

[0003] Imaging mass spectrometry (IMS) is a leading analytical tool for mapping the location of biomolecules (for example, lipids, metabolites and proteins) in biomaterials. Currently, the highest possible resolution for mapping biomolecules is approximately 5 microns, with the standard resolution being used typically ranging between 10 and 100 microns, thus operating at a spatial resolution that precludes intracellular analysis.

[0004] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.SUMMARY OF THE DISCLOSURE

[0005] Systems and methods are provided that facilitate laser-based spatially-resolved ablation and ionization of a material, with low sample volume and little or no fragmentation. Subnanosecond pulses are directed onto a sample with a delay of less than 1 ns. Within an initial 50 fs after the onset of sample irradiation, one of the pulses generates an initial free electron density, and after the initial 50 fs, the other pulse increases the free electron density such that ablation occurs, resulting in an ablation volume having a sub-diffraction-limited lateral area that is smaller than a diffraction-limited 1 / e2spot size of at least one of the first laser pulse and the second laser pulse, and a depth of less than 1 micron. The pulse intensities are provided such that ablation occurs without substantial analyte fragmentation, facilitating, for example, analysis and identification via mass spectrometry.

[0006] According to an aspect, there is provided a method of ablating an analyte from a sample, the method comprising:directing a plurality of laser pulses onto a sample, the plurality of laser pulses including two laser pulses each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including ashorter wavelength pulse and a longer wavelength pulse, the two laser pulses having respective laser pulse properties and being directed onto the sample such that:the two laser pulses are spatially overlapped on the sample;the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

[0007] In another aspect, there is provided a system for ablating an analyte from a sample, the system comprising:a laser system configured to generate two laser pulses, each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including a shorter wavelength pulse and a longer wavelength pulse, the two laser pulses being directed onto a sample location, such that:the two laser pulses are spatially overlapped at the sample location;the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;when a dielectric sample resides at the sample location, one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the dielectric sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the dielectric sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

[0008] A further understanding of the functional and advantageous aspects of the disclosure can be realized by reference to the following detailed description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments are described with reference to the accompanying drawings. In the drawings, like reference numbers can indicate identical or functionally similar elements.

[0010] FIG. 1 schematically illustrates the impact of fragmentation on the mass spectrum signal produced by a mass spectrometer, where (A) shows analyte fragmentation that is too high to facilitate analyte identification, and (B) shows less fragmented analyte, facilitating analyte identification.

[0011] FIG. 2 schematically illustrates the long and short pulse interactions for laser-based material extraction (ablation). Panel A shows a comparison of nanosecond and femtosecond laser ablation processes for femtosecond lasers with a pulse duration longer than 50 fs and typically 100-300 fs. Panel B illustrates the process of nanosecond laser ablation with a heat affected zone caused by the thermal ablation process. Panel C illustrates the process of femtosecond laser ablation, with confinement of electron avalanche effects leading to 10-100 nm ablation depths due to localization of the deposited energy and Coulomb explosion effects on the lattice binding potential.

[0012] FIG. 3 shows a comparison between a two-photon resonant multiphoton ionization (MPI) process and a three-photon non-resonant MPI process.

[0013] FIG.4 schematically illustrates the tunneling ionization process induced by a strong electric field, such as that produced by a high-intensity laser. Panel A shows the system without the strong electric field, while panel B depicts the modification of the system's potential after the field is applied, lowering the potential barrier and thereby increasing the electron tunneling rate.

[0014] FIG.5 schematically illustrates the avalanche ionization process in which liberated electrons gain energy from the applied laser field and can ionize non-liberated electrons from the valance band. These newly liberated electrons can then contribute to the ionization process forming an avalanche affect.

[0015] FIG. 6 is a table listing ionization threshold energy and fragmentation threshold energies for polypeptides, amino acids, and proteins.

[0016] FIG. 7 plots the asymptotic average kinetic energy of conduction band (CB) electrons as a function of wavelength and intensity for ablation threshold intensity (1012W / cm2), close to threshold intensity (1013W / cm2) and well above threshold intensity (1014W / cm2). Values were calculated following the methods outlined in Ref.

[0013] ,

[0017] FIG. 8 presents the mass spectra of Mast-R, a protein with a retinylidene chromophore, obtained after performing ablation with pulses having wavelengths of 1030 nm, 515 nm, and 343 nm, for pulse durations between 100 fs and 170 fs, at a peak intensity near 1013W / cm2, where the intact peak intensity decreases as wavelength decreases. This figure clearly shows how the relative abundance of fragments increases with shorter wavelengths.

[0018] FIG. 9 is an illustration showing the reduction in the feature size produced by a femtosecond laser pulse due to three-photon absorption.

[0019] FIG. 10 schematically illustrates how super-resolution imaging mass spectrometry (IMS) can be used to reduce the size of a laser-ablated volume that is extracted from an analyte structure.

[0020] FIG. 11 shows results from modeling of the dual-pulse ablation setup using a single rate equation approach. A pulse with a wavelength of 1030 nm, a spot size radius of 2.5 pm and pulse duration of 250 fs, irradiates the material first. After a delay of 100 fs, a pulse with a wavelength of 257 nm pulse, a spot size radius of 0.5 pm, follows. Ablation occurs where the electron density exceeds 1O20electrons per cm3, due to both a columbic explosion and a rapid phase change, enabling precise sub-micron material removal. The radius for the volume removed is approximately 445 nm.

[0021] FIG. 12 illustrates an example imaging mass spectrometry modality involving a temporally-overlapping (e.g. simultaneous) dual-wavelength ablation process, with the top panel showing a pulse timing diagram, and the lower panel illustrating the expected physical outcome. It is noted that for this method to be most effective, the wavelength of the shorter-wavelength pulse should be less than 124 nm, however, any wavelength shorter than 1 micron for the shorter-wavelength pulse will see the benefit of this method.

[0022] FIG. 13 illustrates another example imaging mass spectrometry modality involving a temporally-delayed dual-wavelength ablation process, with the top panel showing a pulse timing diagram, and the lower panel illustrating the expected physical outcome.

[0023] FIG. 14 plots the absorption cross section of water, MastR (a protein with a retinylidene chromophore), Hen Egg White Lysozyme (HEWL), myoglobin (mb) and liberated electrons estimated using a Drude model.

[0024] FIG. 15 illustrates an example imaging mass spectrometry modality involving a temporally-delayed dual-wavelength ablation process configured for MPI avalanche seeding, with the top panel showing a pulse timing diagram, and the lower panel illustrating the expected physical outcome.

[0025] FIG. 16 is a table presenting example parameter space ranges for a plurality of nonlimiting example imaging mass spectrometry modalities.

[0026] FIG. 17A illustrates an example system for performing imaging mass spectrometry with sub-micron resolution using multiphoton absorption without external sampling.

[0027] FIG. 17B illustrates an example system for performing imaging mass spectrometry with sub-micron resolution using multiphoton absorption with external sampling.

[0028] FIG. 18 is a flow chart illustrating an example workflow for disease recognition based on imaging mass spectrometry.

[0029] FIG. 19 shows results from modeling of a dual-pulse ablation process using a single rate equation approach. A pulse with a wavelength of 250 nm, a spot size radius of 1 pm and pulse duration of 100 fs, irradiates the material first. After a delay of 100 fs, a pulse with a wavelength of 1030 nm pulse, a spot size radius of 2.5 pm and pulse duration of 250 fs, irradiates the material. Ablation occurs where the electron density exceeds 1O20electrons per cm3due to both a columbic explosion and a rapid phase change.

[0030] FIG. 20 shows results from modeling of a dual-pulse ablation process using a single rate equation approach. A pulse with a wavelength of 250 nm, a spot size radius of 1 pm and pulse duration of 100 fs, irradiates the material simultaneously with a second pulse with a wavelength of 1030 nm, a spot size radius of 2.5 pm and pulse duration of 250 fs. Ablation occurs where the electron density exceeds 1O20electrons per cm3due to both a columbic explosion and a rapid phase change.

[0031] FIG. 21 A illustrates an example sample geometry that can be used to reduce the energy required to drive plasma formation and the resulting ablation when a metal or semiconductor substate is used.

[0032] FIG. 21 B illustrates an another example sample geometry that can be used to reduce the energy and material removal size by using a patterned metal or semiconductor substrate to allow plasma formation and subsequent ablation at lower powers in sub-micron regions of the sample being analyzed.

[0033] FIG. 22 schematically Illustrates the sample geometries referred to as “front illumination” and “back illumination”.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure.However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

[0035] As used herein, the terms “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0036] As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations inproperties, parameters, and dimensions. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25 percent or less.

[0037] It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or sub-groups.

[0038] As used herein, the term "on the order of', when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.

[0039] For the purpose of contextualizing the structure and operation of the systems, devices, and methods disclosed herein, headings are provided. The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology. Embodiments under any one heading may be used in conjunction with embodiments under any other heading.

[0040] For simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the Figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the embodiment or embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the embodiments described herein. It should be understood at the outset that, although exemplary embodiments are illustrated in the figures and described below, the principles of the present disclosure may be implemented using any number of techniques, whether currently known or not. The present disclosure should in no way be limited to the exemplary implementations and techniques illustrated in the drawings and described below.

[0041] Various terms used throughout the present description may be read and understood as follows, unless the context indicates otherwise: “or” as used throughout is inclusive, as though written “and / or”; singular articles and pronouns as used throughout include their plural forms, and vice versa; similarly, gendered pronouns include their counterpart pronouns so that pronouns should not be understood as limiting anything described herein to use, implementation, performance, etc. by a single gender; “exemplary” should be understood as “illustrative” or “exemplifying” and not necessarily as “preferred” over other embodiments.Further definitions for terms may be set out herein; these may apply to prior and subsequentinstances of those terms, as will be understood from a reading of the present description. It will also be noted that the use of the term “a” or “an” will be understood to denote “at least one” in all instances unless explicitly stated otherwise or unless it would be understood to be obvious that it must mean “one”.

[0042] Modifications, additions, or omissions may be made to the systems, apparatuses, and methods described herein without departing from the scope of the disclosure. For example, the components of the systems and apparatuses may be integrated or separated. Moreover, the operations of the systems and apparatuses disclosed herein may be performed by more, fewer, or other components and the methods described may include more, fewer, or other steps. Additionally, steps may be performed in any suitable order. As used in this document, “each” refers to each member of a set or each member of a subset of a set.

[0043] The embodiments described herein are exemplary (e.g., in terms of materials, shapes, dimensions, and constructional details) and do not limit the claims appended hereto and any amendments made thereto. Persons skilled in the art will appreciate that there are yet more alternative implementations and modifications possible, and that the following examples are only illustrations of one or more implementations.

[0044] Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood to one of ordinary skill in the art. Unless otherwise indicated, such as through context, as used herein, the following terms are intended to have the following meanings:

[0045] As used herein, the term “femtosecond” (fs), when employed to characterize a laser pulse, laser system, process or timescale, is intended to refer to a time duration ranging from (and including) 1 femtosecond (1 fs) upto but not including 1 picosecond (1 ps)..

[0046] As used herein, the phrase term “picosecond” (ps), when employed to characterize a laser pulse, laser system, process or timescale, is intended to refer to a time duration ranging from (and including) 1 picosecond (1 ps) up to but not including 1 nanosecond (1 ns).

[0047] As used herein, the phrase term “nanosecond” (ns), when employed to characterize a laser pulse, laser system, process or timescale, is intended to refer to a time duration ranging from (and including) 1 nanosecond (1 ns) up to but not including 1 microsecond (1 ps).

[0048] As used herein, the term “visible” refers to a wavelength range between 400 nm and 750 nm.

[0049] As used herein, the term “infrared” refers to a wavelength range between 750 nm and 20 microns (micrometers).

[0050] As used herein, the term “near-infrared” refers to a wavelength range between 750 nm and 2 microns.

[0051] As used herein, the terms “ultrashort” and ultrafast” when employed to characterize a laser pulse, laser system, process or timescale, is intended to refer to a time duration ranging from 1 fs to 10 ps.

[0052] As used herein, the term “optical penetration depth” refers to a distance, within a given material, over which the intensity of light is attenuated by the given material to 1 / e its original intensity.

[0053] As used herein, the terms “biomolecule” refers to a molecule associated with a living organism or biological system, including but not limited to carbohydrates, lipids, proteins, peptides, metabolites, vitamins, hormones, amino acids, nucleic acids, small molecules of biological origin, neurotransmitters, and any natural, recombinant, synthetic, or chemically modified forms thereof. The term “biomolecule” generally excludes bulk solvents and carrier media, including water, unless expressly stated otherwise.

[0054] As used here, the phrase “phase explosion” refers to the phenomenon where a superheated liquid or solid undergoes an instantaneous phase change to vapor causing a large volume expansion and shockwave.

[0055] As used herein, the term “fragmentation” refers to a molecule or molecular complex (such as but not limited to a protein, metabolite or lipid) that has been significantly changed (for example a bond breakage or dissociation) from its original form such that it does not produce an intact signal through an analytical measurement such as mass spectrometry.

[0056] As used herein, the term “intact”, refers to the original form of a molecule or molecular complex (such as but not limited to a protein, metabolite or lipid).

[0057] As used herein, the term “valence band”, when employed to refer to the electronic properties of molecules such as biomolecules or water, refers to electrons that are localized to a single region, within an extended region having short-range order, periodicity or quasiperiodicity, such as a single water molecule within spatially ordered set of nearby water molecules, of within a single subregion of a molecule within a larger region having short-range order.

[0058] As used herein, the term “conduction band”, when employed to refer to the electronic properties of molecules such as biomolecules or water, refers to electrons that are delocalized beyond a single region into an extended region having short-range order, periodicity or quasiperiodicity, such as an electron delocalized within a spatially ordered set of nearby water molecules, of delocalized beyond a single subregion of a molecule into a larger region having short-range order.

[0059] As used herein, the term “free electron”, sometime referred to in literature as “nearly free electrons” refers to an electron that is delocalized within a host material allowing for independent interactions from the surrounding ions and neutrals. A free electron ischaracterized by its ongoing interaction with the surrounding ions, including the creation of an effective mass which differs from the rest mass due to the electromagnetic potential of the ions surrounding the electron.

[0060] As used herein, the terms “lattice” and “material lattice”, when employed to refer to the electronic properties of water or biomolecules such as proteins, refers to the network of bonds within a region having short-range order that effectively represents a lattice structure.

[0061] As used herein, the term “lattice potential”, when employed to refer to the electronic properties of a lattice, refers to the potential energy barrier formed by the electronic properties of the molecules or molecular subregions within the region having the short-range order.

[0062] As used herein, the term “peak intensity”, often replaced by the term “intensity” in this disclosure when refereeing to a laser pulse, means the total pulse energy divided by the full-width-half-maximum pulse duration (i.e. of the pulse temporal profile), and also divided by the effective cross-sectional area of the beam. For example, a circular gaussian profile, the effective area of the beam would be defined as the area of a circle with radius of one half the 1 / e2optical intensity spot size diameter, while a beam having an elliptical cross-section would have an effective area equal to nwxwywhere wxand wyare the 1 / e2radii along the two principal axes.

[0063] The relatively low spatial resolution of conventional imaging mass spectrometry (IMS), with respect to cellular dimensions, has prevented the use of imaging mass spectrometry to determine the biochemical pathways of single cells, which has the potential to change the understanding of cellular functions, diseases, and the fundamental processes of life. The present inventors thus sought a new approach to imaging mass spectrometry that could facilitate sub-micron spatial resolution IMS-based detection of intact biomolecules, specifically for molecular level characterization of biological samples, whether it be single cells or tissue in relation to the earliest possible detection of disease.

[0064] The present disclosure provides systems and methods involving the controlled use of femtosecond (or, in some implementations, picosecond) laser pulses to achieve submicron-resolution spatially-resolved sampling for laser extraction of a material, well beyond the diffraction limit. In various example embodiments described in detail below, tightly-focused, near-threshold, dual-pulse, femtosecond (or, in some cases, picosecond) laser ablation is employed to extract intact or substantially-intact biomolecules with a submicron spatial resolution.

[0065] The present disclosure is organized as follows. In the first section, imaging mass spectrometry methods are introduced and described, and the utility of minimally destructive sampling on the nanoscale is described, followed by a discussion of mechanisms of laser ablation and ionization, methods of limiting biomolecule fragmentation in laser ablation andionization, and sub-micron focusing and absorption of femtosecond laser pulses. Various example embodiments of the present disclosure are then presented, including systems and methods for achieving near-threshold, dual-pulse femtosecond (or, in some cases, picosecond) laser ablation to extract intact or substantially-intact biomolecules with submicron spatial resolution.Imaging Mass Spectrometry (IMS) and the Need for Small Extraction Volumes to Achieve Sensitive Detection

[0066] Mass spectrometry (MS) is a charged-particle detection technique that, in theory, can achieve single ion detection. However, practical limitations, including inefficiencies in ion collection and detection, prevent this level of sensitivity.

[0067] Loss of molecular signatures and poor detection resolution are driven largely by space charge effects, i.e. the result of Coulomb repulsion between the propagating ions. The localized ion density creates intense repulsive forces as the plume of molecular ions propagates through the MS instrument. These interactions cause spatial distortions and a broadening of the kinetic energy distribution of the plume. The spatial distortions cause deviated ion trajectories which can lead to sample loss, while the kinetic energy distribution broadening ensures ions of the same species (i.e. same m / z) arrive at the detector at varying times. MS is an analytical technique which identifies species based on its precise mobility and its flight time. As such, these spatial and temporal aberrations can significantly decrease the mass resolution and prevent the high-fidelity detection required for single-ion analysis.

[0068] In the case of spatially-resolved (e.g. imaging) mass spectrometry, the present inventors have determined that the volume of charged particles should be kept below a certain charge density to preserve the spatial relationships among analytes during detection. Specifically, single particle detection with time-of-flight mass spectrometry may be performed with extraction volumes (voxels) of about 106nm3or less (e.g. (100 nm)3when approximated as a cube) to keep the total charge sufficiently low to limit transverse Coulomb repulsion which otherwise leads to aberrations in the time of arrival, significantly affecting the mass resolution. While there are several conventional methods to achieve this low extraction volume, all these conventional methods involve highly focused ion or particle beams to obtain small enough spot sizes in the lateral direction. Unfortunately, the interaction of a sample of unknown composition with focused ion beam milling leads to a significant problem: significant fragmentation (molecular breakup) that prevents biomolecule identification.

[0069] In mass spectrometry, detecting sample constituents requires the mass-to-charge ratio of the ionized analyte to be predictable and repeatable. If an analyte is broken (fragmented) in a stochastic manner into too many small components (fragments), then it can be impossible toidentify the original molecule. In general, to identify all the biomolecules in a sample, one aims to limit fragmentation. Ideally, no fragmentation would occur, and the mass spectrometer would detect only a single peak to identify an individual molecule. However, in other cases, a small amount of fragmentation will still permit molecular identification so long as the fragmentation pattern is predictable and repeatable. A depiction of the challenges posed by fragmentation is shown in FIG. 1, which illustrates the impact of fragmentation on the mass spectrum signal produced by a mass spectrometer, where (A) shows analyte fragmentation that is too high to facilitate analyte identification, and (B) shows less fragmented analyte, facilitating analyte identification.

[0070] Without intending to be limited by theory, it may be the case that under high peak powers, multiphoton ionization leads to fragmentation, which can result in complete loss of molecular identity. Similarly, excess heating for extended periods of time (e.g. 1 to 10 ns from long excitation pulses may lead to thermal fragmentation. It therefore can be beneficial to avoid these forms of fragmentation by selecting laser pulse properties that do not predominantly cause excess heating over such extended time periods, thereby improving the laser interaction for material ablation (material removal) to the gas phase for mass spectrometry detection.Applications of Lasers in Mass Spectrometry

[0071] Laser ablation has been used in the field of mass spectrometry since the early 1960s [2], One of the advantages of using lasers for ionizing materials for mass spectrometry is that the laser allows the sampling size to be smaller than the single cell, enabling high-resolution proteomic maps [2,3], The ionization efficiency, fragmentation of molecules and volume of material ablated all depend on the ionizing laser. The key parameters which determine the quality of the ionization source include the laser fluence, wavelength, pulse duration, and the characteristics of the sample being ablated [2,4], Specifically, for biological tissue, the absorption of the constituents determines the wavelength dependence of the ionizing laser. Although nanosecond lasers are the most common lasers used, they tend to result in more fragmentation than ultrafast lasers [2,5,6], Further, ultrashort pulse lasers allow for precision depth control of laser ablation [4,7,8], These advantages make femtosecond laser ablation an important method for desorption and ionization in mass spectrometry. To understand how these processes can be applied for imaging mass spectrometry, it is helpful to first review the fundamentals of ultrafast laser ablation, and a brief summary is provided below before presenting several embodiments of the present disclosure.Mechanisms of Ultrafast Laser Ablation

[0072] Although the average power of a femtosecond laser can be similar to a nanosecond laser, the difference in pulse duration causes the absorption mechanics to be dissimilar

[0010] , FIG. 2 schematically illustrates the long and short pulse interactions for laser ablation.

[0073] In the case of nanosecond laser ablation of a material, the laser energy is absorbed by the surface region of the material through linear or multiphoton absorption (MPA). Due to the relatively low peak intensity of the laser, non-resonant multiphoton absorption is inefficient, necessitating higher pulse energies to drive the heating and material removal process. As the surface temperature rises, thermal vaporization and plasma formation occur. Once the pulse ends, strong shockwaves propagate through the material, potentially damaging the surrounding area as illustrated in FIG. 2, panel B. On a millisecond timescale, a phase explosion leads to the ejection of the ablation plume. In contrast, with a single femtosecond (fs) pulse, these events occur after the pulse has already concluded. Femtosecond lasers have pulses shorter in duration than each of the following times: the electron-to-ion energy transfer time, electron heat conduction time and hydrodynamic expansion time. This difference greatly reduces the thermal damage and heat-affected zone around the ablation site [11,12],

[0074] In contrast, and as shown in FIG. 2, The process of femtosecond laser ablation begins with the ionization of the electrons inside the target material. This process is driven by multiphoton ionization (MPI), tunnelling ionization (Tl) and collisional ionization (Cl) of liberated electrons within the irradiated volume (these ionization methods are described in further detail below). It is noted that MPI differs from MPA in that MPI leads to the liberation of an electron whereas MPA results in the absorption of photons but without the liberation of electrons. The dominance of each ionization type changes the way energy enters the irradiated volume, resulting in significant differences in the ablation process, discussed in detail below.

[0075] In the case of femtosecond laser ablation, the pulse durations are shorter than the major relaxation times for the electron-to-lattice energy transfer, heat conduction and hydrodynamic expansion, confining the excitation energy to the electrons. The resulting high energy density causes ionization to occur, with the electrons of a system reaching extremely high temperatures, while the ions and neutral components of the irradiated are practically unheated. The high charge density of this plasma drives a Coulomb explosion resulting in the first phase of material removal. Only a small fraction of the laser pulse energy is converted to heat when the remaining electrons recombine with the ions, driving a rapid phase change leading to the second phase of material removal. The resulting ablation is referred to as non-thermal.

[0076] Again, in the case of femtosecond processes, because most of the laser energy is confined to be absorbed within the first 100-1000 nm of the sample by plasma shielding and the modification of the lattice potential from the ionization process, the electron temperature, plasma density and pressure become significant. The high density of charged particlesproduces a strong electrostatic field, which ejects a thin layer of ions from the surface via a Coulomb explosion (shown schematically in FIG. 2) [4], It has also been found that the laser fluence threshold for Coulomb explosion is lower than the threshold for complete laser ablation of the excited volume [4],

[0077] After a small amount of material is removed by Coulomb explosion, the time for material removal is governed by the time it takes for the electrons to heat the material to high temperatures. Hot electrons thermalize with the ions and lattice within 10-100 ps, leading to a significant temperature increase within a small volume. Extremely high temperatures and pressures build up beneath the evaporating surface, causing a breakdown of the irradiated volume and a rapid phase change causing thermal vaporization and material ejection from the resulting shockwaves within milliseconds (shown in FIG. 2).Mechanisms of Ultrafast Laser Ionization

[0078] Tunneling ionization (Tl), multiphoton ionization (MPI), and avalanche ionization (Al) dominate under different laser pulse conditions. Specifically, variations in peak pulse intensity, pulse duration and wavelength determine which ionization type occurs or is dominant at the laser-matter interface. In dielectric materials, the avalanche ionization process takes approximately 50 fs to form as the electron densities need to be high enough to allow the liberated electrons to form a plasma with sufficient absorptivity to absorb photons / energy efficiently to further affect the lattice dynamics involved in ablation

[0013] , As such, multiphoton and tunnelling ionization dominate ablation for pulse durations shorter than 50 fs (see FIG. 2) [14,15], The details of the ionization mechanisms are addressed below.Multiphoton Ionization

[0079] Single photon ionization is the most intuitive form of ionization, in which a photon has enough energy to remove an electron from a molecule

[0016] , If a photon does not have enough energy to remove an electron, multiple photons can be absorbed to cause ionization if the peak power is high enough in the quaintly named process of multiphoton ionization (MPI)

[0016] , MPI is highly wavelength dependent, with shorter wavelengths leading to more efficient MPI than longer wavelengths due to resonant enhancement factors.

[0080] When heterogeneous solutions like biomolecules dissolved in water, or tissue in general, are irradiated by an ultrafast laser pulse, multiphoton ionization can occur in multiple constituents. However, within the visible and UV spectral range, the larger absorption cross section of proteins, lipids and metabolites, relative to that of water [8,17], typically results in MPI occurring more readily in the proteins and metabolites, due to resonant enhancement, than in the water of the solution, despite water being in greater abundance. This result is confirmed bythe smaller intact protein signal for UV and visible wavelengths than longer wavelengths when studying intact proteins with femtosecond laser ablation MS, as shown in FIG. 8, which is described further below. This comparison illustrates the importance of limiting MPI for laser based intact protein extraction. The concept of lasers driving MPI in proteins or other components of the cellular matrix holds unless a specific absorption peak of water is targeted (in the deep UV or MIR) [18-20],Tunneling Ionization

[0081] Shown schematically in FIG. 4, particle tunnelling is a quantum mechanical phenomenon in which a particle can move through an energy barrier, which would be impossible in classical physics. In this case, one can consider an electron bound by a Coulomb potential from a molecule. When a strong electric field is applied, the constraining potential is modified, creating a region of potential to which the electron can tunnel without the electron absorbing any energy. Thus, the method prevents fragmentation of analytes as no additional energy is applied to contribute to the fragmentation process. In FIG. 4, panel A, shows a standard Coulomb potential, and in FIG. 4, panel B shows how once a strong electric field, like that from a laser pulse, is applied, the potential can be modified to allow for electron tunneling. As this phenomenon is caused by a strong electric field, it depends only on the field intensity and is wavelength independent

[0015] , This allows for the probability of tunnelling ionization, and the tunneling ionization rate to only depend on the intensity of the incident electric field. When one considers the role of tunnelling ionization in the femtosecond ablation process, it is most dominant when MPI is minimized, and the avalanche has yet to form

[0015] , As such, the effects of tunnelling ionization mostly occur for longer wavelengths, most predominantly in the infrared, with pulse durations shorter than 50 fs [14,15],Avalanche Ionization

[0082] During a multiphoton absorption event, a sufficient electron density to facilitate absorption from the laser pulse by liberated electrons is usually produced after approximately 50 fs (as shown in FIG. 2). The free electrons have a very broad absorption spectrum with stronger absorption at longer wavelengths. This process is much more efficient than tunnelling ionization (Tl) and multiphoton ionization (MPI). As shown in the bottom panel of FIG 2, after a liberated electron gains energy from single-photon absorption, the liberated electron can collide with non-liberated electrons in the valence band, driving more electrons into a free state. This process of electrons colliding with sufficient energy to cause ionization is known as collisional ionization. The newly liberated electrons generated by collisional ionization can undergo absorption to drive more electrons into the plasma, driving an electron avalanche throughadditional collisional ionization. This process is illustrated in FIG. 5. and is known as avalanche ionization. As shown in FIG. 5, free electrons absorb photon energy, gaining enough energy to liberate an additional electron from the valence band while remaining ionized. These new free electrons can then release more electrons, leading to an exponential increase in the number of free electrons in the system, resulting in highly efficient ionization within the material. Once the laser pulse ends, this process will also end.

[0083] The avalanche ionization (Al) process provides a very efficient means of converting laser energy into thermal energy to drive ablation and create a high number of ions and liberated electrons. These electrons cause material ejection through two processes. As shown in FIG. 2, the first (faster) process is the Coulomb explosion. In this process, the electrons in the ablated volume are ionized, resulting in a high density of relatively immobile ions and a higher density region of mobile electrons. The positively charged ions will repel, causing a Coulomb explosion of electrons and ions. The second (slower) process is the phase change and ejection of the ablated volume, which removes significantly more material. This process results from the energetic liberated electrons recombining with the ions, transferring energy to the lattice and causing a fast phase change in the ablated volume (from solid to gas phase or liquid to gas phase).

[0084] In the case of avalanche ionization, the predominant sample constituent that is ionized will be dependent on the scattering cross section of the electrons and the relative abundance of each constituent in the solution. As water is much more abundant than the proteins, lipids or metabolites in the solution of a typical biological tissue sample, and they have a similar electron collisional ionization cross section, most of the avalanche ionization will occur in the water.

[0085] The use of longer wavelengths, e.g. infrared light, leads to a longer interaction time (per cycle) of the laser electric field to accelerate the resulting electrons to drive further tunnel ionization. The use of IR wavelengths provides a means to selectively deposit energy into the matrix for control of the ablation depth (as illustrated in FIG. 2, panel C).Methods of Limiting Biomolecule Fragmentation

[0086] For applications in mass spectrometry, laser-induced ionization should produce a significant yield of ions. When comparing the ionization of femtosecond and nanosecond lasers, the much higher peak power of femtosecond laser pulses results in a much higher number of ions. To reach the fundamental detection limit for mass spectrometry detection, the ablation cloud should be small enough to prevent further ionization in the plume. As a result, it is preferable to limit the pulse energy to encourage Coulomb explosion ionization from femtosecond lasers and limit the role of phase explosion during ablation [4], When analyzing biological tissue using femtosecond laser ablation, it is beneficial to reduce or minimize thedamage of biomolecules, including proteins, during laser extraction. Methods of limiting fragmentation are considered below for different modes of ionization.Fragmentation During Avalanche Ionization

[0087] In conventional mass spectrometry, the use of electrons to drive fragmentation processes is well documented. Some specific examples include electron-induced dissociation (EID), electron capture dissociation (ECD), and electron transfer dissociation (ETD)

[0021] , In EID, electron energies greater than 20 eV are used for efficient top-down protein fragmentation and detection of protein fragments [21 ,22], In these processes, the energy of the electrons is precisely controlled to cause reproducible fragmentation.

[0088] When ablating material using laser pulses with a pulse duration longer than 50 fs, the ionization avalanche causes collisional ionization to be a significant source of ionization. This happens because the electrons have enough energy to drive the fragmentation of biomolecules like proteins. This process of EID, ECD and ETD differs significantly from the avalanche ionization process, which has a wide range of electron energies. As a result, there has been significant concern that the wide distribution of electron energies could cause unpredictable (and therefore unidentifiable) fragmentation

[0023] , However, this concern is unwarranted.

[0089] Previous studies have shown that secondary ionization and fragmentation of proteins occur for electron energies greater than 10 eV

[0022] , To limit biomolecule fragmentation, the goal is to produce an electron cloud with the fewest possible electrons with energy above 10 eV. In general, having less than 20% of free electrons over 10 eV has been found to lead to intact extraction. Previous studies have shown that the energy distribution of the electrons inside the ablation plume can be modeled using rate equation modelling

[0026] , These models indicate that the population of electrons with energy greater than 10 eV changes as a function of wavelength. When considering the ionization threshold and threshold for the fragmentation for proteins and amino acids, only electrons with energies greater than 10 eV contribute to fragmentation. FIG. 6 shows some examples of the energy required to cause ionization and significant fragmentation, which is estimated based on the work by Imasaka et al

[0017] , It is also noted that the 10 eV limit determines the minimum wavelength that can be used in the dual pulse method described below (124 nm, which has a photon energy of 10 eV).

[0090] The avalanche ionization process is well-established to be more efficient at infrared wavelengths, requiring lower peak intensities near 1012W I cm2to drive ablation. At intensities above the ablation threshold, the average energy of the electrons easily exceeds that of the fragmentation threshold of proteins and other large molecules. This is shown, for example, in FIG. 7 (and ref.

[0014] ), which provides results from a method of calculating average energies of electrons as a function of wavelength and intensities). The figure plots asymptotic averagekinetic energy of conduction band (CB) electrons as a function of wavelength and intensity for threshold (1012W / cm2), close to threshold (1013W / cm2) and above well above threshold (1014W / cm2). Values were calculated following the methods outlined in Ref.

[0013] , This figure clearly shows that for intensities above 1014W / cm2the average liberated electron energy is above 10 eV and is enough to cause uncontrolled protein fragmentation, which would prevent intact protein extraction and identification.

[0091] An initial experimental dataset for individual proteins is shown in 9 (and reported in Alexander A.C. Wainwright, Khaled Madhoun, Pei Su, Samuel E. Janisse, Jessica E. Besaw, Harmanjot S. Grewal, Oliver P. Ernst, Jared O. Kafader, Neil L. Kelleher, and R.J. Dwayne Miller, The Journal of Physical Chemistry Letters 2025 76 (34), 8785-8791 DOI:10.1021 / acs.jpclett.5c01440). This dataset presents mass spectra of protein called Mast-R showing post ablation with wavelengths of 1030 nm, 515 nm, and 313 nm for pulse durations between 100 fs and 170 fs, at a peak intensity near 1013W / cm2, where the intact peak intensity decreases as wavelength decreases. This figure clearly shows how the relative abundance of fragments increases with shorter wavelengths and shows successful intact protein extraction for conditions with the laser intensities that minimize the number of electrons with energies exceeding 10 eV.Fragmentation During MPI

[0092] As described above, multiphoton ionization has been used in mass spectrometry for many years to intentionally cause protein fragmentation. Studies by Imasaka et al. have shown that when molecules absorb slightly more than their ionization threshold, the molecule will start to experience fragmentation

[0024] , As such, minimizing the energy absorbed in the multiphoton process to the ionization threshold is ideal.Fragmentation During Tunneling Ionization

[0093] Tunnelling ionization liberates electrons without providing the excess energy required to fragment large organic molecules. The only way that the approach could cause significant fragmentation would be from the formation of a strong Coulomb repulsion across the molecule, which is unlikely as the barrier for tunneling increases significantly for each subsequent ionization which would prevent the formation of the high charge states required to fragment organic molecules. Thus, a process dominated by tunnelling ionization will, by its very nature, limit the fragmentation of proteins.Lateral Submicron Spatial Resolution of Femtosecond Lasers

[0094] For many years, femtosecond lasers have been used to remove material with lateral dimensions smaller than the diffraction-limited spot size, as shown in FIG. 9. The diffractionlimited spot is the smallest diameter to which a laser beam can be focused. FIG. 9 illustrates a scenario where the band gap targeted by multiphoton ionization (MPI) corresponds to a three-photon process. Under this condition, a diffraction-limited, focused Gaussian beam can achieve material removal at a sub-diffraction-limited size. This occurs because only a portion of the beam has sufficient energy to drive the three-photon absorption process responsible for ablation. While sub-diffraction-limited material removal is achievable, the process is limited to material removal just under a micron in diameter when using near-IR lasers, which are most effective for this multiphoton reduction. Consequently, this use of near-IR lasers alone is unsuitable for achieving a 100 nm diameter.Sub-micron Depth Resolution of femtosecond Laser Ablation

[0095] As previously mentioned, the sample ablation volume (“ablation voxel” or “voxel”) desired has a volume of equal to or less than approximately 106nm3to keep the total charge sufficiently low to limit transverse Coulomb repulsion which otherwise leads to aberrations in the time of arrival, significantly affecting the mass resolution. As such, pulse conditions may be selected such that a depth confinement of the extracted volume is approximately 100 nm. It is well documented that femtosecond laser pulses can achieve ablation craters or ablation depths between 0.1 and 1000 nm [10,28-32], This finite ablation depth is possible because of the two-part extraction process which occurs during femtosecond laser ablation as described above: a Coulomb explosion which is followed by a rapid phase change and expansion

[0010] . Notably, the significance of each process is governed by the laser fluence used and the electron lattice recombination rate which is material dependent.

[0096] As described above with reference to FIG. 2, the Coulomb explosion process can best be described in terms of the dynamics of the ionized material [33-35], In a Coulomb explosion, the larger, slower moving positive ions which comprise the lattice are left stationary in the top layers of the sample. These densely packed positively charged ions then repel each other and are assisted in some cases by the pulling force from the liberated electron cloud above the surface. Theoretical calculations have shown that the Coulomb explosion can occur without the additional pull of the electron cloud, provided that the electron mobility is sufficiently low that the electrons cannot recombine with the bulk lattice before the explosion occurs

[0036] , Notably, only the top layers of the material will be affected by this process, allowing for the removal of material on the order of 100 nm in depth

[0037] ,

[0097] In the case of the Coulomb explosion, confinement within the first 100-1000 nm of a sample is made possible by two processes: modification of the lattice potential, and plasmashielding [36,37], Although a lattice is traditionally defined as a periodic arrangement of points in space, this concept can be generalized to liquids, inhomogeneous solids (like tissue) and biomolecules. In liquids, the absence of long-range periodicity means the potential landscape is non-uniform and dynamic; however, on short timescales, atoms, ions, and molecules can be treated as quasi-static. Under these conditions, a free electron experiences an effective potential arising from surrounding ions, which can be modeled as an inhomogeneous “lattice”. Similarly, in biomolecules, the term lattice often refers to the spatial arrangement of atoms within macromolecular structures or transient networks of interactions, which, while not perfectly periodic, still impose constraints that govern molecular dynamics, stability, and electron-photon dynamics. As described above, at the start of a laser pulse, the absorption process is dominated by multiphoton ionization. During this process, the lattice potential of the material will be modified as electrons are removed from the lattice, making it more difficult for secondary ionization to occur from the same molecules within the substrate. In the case of infrared and visible light, after a short period, which can be short as 20 fs

[0036] , but usually is approximately 50 fs (as discussed above and in ref.

[0013] ), the liberated electrons can start to dominate the absorption and ionization process through avalanche ionization. This process leads to significant ionization within the first 100 nm of the irradiated volume

[0036] , As a result, this top surface will eventually have a sufficient Coulomb repulsion from the positive, immobile ions to cause ejection from the surface through a Coulomb explosion, with complete lattice breakdown. Without intending to be limited by theory, it is believed that this change in lattice potential, and the decrease in binding energy due to ionization, is inherently nonlinear, is an effect that greatly localizes the material extracted by the laser action.

[0098] During ultrafast laser ablation, low-density plasma forms between the incoming laser and the target sample. This plasma exhibits extremely high absorptivity from ultraviolet to infrared attenuates the laser energy before it reaches the underlying biomolecules. As a result, direct interaction between the intense laser radiation and the biomolecules is prevented, protecting them from photochemical damage. This phenomenon, where plasma acts as a protective barrier against high-intensity laser exposure, is referred to as plasma shielding. Before the plasma can recombine with the ions in the solution, the extremely high charge density builds up resulting in a columbic explosion before the electrons energy can converted into thermal energy, preventing thermal damage.

[0099] During the phase explosion, the energy left over from the Coulomb explosion is converted into lattice heating from the recombination of the liberated electrons. The result is an ejection of the vaporized bulk lattice, which results in extraction between 10 nm and 1000 nm [10,37],

[0100] For materials with a short electron-lattice recombination time, the Coulomb explosion process is very limited, and in the extreme case of metals, is not reported. As a result, for semi-conductors and metals, only the phase explosion drives the material removal process.

[0101] However, in the case of dielectrics materials, such as biological samples, the recombination time for electrons is much slower. As such, both Coulomb and phase explosion occur. When the fluence is near the threshold fluence, the Coulomb explosion process tends to remove more material than the phase explosion process. As the Coulomb explosion only removes the top surface of the material where there is sufficient charge built up, a very well defined and controlled ablation depth on the order of 100 nm [4] is achieved. Fortunately, this is a suitable depth range for space-charge-free mass spectrometry detection. For applications in biomaterials, this leads to sub-cellular depth control, which is ideal for the next generation of MSI. However, it is noted that the significant change in interstitial fluid pressure at the surface of the cell after ablation can lead to the cell losing structural integrity and collapsing. However, this problem is easily resolved if the cell is ablated when in a frozen state.Super-Resolution, Low-Fragmentation, Nanovoxel and Spatially-Resolved Mass Spectrometry

[0102] The present inventors thus realized, based on understanding of the various ablation and ionization mechanisms described above, in particular, based on an understanding of femtosecond laser ablation when using laser pulses at the ablation threshold, that it is possible, with appropriate femtosecond (or, in some cases, picosecond) laser pulse conditions, to achieve the dual goal of (i) ablation and ionization of biomolecules with little or no fragmentation and (ii) ablation of a volume (“voxel”) with lateral dimensions smaller than the diffraction limit using non-linear absorption associated with femtosecond laser pulses and a sub-1000 nm (or even sub-100 nm) absorption depth. The present example systems and method can therefore provide an ablation and ionization process that can achieve a small voxel volume, such as between approximately 106nm3and 109nm^o keep the total charge sufficiently low to reducer or limit transverse Coulomb repulsion among the ablated ions, thereby facilitating improved resolution and sensitivity when performing mass spectrometry analysis.

[0103] The present inventors understood that with the spatial dependence of the energy absorbed for an n photon process being dependent on n and a spatial coordinate r according to the expression as / (r)n, then for a Gaussian pulse shape, one can expect a reduction in the absorption radius vs the incident beam spot size radius (defined by radius) to be . With this reduction in absorption radius (i.e. the lateral spatial extent over which absorption occurs), it is possible to employ a near infrared laser with a diffraction limited spot size diameter of 1 micron (achievable for a wavelength of 800 nm) to achieve an absorption radius below 500 nm(e.g. via a 7 photon process). However, this spot size reduction does not independently achieve the desired 100 nm voxel objective, nor does it directly achieve the desired absence of significant reduction in fragmentation, since it only provides a lateral spatial constraint without consideration of depth or ablation mechanism.

[0104] To achieve a sub-micron depth, in addition to the aforementioned lateral spatial confinement, the present inventors needed to combine their understanding of the conditions that can achieve a lateral constraint with (i) an understanding of the pulse conditions suitable for obtaining a sub-1000 nm absorption depth, and (ii) an understanding of the pulse conditions suitable for achieving low fragmentation upon ionization. The forthcoming portion of the present disclosure provides a detailed explanation of how this understanding was developed and employed to arrive at improved multi-pulse ablation systems and associated methods for achieving sub-pm3ablation and ionization with little or no fragmentation, thereby enabling, for example, sensitive mass spectrometry detection and other applications. Indeed, with the improved understanding of the femtosecond (fs) and picosecond (ps) laser ablation process for pulse durations exceeding 50 fs that are driven primarily by the electron avalanche effect, the present inventors realized that it is possible to enhance spatial resolution while minimizing biomolecule fragmentation. This can be achieved, for example, by employing at least two pulses: one pulse having a shorter wavelength (e.g. in the ultraviolet or visible range) and another pulse having a longer wavelength (e.g. in the infrared range), with a sub-nanosecond intra-pulse delay.

[0105] In such an example embodiment, the shorter wavelength pulse benefits from a smaller diffraction-limited spot size, allowing for spatial confinement below the diffraction limit of the longer wavelength pulse, and also potentially enabling sub-micron beam overlap on the sample. As described below this approach can be adapted to reduce or avoid fragmentation of ablated molecules (e.g. ablated biomolecules), by (i) employing suitable pulse properties that reduce or minimize the role of MPI, which is the primary contributor to protein fragmentation) in the overall ionization process and (ii) employing suitable pulse properties that avoid a substantial population of electrons with energies above 10 eV, which also contributes to fragmentation.

[0106] The following sections of the present disclosure introduce the concept of an all-optical dual pulse ablation process, where each pulse is either a femtosecond or picosecond pulse, and where the two pulses have different wavelengths and are directed onto a common location to generate ablation and ionization primarily via the Coulomb explosion. The present inventors have found that this two-wavelength dual-pulse ablation method can achieve ablative extraction and ionization of substantially intact biomolecules from bulk material with an extracted volume smaller than 1 pm3, and in some cases, smaller than 100 nm3.

[0107] As will be described below, the example systems and methods disclosed below, and variations thereof contemplated by the present disclosure, exploit multiphoton absorption processes at sufficiently low peak pulse powers to control the electron avalanches typically seen in femtosecond laser “machining.”

[0108] One advantage of the example systems and methods disclosed herein, which employ a dual-pulse ablation scheme involving femtosecond and / or picosecond laser pulses, is that the approach provides a versatile ionization and ablation source that can be employed for the ionization and ablation of a wide range of materials. Even for materials nominally transparent at low laser intensities, such materials become completely opaque at high peak powers that induce plasma formation and extremely strong light absorption.

[0109] The present disclosure thus provides example systems and methods that employ dual (or, more generally, multi) pulse laser ablation and ionization of a biological sample, where the effects of multiphoton ionization, tunnelling ionization and avalanche ionization can be controlled (e.g. balanced, tuned) to reduce (e.g. limit, minimize) biomolecule fragmentation. The example systems and methods disclosed herein also take advantage of the modification of the ion-lattice potential of the sample which results from the surface emission of electrons to confine the depth of ablated material to within a shallow absorption depth, such as the first 1000 nm, or even the first 100 nm. As explained above, the term “lattice” is used to describe the intermolecular forces between molecules in a biological system (tissue, protein solutions, single cells, etc.) in a region with short-range order, following the conventions of the field. The change of the ion-lattice potential, coupled with limiting the multiphoton ionization process leads to the extraction of 100-1000 nm (in some cases sub-100 nm) voxel dimensions with intact biomolecules to be extracted at a scale below the diffraction limit that would normally be expected.

[0110] As described below, the unique nano-voxel (sub 1 pm3ablation volume) and low-fragmentation ablation and ionization that may be achieved by the various system and method embodiments disclosed herein can provide intact (or nearly intact) molecules for use as an injection source in spatially-resolved (e.g. imaging) MS, with low aberrations in the time of arrival, and thus improved mass resolution relative to conventional mass spectrometry systems. This process can facilitate many new applications, such as, for example, the creation of molecular maps of biological processes in cells. This level of sensitivity and resolution can likewise have applications in the earliest possible disease detection and characterization of specific molecular markers to enable objective disease determination at the cellular and subcellular level.

[0111] The example embodiments described herein differ significantly from the thermal processes achieved using dual pulse techniques with longer time delays between the twopulses, such as those described in US Patent No. 11,094,518, which employs two overlapping pulses to drive a thermal ablation process. Such a process requires the use of two nanosecond laser pulses with delays of nanoseconds to microseconds between pulses to drive thermal ablation. It is noted that these lasers disclosed in the 11 ,094,518 patent can operate in a “heat” or “tenderize” modes, where the laser pulses heat the material or breakup the connective tissue of a biological sample to enable ablation with the second pulse. Such a technique can achieve ablation below the diffraction spot size of the IR laser pulse by thermally heating the sample. The shorter pulses of this disclosure, with sub-ns spacing between pulses employ a plasma-mediated process instead of a thermally-mediated process common to all nanosecond laser ablation techniques. Thus, the mechanism and methodology of the aforementioned example embodiments that employ sub-ns pulse delays are fundamentally different, yielding different depth profiles and ablation characteristics.

[0112] It will be understood that the dual pulse example embodiments disclosed herein may be performed with temporally simultaneous pulses (temporally overlapped pulses having a common pulse duration), temporally overlapping pulses with different pulse durations, such that one portion of a first pulse temporally overlaps with at least a portion of the second pulse, and temporally offset pulses, provided that the temporal delay between the two pulses is less than 1 nanosecond. Moreover, while various example implementations described herein involve a dual pulse configuration in which one pulse is UV pulse and another pulse is an IR pulse, it will be understood that such example implementations are not intended to be limiting, and that other combinations of wavelengths (and wavelength regions) may be employed in the alternative.

[0113] FIG. 10 schematically illustrates a non-limiting example implementation involving the temporally overlapped delivery of two pulses, where one pulse is a UV pulse 20 and another pulse is an IR pulse 30 (other pulse wavelength and pulse delay variations may be employed in the alternative, as explained in further detail below). The pulses 20 and 30 are delivered to the sample 10 for spatially-selective ablation, optionally in the form of an array or other spatial distribution that facilitates image generation. The ablated (ablation) plume 40 is collected, for example, via a collection inlet 50 (e.g. having a lower pressure) that transports the ablated molecules to a mass spectrometer for mass analysis. While the figure illustrates an example implementation involving ambient collection, in other example implementations the ablation may be performed within the mass spectrometer directly, as explained in more detail below.

[0114] While FIG. 10 illustrates an example embodiment involving temporally-overlapping pulses, in other example embodiments, sub-diffraction limited intact (nearly-intact, i.e. low-fragmented) biomolecule extraction can be achieved using a pulse sequence that includes (i) an initial laser pulse in the UV range to generate seed electrons through either simultaneous or sequential ionization and (ii) the subsequent delivery of a longer wavelength (e.g. IR) pulse. Theterm “sub-diffraction limited”, as used in the context of the present example embodiment, and other multi-pulse example embodiments disclosed herein, is intended to refer to cases in which the lateral spatial extent of the ablation volume is less than the diffraction-limited spot size of at least one of, and in some cases, both of, the dual laser pulses. Wavelengths (e.g. in the UV and visible) are particularly effective because they ionize more efficiently than longer wavelengths (e.g. in the infrared) before the electron avalanche forms

[0014] , This process is primarily due to the resonant enhancement and hence higher efficiency of multiphoton ionization (MPI) at shorter wavelengths

[0015] , Without intended to be limited by theory, it is believed that an electron avalanche requires an initial electron density of 1016to 1019cm~3

[0014] , Accordingly, in some example embodiments, short wavelengths (e.g. in the UV and visible) can produce this initial electron density with lowest possible electric fields to avoid plasma formation and massive fragmentation, while a longer wavelength pulse, delivered simultaneously or sequentially with the short wavelength pulse, can drive the subsequent electron avalanche. The dual (or, more broadly, multi) pulse excitation process of selected embodiments of the present disclosure can reduce the peak intensity required for ionization and ablation, limiting the degree of MPI (the primary contributor to fragmentation) needed to seed the process, while also minimizing fragmentation caused by avalanche ionization by limiting the energy of the electrons in the ablation cloud. This approach is more effective for sub-diffraction-limited sampling compared to solely relying on multiphoton absorption.

[0115] For example, some embodiments that employ temporally overlapped pulses delivery (e.g. simultaneous) for dual-wavelength ablation, a short-wavelength laser beam and a longer-wavelength beam are directed into the medium simultaneously (e.g. such that they are temporally overlapped, but need not have the same pulse length nor complete temporal overlap). The short-wavelength beam can provide pulses at an intensity below the ablation threshold, but high enough to cause multiphoton ionization to seed the required electron density for an electron avalanche to be driven by the longer wavelength laser source. For example, the short-wavelength beam can have an intensity greater than approximately 109W / cm2(William J. C. Francis, Harmanjot Grewal, Alexander A. C. Wainwright, Xuchun Yang, Massimo Olivucci, R. J. Dwayne Miller; Resonant multiphoton processes and excitation limits to structural dynamics. Struct. Dyn. 1 March 2024; 11 (2): 024301.). In this scenario, the longer-wavelength beam is applied at a peak power below the multiphoton ionization threshold, where the longer-wavelength beam drives the avalanche process most efficiently without leading to further multiphoton ionization that would lead to fragmentation. Without intending to be limited by theory, it is believed that a protein or other molecule of interest can withstand one electron removed to form an ion state but multiphoton ionization leading to multiple charged states of the molecule lead to fragmentation.

[0116] As noted above, the example dual-pulse (and multi-pulse) methods disclosed herein enable control over both spatial confinement and free electron density during ablation and ionization. Indeed, if a single pulse is delivered at a wavelength of 1000 nm, a peak intensity of1012(defined bY FWHM of the pulse duration) is required to drive the ablation process for pulse durations longer than 50 fs. However, by spatially and temporally overlapping a UV laser pulse with an IR laser (with femtosecond or picosecond pulse lengths), the combined ionization generated by the overlapping UV and IR irradiation, resulting from the Coulomb explosion process, is sufficient to enable ablation in the region where the beams intersect, at intensities less than 10% of that required for a single IR pulse (see FIG. 11). This approach allows for exceeding the diffraction-limited spot size of the IR pulse while reducing biomolecule fragmentation during extraction.

[0117] As described above, the example dual-pulse (or multi-pulse) modality generally involves at least two spatially overlapping laser pulses (temporally overlapping or temporally delayed, as described in further detail below) having different wavelengths (e.g. center wavelengths).

[0118] As noted above, in some example embodiments involving a dual- or multi-pulse modality, the first laser pulse (e.g. generated by a first laser or laser system) has a wavelength in the UV range and the second pulse has a wavelength in the near-IR range (the second pulse may be generated by the same or a different laser system). Both laser pulses are delivered onto the material that is to be ablated with at respective peak intensities below 1016-^, that would otherwise lead to run away plasma formation, and at least one of the two beams operates below the ablation threshold. It should be noted that the peak intensity used may vary depending on the implementation. The UV laser is used to achieve, for example, via MPI, a sufficiently high amount of ionization to generate the requisite free electron density to initiate an avalanche ionization process when energy from the second pulse is absorbed, with a substantially lower peak power relative to what would be needed to achieve ablation with a single UV pulse. The IR laser, which is focused onto the same location on the sample such that the UV spot resides within the IR spot, and which is delivered within 1 ns of the UV pulse, in turn, drives the avalanche ionization (Al) process to destabilize the lattice and deposit heat by taking advantage of its longer wavelength and longer interaction time in accelerating free electrons to drive the avalanche process. This combination and cumulative nonlinear coupling to ablation results in effective removal of material involving volumes much below the diffractionlimited spot sizes of the laser pulses. In some example implementations, the IR wavelength can be extended beyond 2 microns, for example, up to 5 pm, or up to 10 pm, or up to 50 pm, or up to 100 pm, although this comes with trade-offs in spatial overlap with the UV beam, as diffraction-limited spot sizes are inversely proportional to wavelength. In practice, a wavelengthof approximately 1 micron is often found to be optimal due to current technology and the relative ease of generating short IR pulses.

[0119] In some example embodiments, both pulses have a pulse intensity above 10A9 W / cmA2 and below 10A14 W / cmA2, or above 10A9 W / cmA2 and below 10A15 W / cmA2, or above 10A9 W / cmA2 and below 10A16 W / cmA2, or above 10A8 W / cmA2 and below 10A14 W / cmA2, or above 10A8 W / cmA2 and below 10A15 W / cmA2, or above 10A8 W / cmA2 and below 10A16 W / cmA2 . When the shorter wavelength is a UV pulse, the resulting outcome is multiphoton ionization generated by the UV pulse, followed by an avalanche ionization causing the electrons to reach a sufficient energy to drive a Coulomb explosion within the overlapped region between the two pulses.

[0120] In some example embodiments, two spatially-overlapped laser pulses irradiate a sample with a temporal delay, for example, from zero to 1 ns between pulses. It should be understood that depending on the implementation the delay between pulses may vary, for example from zero to 0.5 ns between pulses, or from zero to 0.2 ns between pulses, or from zero to 0.1 ns between pulses, or from zero to 0.3 ns between pulses, or from zero to 0.4 ns between pulses, or from zero to 0.5 ns between pulses, or from zero to 0.6 ns between pulses, or from zero to 0.7 ns between pulses, or from zero to 0.8 ns between pulses, or from zero to 0.9 ns between pulses, or a delay of more than zero and less than 1 ns, or a delay from more than zero to 0.5 ns, or a delay from more than zero to 0.6 ns, or a delay from more than zero to 0.7 ns, or a delay from more than zero to 0.8 ns, or a delay from more than zero to 0.9 ns. In some example embodiments, the second pulse has a wavelength that resides within the range of 340 nm and 5000 nm, and the first pulse has a shorter wavelength than the second pulse. In some example embodiments, both pulses used are shorter than 100 ps, and the peak pulse intensity (defined by the total pulse energy divided by the full width half maximum of the pulse temporal profile) of each pulse does not exceed 1016W / cm2.

[0121] Without intending to be being bound by theory, it is the understanding of the present inventors that the plasma-mediated process described herein employs high peak intensities delivered over short timescales to suppress thermally driven ablation mechanisms. In general, this desired effect is best achieved with pulse durations shorter than 10 ps, where the laser pulse is shorter than the thermal relaxation time of the material. Longer pulse durations in the 10-100 ps range can produce similar dynamics, occupying an intermediate regime between ultrafast and nanosecond laser ablation. Pulses in the 100-1000 ps range exhibit ablation behavior with a stronger thermal contribution, although they initiate with plasma dynamics similar to those described above. Consequently, while the ablation efficiency of pulses longer than 100 ps is generally lower than that of shorter pulses, it remains technically feasible that sub-nanosecond laser pulses (shorter than 1 ns) could achieve the effects outlined in thisdisclosure. In general, when two laser pulses are delivered onto the sample, they are provided such that at least one of the laser pulses irradiates the sample more than 50 fs after an onset of irradiation of the sample.

[0122] It will be understood that the wavelength ranges for the two pulses may vary in different example implementations. The following example wavelength ranges pertain to example cases in which the first pulse arrives on the sample before a second pulse, with a subnanosecond delay between the first and second pulses, or the first pulse arrives on the sample after the second pulse, with a sub-nanosecond delay between the second and first pulses, or where the first and second pulses arrive simultaneously on the sample. In one example implementation, the second pulse has a wavelength that resides within the range of 340 nm and 5000 nm, and the first pulse has a shorter wavelength in the UV or visible range. In one example implementation, the second pulse has a shorter wavelength in the UV or visible range, and the first pulse has a longer wavelength that resides within the range of 340 nm and 5000 nm. In one example implementation, the second pulse has a wavelength that resides within the range of 100 nm to 340 nm, with the first pulse is longer in the visible or infrared range. In another example, the second pulse has a wavelength that resides within 100 nm and 340 nm, while the first pulse being longer in the visible or infrared range. In another example, the second pulse has a wavelength that resides within 100 nm and 500 nm, while the first pulse is in the visible or infrared range. In another implementation, the second pulse has a wavelength that resides between 100 nm and 1000 nm, and the first pulse is between 1000 nm and 10 pm. Similarly, the second pulse can have a wavelength between 340 nm and 1000 nm, while the first pulse resides between 100 nm and 340 nm. In another example, the second pulse has a wavelength that resides within the range of 500 nm and 10 pm, and the first pulse resides between 100 nm and 500 nm. In another implementation, the second pulse has a wavelength that resides within the range of 1000 nm to 10 pm, and the first pulse is between 100 nm and 1000 nm. In another example, the second pulse has a wavelength between 1 nm and 340 nm, and the first pulse has a wavelength between 340 nm and the infrared range. The second pulse can also have a wavelength that resides within the range of 340 nm and 10 microns, and the first pulse is in the UV or visible range. In one example, the second pulse has a wavelength that resides within the infrared spectrum, and the first pulse is in the UV or visible range. Another implementation places the second pulse within the near-infrared spectrum, with the first pulse in the UV or visible range. In one example implementation, the second pulse has a wavelength that resides longer than 5000 nm, and the first pulse is in the UV or visible range. Alternatively, the second pulse can have a wavelength that resides longer than 5000 nm, with the second pulse also in the UV or visible range. In one example implementation, the second pulse has a wavelength that resides within the range of visible light, while the first pulse has a wavelength inthe UV or visible range. Conversely, in one example, the first pulse has a wavelength that resides within the range of visible light, and the second pulse has a wavelength in the UV or visible range. In one example implementation, the second pulse has a wavelength that resides within the range of visible or infrared, while the first pulse has a wavelength between 1 nm and 500 nm. Similarly, in one implementation, the first pulse has a wavelength that resides within the range of 1 nm to 500 nm, and the second pulse has a wavelength in the infrared or visible range.

[0123] The preceding example embodiments employ two laser pulses: one with a shorter wavelength that controls the sampling size, and another with a longer wavelength that increases electron density to sufficient levels to drive the ablation process. This approach allows for sub-diffraction-limited biomolecule extraction using, for example, a visible or UV pulse to generate seed electrons through MPI. This first pulse ionizes the sample before the electron avalanche occurs. Shorter wavelengths ionize more efficiently, creating the necessary electron density with minimal electric fields, thus preventing excessive fragmentation. The longer wavelength pulse then drives the Al process only within the region where seeded electrons exist, amplifying the electron density without increasing biomolecule fragmentation. This two-step process reduces the required peak intensity to drive ablation while limiting MPI and fragmentation. This method also controls the energy of the electrons involved in the process to prevent electron collisional fragmentation. The time between the two pulses can be delayed by up to 1 ns without affecting the physical outcomes.

[0124] FIG. 11 shows results from a numerical model of the present method for two 250 fs lasers pulses, one in the UV (257 nm) and one in the IR (1030 nm) and was produced using the single rate equation method outlined in

[0014] , In this example, the 1030 nm laser pulse at an intensity of 10A12 W / cmA2 irradiates a sample of water representing tissue. This IR pulse creates an initial free electron density below that required to drive ablation, but dense enough to be the dominant absorption mechanism. 100 fs after the IR pulse, laser pulse with a wavelength of a 250 nm and a pulse duration of 250 fs pulse duration, irradiates the sample at an intensity of 10A11 W / cmA2 and increases the electron density to 1O20electrons / cm3, driving ablation through a columbic explosion. This figure shows the electron density as a function of radius after both pulses have irradiated the sample. Both laser pulses have a gaussian time and spatial profile. The IR laser pulse is focused on a 5 urn laser 1 / eA2 spot size diameter, while the 250 nm pulse is focused on a 1 urn 1 / eA2 spot size diameter. In this example, only a 1 urn diameter volume will be ablated. Without intending to be limited by theory, it is believed that only the region with electron densities greater than that required for ablation will be extracted (with biomolecules intact), which is much smaller than the diffraction limited spot size of the 1030 nm laser beam.

[0125] Laser ablation via coulombic explosion is contingent upon achieving a charged particle density sufficient to generate electrostatic pressures that overcome the intermolecular forces of the target material. For pure water, this typically corresponds to charge densities equivalent to IO20- 1021electrons per cm3. In contrast, direct vaporization is driven by the transfer of thermal energy from excited electrons to the lattice ions and neutral molecules, thereby inducing a phase transition. The threshold electron density for vaporization, pth, can be approximated by: pthwhere p0is the molecular density, A is the band gap A+Ekinenergy, Fkinis the average electron kinetic energy, andrepresents the energy required for phase change. While this threshold is wavelength and intensity dependent, falling as low as IO20cm-3, the kinetics of these processes differ significantly. Although vaporization may occur at lower densities, the coulombic explosion proceeds several orders of magnitude faster than thermal vaporization. Consequently, the coulombic explosion governs the initial material ejection, with residual energy subsequently driving a secondary phase change. This is followed by a millisecond-scale shock wave that can eject additional mass, the magnitude of which increases proportionally with laser intensity.

[0126] As noted above, in some example embodiments, the second pulse can be temporally delayed relative to the first pulse, with the first pulse providing an MPI seeding process that takes advantage of the higher ionization rate of shorter wavelengths for MPI, to seed to processes of femtosecond laser ablation of longer near IRto MIR wavelengths. The resulting free electrons are deliberately exploited to drive the avalanche ionization process. Specifically, a femtosecond UV or visible laser pulse between 1 fs and 100 ps in pulse duration is used to seed the avalanche ionization process by using below ablation threshold intensities. Then before the electrons have time to recombine with the lattice (e.g. within 100 fs after the start of the shorter wavelength pulse, or earlier), a longer wavelength laser with a pulse duration between 1 fs and 100 ps is applied to cause an electron avalanche process by which additional energy is deposited into the lattice to drive ablation faster than any fragmentation process. In this method the longer wavelength pulse will be just below the ablation threshold for conventional femtosecond laser ablation. As a result, only the overlapped region of the system will be ablated. This pulse combination is specifically designed to prevent fragmentation of proteins but harnessing collisional ionization to provide the driving force for the ablation process. This combination enables the use of lower intensity of UV or visible laser light than otherwise needed alone to drive ablation as discussed above, which in turn minimizes the fragmentation otherwise caused by using short wavelengths.

[0127] An example of such a system would be a 200 fs UV laser pulse focused tightly with a peak intensity of 1012-^ striking a sample, and then 100 fs after the start of the UV pulse, alOH- IR pulse with a pulse duration of 200 fs, centered on the same location to drive the cmzelectron avalanche and ablation process. Thus, one can remove a smaller area than the diffraction-limited spot of the IR pulse while simultaneously limiting the fragmentation of the biomolecules extracted.

[0128] In general, this method of ablation is characterized by using two spatially overlapped, and temporally sequential laser pulses of different wavelength. In one example implementation, the first pulse has a wavelength below 800 nm, ideally in the 200 - 300 nm range, and a pulse duration less than 10 ps. Before twice the electron recombination time for a given excitation level, another pulse with a wavelength above 340 nm, ideally between 800 nm and 2 microns, and a pulse duration less than 10 ps is applied to the same location. Both lasers have a peak intensity below 1016-^, with at least one of the two beams being below the ablation threshold and the shorter wavelength is applied first to allow for the seeding of the ablation process.

[0129] In some example implementations, the two pulses overlapped on the sample can have pulse properties (e.g. respective peak intensities and / or fluences) selected such that when the threshold electron density for ablation is achieved, 50% of the free electrons have an energy less than 10 eV, or such that when the threshold electron density for ablation is achieved, 70% of the free electrons have an energy less than 10 eV, or such that when the threshold electron density for ablation is achieved, 80% of the free electrons have an energy less than 10 eV, or such that when the threshold electron density for ablation is achieved, 90% of the free electrons have an energy less than 10 eV, or such that when the threshold electron density for ablation is achieved, 95% of the free electrons have an energy less than 10 eV, thereby limiting fragmentation.

[0130] FIG. 12 illustrates a timing diagram for an example implementation of the dualpulse method using temporally overlapping laser pulses. In this embodiment, a shortwavelength pulse (e.g., UV) and a longer-wavelength pulse (e.g., IR) are applied simultaneously to the sample. The UV pulse initiates multiphoton ionization, generating an initial electron density that is insufficient to cause ablation. Concurrently, the IR pulse accelerates these free electrons, triggering collisional ionization and leading to an electron avalanche. This process continues until the IR pulse terminates, at which point the electron density is high enough to drive laser ablation.

[0131] FIG. 13 shows the timing diagram of one example implementation of the dual pulse method involving temporal delay between the two irradiating pulses. In this example embodiment, a short wavelength UV pulse irradiates the sample first, 200 ps before the longer wavelength, IR pulse, is applied to the sample at the same location as the shorter wavelength pulse. Both laser pulses have a pulse energy below 1016W / cm2. The resulting outcome is multiphoton ionization generated by the UV pulse, followed by a strong avalanche ionizationproduced by the IR pulse, causing the electrons to reach a sufficient energy to drive a Coulomb explosion within the overlapped region between the two pulses. This implementation would result in sub-micron3extraction of the dual irradiated volume.

[0132] Although reducing the role of MPI improves the yield of intact biomolecules during extraction, some of the preceding example embodiments can rely heavily on MPI to initiate the ablation process. Recent findings by the present inventors suggest that MPI is a primary cause of biomolecule fragmentation. Therefore, minimizing the contribution of MPI to the ablation process can be beneficial for preventing fragmentation.

[0133] To address this issue and achieve sub-diffraction-limited ablation areas, one can leverage the fact that, across all wavelengths, free electrons absorb energy more efficiently than biomolecules or water. This property is illustrated in FIG. 14, where the absorption cross section of water, MastR (a protein with a retinylidene chromophore), Hen Egg White Lysozyme (HEWL), myoglobin (mb) and liberated electrons estimated using a Drude model are shown for wavelengths from 250 nm to 1100 nm. The free electrons are shown to have the largest absorption cross section for all wavelengths shown, and this trend holds for the entire IR range.

[0134] Most biomolecules and surrounding water are effectively transparent in the visible to IR range (400 nm to 2 micron) being exploited, whereas the free electrons strongly absorb in this spectral region. Accordingly, one can use a long-wavelength pulse, such as a pulse having a wavelength as short as 340 nm to as long as 5 pm, to drive an MPI process to achieve a suitable electron density to support avalanche ionization, generating the required electron density for ablation without MPI dominating the overall process. Within 50 fs, the electron density can reach a level where a subsequent short-wavelength pulse (in the UV or visible range) can further increase the electron density via avalanche ionization (Al), surpassing the threshold required for ablation while reducing the material removal size. This approach protects biomolecules from fragmentation by utilizing the resulting electrons to shield the biomolecules (via their much stronger absorption of the incident light) during the process, enabling the intact extraction of biomolecules. To ensure that these electrons do not contribute to fragmentation, their energies must remain below 10 eV.

[0135] One example implementation of such an IR-seeding dual pulse ablation and ionization method is shown in FIG. 15. This figure shows the timing diagram of one example implementation of the dual pulse method involving temporal delay between the two irradiating pulses. In this embodiment, a longer, IR, wavelength pulse irradiates the sample first, 200 ps before the shorter wavelength, UV pulse, is applied to the sample at the same location as the shorter wavelength pulse. Both laser pulses have a pulse energy below 1014W / cm2. The resulting outcome is multiphoton ionization driven by the IR pulse, followed by an avalanche ionization produced by the UV pulse, causing the electrons to reach sufficient energy to drive aCoulomb explosion within the overlapped region between the two pulses. Although the ionization efficiency is lower in this implementation, the lower rate of MPI would lead to a higher yield of intact biomolecules from a sub-micron voxel.

[0136] FIG 16. provides various non-limiting example parameter space ranges for some of the example dual-pulse delivery modalities described above.

[0137] It is noted that while many aspects of the present disclosure relate to imaging mass spectrometry in which an image can be generated based on the collection of a spatial array of mass spectrometry measurements via the submicron multiphoton-absorption-based ablation and ionization methods described herein, it will be understood that in some example implementations, mass spectrometry measurements may be made according to the methods described above at one or more spatial locations in a sample that need not be defined in an array suitable for forming an image.

[0138] While many of the preceding examples refer to the need to achieve an ablation volume of less than 106nm3, in other example implementations, the pulse conditions can be selected such that the ablation volume is between 0.5x10A6 nmA3 and 1x10A6 nmA3, or between 0.5x10A6 nmA3 and 1x10A7 nmA3, or between 1x10A6 nmA3 and 1x10A7 nmA3, or between 0.5x10A6 nmA3 and 2.5x10A6 nmA3, or between 1x10A6 nmA3 and 2.5x10A6 nmA3, or between 0.5x10A6 nmA3 and 5x10A6 nmA3, or between 1x10A6 nmA3 and 5x10A6 nmA3, or between 0.5x10A6 nmA3 and 10x10A6 nmA3, or between 1x10A6 nmA3 and 10x10A6 nmA3, or between 0.5x10A6 nmA3 and 100 pmA3, or between 1x10A6 nmA3 and 100 pmA3, or between 0.5x10A6 nmA3 and 10 pmA3, or between 1x10A6 nmA3 and 10 pmA3, or between 0.5x10A6 nmA3 and 1x10A8 nmA3, or between 1x10A6 nmA3 and 1x10A8 nmA3, or between 0.5x10A6 nmA3 and 1x10A9 nmA3, or between 1x10A6 nmA3 and 1x10A9 nmA3, or between 0.5x10A6 nmA3 and 10A7 nmA3, or between 1x10A6 nmA3 and 10A7 nmA3, or between 0.5x10A6 nmA3 and 10A8 nmA3, or between 1x10A6 nmA3 and 10A8 nmA3.

[0139] Moreover, while many of the preceding examples refer to the use of pulse conditions that can lead to, via nonlinear absorption, an absorption depth of less than 100 nm, in other example implementations, the pulse conditions may be selected such that the absorption depth is between; 50 nm and 100 nm or between 50 nm and 250 nm, or between 50 nm and 500 nm, or between 50 nm and 1000 nm, or between 50 nm and 5000 nm, or between 50 nm and 10000 nm, or between 100 nm and 250 nm, or between 100 nm and 500 nm, or between 100 nm and 1000 nm, or between 250 nm and 500 nm, or between 250 nm and 1000 nm, or between 500 nm and 1000 nm, or between 500 nm and 5000 nm, or between 500 nm and 10000 nm, or between 1000 nm and 5000 nm, or between 1000 nm and 10000 nm, or between 5000 nm and 10000 nm.

[0140] Furthermore, while many of the preceding examples refer to the multiphoton ablation based on femtosecond pulses, in some example implementations, one or both of the pulses may be picosecond pulses having a pulse duration between, for example, 1 ps and 2.5 ps, 1 ps and 5 ps, 1 ps and 10 ps, 1 ps and 25 ps, 1 ps and 50 ps, 1 ps and 75 ps, and 1 ps and 100 ps, and 1 ps and 1000 ps.

[0141] Furthermore, while many of the example embodiments disclosed above refer to dualpulse excitation, in other example embodiments, three or more pulses may be employed, provided that they facilitate ablation via multiphoton absorption and ionization involving the generation of free electrons having sufficiently low energies to avoid or mitigate fragmentation. In other example implementations, the pulses may be delivered in a burst mode. For example, UV pulse at an intensity of less than 1014W / cm2, and a pulse duration shorter than 10 ps would irradiate a sample. Within 1 ns prior or after the UV pulse, a burst of IR pulses, each pulse less than 100 ps, with a burst envelope of less than 1 ns arrive at a sample at a repetition rate greater than 2GHz to drive the formation of an electron avalanche, such that the electrons cannot fully recombine with the lattice before the next pulse arrives. As such, the ionization avalanche drives that ablation process to remove substantially intact biomolecules. To prevent fragmentation, the energy of the electrons within the plasma should remain as low, preferably below 10 eV as previously described. As a result, the intensity of each pulse in the train should not exceed 1016W / cm2.

[0142] The skilled artisan will readily understand that the various example embodiments of the present disclosure can be implemented using different systems, and the skilled artisan will also understand how to adapt such laser systems for the ablation of various different types of materials according to the ablation methods disclosed herein. For example, in one example implementation, a person skilled in the art can employ a laser system, such as a 1030 nm laser system with a pulse duration of 200 fs split into two paths, each with independent power control. In the present example, a non-linear process can be employed to generate, from one of the paths, pulses with a different wavelength than the other path. For example, laser pulses in one path can be frequency converted to a harmonic (e.g. fourth harmonic) using known nonlinear optical techniques. One beam path can be delayed relative to another, within 1 ns, for example by a retroreflector-based delay stage, enabling precise control of the time difference between the two paths. The beams are then combined, for example, collinearly using a dichroic mirror and focused with a conventional optical element such as a lens or curved mirror, or separately focused along angled beam paths (e.g. using two separate lenses and beam paths to focus the pulses in an overlapped fashion). The fluence and intensities of each beam path can be controlled, for example, using one or more attenuators, enabling controlled delivery of the pulses with suitable focused pulse properties to achieve, via spatial overlap of the laser pulses,the ablative processes described herein, for example, in which an initial free electron density of a plasma is seeded with one pulse and increased by the pulse to achieve a threshold electron density for ablation within a volume that has a lateral extent that is less that the 1 / e2diffractionlimited spot size of at least one of the beams, and a submicron depth.

[0143] To determine the conditions under which such dual-pulse irradiation produces plasma within 50 fs of onset, the power of each beam can be controlled such that each beam alone is insufficient to cause ablation, and such that ablation occurs only when both pulses overlap within a delay of <1 ns. Verification can be performed by ablating a suitable sample (e.g. a sample containing an analyte of interest, such as a protein standard) and analyzing the plume with a mass spectrometer to confirm the ablation of the protein signals, and optionally varying one or more pulse properties such that the mass spectrometer signals are indicative of intact analyte and / or . a sufficiently low (e.g. sufficiently low for analyte identification) number of analyte fragments.

[0144] The skilled artisan will be aware of various analytical techniques for confirming that the pulse conditions are suitable for the formation of the plasma and the generation of plasma-mediated ablation, optionally with suitable electron energies to avoid substantial fragmentation, as described herein. For example, plasma mediation can be assessed by comparing emission spectra for single-pulse and dual-pulse irradiation, and observation of ablation accompanied by characteristic plasma emission spectra can be employed to confirm and / or characterize a plasma mediated process. The plasma characteristics and time evolution can be assessed, for example, using pump-probe spectroscopy to determine the density and energy of the plasma overtime. Further example methods of characterization include measuring ablation depth (<1 pm) and area (smaller than the diffraction-limited spot size of the longer wavelength), which can be measured, for example, using optical techniques, scanning electron microscopes, or atomic force microscopes. Furthermore, rate-equation modeling can predict optimal delay, pulse energy, and pulse width for achieving the desired plasma density and material removal, which can then be validated experimentally. The skilled artisan will appreciate that the analytical methods listed above, and other suitable known analytical methods, can be employed to facilitate the experimentation tuning and / or confirmation of suitable pulse properties for carrying out the methods disclosed herein.

[0145] Based on the discussion and discoveries outlined above, various example methods and applications are disclosed below for enhancing the spatial resolution of intact biomolecular extraction with minimal fragmentation for applications in mass spectrometry in ambient or vacuum conditions.Examples of Imaging Mass Spectrometry Systems

[0146] Referring now to FIG. 17A, an example imaging mass spectrometer system is shown. A laser ablation and scanning subsystem 100 is employed to scan a sample 105 and generate an ablated ionized sample according to the methods disclosed above, e.g. with little or no fragmentation, and / or with extraction volumes (voxels) of about 106nm3or less. In the example embodiment shown, the laser-ablated aerosol is generated in vacuum within the mass spectrometer 130. This can be achieved by placing a vacuum stable sample within the mass spectrometry chamber and then sending the laser light through an optical port into the MS. The two-laser pulses are then focused using a curved mirror system to create a laser spot on the sample. In this configuration, the laser ablation process will generate the charged aerosol within the MS. To improve ionization efficiency, secondary ionization sources like UV light with a wavelength longer than 300 nm can be used.

[0147] A wide variety of femtosecond laser systems may be employed according to the methods disclosed herein, provided that the emitted pulses, when focused onto the sample, satisfy the aforementioned criteria for achieving submicron ablation and little or no fragmentation. For example, titanium-sapphire, neodymium-doped or ytterbium-doped laser gain media-based systems can be used to create the dual pulse apparatus. With the fundamental IR beam serving as the longer wavelength, and a harmonic of the fundamental beam used as the shorter wavelength pulse. Other examples of ways to produce the shorter wavelength pulse include Nonlinear optical parametric amplification (NOPAs), Optical parametric amplifiers (OPAs) or any variety of two wave mixing nonlinear optical process could be used to generate the shorter wavelength pulse. There are also methods to use the fundamental as the short wavelength and use of parametric amplification to convert the aforementioned fundamentals to wavelengths in the 1 - 20 micron range by mixing the fundamental as the pump field and "white" light continua as the signal field to generate idler outputs continuous in the IR range from 1-20 microns with enough pulse energy to reach the critical peak power to use the dual wavelength approach for coupling laser fields into ablation processes without fragmentation and exploiting the short wavelength pulse of the 2 pulse interaction and its associated nonlinearity to goto ablation, material extraction, well below the diffraction limit of the corresponding linear field interaction. By adding a delay stage to one of the paths to ensure the beams arrive at the sample within the required delay time, for example, 200 ps or 1000 ps, it is possible to create the required dual pulse setup.

[0148] The laser pulses may be scanned relative to a sample to facilitate the desired spatially resolved focusing of the incident beam according to many different scanning mechanisms, including, for example, galvanometric (galvo) mirror scanners, polygon mirror scanners, and acousto-optic deflectors. Alternatively, the sample can be scanned relative to anincident beam using, for example, a motor-controlled translation stage movable in 1 , 2 and / or 3 dimensions.

[0149] Many different types of mass spectrometers may be employed to perform mass-resolved detection, including, for example, implementation with industry standard and custom-built ion-mobility, quadrupole, time of flight, magnetic sector, electrostatic sector, quadrupole ion trap, and ion cyclotron resonance mass analyzer systems as well as isotope ratio mass spectrometers and any system coupled with a reflectron. Examples of such systems include Thermo Scientific Orbitrap Velos Pro, Q-exactive series, QstarXL, GC-TOF, ZQ-MS, EI-GC-TOF, Delta Plus, 253 Plus, BrukerTimsTOF series, MRM, QTOF, triple Quad Ion Trap, and LC-MS based systems. To improve the mass spectrometer ion collection efficiency, the sample can be loaded directly into the vacuum chamber of the mass spectrometer, and brought to a vacuum stable state before the system is brought to ultra-high vacuum, or more generally vacuum condition. Examples of a vacuum stable state for biological material would be a frozen condition which could be achieved by a cryogenic sample stage. After being brought to a high vacuum, the sample can be ablated using the dual pulse method, with the generated ions being guided directly to one of the mass analyzers outlined above.

[0150] FIG. 17B illustrates an alternative example implementation in which the ablation plume is generated in ambient conditions and introduced into the mass spectrometer 130 by ablating the sample ahead of the inlet capillary so that the ablation plume is carried into the mass spectrometry by the gas flow driven by the vacuum of the MS. An alternative to this method includes the use of electrospray ionization (ESI) which mixes with the ablated plume to improve ionization efficiency.

[0151] The example control and processing circuitry 200 may include a processor 210, a memory 215, a system bus 205, one or more input / output devices 220, and a plurality of optional additional devices such as communications interface 225, external storage 230, and a data acquisition interface 235. In one example implementation, a display (not shown) may be employed to provide a user interface for facilitating input to control the operation of the system 200. The display may be directly integrated into a control and processing device (for example, as an embedded display), or may be provided as an external device (for example, an external monitor). The control and processing system 200 may include or be connectable to a console that provides an interface enabling an operator to control the system. The console may include, for example, one or more input devices, such, but not limited to, a keypad, mouse, joystick, touchscreen, and may optionally include a display device.

[0152] The methods described herein, such as methods of dual-femtosecond-pulse delivery to achieve sub-micron spatially-resolved ablation and ionization for mass spectrometry with little or no fragmentation (and optionally with extraction volumes (voxels) of about 106nm3or less) as described herein, can be implemented via processor 210 and / or memory 215. As shown in FIG. 17A, executable instructions represented as control module 250 are processed by control and processing circuitry 200. Such executable instructions may be stored, for example, in the memory 215 and / or other internal storage.

[0153] The methods described herein can be partially implemented via hardware logic in processor 210 and partially using the instructions stored in memory 215. Some embodiments may be implemented using processor 210 without additional instructions stored in memory 215. Some embodiments are implemented using the instructions stored in memory 215 for execution by one or more microprocessors. Thus, the disclosure is not limited to a specific configuration of hardware and / or software.

[0154] It is to be understood that the example system shown in the figure is not intended to be limited to the components that may be employed in each implementation. For example, the system may include one or more additional processors. Furthermore, one or more components of control and processing circuitry 200 may be provided as an external component that is interfaced to a processing device. Furthermore, although the bus 205 is depicted as a single connection between all of the components, it will be appreciated that the bus 205 may represent one or more circuits, devices or communication channels which link two or more of the components. For example, the bus 205 may include a motherboard. The control and processing circuitry 200 may include many more or less components than those shown.

[0155] Some aspects of the present disclosure can be embodied, at least in part, in software, which, when executed on a computing system, transforms an otherwise generic computing system into a specialty-purpose computing system that is capable of performing the methods disclosed herein, or variations thereof. That is, the techniques can be carried out in a computer system or other data processing system in response to its processor, such as a microprocessor, executing sequences of instructions contained in a memory, such as ROM, volatile RAM, non-volatile memory, cache, magnetic and optical disks, or a remote storage device. Further, the instructions can be downloaded into a computing device over a data network in the form of compiled and linked version. Alternatively, the logic to perform the processes as discussed above could be implemented in additional computer and / or machine-readable media, such as discrete hardware components as large-scale integrated circuits (LSI's), application-specific integrated circuits (ASIC's), or firmware such as electrically erasable programmable read-only memory (EEPROM's) and field-programmable gate arrays (FPGAs).

[0156] A computer readable storage medium can be used to store software and data which when executed by a data processing system causes the system to perform various methods. The executable software and data may be stored in various places including for example ROM, volatile RAM, nonvolatile memory and / or cache. Portions of this software and / or data may bestored in any one of these storage devices. As used herein, the phrases “computer readable material” and “computer readable storage medium” refers to all computer-readable media, except for a transitory propagating signal perse.

[0157] The systems and methods disclosed herein may be employed to interrogate a wide variety of samples. Non-limiting examples of sample include tissue samples, microbiological cultures, biological cells, subcellular components, cytoplasm, organelles, extracellular components, extracellular vesicles, blood, urine, biofluids, biopsies, cultural artifacts, archeological artifacts, microplastics, soil and environmental samples, plant materials, food products, pharmaceutical products, nano-materials, microelectronics, mineral samples, geological samples, forensic samples, protein and peptides solutions, tissue engineered constructs, synthetic polymers, biofilms, and cellular microenvironments.

[0158] It will be understood that the systems and methods disclosed above can be employed for a wide variety of uses and applications. A non-limiting list of example applications includes biomedical research, microbiological identification, drug distribution studies, tissue engineering and regenerative medicine, pharmaceutical drug development, pathology, quality control, material science, surface and interface studies including thin film analysis, environmental studies, microplastic analysis, forensic science, toxicology, agricultural analysis, metabolomics, proteomics, lipidomics, transcriptomics, geological and mineral investigations, ore testing, space exploration, explosive and chemical weapon analysis, archeological and paleontological studies, semiconductor testing, micro and nano-electronic failure analysis, bioprinting, and enhanced methods for drug delivery from surrounding fluids or direct injection into the mass spectrometer inlet.

[0159] For example, in some implementations, the methods disclosed herein may be employed for the spatially-resolved intracellular detection of intact biomolecules or biomolecules that undergo sufficiently low fragmentation to permit identification via a given mass spectrometry system.

[0160] To give a specific application of the above concept, one can consider the application for spatially imaging cells. In this regard, it should be noted that cell functions are inherently robust, given the unerring process of cell division and differentiation. The macroscopic structure of cells is well understood. However, relatively little information is available on the spatial relationships and correlations by which biochemicals perform the functions common to all life. This understanding is crucial as it forms the basis for all biological functions. To comprehend the basic physics of these strongly correlated biochemical pathways, one must go inside the cell, extract submicron voxels, and measure all the molecules within each volume element [1], A subcellular map of biomolecules will not only significantly enhance one’s comprehension ofcellular processes such as signaling, differentiation, proliferation, mortality, programmed cell death, and breakdowns leading to mutations but also pave the way for identifying new disease markers. This breakthrough could open a new field of subcellular medicine, revolutionizing disease diagnosis and treatment. For example, by understanding the spatial relationship of biomolecules between healthy and diseased tissue, it may be possible to use artificial intelligence in combination with this molecular level information to detect diseases more effectively than is currently possible by medical professionals. This could be done using spatially resolved biomarkers to train artificial intelligence in disease image recognition.

[0161] An example workflow of such a system is proposed in FIG. 18. The process begins by creating a training set composed of healthy and diseased tissue samples, identified using conventional histological techniques. Once this reference set is established, mass spectrometry imaging (MSI) is employed to map the spatial distribution of proteins within both healthy and diseased tissues. The resulting MSI datasets are then used to train an Al algorithm. After training, the algorithm analyzes new tissue samples and predicts regions likely to be diseased. These predicted areas are subsequently validated through MSI to confirm whether they exhibit the characteristic protein expression patterns associated with disease. The new MSI data is fed back into the Al model to refine its accuracy. This iterative cycle continues until the algorithm achieves near-perfect performance (e.g., 99.9% accuracy), at which point it can be deployed as an Al-powered imaging tool for disease recognition. This workflow is adaptable to any known or emerging disease, leveraging MSI technology to enhance diagnostic precision. Furthermore, higher MSI resolution directly improves the quality of data available for Al training, making the system increasingly robust and valuable.

[0162] In other example embodiments, the high resolution of the ablation can be used to precisely sample individual cells in a complex mixture of cells or cell culture for purposes such as microbial identification. Current techniques such as the MALDI-based microbial identification (e.g. Kostrzewa, M. (2018). Application of the MALDI Biotyper to clinical microbiology: progress and potential. Expert Review of Proteomics, 15(3), 193-202) require culturing microbial samples, whereas the described method is capable of sampling individual cells from a sample with minimal sample preparation, and would be capable of maintaining the native properties of the original sample such as cell variability, and distribution. This information can be used for biomedical research and clinical applications to study cells in areas such as but not limited to oncology, infectious diseases, immunology, hepatology, neurology, dermatology, cardiology, gastroenterology, hematology, ophthalmology, and obstetrics.

[0163] Various methods, including MS, have been developed to study proteins within a cell. Current mass spectrometry systems lack the spatial resolution for subcellular biomolecule mapping and often cannot detect all the biomolecules from a single sampling location. Oneneeds to detect all the molecules from each sampling spot to understand the whole molecular picture (i.e., free energy relationships) and the biomolecule processes essential to biological functions. This one non-limiting example of where the present example methods of IMS may be applied; e.g. in leveraging near-damage threshold femtosecond laser ablation to create subdiffraction limited sampling volumes of the analyte of interest. This level of detail will give the first full molecular map of sufficient detail to understand the highly orchestrated biochemistry involved in cell division. Furthermore, this technique demonstrates a method that can be used for ultrasmall volume biopsies on the order of femtolitres.

[0164] Further applications involving the precise structural modification of a substrate for additive or subtractive manufacturing can be performed using the current disclosure. One example is semiconductor processing for the purpose of circuit prototyping or manufacturing via the direct-write subtraction of conductive traces and the precise creation of sub-micron contact openings or interconnecting vias. This extends to the modification, repair, or manufacture of integrated circuits and microelectronic devices, including but not limited to logic processing architectures (such as central processing units and graphics processing units), memory architectures (such as volatile and non-volatile memory), and application-specific integrated circuits. In another example, modifications or repairs of sub-wavelength features in lithography photomasks can be performed. In another example, biocompatible texturing of titanium ora polymer surface for biomedical purposes such as the adhesion or growth of tissue can be performed. In another example, the method can be used for the precise manufacture and modification of nanostructures of lab-on-a-chip or organ-on-a-chip or organoid technologies.Fragmentation During MPI Methods of Enhancing Superresolution MSI

[0165] The examples disclosed above can be adapted to include or employ any matrix assisted laser desorption ionization (MALDI) technique to enhance the spatial resolution obtained by the laser during the desorption process. For example, the presence of a matrix material such as but not limited to sinapinic acid, caffeic acid or2,5-dihydroxybenzoic acid may be used with the sample. The presence of water or any other solvent in gas, liquid, solid, or supercritical phase can also be used as a sample matrix or as part of the matrix preparation process (for example, as disclosed in “The Desorption Process in MALDI. Klaus Dreisewerd, Chemical Reviews 2003103 (2), 395-426, DOI: 10.1021 / cr010375i).

[0166] Some of the advantages of the MALDI method can also be achieved by using a specific arrangement of metal or semiconductor substrate. Metals differ fundamentally from dielectric materials because their electrons are only weakly bound to the atomic lattice. This makes metals significantly easier to ionize compared to dielectrics. Leveraging this property, one could deposit an extremely thin layer of an analyte of interest onto a metallic substrate,such that incident light can pass through the analyte without substantial attenuation. In other words, the analyte layer is thinner than its optical penetration depth.

[0167] In such a configuration, when the system is irradiated with a strong, ultrafast laser pulse, the light interacts with both the analyte and the underlying metal. Under these conditions, both materials undergo strong-field (either tunneling and / or multiphoton ionization depending on the laser intensity used) and avalanche ionization. Because the energy required for strong-field ionization is lower for the metal substrate, and because both the analyte and the metal contribute to the overall plasma density, the threshold for laser-induced ablation and material ejection is reduced.

[0168] Accordingly, a thin analyte layer can be ablated at lower laser intensities using a dual-pulse irradiation technique. Such an approach offers several advantages: for instance, nano-scale metal spots could be patterned on the substrate to further minimize the ablation spot size (see FIG. 21 B), or the plasma formation process could be driven more efficiently, reducing the total energy required for ablation (see FIG. 21 A).

[0169] In principle, many of these advantages could also be achieved using a semiconductor substrate, provided the substrate is tuned to the laser wavelength to enable resonantly enhanced multiphoton absorption processes. This optimization would allow for more precise nano-scale material removal by introducing a nano-pattern of dopants, which would localize the resonant enhancement effects to specific regions. As with the metal-based approach, the analyte layer deposited on the semiconductor should be extremely thin.Specifically, the sample being ablated must be thinner than the optical penetration depth of the analyte, so that the incident light can pass through without significant attenuation. Under these conditions, the laser interacts with both the analyte and the semiconductor substrate, enabling efficient energy coupling. Such a process would facilitate the ejection of nano-scale ablation spots, combining the benefits of resonant enhancement with controlled layer thickness for improved precision.

[0170] To improve efficiency of collection, under some circumstances, front illumination, where the laser and the ejected material are on the same side of the sample, will lead to a stronger signal. In other circumstances, back illumination, where the laser and the ejected material are on either side of the sample will lead to a stronger signal. As such, all examples should be understood to apply for both back and front illumination geometries as shown in FIG.22.Enumerated Embodiments

[0171] Embodiment 1. A method of ablating an analyte from a sample, the method comprising:directing a plurality of laser pulses onto a sample, the plurality of laser pulses including two laser pulses each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including a shorter wavelength pulse and a longer wavelength pulse, the two laser pulses having respective laser pulse properties and being directed onto the sample such that:the two laser pulses are spatially overlapped on the sample;the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

[0172] Embodiment 2. The method according to embodiment 1 wherein at least a portion of the analyte within the ablation plume ionized.

[0173] Embodiment 3. The method according to embodiment 1 or 2 wherein the two laser pulses having respective laser pulse properties and being directed onto the sample such that the volume over which the free electron density of the plasma is increased above the threshold electron density for ablation has a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of both the shorter wavelength pulse and the longer wavelength pulse.

[0174] Embodiment 4. The method according to any one of embodiments 1 to 3 wherein the two laser pulses are delivered onto the sample with pulse properties such that a Coulomb explosion mechanism is the dominant mechanism of ablation of the sample caused by the two laser pulses.

[0175] Embodiment 5. The method according to any one of embodiments 1 to 3 wherein the volume is a first volume, and wherein, after a Coulomb explosion mechanism occurs, an additional volume of the sample is ablated via thermal vaporization mechanism, wherein the two laser pulses are delivered onto the sample with pulse properties such that the first volume exceeds the additional volume.

[0176] Embodiment 6. The method according to any one of embodiments 1 to 5 wherein the shorter wavelength pulse has a respective pulse intensity configured to seed the generation of the initial free electron density by multiphoton ionization.

[0177] Embodiment 7. The method according to any one of embodiments 1 to 6 wherein the wavelength of the longer wavelength pulse exceeds 340 nm.

[0178] Embodiment 8. The method according to any one of embodiments 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are unfragmented.

[0179] Embodiment 9. The method according to any one of embodiments 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into only two fragments.

[0180] Embodiment 10. The method according to any one of embodiments 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into three or fewer fragments.

[0181] Embodiment 11. The method according to any one of embodiments 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into five or fewer fragments.

[0182] Embodiment 12. The method according to any one of embodiments 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least half of the electrons in the plasma have an energy less than 10 eV.

[0183] Embodiment 13. The method according to any one of embodiments 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least 80% of the electrons in the plasma have an energy less than 10 eV.

[0184] Embodiment 14. The method according to any one of embodiments 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least 90% of the electrons in the plasma have an energy less than 10 eV.

[0185] Embodiment 15. The method according to any one of embodiments 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulsefluences such that when the threshold for ablation is reached, at least 95% of the electrons in the plasma have an energy less than 10 eV.

[0186] Embodiment 16. The method according to any one of embodiments 1 to 15 wherein the shorter wavelength pulse has a wavelength in the ultraviolet portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the infrared portion of the electromagnetic spectrum.

[0187] Embodiment 17. The method according to any one of embodiments 1 to 15 wherein the shorter wavelength pulse has a wavelength within the visible portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the infrared portion of the electromagnetic spectrum.

[0188] Embodiment 18. The method according to any one of embodiments 1 to 15 wherein the shorter wavelength pulse has a wavelength within the ultraviolet portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the visible portion of the electromagnetic spectrum.

[0189] Embodiment 19. The method according to any one of embodiments 1 to 18 wherein the shorter wavelength pulse is temporally overlapped with the longer wavelength pulse.

[0190]

[0191] Embodiment 20. The method according to any one of embodiments 1 to 18 wherein the shorter wavelength pulse and the longer wavelength pulse arrive simultaneously on the sample.

[0192] Embodiment 21. The method according to any one of embodiments 1 to 18 wherein the longer wavelength pulse is temporally delayed relative to the shorter wavelength pulse.

[0193] Embodiment 22. The method according to any one of embodiments 1 to 18 wherein the shorter wavelength pulse is temporally delayed relative to the longer wavelength pulse.

[0194] Embodiment 23. The method according to any one of embodiments 1 to 18 wherein the two laser pulses are absent of temporal overlap.

[0195] Embodiment 24. The method according to any one of embodiments 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 10 ps.

[0196] Embodiment 25. The method according to any one of embodiments 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 1 ps.

[0197] Embodiment 26. The method according to any one of embodiments 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 100 fs.

[0198] Embodiment 27. The method according to any one of embodiments 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 10 fs.

[0199] Embodiment 28. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A6 umA3.

[0200] Embodiment 29. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A7 umA3.

[0201] Embodiment 30. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A8 umA3.

[0202] Embodiment 31. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A9 umA3.

[0203] Embodiment 32. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such an ablation crater resulting from ablation of the volume has a depth less than 1000 nm.

[0204] Embodiment 33. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such an ablation crater resulting from ablation of the volume has a depth less than 100 nm.

[0205] Embodiment 34. The method according to any one of embodiments 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such when the threshold electron density for ablation of the volume is achieved, a lateral spatial region associated with the plasma has an area of less than 10A4 umA2, and a depth of less than 100 nm.

[0206] Embodiment 35. The method according to any one of embodiments 1 to 34 wherein the analyte comprises a biomolecule.

[0207] Embodiment 36. The method according to embodiment 35 wherein the biomolecule is selected from the group consisting of lipids, metabolites, and proteins.

[0208] Embodiment 37. The method according to any one of embodiments 1 to 36 wherein the sample is a biological sample.

[0209] Embodiment 38. The method according to embodiment 37 wherein the biological sample comprises at least one of blood, animal tissue, and plant tissue.

[0210] Embodiment 39. The method according to any one of embodiments 1 to 38 wherein the ablation plume is analyzed at a location that is spatially remote from the sample.

[0211] Embodiment 40. The method according to any one of embodiments 1 to 38 wherein the ablation plume is analyzed after a time delay during which the ablation plume is transported for subsequent analysis.

[0212] Embodiment 41. The method according to any one of embodiments 1 to 40 further comprising employing secondary ionization source to increase an ionization yield.

[0213] Embodiment 42. The method according to embodiment 41 wherein the secondary ionization source comprises an optical apparatus for directing a vacuum-ultraviolet beam onto the ablation plume within an evacuated region, wherein the vacuum-ultraviolet beam is configured to photoionize non-ionized analyte within the ablation plume.

[0214] Embodiment 43. The method according to any one of embodiments 1 to 42 wherein a metal, alloy or semiconductor substrate is provided beneath the sample, and the sample is provided with a thickness that is less than an absorption depth of at least one of the shorter wavelength pulse and the longer wavelength pulse, the metal, alloy or semiconductor substrate thereby increasing the free electron density of the plasma and lowering a respective fluence of at least one of the shorter wavelength pulse and the longer wavelength pulse needed to achieve the threshold electron density.

[0215] Embodiment 44. The method according to any one of embodiments 1 to 43 wherein the sample comprises a cell, and wherein the two laser pulses are directed onto the cell, such that the volume resides within the cell.

[0216] Embodiment 45. The method according to any one of embodiments 1 to 44 wherein the ablation plume is analyzed using an analytical device other than a mass spectrometer.

[0217] Embodiment 46. The method according to embodiment 45 wherein the analytical device is configured to perform analysis according to an analytical modality selected from the group consisting of high-performance liquid chromatography, Gas Chromatography and nuclear magnetic resonance.

[0218] Embodiment 47. The method according to any one of embodiments 1 to 43 further comprising:employing a mass spectrometer to measure the ablation plume; anddetecting the analyte via mass analysis of one or more mass peaks associated with the analyte.

[0219] Embodiment 48. The method according to embodiment 47 wherein the sample is positioned such that the ablation plume is directed through the inlet of the mass spectrometer.

[0220] Embodiment 49. The method according to embodiment 47 or 48 wherein the two laser pulses are scanned relative to the sample and wherein the mass spectrometer is employed to generate a mass spectrometry image of the analyte within the sample.

[0221] Embodiment 50. The method according to any one of embodiments 47 to 49 wherein the sample comprises a MALDI matrix material.

[0222] Embodiment 51. The method according to any one of embodiments 47 to 50 wherein the sample comprises a cell, and wherein the two laser pulses are directed onto the cell, such that the volume resides within the cell.

[0223] Embodiment 52. The method according to embodiment 51 wherein the cell is a microbial cell, and wherein the mass spectrometer is employed to identify the microbial cell.

[0224] Embodiment 53. The method according to any one of embodiments 47 to 50 wherein the mass spectrometer is configured to sample under ambient atmospheric conditions.

[0225] Embodiment 54. The method according to any one of embodiments 47 to 50 wherein the mass spectrometer is configured to sample under vacuum conditions.

[0226] Embodiment 55. A system for ablating an analyte from a sample, the system comprising:a laser system configured to generate two laser pulses, each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including a shorter wavelength pulse and a longer wavelength pulse, the two laser pulses being directed onto a sample location, such that:the two laser pulses are spatially overlapped at the sample location; the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;when a dielectric sample resides at the sample location, one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the dielectric sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the dielectric sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

[0227] Embodiment 56. The system according to embodiment 55 further comprising a mass spectrometer configured to perform mass analysis of the ablation plume.

[0228] Embodiment 57. A method of detecting an analyte from a sample via mass spectrometry, the method comprising:directing a first laser pulse and a second laser pulse onto a sample, the first laser pulse having a wavelength between 340 nm and 5000 nm and the second laser pulse having a shorter wavelength than the first laser pulse, and the first laser pulse and the second laser pulseeach having a respective pulse duration less than 100 ps, the first laser pulse and the second laser pulse being directed onto the sample such that:the first laser pulse and the second laser pulse are spatially overlapped on the sample;the first laser pulse and the second laser pulse are provided with a temporal delay of less than 1 ns;the first laser pulse and the second laser pulse are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;one of the first laser pulse and the second laser pulse produces, within an initial 50 fs after the onset of irradiation of the sample, an initial free electron density, and after the initial 50 fs, the other of the first laser pulse and the second laser pulse increases the free electron density such that ablation predominantly occurs via a Coulomb explosion mechanism, resulting in an ablation volume having a sub-diffraction-limited lateral area, within a lateral spatial region that is smaller than a diffraction-limited 1 / eA2 spot size of at least one of the first laser pulse and the second laser pulse, and a depth of less than 1 micron, without substantial fragmentation of ionized analyte in an ablation plume;employing a mass spectrometer to measure the ablation plume; anddetecting the analyte via mass analysis of one or more mass peaks associated with the analyte.

[0229] Embodiment 58. The method according to embodiment 57 wherein the first laser pulse and the second laser pulse are directed onto the sample such that the lateral area of the ablation volume is smaller than a diffraction-limited 1 / eA2 spot size of the first laser pulse and the second laser pulse.

[0230] Embodiment 59. The method according to embodiment 57 or 58 wherein the first laser pulse and the second laser pulse are directed onto the sample such that a lateral spatial region associated with the increased free electron density has an area of less than 10A4 nmA2, and an ablation depth of less than 100 nm.

[0231] Embodiment 60. The method according to embodiment 59 wherein the sample is a cell.

[0232] Embodiment 61. The method according to any one of embodiments 57 to 60 wherein a secondary ionization source is employed to increase an ionization yield.

[0233] Embodiment 62. The method according to any one of embodiments 57 to 61 wherein sample ablation and input of the ablation plume into the mass spectrometer are separated in time and / or space.EXAMPLES

[0234] The following examples are presented to enable those skilled in the art to understand and to practice embodiments of the present disclosure. They should not be considered as a limitation on the scope of the disclosure, but merely as being illustrative and representative thereof.Example 1: IR First, UV Second

[0235] In this first example, a numerical simulation is used to illustrate the example of a 100 fs, 1030 nm laser pulse at an intensity of 10A12 W / cmA2 irradiates a sample of water representing tissue and is followed after 100 fs by a 250 nm laser pulse, with 250 fs pulse duration at an intensity of 10A11 W / cmA2. Both laser pulses have a gaussian time and spatial profile. The IR laser pulse is focused on a 5 urn laser 1 / eA2 spot size diameter, while the 250 nm pulse is focused on a 1 urn 1 / eA2 spot size diameter. To demonstrate the role of plasma in the material removal process, a standard rate equation modeling approach can be used (see Liang, X.-X.; Zhang, Z.; Vogel, A. Multi-Rate-Equation Modeling of the Energy Spectrum of Laser-Induced Conduction Band Electrons in Water. Opt. Express, OE 2019, 27 (4), 4672-4693 and Linz, N.; Freidank, S.; Liang, X.-X.; Vogel, A. Wavelength Dependence of Femtosecond Laser-Induced Breakdown in Water and Implications for Laser Surgery. Phys. Rev. B 2016, 94 (2), 024113.) The numerical model used is described in more detail in Example 4. In this example, only a 1 urn diameter volume will be ablated, as shown in FIG. 11. It is noted that the ablated volume is smaller than the diffraction limited spot size of the IR laser.Example 2: UV First IR Second

[0236] In this second example, a numerical simulation is used to illustrate the example of a 250 nm laser pulse, with 250 fs pulse duration at an intensity of 10A11 W / cmA2 irradiates a sample and is followed after 100 fs by a 1030 nm laser pulse at an intensity of 10A12 W / cmA2. Both laser pulses have a gaussian time and spatial profile. The IR laser pulse is focused on a 5 urn laser 1 / eA2 spot size diameter, while the 250 nm pulse is focused on a 1 urn 1 / eA2 spot size diameter. In this example, only a 1 urn diameter will be ablated, as shown in FIG. 19. It is noted that the diameter of the ablated volume shrinks by about 100 nm compared to the results shown in FIG. 11, however the overall performance is comparable, indicating the robustness of the disclosed invention.Example 3: Simultaneous Irradiation

[0237] In this third example of the embodiment, a numerical simulation is used to illustrate the example of a 250 nm laser pulse, with 250 fs pulse duration at FIG. 10A11 W / cmA2irradiates a sample at the same time as a 1030 nm laser pulse at FIG. 10A12 W / cmA2, with a 250 fs pulse duration. Both laser pulses have a gaussian time and spatial profile. The IR laser pulse is focused on a 5 urn laser 1 / eA2 spot size diameter, while the 250 nm pulse is focused on a 1 urn 1 / eA2 spot size diameter. In this example, only a 1 urn diameter will be ablated, as shown in FIG. 20. In this case, the ablated diameter is within 100 of the diameters removed in Example 1, showing the efficacy of the disclosed method.Example 4: Numerical Modeling with Pulse Delays

[0238] In this fourth example of the embodiment, the general model used to predict the material removal is described. Using a single rate equation approach, the time dependent plasma density can be determined by adding together the time dependent rate of strong field ionization (ns), avalance ionization^,) , and the electron-ion recombination rate (j]rec), which can be written as:recTT-totai^'^- Where ntotaiis the total number of liberated electrons from strong field and avalanche ionization and the intensity and wavelength dependence is included in thet), )ntotal(r,t)]. To model the multi-pulse irradiation, the intensity contributions of sequential pulses which have gaussian spatial and temporal profile are included and the equation is solved using a Runge-Kuta model. Ablation does not occur unless the plasma density ( ntotal(r,t)) exceeds 10A20 electrons per cmA3. As such, one can easily estimate the ablation volume and required pulse durations for a pulse system, where the pulses can have any delay. However, one will note that the dual pulse system is only more efficient at plasma generation when the two pulses arrive within 1 ns, as the plasma density dissipates quickly at the recombination rate. This model can be used to verify the examples discussed above and provides a basis fortesting the conditions under which the disclosed method can be successfully employed.

[0239] The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.REFERENCES1. J. A. Christopher, C. Stadler, C. E. Martin, M. Morgenstern, Y. Pan, C. N. Betsinger, D. G. Rattray, D. Mahdessian, A.-C. Gingras, B. Warscheid, J. Lehtid, I. M. Cristea, L. J. Foster, A. Emili, and K. S. Lilley, "Subcellular proteomics," Nat. Rev. Methods Primer 1, 32 (2021). 2. X. Ding, K. Liu, and Z. Shi, "Laser Desorption / Ablation Postionization Mass Spectrometry: Recent Progress in Bioanalytical Applications," Mass Spectrom. Rev. 40, 566-605 (2021).3. M. Niehaus, J. Soltwisch, M. E. Belov, and K. 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Claims

1. CLAIMS1. A method of ablating an analyte from a sample, the method comprising:directing a plurality of laser pulses onto a sample, the plurality of laser pulses including two laser pulses each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including a shorter wavelength pulse and a longer wavelength pulse, the two laser pulses having respective laser pulse properties and being directed onto the sample such that:the two laser pulses are spatially overlapped on the sample;the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

2. The method according to claim 1 wherein at least a portion of the analyte within the ablation plume ionized.

3. The method according to claim 1 or 2 wherein the two laser pulses having respective laser pulse properties and being directed onto the sample such that the volume over which the free electron density of the plasma is increased above the threshold electron density for ablation has a lateral extent that is less than a diffraction-limited 1 / eA2 spot size of both the shorter wavelength pulse and the longer wavelength pulse.

4. The method according to any one of claims 1 to 3 wherein the two laser pulses are delivered onto the sample with pulse properties such that a Coulomb explosion mechanism is the dominant mechanism of ablation of the sample caused by the two laser pulses.

5. The method according to any one of claims 1 to 3 wherein the volume is a first volume, and wherein, after a Coulomb explosion mechanism occurs, an additional volume of the sample is ablated via thermal vaporization mechanism, wherein the two laser pulses aredelivered onto the sample with pulse properties such that the first volume exceeds the additional volume.

6. The method according to any one of claims 1 to 5 wherein the shorter wavelength pulse has a respective pulse intensity configured to seed the generation of the initial free electron density by multiphoton ionization.

7. The method according to any one of claims 1 to 6 wherein the wavelength of the longer wavelength pulse exceeds 340 nm.

8. The method according to any one of claims 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are unfragmented.

9. The method according to any one of claims 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into only two fragments.

10. The method according to any one of claims 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into three or fewer fragments.

11. The method according to any one of claims 1 to 7 wherein the analyte is a molecular analyte comprising analyte molecules, and wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that at least a portion of the analyte molecules within the ablation plume are each fragmented into five or fewer fragments.

12. The method according to any one of claims 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least half of the electrons in the plasma have an energy less than 10 eV.

13. The method according to any one of claims 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least 80% of the electrons in the plasma have an energy less than 10 eV.

14. The method according to any one of claims 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold electron density for ablation is reached, at least 90% of the electrons in the plasma have an energy less than 10 eV.

15. The method according to any one of claims 1 to 11 wherein the two laser pulses are delivered with respective pulse intensities and respective pulse fluences such that when the threshold for ablation is reached, at least 95% of the electrons in the plasma have an energy less than 10 eV.

16. The method according to any one of claims 1 to 15 wherein the shorter wavelength pulse has a wavelength in the ultraviolet portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the infrared portion of the electromagnetic spectrum.

17. The method according to any one of claims 1 to 15 wherein the shorter wavelength pulse has a wavelength within the visible portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the infrared portion of the electromagnetic spectrum.

18. The method according to any one of claims 1 to 15 wherein the shorter wavelength pulse has a wavelength within the ultraviolet portion of the electromagnetic spectrum and the longer wavelength pulse has a wavelength in the visible portion of the electromagnetic spectrum.

19. The method according to any one of claims 1 to 18 wherein the shorter wavelength pulse is temporally overlapped with the longer wavelength pulse.

20. The method according to any one of claims 1 to 18 wherein the shorter wavelength pulse and the longer wavelength pulse arrive simultaneously on the sample.

21. The method according to any one of claims 1 to 18 wherein the longer wavelength pulse is temporally delayed relative to the shorter wavelength pulse.

22. The method according to any one of claims 1 to 18 wherein the shorter wavelength pulse is temporally delayed relative to the longer wavelength pulse.

23. The method according to any one of claims 1 to 18 wherein the two laser pulses are absent of temporal overlap.

24. The method according to any one of claims 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 10 ps.

25. The method according to any one of claims 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 1 ps.

26. The method according to any one of claims 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 100 fs.

27. The method according to any one of claims 1 to 23 wherein at least one of the two laser pulses has a pulse width of less than 10 fs.

28. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A6 umA3.

29. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A7 umA3.

30. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A8 umA3.

31. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such that the volume is less than 10A9 umA3.

32. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such an ablation crater resulting from ablation of the volume has a depth less than 1000 nm.

33. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such an ablation crater resulting from ablation of the volume has a depth less than 100 nm.

34. The method according to any one of claims 1 to 27 wherein the two laser pulses have respective laser pulse properties and are directed onto the sample such when the threshold electron density for ablation of the volume is achieved, a lateral spatial region associated with the plasma has an area of less than 10A4 umA2, and a depth of less than 100 nm.

35. The method according to any one of claims 1 to 34 wherein the analyte comprises a biomolecule.

36. The method according to claim 35 wherein the biomolecule is selected from the group consisting of lipids, metabolites, and proteins.

37. The method according to any one of claims 1 to 36 wherein the sample is a biological sample.

38. The method according to claim 37 wherein the biological sample comprises at least one of blood, animal tissue, and plant tissue.

39. The method according to any one of claims 1 to 38 wherein the ablation plume is analyzed at a location that is spatially remote from the sample.

40. The method according to any one of claims 1 to 38 wherein the ablation plume is analyzed after a time delay during which the ablation plume is transported for subsequent analysis.

41. The method according to any one of claims 1 to 40 further comprising employing secondary ionization source to increase an ionization yield.

42. The method according to claim 41 wherein the secondary ionization source comprises an optical apparatus for directing a vacuum-ultraviolet beam onto the ablation plume within an evacuated region, wherein the vacuum-ultraviolet beam is configured to photoionize nonionized analyte within the ablation plume.

43. The method according to any one of claims 1 to 42 wherein a metal, alloy or semiconductor substrate is provided beneath the sample, and the sample is provided with a thickness that is less than an absorption depth of at least one of the shorter wavelength pulse and the longer wavelength pulse, the metal, alloy or semiconductor substrate thereby increasing the free electron density of the plasma and lowering a respective fluence of at least one of the shorter wavelength pulse and the longer wavelength pulse needed to achieve the threshold electron density.

44. The method according to any one of claims 1 to 43 wherein the sample comprises a cell, and wherein the two laser pulses are directed onto the cell, such that the volume resides within the cell.

45. The method according to any one of claims 1 to 44 wherein the ablation plume is analyzed using an analytical device other than a mass spectrometer.

46. The method according to claim 45 wherein the analytical device is configured to perform analysis according to an analytical modality selected from the group consisting of high-performance liquid chromatography, Gas Chromatography and nuclear magnetic resonance.

47. The method according to any one of claims 1 to 43 further comprising:employing a mass spectrometer to measure the ablation plume; anddetecting the analyte via mass analysis of one or more mass peaks associated with the analyte.

48. The method according to claim 47 wherein the sample is positioned such that the ablation plume is directed through the inlet of the mass spectrometer.

49. The method according to claim 47 or 48 wherein the two laser pulses are scanned relative to the sample and wherein the mass spectrometer is employed to generate a mass spectrometry image of the analyte within the sample.

50. The method according to any one of claims 47 to 49 wherein the sample comprises a MALDI matrix material.

51. The method according to any one of claims 47 to 50 wherein the sample comprises a cell, and wherein the two laser pulses are directed onto the cell, such that the volume resides within the cell.

52. The method according to claim 51 wherein the cell is a microbial cell, and wherein the mass spectrometer is employed to identify the microbial cell.

53. The method according to any one of claims 47 to 50 wherein the mass spectrometer is configured to sample under ambient atmospheric conditions.

54. The method according to any one of claims 47 to 50 wherein the mass spectrometer is configured to sample under vacuum conditions.

55. A system for ablating an analyte from a sample, the system comprising:a laser system configured to generate two laser pulses, each having a respective pulse duration less than 100 ps and a respective wavelength between 124 nm and 5000 nm, the two laser pulses including a shorter wavelength pulse and a longer wavelength pulse, the two laser pulses being directed onto a sample location, such that:the two laser pulses are spatially overlapped at the sample location;the two laser pulses are provided with a temporal delay therebetween of less than 1 ns;the two laser pulses are each focused with a respective intensity between 10A9 W / cmA2 and 10A16 W / cmA2;when a dielectric sample resides at the sample location, one of the two laser pulses produces, within an initial 50 fs after the onset of irradiation of the dielectric sample, a plasma having an initial free electron density; andafter the initial 50 fs, the other of laser pulse increases the free electron density of the plasma above a threshold electron density for ablation within a volume of the dielectric sample having a lateral extent that is less than a diffraction-limited 1 / eA2 spot sizeof at least the longer wavelength pulse and a depth that is less than 1 micron, resulting in ablation of the volume and generation of an ablation plume comprising the analyte.

56. The system according to claim 55 further comprising a mass spectrometer configured to perform mass analysis of the ablation plume.

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