Systems and methods for imaging and ablating a sample

Abstract

Disclosed herein are systems for imaging and ablating a sample. An imaging / ablation device includes an optical assembly, a sample stage, and a receiver. The optical assembly is capable of ablating a region of interest within the sample. Laser light propagating from the optical assembly during ablation propagates substantially in the same direction as the direction of travel of an ablation plume toward the receiver. A laser focus detection unit, including at least one reference laser and a photodetector, generates at least one real-time detection signal indicative of one or more properties of the sample during ablation and / or indicative of a distance from an objective lens to a surface of the sample stage or the sample. A controller coupled with the laser focus detection unit dynamically controls one or more parameters of an ablation laser and / or a position of the objective lens and / or a position of the receiver relative to the sample in real-time to improve MS imaging quality.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to systems, devices, and methods for imaging and ablating a sample. In particular, the present disclosure relates to systems, devices, and methods for imaging and ablating a biological sample that are capable of getting into the fine details of the subcellular level and are capable of transferring the ablated target to a receiver without excessive degradation of the target to allow additional downstream analysis. BACKGROUND

[0002] The field of "omics" seeks to characterize, quantify, or otherwise analyze a set of biological molecules related to the structure, function, or kinetics of a target organism or population of organisms. Each omics field is related to the study of a relevant "ome." The omics fields include the following: genomics, which is the study of a genome; epigenomics, which is the study of the support structure of a genome, including DNA binders and chemical modifications of DNA; transcriptomics, which is the study of a set of RNA molecules produced by a target organism, including mRNA, miRNA, rRNA, and tRNA; proteomics, which is the study of the complement of proteins produced by a target organism; and metabolomics, which is the study of a set of metabolites produced by cellular processes of a target organism.

[0003] "Multi-omics" (sometimes also referred to as integrative omics) involves the analysis of one or more such omes. The goal of multi-omics analysis is to collect and / or analyze complex biological data, for example, to find associations between various omes in a way that better enables the determination of disease markers, better understanding of the partitioning between genotype, phenotype, and environmental influences under specific conditions, and a deeper understanding of the ways in which various omes are inter-regulated and influenced.

[0004] However, there remain some challenges in further advancing the field of multi-omics. In particular, as one moves along the genotype-to-phenotype path from genome to transcriptome to proteome and metabolome, the level of chemical diversity and complexity increases exponentially. As the complexity increases, the difficulty of obtaining and analyzing the relevant biological molecules also increases.

[0005] Further, the relevant region of interest in which the biomolecule of interest resides is often at a subcellular level. Thus, obtaining the biomolecule of interest in a manner that allows for effective downstream analysis of the biomolecule is challenging. However, conventional methods such as laser ablation electrospray ionization (LAESI), including picosecond infrared LAESI (PIR LAESI), do not allow for collection of biomolecules at a subcellular level. In LAESI, laser ablation of a material is ionized by nanodroplets from an electrospray ion source and then transmitted to a mass spectrometer for analysis. However, conventional LAESI systems have inherent spatial limitations that result in low numerical aperture (NA) optics. The low numerical aperture optics used for sample ablation result in large ablation spot sizes. For example, the minimum spot size is typically much larger than an entire living cell and is simply not small enough to allow for targeted ablation of a subcellular region of interest.

[0006] Direct access to intracellular and / or intercellular biological material of a living cell is also key to effectively analyzing biochemical aspects of such cells under real-time conditions. However, conventional methods of obtaining cell material of interest for further downstream analysis typically rely on fixed and often dried samples. For example, matrix assisted laser desorption / ionization time-of-flight (MALDI TOF) uses a matrix to ionize molecular components. To fix a sample, MALDI TOF is incompatible with living cell monitoring.

[0007] Thus, there is an ongoing need for systems, devices, and methods of obtaining cell material from a living cell at a subcellular level in a manner that allows for effective downstream analysis of the obtained material. Various methods of laser ablation are found in the development of “Laser Ablation Direct Analysis in Real Time Imaging,” Spatial Distribution Mapping of Metabolites Along a Biosynthetic Cascade Leading to Atropine and Hyoscyamine Synthesis in Plant Tissues, Anal. Chem. 2017, 89, 6, 3421-3429 and Y. Cui et al., Depth profiling and imaging capabilities of an ultrashort pulsed laser ablation time-of-flight mass spectrometer, Rev. Sci. Instrum. 83, 093702 (2012), but these methods suffer from one or more drawbacks. US2021 / 0118661A1, US2015 / 0018807A1, US8199321B2, WO2014 / 079802A2, W. Muller et al., Initial performance criteria of a newly custom-designed ArF excimer LA-ICPMS system coupled to a two-volume laser ablation cell, J. Anal. At. Spectrom., 2009, 24, 209-214, K. Fowble et al., Figure 1 Figure 2 “Laser Ablation Direct Analysis in Real Time Imaging,” Spatial Distribution Mapping of Metabolites Along a Biosynthetic Cascade Leading to Atropine and Hyoscyamine Synthesis in Plant Tissues, Anal. Chem. 2017, 89, 6, 3421-3429 and Y. Cui et al., Depth profiling and imaging capabilities of an ultrashort pulsed laser ablation time-of-flight mass spectrometer, Rev. Sci. Instrum. 83, 093702 (2012), but these methods suffer from one or more drawbacks. SUMMARY

[0008] Embodiments described herein can enable collection of target biological material from a sample in a manner that does not overly degrade the biological material and allow for efficient downstream analysis of the obtained material. In certain embodiments, the sample can include live cells, and the target biological material can be obtained under normal environmental conditions (e.g., without pressure control, humidity control, etc.). In certain embodiments, a target region of interest of the sample can have a subcellular size. In certain embodiments, the target biological material can be removed from the cells in a manner that minimizes impact on the remaining cellular structure that can even allow the cells to survive for optional use in further testing. In certain embodiments, the characteristics of the sample ablation are monitored in real-time during the ablation and contain spatial and temporal information. Accordingly, this enables the parameters of the ablation device to be dynamically adjusted in real-time to allow for optimization of the ablation and / or to maintain constant conditions for sample analysis, such as mass spectrometry imaging, which can improve MS imaging quality.

[0009] In one embodiment, a device for imaging and ablating a sample that allows for analysis of the ablated portion of the sample is provided, the device including a sample stage having a first side (e.g., an upper side) configured for placement of a sample thereon and a second side (e.g., a lower side) disposed opposite the first side. The device includes an optical assembly including an objective lens disposed at the second side of the sample stage and configured to enable microscopic imaging of a sample placed on the sample stage, the optical assembly further including an ablation laser. The ablation laser is disposed at the second side of the sample stage, the ablation laser configured to direct laser light through the objective lens, through the sample stage, and into the sample to selectively ablate at least a portion of the sample (e.g., a target region of interest of the sample). The sample stage generally has a transparent window that allows the laser light and other light to pass through. The sample can be placed on an upper surface of the transparent window. For example, the transparent window can be made of glass or plastic. For example, the transparent window can be a glass slide or a coverslip. The transparent window generally defines a field of view (FOV) of the optical assembly, e.g., the objective lens. A receiver is disposed at the first side of the sample stage, the receiver configured to receive ablated material ejected from the sample to enable further analysis of the ablated material.

[0010] In one embodiment, the optical assembly further comprises a laser focus detection unit (FDU) comprising at least one reference laser arranged at the second side of the sample stage and configured to direct laser light through the objective lens, through the sample stage into the sample. The FDU can be used to detect or observe the interface between media with different refractive indices. In this way, the axial (z) position of one or more interfaces, such as the atmosphere / sample stage interface, the sample stage / sample interface or the sample / atmosphere interface. This enables both the relative position (distance) of the objective lens to the sample and the sample thickness to be monitored in real-time. The laser focus detection unit further comprises at least one photodetector configured to generate at least one detection signal by detecting laser light from the at least one reference laser, the detection signal being indicative of one or more properties of the sample when at least a portion of the sample is ablated, and / or indicative of the distance from the objective lens to the sample stage, and / or indicative of the distance from the objective lens to the surface of the sample. In terms of the sample, one or more of the following can be observed: the size of a cavity in the sample (and its delay relative to the ablation laser pulse); the height or position of the sample / atmosphere interface, for example relative to the sample stage or the objective lens; and the axial plume formation from the sample to the atmosphere.

[0011] The laser focus detection unit is further configured for sending the at least one detection signal to a controller configured to dynamically control one or more parameters of the ablation laser and / or the position of the objective lens and / or the position of the receiver based on the at least one detection signal. Thereby the position of the objective lens and / or the position of the receiver is adjusted relative to the position of the sample.

[0012] In one embodiment, the laser focus detection unit is configured to detect the laser light from the at least one reference laser continuously or at a high frequency using the at least one photodetector and send the at least one detection signal to the controller. In this way, the at least one detection signal can allow the sample and / or the position of the objective lens and / or the receiver relative to the sample to be monitored in real-time during ablation. The at least one photodetector preferably has a temporal resolution of 100 microseconds or less. In one embodiment, the at least one reference laser can be modulated on and off to avoid conflicts with the detection of other light or excitation sources and images from the sample. The at least one photodetector preferably has a nanosecond temporal resolution or sub-nanosecond temporal resolution (temporal resolution at which light from the at least one reference laser is detected), for example 1-10 nanoseconds or less, for example 1 nanosecond or less than 1 nanosecond. A photodetector with sub-nanosecond temporal resolution can be used to probe the dynamics of the ablation plume expansion in a continuous manner. For this function, the photodetector with sub-nanosecond temporal resolution can be on, i.e. active, all the time. However, the at least one reference laser detected by the photodetector with sub-nanosecond temporal resolution can be modulated on and off.

[0013] In one embodiment, the one or more properties of the sample include one or more of the following: formation of a cavity in the sample caused by the laser light from the ablation laser, formation of an ablation plume caused by ablation material ejected from the sample, and a change in thickness of the sample. In many cases, the change in thickness of the sample due to surface tension of water present on or in the sample disappears after a millisecond time range. In the case of certain very thin samples (single layer of cells), a "dimple" can be left on the surface after ablation.

[0014] In one embodiment, the controller is configured to control one or more parameters of the ablation laser based on the at least one detection signal comprising the laser pulse energy and / or the laser pulse frequency. In one embodiment, the at least one detection signal can be used to monitor whether an ablation plume is formed at the sample surface (i.e. sample-air surface). If no ablation plume is formed at the sample surface, the controller can adjust the laser pulse energy and / or the laser pulse frequency. Preferably, the laser pulse energy can be increased and / or the laser pulse frequency can be increased until an ablation plume is formed at the surface.

[0015] In one embodiment, the controller is configured to continuously (i.e. in real-time at a high numerical frequency) adjust the position of the objective and / or the position of the receiver based on the at least one detection signal. One or more actuators can drive the objective. Similarly, one or more actuators can drive the receiver stage. The actuators can be controlled in real-time by the controller. The actuators can have a response time in the sub-millisecond range, for example a response time of 1 millisecond or less, allowing for real-time control of the position. The adjusted objective position and / or receiver position can be an axial position (referred to herein as a z-position) which changes the distance between the objective and the sample stage or between the receiver stage and the sample stage. In this way, for example, the focal depth of the laser light from the ablation laser in the sample can be continuously adjusted by adjusting the position of the objective. This is advantageous because the thickness of the sample changes over time. Furthermore, the distance of the receiver from the sample surface can be kept constant and / or within an optimal range.

[0016] In one embodiment, the at least one photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that has been reflected from at least one surface or interface of the sample stage and / or the sample and / or transmitted through the at least one surface or interface. In one embodiment, the at least one photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that has been reflected from a surface or interface of the sample stage and directed back through the objective lens. In one embodiment, the at least one photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that has been reflected from a surface or interface of the sample facing the receiver and directed back through the objective lens. In such embodiments, the photodetector can comprise an array detector. The array detector can enable peak detection of the at least one reference laser. These peaks are objects observable in the FDU and originate from the (axial) interfaces of the sample with different refractive indices. The array detector can comprise a plurality of individual photodetectors or pixels. The array detector can be combined with a field programmable gate array (FPGA) and a controller to enable fast signal analysis (e.g. peak detection in the microsecond time range).

[0017] In one embodiment, the at least one photodetector comprises a photodetector disposed on a first side of the sample and configured to detect laser light from the at least one reference laser that has been transmitted through the sample. Preferably, this photodetector signal is monitored simultaneously with the ablation of the sample.

[0018] In one embodiment, the at least one photodetector is configured to resolve the at least one detection signal with sub-nanosecond temporal resolution. This enables monitoring of the position of a cavity formed in the sample over time by the laser focused into the sample by the objective lens. Such a cavity typically travels at the speed of sound in the sample medium.

[0019] In one embodiment, the at least one reference laser is a diode laser, preferably in the form of a photonic integrated circuit. In one embodiment, the at least one reference laser comprises a plurality of reference lasers, i.e. at least a first reference laser and a second reference laser. A plurality of reference lasers can be provided as laser output apertures of a photonic integrated circuit (PIC).

[0020] In one embodiment, the at least one reference laser comprises at least a first reference laser configured to direct a first laser light along an axis deviating from the optical axis of the objective to the objective, and the at least one photodetector is configured to detect the first laser light reflected back through the objective along the axis deviating from the optical axis of the objective. The detected first laser light can provide information about the distance of the objective to the sample stage and / or the distance of the objective to the sample-air interface. These distances can be determined using a triangulation method. This method further allows monitoring the interface of the sample medium / air with high temporal resolution. For example, using a fast photodiode, the ablation time, the propagation of the cavity to the sample surface, and the discharge of the cavity plume into the atmosphere can be monitored.

[0021] In one embodiment, the at least one reference laser comprises at least a second reference laser configured to direct a second laser light along the optical axis of the objective to the objective, and the at least one photodetector is configured to detect the second laser light reflected back through the objective along the optical axis of the objective. The at least one photodetector configured to detect the second laser light reflected back along the optical axis of the objective can form part of an interferometer, which itself can be part of an optical coherence tomography (OCT) device. A suitable OCT technique is described by Wang et al. in Optics Letters, Vol. 42, Issue 17, 2017, pp. 3466-3469. Thus, the second laser light reflected back along the optical axis of the objective can for example simultaneously provide an interference signal from a cavity in the sample formed by the laser light from the ablation laser. The interferometer or OCT setup preferably allows the interference signal to be generated by the laser-induced cavity in the sample and / or the first laser light reflected from the ablation plume / sample interface. The interference signal can provide precise, time-resolved positional information about the cavity in the medium and / or the plume formation in the atmosphere. Information about the cavity and / or plume formation can be provided in real time with sub-nanosecond resolution (1 ns or less). Using the interferometer, the observation volume can be extended from the sample medium only to the plume volume of the ablation material leaving the sample in the atmosphere. As part of the OCT setup, the interference signal can provide tomographic measurements of the cavity and / or plume formation.

[0022] In one embodiment, the apparatus can further comprise a light source arranged at the first side of the sample, the light source being configured for transillumination of the sample. For this purpose a flash lamp or a lamp can be used. The flash lamp or lamp can preferably emit light pulses of nanosecond (e.g. 1-10 ns) or sub-nanosecond (less than 1 ns) width. Thus, snapshot imaging can be obtained using the flash lamp / lamp, i.e. a snapshot image of the cavity at a time delay t after the sample is pulsed with the ablation laser. A detector such as a camera can be arranged at the second side of the sample to detect the transillumination light and form an image of the sample. The transillumination light can be used for cavity sizing in the sample, e.g. at any axial position of the working objective focal depth.

[0023] In one embodiment, the apparatus can further comprise a fluorescence excitation source configured for epi-fluorescence imaging and / or analysis of the sample. The fluorescence excitation source is preferably arranged at the second side of the sample. The fluorescence excitation can be applied with a delay after the ablation laser. The fluorescence excitation source can comprise a pulsed light source, preferably with a pulse length of about ns, to probe the number of fluorescent (labeled) molecules of interest in the sample at a matching fluorescence excitation wavelength.

[0024] A detector, e.g. a camera, or a spectrometer with (fast) photodetector, can be provided which is configured to spectrally and / or time-resolvedly (i.e. real-time detection) provide a fluorescence image or signal from the sample in response to auto-fluorescence after application of the ablation laser (with the fluorescence excitation source switched off) or at a time delay t' after illumination by the fluorescence excitation source after application of the ablation laser. The fluorescence emission detection can occur within sub-millisecond exposure times. The detector for fluorescence emission is preferably arranged at the second side of the sample. The detector, e.g. camera, for detecting the fluorescence emission can be the same as the detector or camera for detecting the transillumination. Using this system, cavity formation in the sample medium can be imaged by transillumination using the flash lamp at a time t after the ablation laser pulse and by fluorescence imaging at a later time t' after fluorescence excitation. Thus, time-lapse snapshot imaging information can also be obtained from the different light sources of transillumination and fluorescence. With a camera having a sufficient dynamic intensity range, the image can show both contrasts. Typically, the contrast regions of the transillumination and fluorescence images are not the same. High contrast can be seen from the transillumination image and weak (less strong) signals can be identified as fluorescence.

[0025] The detected epi-fluorescence signal or image is preferably time-resolved and can be used to provide information on the cavity properties and on the molecular cleavage in the sample, i.e. at the focal position of the objective. A spectral image splitter can be used to direct the fluorescence onto a fast (faster) photodetector (e.g. array) in combination with a spectrometer to spectrally separate the transillumination and fluorescence images.

[0026] In one embodiment, the measured depletion of fluorescence indicates that molecules in the sample are split by the ablation, typically because the laser pulse energy was too high. In this case, the next laser pulse needs to be applied with lower energy. Thus, the fluorescence signal can be used to adjust the laser pulse energy. Quantitative fluorescence intensity analysis enables estimation of the transfer efficiency of the material to the receiver and hence to the analyzer (e.g. mass spectrometer).

[0027] In one embodiment, Raman signals from the focus of the ablation laser can additionally or alternatively be detected. Raman signals provide complementary information to the fluorescence signals. While the sensitivity of the Raman signal is typically lower than that of the fluorescence, it has the advantage that the sample does not need to be modified with a fluorophore label. The Raman signals from the ablated material can be detected, for example, by the same detector or spectrometer as used for detecting the fluorescence. The Raman signals provide vibrational and rotational bands of the ablated molecules. For example, the presence of Raman signals reveals information about the expected mass of the molecules sent to the analyzer (e.g. mass spectrometer), while the intensity distribution of the vibrational / rotational bands indicates the thermal state (internal energy) of the molecules. The Raman signals can be correlated to the peak intensities of the molecules detected in the analyzer (mass spectrometer). Thus, optimizing the Raman signals can be used to make a more quantitative calibration of the molecules detected in the analyzer (mass spectrometer).

[0028] Various further features of the ablation device and system are now described.

[0029] In one embodiment, the ablation laser and objective are configured such that the laser light is directed through the objective and is oriented such that the laser light propagates substantially in the expected direction of motion of the ablation plume resulting from ablation of the sample. This advantageously allows the expanding ablation plume to effectively travel towards the receiver along a direction substantially parallel to the propagating laser light rather than against the propagating laser light.

[0030] The optical assembly can be configured to enable brightfield imaging, slicing (e.g. by confocal microscopy), epi-fluorescence imaging, two-photon imaging, or a combination thereof. The objective can have a numerical aperture (NA) of about 0.5 or greater, or about 0.65 or greater, or about 0.75 or greater, or about 0.8 or greater.

[0031] The ablation laser is preferably a pulsed laser. The ablation laser is preferably a femtosecond infrared laser. The laser and other optical assembly components can be configured to deliver a pulse energy of about 1 nJ to about 10 μJ per μm 3 The sample delivers a pulse energy of about 1 nJ to about 10 μJ. The target region of interest to be ablated can have a "spot size" diameter of about 50 μm or less, or about 30 μm or less, or about 10 μm or less, or about 5 μm or less, or about 3 μm or less, or about 1.5 μm or less, or about 1 μm or less. In terms of volume, the target region of interest to be ablated can have a volume of about 500 μm3 or less, about 250 pm 3 or less, about 100 pm 3 or less, about 50 pm 3 or less, about 25 pm 3 or less, about 10 pm 3 or less, about 5 pm 3 or less, or about 2 pm 3 or less. Thus, the optical assembly can be used to ablate multiple whole cells, a single whole cell, or a sub-cellular volume, e.g., targeting organelles or other intracellular structures, or an extracellular volume outside the cell.

[0032] In one embodiment, the receiver comprises a medium configured for non-overlapping spatial partitioning of individual subsamples of ablated material, e.g., microwell plates or chips. In one embodiment, the receiver comprises an array of nanodroplets. In one embodiment, the receiver comprises an electrospray probe configured to collect the ablated subsamples and transport them to the inlet of a mass spectrometer in the form of ionized droplets. The electrospray probe can be associated with a capillary that provides a solvent for wetting the outer surface of the electrospray probe.

[0033] In one embodiment, a system for ablating and analyzing a target region of a sample comprises an imaging and ablation device and an analyzer configured to receive and analyze at least a portion of ablated material received by a receiver. The analyzer can comprise, e.g., one or more PCR machines, sequencing machines, spectrometers, nuclear magnetic resonance (NMR) spectrometers, mass spectrometers, chromatography devices, centrifuges, electrophoresis devices, radioactive labels and radioactive label detection devices, other analytical biochemical devices, or combinations thereof. The system can also comprise an upstream processor, e.g., an electrically operated droplet sorting machine, a sorting centrifuge, etc., configured for sorting or other processing of the sample prior to positioning the sample on the sample stage.

[0034] In one embodiment, a method of imaging and ablating a sample to enable analysis of an ablated portion of the sample comprises the steps of providing an imaging and ablation device, acquiring an image of the sample, selecting a region of interest within the sample, delivering a laser to the region of interest to ablate at least a portion of the region of interest, and capturing at least a portion of the ablated material on a receiver.

[0035] In one embodiment, the method comprises dynamically adjusting one or more parameters of the ablation laser and / or the position of the objective and / or the position of the receiver based on at least one detection signal from a laser focus detection unit, e.g., in real time with millisecond or higher temporal resolution.

[0036] In one embodiment, ablation is performed in an ambient atmosphere. In one embodiment, the sample comprises living cells. In one embodiment, an ablation sub-sample is removed from a target cell without killing the target cell.

[0037] In one embodiment, a plurality of laser pulses is applied to the sample. The plurality of laser pulses can be applied to ablate one sub-sample. A plurality of ablation sub-samples can be formed from a plurality of ablation events and can be collected at the receiver in non-overlapping spatially distinct locations. One or more of laser pulse frequency, laser pulse energy level, or laser pulse depth can be dynamically varied across the plurality of laser pulses. For example, in one mode of operation, the laser pulse intensity is set to an initially high level to remove material in the sample that overlies a region of interest, and then the laser pulse intensity is set to a lower level to ablate at least a portion of the region of interest.

[0038] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0039] Additional features and advantages of the disclosure will be set forth in the description that follows, and in part will be apparent from the description, or can be learned by practice of the present disclosure. The features and advantages of the present disclosure can be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims, or can be learned by the practice of the present disclosure as set forth hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to describe the manner in which the above-recited and other advantages and features of the present disclosure can be obtained, a more particular description will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the present disclosure and are not therefore to be considered to be limiting of its scope, the present disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0041] Figure 3 A conventional laser assisted electrospray ionization (LAESI) system is shown;

[0042] Figure 4 A schematic of a system for imaging and ablating a sample is shown, the system having one or more advantages over conventional imaging and ablation systems, the system comprising an imaging / ablation device having an optical assembly, a sample stage, and a receiver;

[0043] Figure 5An exemplary upstream processor in the form of an upstream electrodynamic droplet sorter is shown;

[0044] Figure 6A An example of an optical assembly that can be used in an imaging / ablation device as described herein is shown;

[0045] Figure 6B An example of a receiver comprising a spatially partitioned medium such as a micro well plate is shown;

[0046] Figure 7 An example of a receiver comprising an array of nanodrops is shown;

[0047] Figure 8 An example process for forming an array of nanodrops is shown;

[0048] Figure 9 An embodiment of a receiver comprising an electrospray probe and configured for producing ionized droplets containing ablated subsamples for transmission to an inlet of a mass spectrometer is shown;

[0049] Figure 10 An example of a downstream process in which one or more reagents can be added to spatially separated compartments of a receiver and / or one or more subsamples can be transmitted to a liquid chromatography column coupled to an electrospray probe for transmission of ionized samples to a mass spectrometer is shown;

[0050] Figure 11 An exemplary method for associating locations of ablated subsamples on a receiver with ablation events and target regions of interest on a sample slide is shown;

[0051] Figure 12 An example imaging / ablation device operating mode in which a rectangular xy region of a sample is scanned and ablated is shown graphically; and

[0052] Figure 13 An example imaging / ablation device operating mode is shown graphically that can be used to remove a layer of tissue, medium or other obstructing material covering a target region of interest by dynamically adjusting the position and pulse energy level of applied laser pulses.

[0053] Figure 14 An exemplary imaging / ablation device comprising a laser focus detection unit is shown.

[0054] Figure 16 An exemplary optical system comprising an imaging / ablation device and a laser focus detection unit is shown schematically.

[0055] Figure 17Further details of an exemplary optical system containing a laser focus detection unit comprising an interferometer are shown.

[0056] Figure 15 shows in A the position of the reference laser beam received at the array detector of the laser focus detection unit and in B one of the passing beams is reflected by the beamsplitter and detected by a fast photodetector.

[0057] Figure 18 Another exemplary optical system containing a laser focus detection unit is shown.

[0058] Figure 19 Exemplary data of the pixel intensity on the array detector from the laser beams reflected from the lower surface of the sample stage (ΔZ1) and the upper surface (ΔZ1') are shown.

[0059] Figure 20 Calibration of the detection pixels of the array detector for a known objective position is shown.

[0060] Figure 19 Exemplary data of the pixel intensity on the array detector due to the beams reflected from the sample stage (ΔZ1) and from the sample surface (ΔZ2) for different water levels or sample heights a, b, c, d are shown.

[0061] Figure 21 Data of Figure 22 and additional exemplary data of the pixel intensity on the array detector when the sample is drying on the sample stage are shown.

[0062] Figure 24 The time (t) dependence of the interference signal (I) from the reference laser at the photodetector of the laser focus detection unit is shown, which results from the reflection of the laser light from the cavity formation and the plume-sample interface.

[0063] Figure 1 The operation of the optics for ablation monitoring is schematically shown.

[0064] Figure 23 shows the effect of real-time (t) calibration or control of the ablation parameters: (i) the pulse energy E0per pulse, (ii) the expansion of the cavity, (iii) the transition position from the sample medium to the atmosphere, (iv) the epi-fluorescence signal.

[0065] Figure 2 A flow chart of the procedure for real-time optimization of the control parameters of the imaging / ablation device is shown. DETAILED DESCRIPTION

[0066] INTRODUCTION

[0067] Before describing various embodiments of the disclosure in detail, it is to be understood that the disclosure is not limited in scope to the particular examples described herein, which are intended as illustrations of one or more aspects of the disclosure. Other embodiments are contemplated within the scope of the disclosure, which is set forth with particularity by the claims. Furthermore, a particular embodiment can have as many units as desired, in other embodiments, further combinations and sub-combinations of the described features and / or functions can be used, alone or in combination with other features and functions, and some features and functions can be utilized independently from others.

[0068] Furthermore, it is to be understood that any given component or embodiment described herein can be used with any other component or embodiment described herein, unless otherwise explicitly stated or understood from the context. Additionally, it is to be understood that any list of such candidates or alternatives is merely exemplary, and not a limitation, unless otherwise implicitly or explicitly stated or understood from the context.

[0069] Additionally, unless otherwise indicated, the use of ordinal terms such as "first", "second", "third", etc., in the description and / or claims to denote certain steps, can not be construed to be a strict temporal sequence. Rather, such ordinal terms can be used merely for purposes of nomenclature. Moreover, unless otherwise indicated, the use of relational terms such as "top", "bottom", "front", "back", "rear", "side", etc., in the description and / or claims is used for

[0070] The headings and sub-headings used herein are for organizational purposes only and are not to be used to limit the scope of the specification or claims.

[0071] Figure 3A conventional LAESI system 60 is shown. As shown, a laser 40 is directed at a sample 10 disposed on a sample slide 30. The laser 40 is tuned to ablate a portion of the sample 10, causing an ablation plume 20 to propagate upward in a direction opposite the direction of laser propagation. An electrospray needle 50 is disposed above the sample slide 30 at a height between the sample slide 30 and the optics through which the laser 40 passes. The electrospray needle 50 emits electrospray droplets 52 through the path of the ablation plume 20. Some of these droplets 52 will interact with droplets of the ablation plume 20 to form ionized sample droplets 54. A mass spectrometer inlet 56 is generally aligned with the electrospray needle 50 and positioned to receive some of the ionized sample droplets 54 for analysis.

[0072] While conventional LAESI systems such as system 60 are capable of collecting and analyzing an ablated portion of a sample 10, there are still some limitations. In particular, the inherent spatial limitations severely limit the resolution with which the laser 40 can be applied to the sample 10, meaning that the spot size is relatively large, often much larger than an entire cell. In order for the stream of electrospray droplets 52 to pass through the ablation plume 20, the electrospray needle 50 and the mass spectrometer inlet 56 must be positioned between the sample slide 30 and the optical components through which the laser 40 propagates. This limits the focusing potential of the system and results in a less than ideal NA for the optics. In addition to the spatial limitations, it can also be necessary for the optics to be focused through the stream of electrospray droplets 52 to focus on the sample 10.

[0073] Another drawback of such conventional LAESI systems 60 is that in order to reach the intersecting path of the electrospray droplets 52, the direction of propagation of the laser 40 (i.e., the k-vector of the laser) must be opposite the direction in which the ablation plume 20 must travel. This reduces the efficiency of transporting the ablated material to the mass spectrometer inlet 56. In addition, the spatial positioning of the components means that debris from the ablation plume 20 will dirty the optics and other overlying components, further reducing the performance of the system and increasing the operational cost of cleaning and / or component replacement.

[0074] Imaging and ablation system overview

[0075] Figure 2 A schematic of a system 100 for imaging and ablating a sample is shown. The illustrated system 100 can improve one or more of the limitations of the conventional LAESI system 60 described above. The system 100 includes an imaging / ablation device 110. The imaging / ablation device 110 includes an optical assembly 112, a sample stage 114, and a receiver 116. As shown, the optical assembly 112 is disposed upside down below the sample stage 114 such that light propagates upward through the optical assembly 112 and into the sample stage 114. A first side of the sample stage 114 is an upper side and a second side of the sample stage 114 is a lower side such that an objective lens of the optical assembly 112 is disposed upside down below the sample stage.

[0076] In operation, light for imaging and / or ablation passes through optical train 112, then through sample stage 114 and into sample 10 (e.g., on a sample slide itself on sample stage 114) located on sample stage 114. The same optical train 112 can be used for both imaging of sample 10 and ablation of a target region of interest within sample 10. During ablation, a region of interest within sample 10 is targeted and laser light is directed through optical train 112 and into the region of interest. The directed laser light can be tuned to ablate the region of interest and form an ablation plume 20 extending from sample stage 114 toward receiver 116. Receiver 116 is located above sample stage 114 such that at least a portion of the extending ablation plume 20 can be captured at receiver 116. Sample stage 114, receiver 116, or both can include positioning systems that allow them to be selectively moved in at least two axial directions, and preferably in all three axial directions.

[0077] In contrast to conventional LAESI system 60, the illustrated imaging / ablation apparatus 110 provides an inverted optical train 112. Beneficially, optical train 112 can be more freely positioned relative to sample stage 114, allowing for the use of higher-NA optics. In turn, higher-NA optics are capable of focusing on smaller regions of interest and smaller ablation spot sizes. As explained in greater detail below, some embodiments can achieve sub-cellular level ablation spot sizes.

[0078] The illustrated imaging / ablation apparatus 110 is also configured to direct laser light in the direction in which ablation plume 20 extends. In the illustrated imaging / ablation apparatus 110, ablation plume 20 is intended to extend in the same direction as the propagating laser light, rather than in the opposite direction as in conventional LAESI system 60. The illustrated imaging / ablation apparatus 110 is thus able to efficiently transfer ablated sample material to receiver 116 without requiring ablation plume 20 to travel against the direction of propagating laser light.

[0079] Furthermore, the configuration of the illustrated imaging / ablation apparatus 110 removes optical train 112 from the path of extending ablation plume 20. Beneficially, this limits optical degradation and / or component damage caused by ablation debris contacting or accumulating on optical train 112.

[0080] Receiver 116 can be configured to spatially and / or temporally distinguish individual subsamples (i.e., material corresponding to each ablation event, respectively) of received ablation material. In some embodiments, receiver 116 includes a medium configured for spatial distinction of individual subsamples, such as a microwell plate or nanodroplet array. Such a medium allows for subsequent analysis of the collected and spatially distinguished subsamples, such as PCR of nucleic acids within the ablation material or sequencing of nucleic acids within the ablation material.

[0081] The receiver 116 can additionally or alternatively include an electrospray probe. The electrospray probe can be used to generate ionized sample droplets for transmission to the mass spectrometer inlet. The receiver 116 can also be configured as a solvent-wetted surface. The solvent can have a flow rate such that the received ablated subsamples are spatially distinguished based on when they were ablated and received.

[0082] In some embodiments, the electrospray probe and wetted surface are combined. For example, as explained in greater detail below, the electrospray probe can be partially disposed within a capillary tube, with an exposed distal portion that extends from the capillary tube and terminates at a tip. The exposed distal portion is positioned to receive the ablated subsamples from the sample stage (i.e., positioned above the sample stage). The capillary tube is configured to apply solvent to the outer surface of the electrospray probe such that the solvent flows along the outer surface of the exposed distal portion to the tip of the electrospray probe. In this way, when the ablated subsamples are captured by the exposed distal portion, they then flow to the tip of the probe, where they are ionized and transmitted to the mass spectrometer inlet.

[0083] The receiver 116, particularly the portion of the receiver that initially contacts and receives the ablated plume 20, can be spaced apart from the upper surface of the sample stage by a distance of about 1 mm or less, or about 500 pm or less, or about 350 pm or less, or about 250 pm or less, or about 200 pm or less, or about 150 pm or less. It has been found that dimensions within the above ranges can effectively collect the ablated material by the receiver.

[0084] The distance between the sample and the portion of the receiver 116 that initially receives the ablated plume 20 can also be adjusted to effectively transmit the ablated material to the receiver 116. The ablated material will transfer from the sample with kinetic energy (quadratic with velocity), but will be subject to drag (quadratic with velocity) that slows the material down and eliminates the kinetic energy. All of the kinetic energy will be removed within a distance of about L:

[0085]

[0086] where h is the thickness / height of the ablated portion of the sample, ps is the density of the sample, pg is the density of the gas, and C is the drag coefficient (typically about 1). Typically, L is calculated to be about 700 hours. Thus, the distance between the upper surface of the sample and the receiver is preferably less than L, or in other words, preferably less than about 700 times the height of the ablated portion of the sample.

[0087] In some embodiments, the imaging / ablation device 110 includes an incubation vessel (not shown) that is configured in size and shape to be disposed between the sample stage 114 and the receiver 116, and is configured to provide an incubation environment to the sample 10 placed on the sample stage 114.

[0088] The illustrated system 100 may further include an upstream processor 120 configured to classify, spatially orient, and / or otherwise process the sample before positioning it on the sample stage 114. For example, the upstream processor 120 may include a classification device, such as an electrokinetic droplet classifier, for classifying cells or other sample components onto a sample slide configured for subsequent placement on the sample stage 114. The upstream processor 120 may additionally or selectively include a centrifuge, such as a Cytospin centrifuge. TM Centrifuge. The centrifuge may be configured, for example, to spin a cell suspension onto a glass slide, which is configured for subsequent placement on a sample stage 114. Other upstream processing components known in the art for sorting and positioning samples and / or cells may be additionally or selectively included in the upstream processor 120.

[0089] An example of an upstream processor is as follows Figure 2 The illustrated electrodynamic droplet classifier 220. A series of droplets 11 can pass near a deflector 222 (e.g., one or more electrodes) used to selectively deflect droplets of interest, such as droplets containing cells 12. Cells 12 can be guided to an imaging slide 215 located on a response stage 213. As additional droplets are classified on the slide 215, the response stage 213 can move sequentially to provide space on the slide 215. The slide 215 may have reference markers allowing reference to the spatial position of the individual classified droplets. A camera 224 allows recording of the position of the individual classified droplets, which can also be correlated with droplet flow data (e.g., timing data indicating when each individually classified droplet is classified from the droplet flow).

[0090] After the required number of droplets have been sorted onto the slide 215, the slide 215 can be transferred to, for example... Figure 4 The sample stage 114 is shown. The positions of the individual classified droplets on the slide 215, recorded by the camera 224, can be correlated with the images obtained using the imaging / ablation device 110. Therefore, the ablated subsamples can be traced back to the images obtained using the imaging / ablation device 110, then to their spatial positions on the slide 215, and finally to the droplet flow data.

[0091] Additional or optional upstream processing steps may include fixing cells onto a glass slide. However, as mentioned above, the systems, apparatus, and methods described herein are capable of performing live cell imaging and ablation under ambient conditions, therefore cell fixation is not a necessary pretreatment step. Other additional or optional upstream processing steps may include staining the sample and / or adding tags to the sample.

[0092] Refer againFigure 11 The illustrated system 100 can also include a downstream analyzer 130 configured to receive the ablated sample from the receiver 116 for further analysis. The downstream analyzer 130 can include, for example, a PCR machine, a sequencing machine, a spectrometer, a mass spectrometer, or a combination thereof. Other biomolecule analysis devices known in the art can additionally or alternatively be included. In the case of a mass spectrometer, the analyzer 130 can include, for example, one or more of a time-of-flight (TOF) mass spectrometer, an Orbitrap TM a linear ion trap mass spectrometer, a quadrupole mass spectrometer, a quadrupole ion trap mass spectrometer, a sector magnetic mass spectrometer, or a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.

[0093] The illustrated system 100 can also include a controller 140 communicatively coupled to one or more other components of the system to provide control and / or feedback of the system 100. The controller 140 includes one or more processors 142, memory 144 (e.g., on one or more hardware storage devices), and a communication module 146 for controlling the sending and receiving of data between the controller and various components of the system 100 to which the controller 140 is coupled. The controller 140 can also include input / output hardware 148 known in the art for receiving input from a user and / or for displaying information to a user.

[0094] Additional details and embodiments relating to the system 100 are described below. It should be understood that the embodiments described below can be provided in any combination and used in conjunction with the entire system 100 as described above. In the embodiments described below, like numbers can be used to refer to like components.

[0095] Optical assembly

[0096] Figure 5 One example of an optical assembly 312 that can be used in an imaging and ablation system such as the system 100 described above is schematically illustrated. In some embodiments, the optical assembly 312 is configured to provide epi-fluorescence imaging. The optical assembly 312 can include an objective lens 318, which preferably has a NA of about 0.5 or greater, or about 0.65 or greater, or about 0.75 or greater, or about 0.8 or greater. The optical assembly 312 also includes an imaging light source 356, an ablation light source 354 (i.e., an ablation laser source 354), and a camera 352 (e.g., a charge-coupled device (CCD) or CMOS camera).

[0097] Optical assembly 312 can also include one or more dichroic beam splitters / mirrors, such as dichroic mirrors 355 and 357. For example, dichroic mirror 357 is configured to reflect excitation light or a portion thereof from imaging light source 356 toward objective lens 318 and to allow emission light emitted by the sample back toward objective lens 318 to pass through. The emitted light can then be reflected by dichroic mirror 355 toward camera 352. One or more optical filters can also be positioned along the optical path in order to filter / block source light and / or reflected excitation light as needed for a particular application. For example, one or more excitation filters can be used to suppress unwanted background from the excitation light source and one or more emission filters can be used to suppress unwanted fluorescent background from the sample and / or other known filtering elements as needed. Dichroic mirror 355 can also be used to allow ablation light from ablation light source 354 to pass toward objective lens 318. One or more mirrors / filters 353 can also be included for steering the laser light along the optical path between source 354 and objective lens 318.

[0098] For example, imaging light source 356 can include a xenon arc lamp, a mercury lamp, or an LED. Ablation light source 354 preferably includes an infrared laser (e.g., near infrared or “near infrared (NIR)”). The ablation laser is also preferably a femtosecond laser. The laser can also be configured to enable two-photon imaging using objective lens 318. Ablation light source 354 can be used in addition to or in place of light source 356 for both imaging and ablation purposes. For example, as described below, a NIR source can be used at low pulse energies for obtaining imaging information and at high pulse energies for ablation.

[0099] The use of NIR from ablation light source 354 can be particularly advantageous for the intended ablation operation, particularly as compared to the use of ultraviolet (UV) light. For example, the meaningful interaction of the ablation light with the target biological sample is limited by the scattering of the light at the structures of the biological material (e.g., changes in the refractive index at membranes, nuclei, vesicles, etc.). This results in a loss of information and makes it difficult to concentrate the target information in a meaningful way on the detector. The applied ablation light will also be affected by phase shifts and losses on the biological structures and by absorption of the light by the biological structures. Because these limitations are primarily a function of the wavelength of the applied light, the limitations are more relaxed when NIR light is used. Thus, configuring ablation light source 354 as a NIR source has higher resolution and sample penetration depth as compared to UV applications such as UV matrix assisted laser desorption ionization (MALDI) applications.

[0100] Furthermore, to achieve super-resolution better than about 20 nm through localization, a penetration depth is limited to about 1 μιη for a typical fixed biological sample thickness. For standard visible (visual identification system (VIS)) range resolution or about 250 nm, the penetration depth is at most about 10 to 20 μιη. In the case of NIR, the two-photon excitation penetration depth is much higher, e.g., up to about 100 μιη. Thus, the use of NIR advantageously allows for deeper penetration into the target sample tissue and even enables ablation of relatively complex tissue (e.g., brain). The enhanced depth penetration and resolution also increase the chance that the remaining material survives and / or retains structural information after the laser operation. Furthermore, while imaging of thicker samples is not feasible in the VIS range, two-photon imaging using low NIR pulse energies advantageously allows for imaging at greater depths, thus enabling better volumetric information.

[0101] While an exemplary optical system 312 is shown here, it should be understood that other optical components for imaging and / or ablation, including other dichroic mirrors, filters, mirrors, light sources, cameras, and / or other optical components known in the art, can be additionally or alternatively included to provide other imaging modalities. The optical assembly 312 can also be configured, e.g., to enable brightfield imaging and / or sectioning (e.g., using a confocal imaging assembly). The optical assembly described herein can be configured to provide independent focus control for each of imaging and ablation. That is, one or more adaptive optical assemblies can be included to provide, e.g., dynamic control over the ablation spot size within a given imaging field of view.

[0102] When used to ablate a region of interest, the laser can be configured to deliver about 1 nJ to about 20 μΐ of pulse energy for a given 1 μιη by 1 μιη by 2 μιη voxel. Larger regions of interest are able to absorb more energy, and thus are able to withstand greater absolute pulse energies. In other words, the laser can be configured to deliver up to about 20 μΐ per μιη 3 about 0.5 nJ to about 10 μΐ of pulse energy.

[0103] about 0.5 nJ to about 10 μΐ of pulse energy. 3 The upper limit of 10 μΐ represents an upper limit before excessive fragmentation and ionization is expected to occur, and thus represents an upper limit at which it is desirable to preserve the ablated region of interest. However, as explained in greater detail below, in some embodiments it can be desirable to fragment / ionize the target region by delivering one or more pulses above the per μιη 3 10 μΐ upper limit. In short, for example, it can be desirable to focus the laser on a large amount of media above the region of interest within a cell and blow away the overlying media before subsequently ablating the region of interest within the cell. This process can provide a greater gap for the ablated region of interest to travel from the sample stage to the receiver (see corresponding to FIG. 4B). In other words, in some embodiments it can be desirable to deliver one or more pulses above the per μιη Figure 6A(see discussion above regarding other embodiments).

[0104] As noted above, the imaging / ablation devices described herein are advantageously capable of targeting relatively small regions of interest. The optical assembly can be configured to provide a spot size diameter of the target region of, for example, about 50 pm or less, or about 30 pm or less, or about 10 pm or less, or about 5 pm or less, or about 3 pm or less, or about 1.5 pm or less, or about 1 pm or less. 3 or less, about 250 pm 3 or less, about 100 pm 3 or less, about 50 pm 3 or less, about 25 pm 3 or less, about 10 pm 3 or less, about 5 pm 3 or less, or about 2 pm 3 or less.

[0105] In some embodiments, the optical assembly can be configured to ablate an entire cell or a collection of multiple cells. In other embodiments, the optical assembly can be configured for ablating sub-cellular sized target regions, such as specific organelles or other intracellular regions, or specific extracellular regions.

[0106] ablation sample receiver

[0107] Figure 6B An example of a receiver 416 is shown, which includes a spatially differentiating medium, such as a microwell plate, a microwell chip, a nanodroplet array, or other structure capable of receiving individual ablation subsamples and holding different subsamples in different, spatially separated compartments. The receiver 416 can be attached to a receiver stage 417, which is capable of selectively moving in at least two axial directions, more preferably in all three axial directions.

[0108] As described above with respect to other embodiments, the optical assembly 412 is configured to provide imaging and / or ablation of a sample 10 placed on the sample stage 414. During ablation, the resulting ablation plume 20 extends upward toward the receiver 416, where it is collected and spatially differentiated from other ablation subsamples. When a desired number of subsamples have been collected, or when the receiver 416 has been filled, it can be removed from the receiver stage 417 and passed to an analyzer for further processing and / or analysis of the collected subsamples.

[0109] Figure 7A particular embodiment is shown in which the receiver 516 is configured as an array of nanodroplets. As with other embodiments, the optical assembly 512 is configured to provide imaging and / or ablation of a sample 10 placed on the sample stage 514. During ablation, the resulting ablation plume 20 extends upward toward the receiver 516, where it can be collected into a respective nanodroplet 519 or series of such nanodroplets 519. Different nanodroplets 519 thus form different compartments that serve to spatially distinguish different ablation subsamples from different ablation events. As described above, when a desired number of subsamples has been collected, or when the receiver 516 has been filled, it can be removed from the receiver stage and passed to an analyzer for further processing and / or analysis of the collected subsamples.

[0110] Figure 8 An exemplary process for forming an array of nanodroplets, such as included in the receiver 516, is shown. An acoustic transducer 562 can be used to apply acoustic energy to separate a solution of barcodes in a barcode array 560. The resulting nanodroplets are transported from the barcode array 560 to the overlying coverslip 515. The nanodroplets 519 can include different barcodes and thus be ready for subsequent analysis of the ablation subsamples captured by the nanodroplets, such as subsequent PCR or sequencing of the captured nucleic acids. The barcodes can be associated with the spatial locations of the nanodroplets 519 on the coverslip 515.

[0111] Figure 8 An embodiment is shown in which the receiver 616 includes an electrospray probe 670. This type of receiver can be particularly useful for generating ionized droplets containing ablation subsamples for analysis by mass spectrometry. The electrospray probe 670 passes through a capillary 674. An exposed distal portion 676 of the probe 670 extends beyond the distal end of the capillary 674. A solvent 672 is disposed within the capillary 674 and flows out to wet the surface of the exposed distal portion 676. The capillary 674 is configured to apply the solvent 672 so that it flows along the outer surface of the exposed distal portion 676 toward the tip of the electrospray probe 670. The tip of the electrospray probe 670 forms electrospray droplets and directs them toward a mass spectrometer inlet 678. The solvent can include, for example, water and / or one or more volatile organic compounds, such as methanol, acetonitrile, acetic acid, etc.

[0112] As shown, the wetted surface of the exposed distal portion 676 can be positioned above the ablation plume 20 so that the subsamples to be ablated are collected on the wetted surface during ablation of the sample 10 using the optical assembly 612. The positioning systems of the sample stage 614 and the receiver stage 617 can be coordinated to align the ablation plume 20 with the exposed distal portion 676. The flow rate of the solvent 672 can be controlled according to the ablation frequency to ensure effective spatial separation of successive subsamples captured by the flowing solvent 672 on the wetted surface of the exposed distal portion 676.

[0113] The illustrated configuration has been found to ionize ablated material with an efficiency of about 20% or greater, about 35% or greater, about 50% or greater, about 65% or greater, about 80% or greater, about 90% or greater, about 95% or greater, or about 99% or greater, as compared to conventional LAESI systems that ionize a small fraction of the ablated material.

[0114] Ablated sample analyzer

[0115] As noted above, a downstream analyzer can be used to further process and / or analyze the ablated subsample collected by the receiver. Depending on the type of processing and / or analysis desired, the downstream analyzer can include one or more, for example, PCR machines, sequencing machines, spectrometers, nuclear magnetic resonance (NMR) spectrometers, mass spectrometers, chromatography devices, centrifuges, electrophoresis devices, radioactive labeling and detection devices, other analytical biochemical devices, or combinations thereof.

[0116] In cases where a mass spectrometer is included, the analyzer can include one or more, for example, time-of-flight (TOF) mass spectrometers, Orbitrap mass spectrometers, linear ion trap mass spectrometers, quadrupole mass spectrometers, quadrupole ion trap mass spectrometers, sector magnetic mass spectrometers, or Fourier transform ion cyclotron resonance (FTICR) mass spectrometers.

[0117] In cases where sequencing is applied, the sequencing machine can be configured to perform next generation sequencing (NGS), sometimes also referred to as high-throughput sequencing. Suitable sequencing modalities include 454 pyrosequencing, ion torrent sequencing, nanopore sequencing, sequencing by synthesis (i.e., Illumina sequencing), and / or other sequencing methods known or to be developed in the art. More traditional chain termination methods (e.g., Sanger sequencing) can also be used.

[0118] Figure 9 One example of a downstream process is shown, in which one or more reagents can be added to the spatially separated compartments (nanodroplets 719 in this example) of the receiver 716. One or more reagents can be added to perform, for example, cell lysis, protein extraction, reduction, alkylation, fragmentation, and / or other desired reactions to prepare the collected subsample.

[0119] Figure 3 It is also shown that the subsample can additionally or alternatively be transferred to a liquid chromatography mass spectrometry (LC MS) system. For example, the subsample can be passed to a liquid chromatography column 780, which is coupled with an electrospray probe 770 for delivering ionized sample to a mass spectrometer 730.

[0120] Modes of operation:

[0121] The devices and systems described herein can be configured to perform various imaging and / or ablation procedures.Figure 10 An exemplary method 800 for associating the location of an ablation subsample on a receiver with an ablation event and a target region of interest on a sample slide is shown. As described above, a region of interest on a sample slide can be further associated with upstream classification / streaming data (see, e.g., Figure 11 and related descriptions). The method 800 can be performed using a controller communicatively coupled to certain components of the system, such as the controller 140 described above with respect to the system 100.

[0122] In the illustrated method, the controller can first record the spatial location of a region of interest using collected image data of the sample (step 810). For example, the imaging can be accomplished using one or more of brightfield imaging, sectioning, epi-fluorescence imaging, and / or two-photon imaging. Further, the imaging can be accomplished using the same objective lens through which the ablation laser pulses subsequently pass for ablating the target region of interest.

[0123] The controller can then associate the spatial location of the region of interest with an ablation event that caused at least a portion of the region of interest to ablate, the ablation event forming an ablation plume that carried an ablation subsample of ablated material from the region of interest to a receiver (step 820). Thus, this step can associate temporal information of the ablation event with the spatial location of the region of interest on the sample slide.

[0124] The controller can then record the location of the ablation subsample on the receiver (step 830), and then associate the ablation event with the location of the ablation subsample on the receiver, which is thereby associated with the ablation event and the spatial location of the region of interest (step 840). Thus, these steps can associate the spatial location of the ablation subsample on the receiver with the corresponding temporal information of the ablation event and the spatial location of the region of interest on the sample slide. Thus, this set of associations allows for subsequent tracing of the subsample data back to the corresponding temporal event and spatial location that caused the subsample data.

[0125] During ablation, the laser pulse frequency, the laser pulse energy level, and the laser pulse depth can be varied independently to provide the desired operational capabilities. For example, in a standard implementation, a single pulse can be directed to a fixed focal point location. The pulse energy can be selected to optimize ablation, to optimize plume formation, and / or to minimize degradation of the transferred subsample.

[0126] In other implementations, the depth and / or the pulse energy level of the applied laser pulses can be varied dynamically to provide the desired effects. Figure 11An example mode of operation is shown graphically in which a rectangular xy region is scanned and ablated. The laser pulse frequency can be tuned to balance dwell time and pulse-to-pulse overlap. This type of implementation can be used to ablate an entire structure (e.g., various organelles within a cell) or a desired portion thereof in a spatially coordinated manner. Note that the units along the chart axes are for illustrative purposes only and are not necessarily to scale.

[0127] Figure 11 Another example of a mode of operation that can be used in the case where a target region of interest is covered by a layer of tissue, media, or other obstructive material is shown graphically. The "z-drive" line represents the movement of the applied laser pulses along the axial / vertical channel (i.e., along the z-axis, perpendicular to the sample stage plane defined by the x- and y-axes). The "focal position" line represents the depth at which the target region of interest lies. As shown, the upper layer will initially experience higher energy laser pulses, which can be better tailored on-demand for removing the covering material and opening the axial channel. Once the dynamically moving laser pulses reach the depth of the region of interest, the laser pulse energy level can be reduced to a level more suitable for ablation of the region of interest. The ablated material can then exit the sample and travel toward the receiver before the formed channel collapses back to the region of interest. Note that the units along the chart axes are for illustrative purposes only and are not necessarily to scale.

[0128] An operating mode as shown in Figure 11 can advantageously ablate a target region within a sample that is deeper than measured from the upper surface. For example, in some cases, ablation can be limited to the upper 2-10 pm of a sample without first removing some covering material, as the resulting plume would need to be transported up through any remaining covering material to the receiver. Dynamically configuring the operating mode as in Figure 12 can remove or reduce the amount of covering tissue, thus allowing the ablation plume from a deeper region of interest to be effectively transported to the receiver.

[0129] The imaging and ablation apparatus as described herein can also operate to focus the ablation laser at a depth from the upper surface of the sample that is associated with a value of spatial resolution (i.e., the shortest distance between two points on the sample that can still be distinguished). That is, when the value of spatial resolution required is small, the maximum depth of focus of the ablation laser is also small, and when the value of spatial resolution required and / or utilized is large, the maximum depth of focus of the ablation laser is also larger. This approach allows for greater depth of focus in cases where the value of spatial resolution required for effective targeting of the region of interest is large enough, but limits the depth of focus in cases where the value of spatial resolution required is small, thus increasing the likelihood that the resulting ablation plume can successfully be transported from the sample to the receiver.

[0130] For example, the ablation laser can be focused at a depth from the upper surface of the sample that is no more than R times the spatial resolution, where R is a value of about 5 to about 30, such as a value of about 10, 15, 20, or 25. However, it will be appreciated that at depths greater than that specified by the value of R, ablation can occur in at least some applications, such as in the case of using dynamic laser operation, as Figures 2 to 11 shown in the operation.

[0131] At least a portion of the sample disposed between the bottom surface of the sample and the laser focal point can remain un-ablated. Thus, a particular subsample volume at a particular depth can be targeted for ablation in a manner that allows successful transmission of the resulting ablation plume to a receiver.

[0132] Laser focus detection unit

[0133] Figure 12 An exemplary imaging / ablation apparatus is shown that includes a laser focus detection unit (FDU) 1000. The FDU can be used with any of the imaging / ablation apparatuses mentioned above with reference to Figure 12 the selected objective 1020 (the apparatus can be equipped with multiple different objectives, such as up to five objectives) focuses light through a transparent window of the sample stage 1001 and into the sample 1002. The optical axis of the system is indicated by arrow 1008. The sample stage 1001 includes a transparent window, such as a glass or plastic window, such as a transparent coverslip or coverslips, or other transparent glass or plastic support on which the sample is mounted. The transparent window can protect the sample from the atmosphere of the microscope or optical components. The sample 1002 can be a sample of one or more cells. The sample 1002 has a top layer of water 1002b, i.e., covers the cells. An atmosphere 1007, such as air, surrounds the sample. The atmosphere can include humidity / C02 flow control for live cell experiments.

[0134] Arrow C represents the path or diameter of an incident light beam, which can be an epi-illumination beam, a laser pulse for ablation, or an excitation light for fluorescence emission. The light beam C, e.g., a laser for ablation, is focused by the objective at a focal point 1003 in the sample. A cavity having a diameter 1009 is initially created in the sample at the focal point 1003 by a pulse of the ablation laser. A subsequent ablation plume 1004 containing ablated sample material is created, which expands from the sample at its surface with the atmosphere, typically having a cos 2 distribution. The ablation plume 1004 expands toward a receiver 1005 located above the sample, such as a receiver plate, an electrospray probe, or an inlet of a mass spectrometer. The region 1006 represents the projection of the plume 1004 on the receiver 1005.

[0135] A light source 1011, such as a flash lamp, that allows (pulsed) trans-illumination snapshot imaging is positioned above the sample. A lens 1014 is provided for collimating light from the light source into the sample 1002.

[0136] Several optical paths are indicated. Laser light from the ablation laser is indicated by wavefront A and a pulsed waveform with energy P, which follows path C. Path C generally represents the down-fall illumination beam path of light from below toward the sample, including light from the ablation laser and excitation light for fluorescence emission. The reference laser beam of the focus detection unit (FDU) is represented by beam path B. Path D represents the optical path of back-reflected light and fluorescence emission. Figure 13 A camera (e.g., CCD) or spectrometer not shown in receives light from path D to produce information that can be time-resolved and / or spectrally resolved.

[0137] Laser light from the reference laser of the FDU is reflected back from the surface 1010 of the sample. The transmitted laser light B from the reference laser of the FDU is shown / / has been transmitted through the sample 1002. A fast photodetector 1012 is positioned to detect the transmitted laser light B / / .

[0138] includes Figure 13 The ablation system of the FDU schematically shown in Figure 4 may be implemented using the exemplary optical system shown in Figure 13 The optical system shown in includes many of the same components as the optical system schematically shown in Figure 12 and these same components use the same reference numerals. Figure 14 The optical system shown also includes an FDU that includes at least one reference laser 2002. Two reference lasers are used Figure 13 The system shown in. The reference laser beam, generally shown by arrow B, is reflected by the FDU dichroic beamsplitter / mirror 2004 to enter the objective 318. The reference laser of the FDU is preferably a near-infrared or “NIR” laser. In the embodiment shown, the reference laser beam consists of 905 nm laser light. The reference laser is focused in the back focal plane (BFP) of the objective, forming a collimated light in the sample space. The dichroic mirror 2004 reflects only the 905 nm laser light, other wavelengths of light (e.g., UV, visible, other NIR, etc.) pass through the dichroic mirror. Thus, the reference laser wavelength, together with the dichroic mirror 2004, makes the epi-illumination-microscopy setup independent of the FDU. The reflected reference laser beam that has been back-reflected from the sample stage and / or sample, which can include reflections from a generated cavity or ablation plume, is detected by photodetector 2006. Further details of the FDU reference laser and detection are described in reference to Figure 12 and 15 below. Figure 12The optical system in the system is controlled by a controller 2050, which like the controller 140 described for the system 100 above, comprises one or more processors, memory (e.g., on one or more hardware storage devices), and communication modules for controlling the sending and receiving of data between the controller and the various components of the system coupled with the controller. The sending / receiving of data to / from the controller is indicated by the arrows. The controller can also include input / output (I / O) hardware for receiving input from a user and / or for displaying information to a user, as known in the art.

[0139] Referring to Figure 14 , several distances in the system are shown, which will be monitored during the ablation process: ΔZ1 is the distance from the objective 1020 to the sample stage 1001 (i.e., its transparent window), ΔZ2 is the distance from the objective 1020 to the sample reflecting surface 1010 at the sample / atmosphere interface, and ΔZ3 is the distance from the sample stage 1001 to the receiver 1005.

[0140] Referring to Figure 14 , F1, F2, F3 denote the reference laser beams of the FDU. A first reference laser beam F1 from a first reference laser of the FDU (laser #1) is directed into the objective of the optical system off the optical axis. The objective 1020 focuses the first reference laser beam F1 towards the sample stage at an angle a with respect to the objective. The angle a depends on the objective used. The first reference laser beam F1 is focused at the objective to form parallel light in the objective space at the angle a. The angle a can be used for triangulation to measure and / or control the relative distance ΔZ1 of the objective 1020 to the sample stage 1001 and the relative distance ΔZ2 of the objective 1020 to the sample surface 1010.

[0141] A portion of the first reference laser beam F1 is reflected from the sample stage (i.e., from the transparent window of the sample stage), resulting in a first reflected reference laser beam F'2. The reflected reference laser beam F'2 is radially displaced within the objective by a radial distance R1 with respect to the incoming first reference laser beam F1. Referring to Figure 14 and 15, the array detector of the photodetector 2006 detects the beam positions of the first reference laser beam F1 and the reflected reference laser beam F'2 and thus the distance R1 therebetween in more detail. The distance R1 between the reference beams is a function of the distance ΔZ1 so for measuring the distance of the objective 1020 to the sample stage 1001.

[0142] A portion of the first reference laser beam F1 is also reflected from the sample surface with the atmosphere, which can be the water level above the cell sample, resulting in a second or further reflected reference laser beam F3. The reflected reference laser beam F3 is radially displaced within the objective by a radial distance R2 with respect to the incoming first reference laser beam F1. Referring to Figure 14And 15 show in more detail that the array detector of the photodetector 2006 detects the beam positions of the first reference laser beam F1 and the reflected reference laser beam F3 and thus the distance R2 between them. The distance R2 between the reference beams is a function of the distance ΔZ2 so that it is used to measure the distance of the objective 1020 to the sample surface 1010 with respect to the atmosphere.

[0143] The second reference laser beam F2 from the second reference laser of the FDU (laser #2) is directed in and out of the objective along the optical axis 1008 (i.e. perpendicular to the objective). The second reference laser beam F2 is directed to the objective by a beam splitter which forms part of the interferometer (reference Figure 14 , 15 and 16 described below). The first and second reference lasers of the FDU can be applied to each other simultaneously (without a time delay) or sequentially (i.e. with a time delay). The first and second reference lasers of the FDU can be applied simultaneously or sequentially with respect to the timing of the ablation laser pulse, preferably simultaneously with the ablation laser pulse. In other words, the measurement of ΔZ1 and / or ΔZ2 and / or ΔZ3 can occur simultaneously or sequentially (preferably simultaneously) with respect to the timing of the ablation laser pulse. The measurement of ΔZ1 and / or ΔZ2 and / or ΔZ3 can preferably start before and preferably continue simultaneously with the ablation laser pulse. The measurement of ΔZ1 and / or ΔZ2 and / or ΔZ3 can be performed substantially continuously over a time period including the ablation laser pulse.

[0144] Figure 15 shows in A the positions of the reference laser beams F2, F2’ and F3 received at the array detector 2006. For simplicity, the reference laser beam F1 is not shown. The positions of the reference beams F2’ and F3 on the array detector with respect to the position of beam F1 are a measure of the z-axis position of the sample stage and sample surface, respectively, with respect to the objective. As Figure 14 shown, the reference beams pass through a beam splitter 2010 to the array detector 2006. The beam splitter 2010 is a 50:50 beam splitter. In other embodiments, a beam splitter can be used which separates the light in different ratios, for example 10:90 (transmission:reflection). In Figure 16 , for simplicity only the second reference beam F2 is shown.

[0145] The array detector of photodetector 2006 is a photodetector with real-time peak detection, i.e. operating at a frame rate of at least kHz (>1 kHz). The time resolution of the array detector is preferably 1 ms or higher, or 1 ps or higher, e.g. in the range of 1 ms to 1 ps, or 1 ms to 1 ns, or 1 ms to 1 ps, or 1 ps to 1 ns. In one embodiment, the photodetector of the FDU comprises an array detector as described and one or more single-element photodetectors. The single-element photodetector is used to achieve the highest possible time resolution, e.g. 1 ns or higher or 1 ps or higher. In one embodiment, the light beam F2 in and out of the objective along the central optical axis is detected by a (single-element) detector or a pixel of an array with a higher time resolution than the array detector, e.g. a time resolution of at least 10 ns, or at least 1 ns, or at least 1 ps, e.g. a resolution of 1 ns to 1 ps. Fig. 15 in B schematically shows the light beam F2 passing through and then being reflected by beamsplitter 2010 to be detected by a single-element fast photodetector Z. Also shown is the interference pattern 2011 resulting from this detection of the reflected F2 light beam. This can detect the sample surface, e.g. the water level, in real time.

[0146] As Figure 17 schematically shown, the detector 2006 forms part of an interferometer 2014, which further comprises beamsplitter 2010 and mirror 2016, as well as light sources in the form of a first reference laser (#1) 3002 and a second reference laser (#2) 3004 of the FDU (laser #2 only shown for simplicity). Another view of the FDU is schematically shown in Figure 18 , which again shows the array detector 2006, beamsplitter 2010, dichroic mirror 2004, as well as first reference laser (#1) 3002 and second reference laser (#2) 3004.

[0147] As mentioned above, the reference laser #1 is used for triangulation to determine the distances AZ1 and AZ2 in the system. In Figure 19 , pixel intensity data on the array detector is shown due to the reflection of the light beam from the sample stage (e.g. a glass slide or coverslip) surface AZ1 (lower surface) and AZ1'(upper stage surface). Typically, the AZ1 measurement is used to adjust or control the z-drive mechanism positioning the objective relative to the sample stage, thereby controlling the laser focal point depth of the ablation laser. The pixel distance AZ 1,1' is a function of the magnification and NA of the objective used. As Figure 20 shown, the detection pixels of the array detector can be calibrated against known objective positions (AZ1 values, as well as AZ2 values). The calibration provides a linear correlation of the pixels of the array detector and the AZ1 values.

[0148] As mentioned above, the reference laser #1 is also used for triangulation to determine the distance ΔΖ2 in the system, e.g. with sub-micron precision, depending on the type of objective used. In Figure 21 In Figure 12 The same data is shown in

[0149] The reference laser #2 (beam F2) of the FDU is used for interferometric measurement of the ablation cavity and plume propagation, i.e. in the z-axis direction. Figure 22 The time (t) dependence of the interference signal (I) from laser #2 at a photodetector is shown, which originates from reflections at the cavity formation and plume-sample interface. The interference signal provides precise position and time resolved information about the cavity and plume formation. The constructive and destructive extrema in the signal define the trajectory coordinates from the function X(t, n. λ / 2). ΔΧ(ί) / Δί), which allows to precisely monitor the velocity of the cavity towards the receiver. The extrema occur at n*λ / 2 (n is an integer), and since the direction of the plume motion is known, the data point at each λ / 2, the extremum X, ΔΧ(ί) / Δί at time t is the velocity.

[0150] As can be seen from the described embodiments, the FDU can comprise at least a first reference laser configured to direct a first laser along an axis that deviates from an optical axis of the objective towards the objective, and the at least one photodetector is configured to first detect the first laser reflected back through the objective along the axis that deviates from the optical axis of the objective. As can also be seen from the described embodiments, the FDU can comprise at least a second reference laser configured to direct a second laser along the optical axis of the objective towards the objective, and the at least one photodetector is configured to detect the second laser reflected back through the objective along the optical axis of the objective. Furthermore, in one embodiment, the at least one photodetector configured to detect the second laser reflected back along the optical axis of the objective forms part of an interferometer. In one embodiment, the second laser reflected back along the optical axis of the objective simultaneously provides an interference signal from a cavity in the sample formed by the laser from the ablation laser. In one embodiment, the second laser reflected back along the optical axis of the objective simultaneously provides an interference signal from a cavity in the sample formed by the laser from the ablation laser.

[0151] Referring again to Figure 4The transmitted reference laser beam B" is located above the sample and the angle a corresponds to the NA of the objective. A fast photodetector 1012, located above the sample, monitors the cavity as it propagates through the sample surface. When the cavity propagates to the surface, it interferes with the transmitted reference laser beam B" and thus the signal on the photodetector 1012 changes, providing dynamic information about the cavity formation and the cavity travel to the sample surface. This monitoring of the ablation cavity and plume propagation can be used in addition to or in place of the reference laser #2 to monitor ΔΖ2.

[0152] Dynamic detection of the ablation cavity and plume formation is performed by a fast photodetector (sub-nanosecond (ns) resolution) to monitor the surface reflection properties of the sample, i.e. the sample height H(t) as a function of time. The detector 1012 is used to monitor the detection signal D(H t ) which indicates when the cavity passes through the sample / atmosphere surface at time t'. From time t' to t", the plume is traversing the surface. The axial length of the cavity / plume is defined by Δt = t" - t' and the speed of sound, which is influenced by the sample medium and the atmosphere and can be estimated at this time. Figure 4 The operation of the optics for ablation monitoring is shown schematically. The height H of the sample surface is shown developing over time as the cavity propagates, ti < t2< t3< t4, until a plume is formed at t". As described above, the monitoring of the cavity propagation and plume formation using the reference laser beams Fl, F2 and F3 of the FDU is shown.

[0153] As can be seen from the above, in some embodiments, the at least one photodetector comprises a fast photodetector configured to detect laser light from the at least one reference laser that has been reflected from and / or transmitted through at least one surface of the sample stage and / or the sample. The fast photodetector here preferably has a sub-nanosecond time resolution, i.e. a resolution of at least 1 ns, e.g. 1 ns - 1 ps. Moreover, in some embodiments, the at least one fast photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that originates from an (axial) interface of the sample having a different refractive index, e.g. reflected from the sample stage surface and directed back through the objective. The at least one photodetector can comprise a photodetector configured to detect laser light from the at least one reference laser that has been reflected from a surface of the sample facing the receiver and the atmosphere and directed back through the objective. The photodetector can comprise an array detector for peak detection. In one embodiment, the at least one photodetector comprises a photodetector disposed at a first (upper) side of the sample and configured to detect laser light from the at least one reference laser that has been transmitted through the sample. The at least one fast photodetector is preferably configured to resolve the at least one detection signal with sub-nanosecond time resolution. The at least one fast photodetector is preferably at least one photodiode.

[0154] Pulsed transillumination of the sample from the light source 1011, in particular configured as a flash lamp, and detection of the transillumination by the CCD detector 352 in the camera (e.g. Figure 22 and 13 enables snapshot imaging of the dynamics of cavity formation, propagation of the cavity towards the surface medium, and plume formation. The transillumination images can also determine the cavity size.

[0155] Fluorescence emission, e.g. epi-fluorescence and / or Raman emission, can also be measured using the imaging / ablation device. The fluorescence excitation source (e.g. Figure 24The light of the light source 356) in 13 can be applied, e.g., after the ablation laser with or without delay. At a delay t=0 (i.e., no delay relative to the ablation laser), the nonlinear effects of the ablation laser can be applied to the fluorescence, e.g., as described at: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6788598 (Lee et al., Simultaneous label-free autofluorescence and multi-harmonic imaging reveals in vivo structural and metabolic changes in mouse skin, Biomed Opt Express. 2019, 10(10), 5431-5444; https: / / www.nature.com / articles / s41467-018-04470-8 (You et al., In vivo imaging by simultaneous label-free autofluorescence multi-harmonic microscopy, Nature Communications 9, Article number: 2125 (2018); and https: / / aip.scitation.org / doi / full / 10.1063 / 1.5098349 (S A Boppart et al., Simultaneous label-free autofluorescence-multi-harmonic microscopy and beyond. For Raman emission, a separate excitation laser (not shown) can be used. The emission can be spectrally and / or time-resolved. Such emission can be registered as a signal from the focus of the ablation laser and e.g., obtained from a snapshot image. The same detector, e.g., a camera for detecting the transillumination, can be used to detect the fluorescence emission image. Preferably, however, the fluorescence emission or Raman emission (at least Raman) is directed to a fast photodetector (e.g., an array photodetector) with a spectrometer to enable time- and spectrally-resolved detection of the fluorescence or Raman emission from the ablation volume of the sample. Using such a system, the cavity formation in the sample can be imaged by transillumination using the flash lamp 1011 at a time t after the ablation laser pulse and can be imaged by fluorescence at a later time t' after the material excitation. Thus, also a time resolution can be obtained by using different transillumination and fluorescence light sources. With reference to ​ , the epi-illumination signal (epi-illumination fluorescence and / or Raman signal) from the laser focus 1009 is directed by a beamsplitter to a fast photodetector (e.g., an array photodetector) in combination with a spectrometer to enable time- and spectrally-resolved detection of the fluorescence spectrum or Raman spectrum, e.g., I(A).

[0156] The detected epi- fluorescence signal or image is preferably time resolved and can be used to provide information about the cavity properties and the molecular dissociation at the focal position of the objective. The detection of a fluorescence depletion can indicate that molecular dissociation is occurring, e.g. if the ablation laser pulse energy is too high. In one embodiment, the next ablation laser pulse is subsequently applied at a lower energy. A quantitative fluorescence intensity analysis enables to estimate the transport efficiency of the ablated material to the analyzer, e.g. mass spectrometer. On the other hand, an analysis of the vibrational / rotational bands of the Raman signal reveals information about the expected mass of the molecules sent to the analyzer, e.g. mass spectrometer, while the intensity distribution of the vibrational / rotational bands indicates the thermal state (internal energy) of the molecules. The Raman peak intensities can be correlated to the peak intensities of the molecules detected by the mass spectrometer.

[0157] Fig. 23 shows how the calibration of the ablation parameters and the ablation laser pulse energy E0is performed. The pulse energy E0and the sample height ΔZ2from the objective determine whether a cavity can pass through the sample / air interface and form an ablation plume. At a threshold pulse energy E max The ablated molecules are denatured, i.e. dissociated, which is detected as a fluorescence depletion. Thus, if the effective pulse energy E0> E max , the pulse energy EΔZ2needs to be reduced. The value of Δz2decreases due to evaporation. However, this takes time. Alternatively or additionally, Δz3may decrease due to the cos2divergence of the plume concentration. The change of the signal D on the detector 1012is a function H(t). In one embodiment, as shown in (i), the pulse energy E0of each pulse and / or the pulse frequency is increased until, as shown in (ii), the detection signal D(H(t)) indicates that the cavity expands at a time t'. The time t' is a function of the speed of sound and Δz3. From t'-t", the axial height of the plume through the interface medium / air can be found. As shown in (iii), the reference beam F3(H t ) measures the exact position of the transition (interface) from the medium to the air and the propagation of the particles in the air to the receiver, which can be, e.g., the ionization module of a mass spectrometer. The (time) resolved epi- fluorescence signal shown in (iv) provides information about the cavity properties and the molecular dissociation at the focal position of the objective.

[0158] As can be taken from the above, in embodiments, the optical assembly comprises a laser focus detection unit, the laser focus detection unit comprising at least one reference laser arranged at the second side of the sample stage and configured to direct laser light through the objective, through the sample stage and into the sample, the laser focus detection unit further comprising at least one photodetector configured to generate at least one detection signal by detecting the laser light from the at least one reference laser, which is indicative of one or more properties of the sample when at least a portion of the sample is ablated, and / or which is indicative of the distance from the objective to the sample stage, and / or which is indicative of the distance from the objective to the surface of the sample.

[0159] In embodiments, the laser focus detection unit is further configured to send the at least one detection signal to a controller, the controller being configured to dynamically control one or more parameters of the ablation laser and / or the position of the objective and / or the position of the receiver based on the at least one detection signal. The controller can be the controller 140 or the controller 2050, or a similar controller. In general, the controller comprises a microcontroller and an FPGA or a computer system and is communicatively coupled to one or more other components of the system to provide control and / or feedback of the system, as further described below. The controller is communicatively coupled to the laser focus detection unit to receive the one or more detection signals. In one embodiment, the controller is communicatively coupled to the objective (e.g., to control the position of the objective in the z-axis direction via a driver). In one embodiment, the controller is further communicatively coupled to the receiver stage (e.g., to control the position of the receiver in the x, y, and / or z-axis direction via a driver). In one embodiment, the controller is further communicatively coupled to the ablation laser (e.g., to control the laser pulse energy and / or the pulse frequency).

[0160] The controller can comprise one or more processors, memory (e.g., on one or more hardware storage devices), and a communication module for controlling the sending and receiving of data between the controller and various components of the system to which the controller is coupled. The controller can further comprise input / output hardware known in the art for receiving input from a user and / or for displaying information to a user.

[0161] The described apparatus and system can be used to provide a means for continuously or real-time adjusting or controlling the ablation process, in particular the laser pulse energy, the pulse frequency, and / or the laser focus position in the sample. In one embodiment, the controller is configured to dynamically control one or more parameters of the ablation laser and / or the position of the objective and / or the position of the receiver based on the at least one detection signal from the FDU. In one embodiment, the controller is configured to continuously adjust the position of the objective and / or the position of the receiver based on the at least one detection signal. Thus, the at least one detection signal from the FDU forms the basis of a control loop for continuously, real-time controlling the ablation process.

[0162] In one embodiment, the axial (z) distance between the objective and the sample stage is continuously adjusted in real-time with high frequency as the laser focus within the sample is moved, based on ΔΖ1(and ΔΖ2) measured by the FDU. The adjustment of the axial position of the objective can be achieved by a movable driver / actuator controlled by the controller in response to the feedback of ΔΖ1(and ΔΖ2). The distance between the laser focus and the receiver or between the sample surface and the receiver is continuously corrected based on ΔΖ1and ΔΖ2. The adjustment of the axial position of the receiver can be achieved by a movable receiver stage controlled by the controller in response to the feedback of ΔΖ1(and ΔΖ2).

[0163] In one embodiment, the detection signal from the fast photodetector detecting the transmitted reference beam B" and / or the detection signal from the fast photodetector detecting the reference beam F2 along the optical axis of the objective is preferably used to control the number of pulses in the pulse sequence (pulse frequency) of the ablation laser to ensure laser-induced cavity feedback into the sample medium. Both the number of pulses and the pulse intensity can be control loop parameters. The interference in the detection signal from the fast photodetector detecting the reference beam F2 is used to monitor the relative advancement of the cavity in the axial (z) direction.

[0164] In one embodiment, the detection signal (D) from the fast photodetector detecting the transmitted reference beam B" is preferably used to control the pulse energy of the ablation laser. In addition, the fluorescence / Raman signal can also or alternatively allow monitoring and control of the dissociation of molecules in the sample by correspondingly controlling the laser power.

[0165] In one embodiment, snapshot epi-fluorescence imaging can account for plume formation and thermal detection. In this way, the ablated region of interest (ROI), the laser power and / or the pulse sequence characteristics can be adjusted accordingly.

[0166] In one embodiment, the controller is communicatively coupled to a mass spectrometer analyzing the ablated portion of the sample such that one or more control parameters of the system controlled by the controller are correlated to the mass spectrometer signal. The correlation of the observable of the FDU to the MS signal enables a qualitative correction of the mass spectrometer response. Snapshot imaging of the mass spectrometer signal keeps the mass spectrometer signal constant for MS detection.

[0167] Reference ​ Fig. 6 shows a flowchart of a process for real-time optimization of control parameters of an imaging / ablation device.

[0168] Using the imaging capability as described above, a region of interest, ROI, having x, y, z coordinates in the sample located on the sample stage is selected in step 4010. The minimum distance from the sample stage to the receiver is set in step 4020 by means ensuring no contact between the receiver and the sample. In step 4030, the thickness of the sample medium above the sample stage can be measured using the described method of triangulating ΔZ1, ΔZ2, ΔZ3 using the focus detection unit (FDU) and, by feedback in a loop by the controller, the distance from the sample medium to the receiver can be kept constant during the mass spectrometer measurement, for example to compensate for any condensation or evaporation affecting the sample. In step 4040, the objective focal point position is continuously corrected together with the distance from the sample medium to the receiver.

[0169] In step 4050 an ablation laser pulse is applied with energy E0. Then in step 4060 the signal of the reference FDU laser beam F1(B") emitted at the fast photodetector is monitored together with the interference signal of the reflected FDU laser beam F2. From these signals it can be determined whether the ablation plume has passed the interface of the sample with the atmosphere. If "no", then step 4050 is repeated, adjusting the ablation laser pulse energy to E 0,i+1 = E0+ ΔE. If "yes", then in step 4070 an image is captured with the transillumination light source at a delay time t' after the measurement of the ablation pulse and / or fluorescence emission and / or Raman signal. At the same time, in step 4080 a mass spectrometer measurement of the ablated material introduced through the receiver into the mass spectrometer is taken. In step 4090 the fluorescence emission intensity is compared with the imaging to check whether the degree of molecular fragmentation in the sample by the laser pulse is at an acceptable level. If "no", then ΔZ3 and the laser pulse energy E0 are lowered, or a new focal point position and / or ROI is selected. If "yes", then in step 4100 a mass spectrometer measurement of the ablated sample material is taken for the full ROI. In step 4200 the data from steps 4060, 4070, 4080 and 4100 is provided for quantitative interpretation of the transport of molecules from the sample detected with the mass spectrometer using input parameters such as the cavity volume and the fluorescence signal.

[0170] Computer / controller system

[0171] It should be appreciated that computer systems increasingly take a wide variety of forms. In this description and in the claims, the term "controller," "computer system" or "computing system" is intended to encompass any device or system, or combination thereof, that includes at least one physical and tangible processor and physical and tangible memory capable of having stored thereon computer-executable instructions that can be executed by the processor. As used in this description and in the claims, the term "computer system" or "computing system" is intended to encompass a personal computer, desktop computer, laptop, tablet, handheld device (e.g., mobile telephone, palmtop, pager), microprocessor-based or programmable consumer electronics, minicomputer, mainframe computer, multiprocessor system, networked personal computer, distributed computing system, data center, message processor, router, switch, and even devices that have not traditionally been considered a computing system, such as a wearable (e.g., glasses).

[0172] The memory can take any form and can depend on the nature and form of the computing system. The memory can be physical system memory, including volatile memory, non-volatile memory, or some combination of the two. The term "memory" can also be used herein to refer to non-volatile mass storage such as physical storage media.

[0173] The computing system also has on it a number of structures, often referred to as "executable components." For example, the memory of the computing system can include executable components. The term "executable component" is the name for a structure that a person of ordinary skill in the computing arts understands can be a structure of software, hardware, or a combination thereof.

[0174] For example, when implemented in software, a person of ordinary skill in the art will understand that the structure of an executable component can include software objects, routines, methods, etc., that can be executed by one or more processors on the computing system, whether such executable component exists in the heap of the computing system, or whether the executable component exists on a computer-readable storage medium. The structure of an executable component exists on a computer-readable medium in this form so that it is operable when executed by one or more processors of the computing system to cause the computing system to perform one or more functions, such as the functions and methods described herein. Such structure can be directly executable by the processor as in the case of executable components that are binary. Alternatively, the structure can be structured to be interpretable and / or compilable (whether in a single stage or in multiple stages) in order to generate such binary that is directly interpretable by the processor.

[0175] The term "executable component" is also fully understood by the person of ordinary skill in the computing arts to include structures that are implemented in dedicated or near-dedicated hardware logic components, such as within programmable gate arrays (FPGA), application-specific integrated circuits (ASIC), program- specific standard products (ASSP), system-on-a-chip (SoC), complex programmable logic devices (CPLD), or any other dedicated circuitry. Thus, the term "executable component" is a term of art for structures that are fully understood by the person of ordinary skill in the computing arts, whether implemented in software, hardware, or a combination thereof.

[0176] The terms "component," "service," "engine," "module," "control," "generator," and the like can also be used in the present description. As used in this description and in this context, these terms— whether expressed with or without the modifier clause— are also intended to be synonymous to the term "executable component," and thus also have the structure that is fully understood by the person of ordinary skill in the computing arts.

[0177] While not all computing systems require a user interface, in some embodiments, a computing system includes a user interface for communicating information from / to a user. A user interface can include output mechanisms as well as input mechanisms. The principles described herein are not limited to precise output or input mechanisms, as these will depend on the nature of the device. However, output mechanisms can include, for example, speakers, displays, tactile outputs, projections, holograms, and the like. Examples of input mechanisms can include, for example, microphones, touchscreens, projections, holograms, cameras, keyboards, stylus, mouse or other pointer input, any type of sensor, and the like.

[0178] Accordingly, embodiments described herein can include or utilize special-purpose or general-purpose computing systems. Embodiments described herein also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media can be any available media that is accessible by a general- or special-purpose computing system. Computer-readable media that store computer-executable instructions are physical storage media. Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example, and not limitation, embodiments disclosed or

[0179] Computer-readable storage media includes RAM, ROM, EEPROM, solid state drives ("SSDs"), flash memory, phase-change memory ("PCM"), CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other physical and tangible storage medium which can be used to store desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computing system after being stored. For example, a computer-executable instruction can be embodied on one or more computer-readable storage media to form a computer program product.

[0180] Transmission media can include a network and / or data links which can be used to carry desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computing system. Combinations of the above should also be included within the scope of computer-readable media.

[0181] Further, upon reaching various computing system components, program code means in the form of computer-executable instructions or data structures can be transferred automatically from transmission media to storage media (or vice versa). For example, computer-executable instructions or data structures received by way of network or data link can be buffered in RAM within a network interface module (e.g., a "NIC"), and then eventually transferred to computing system RAM and / or to less volatile storage media at computing system. Thus, it should be understood that storage media can be included in computing system components that also (or even primarily) utilize transmission media.

[0182] Those skilled in the art will further appreciate that a computing system can also contain communication channels that allow the computing system to communicate with other computing systems over, for example, network. Thus, the methods described herein can be practiced in a network computing environment that includes many types of computing systems and computing system configurations. The disclosed methods can also be practiced in distributed system environments where local and / or remote computing systems, which are linked through a network (by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) both perform tasks and / or share data. In a distributed system environment, processing, memory, and / or storage capabilities can be distributed among local and / or remote computing systems.

[0183] Those skilled in the art will further appreciate that the disclosed methods can be practiced in a cloud computing environment. Cloud computing environments can be distributed and / or virtualized, but this is not required. When distributed, cloud computing environments can be distributed internationally within an organization and / or have components possessed across multiple organizations. In this specification, and in the following claims, "cloud computing" is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources (e.g., networks, servers, storage, applications, and services). The definition of "cloud computing" is not limited to any of the numerous advantages that can be obtained from such a model when properly deployed.

[0184] Cloud computing models can be composed of various characteristics, such as on-demand self-service, broad network access, resource pooling, rapid elasticity, measured service, and the like. Cloud computing models can also come in the form of various service models, such as Software as a Service (“SaaS”), Platform as a Service (“PaaS”), and Infrastructure as a Service (“IaaS”). Cloud computing models can also be deployed using different deployment models, such as private cloud, community cloud, public cloud, hybrid cloud, and the like.

[0185] List of Abbreviations for Defined Terms

[0186] To aid in the understanding of the scope and content of the present written description and the appended claims, a few selected terms are defined directly below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0187] The terms “approximately,” “about,” and “substantially” as used herein represent an amount or condition close to the stated amount or condition that the specified recitation still performs a desired function or achieves a desired result. For example, the terms “approximately,” “about,” and “substantially” can refer to an amount or condition that is within less than 10%, or within less than 5%, or within less than 1%, or within less than 0.1%, or within less than 0.01% of a stated amount or condition.

[0188] As used herein, the term “ablation” refers to the selective application of energy to a target region in order to release biological molecules within the target region from surrounding structures. Ablated material typically forms a “plume” of material that travels away from the initial location of the target region.

[0189] As used herein, the term “region of interest” is intended to be understood as any region within the field of view of an imaging / ablation device in which one or more biological molecules are present that need to be collected, processed, and / or analyzed. The region of interest can include the entire field of view, but is more typically a portion of the field of view and can be, for example, a cell or collection of cells, an intracellular / subcellular region such as an organelle within a cell, or an extracellular region.

[0190] As used herein, the term “subsample” is intended to refer to individual portions of ablated material that are spatially and / or temporally separated from one another, for example by being spatially separated from one another on a receptacle and / or by being received by the receptacle at different times from different ablation events. Thus, the term subsample is intended to distinguish individual portions of collected ablated material from the larger overall “sample” that is located on the slide / stage that can be imaged and selectively ablated.

[0191] Various aspects of the disclosure, including devices, systems, and methods, can be described with reference to one or more exemplary embodiments or implementations. As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein. Furthermore, reference to "the implementation" or "the implementation" of the present disclosure includes reference to one or more embodiments of the present disclosure, and vice versa, and that such references mean that the specification is to be interpreted as illustrative only and not to be construed in a limiting sense of the scope of the present disclosure, the scope of which is indicated by the appended claims, not the following description.

[0192] As used in the specification, a word appearing in a singular form encompasses its plural counterpart and a word appearing in a plural form encompasses its singular counterpart unless the singular or plural connotation is clearly indicated by the context. Thus, it is to be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. By way of example, a reference to a single item, e.g., "a widget," includes one, two, or more items unless the context clearly dictates otherwise. Similarly, a reference to a plurality of items, e.g., "widgets," is to be construed as including a single item and / or a plurality of items, unless the content and / or context clearly dictates otherwise. For example, a reference to a plurality of items, e.g., "widgets," does not necessarily require a plurality of such items unless otherwise indicated. Rather, one or more items are contemplated herein independent of the number of items inferred.

[0193] As used herein, directional terms such as "top," "bottom," "left," "right," "upper," "lower," "upward," "downward," "proximal," "distal," "adjacent," and the like are used herein only to denote relative directions and are not intended to limit the scope of the disclosure and / or the claimed invention in any manner.

[0194] CONCLUSION

[0195] The terminology and phraseology adopted herein is for descriptive purposes only and not intended to be limiting, and the use of such terminology and phraseology does not exclude the equally effective incorporation of the features or parts of the features so exhibited and described, but rather, it is recognized that various modifications are possible within the scope of the claimed invention. Therefore, it is to be understood that, even though a number of embodiments of the invention have been described specifically, those skilled in the art will be able to make modifications and alterations to the concepts disclosed herein without departing from the spirit and scope of the invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it is not intended that the invention be limited to the specifics of the embodiments described. Various modifications and / or alterations to the described embodiments and / or the features of the invention described herein will be apparent to one of ordinary skill in the art and are considered to be within the scope of the present disclosure, and it is intended that the scope of the present disclosure be limited only by the appended claims.

[0196] It should also be appreciated that systems, apparatuses, products, kits, methods, and / or processes in accordance with certain embodiments of the present disclosure can include, incorporate, or otherwise comprise the properties or features (e.g., components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Thus, various features of certain embodiments can be compatible with, combined with, included in, and / or incorporated into other embodiments of the present disclosure. Accordingly, the disclosure of certain features with respect to a particular embodiment of the present disclosure should not be interpreted as limiting the application or inclusion of the features to the particular embodiment. Rather, it should be appreciated that other embodiments can also include the features, members, elements, parts, and / or portions, without necessarily departing from the scope of the present disclosure.

[0197] Furthermore, any of the features herein can be combined with any other feature or features disclosed herein, unless the contrary is explicitly noted by the context. Moreover, various well-known aspects, such as illustrative systems, methods, apparatuses, and the like, have not been described in detail in order to avoid obscuring aspects of the example embodiments. It will be appreciated that the various embodiments can include aspects not expressly described above but inherent in the systems, methods, apparatuses, and the like.

[0198] All references cited in this application, to the extent that they are inconsistent with the disclosure provided herein, are expressly incorporated herein by reference in their entirety. It will be apparent to those of ordinary skill in the art that methods, device elements, materials, procedures and techniques other than those specifically described herein can be employed in the practice of the present invention as broadly disclosed herein without resort to undue experimentation, and all such alternative methods, device elements, materials, procedures and techniques are expressly contemplated as falling within the scope of the present invention. All functions of the methods, device elements, materials, procedures and techniques described herein are intended to be encompassed by the present invention.

[0199] When a group of materials, compositions, components, or compounds are disclosed herein, it is understood that all individual members of those groups and all subgroups and combinations of those groups are disclosed separately. When Markush groups or other groupings are used herein, all individual members of the groups and all possible combinations and subcombinations of the groupings are intended to be individually included in the disclosure. Unless otherwise specified, each formulation or combination of components described or exemplified can be used to practice the application, as are any of the individual formulations or components. Whenever a range is given in the specification, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.

[0200] All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. An apparatus for imaging and ablating a sample, which allows for analyzing an ablated portion of the sample, the apparatus comprising: a sample stage having a first side configured for placing a sample thereon and a second side disposed opposite the first side; an optical assembly comprising an objective lens disposed at the second side of the sample stage and configured to enable microscopic imaging of the sample placed on the sample stage, the optical assembly further comprising an ablation laser, wherein the ablation laser is disposed at the second side of the sample stage, the ablation laser being configured to direct a laser light through the objective lens, through the sample stage and into the sample to selectively ablate at least a portion of the sample; and a receiver disposed at the first side of the sample stage, the receiver being configured to receive ablated material ejected from the sample to enable further analysis of the ablated material; wherein the optical assembly further comprises a laser focus detection unit, the laser focus detection unit comprising: at least one reference laser disposed at the second side of the sample stage and configured to direct a laser light through the objective lens, through the sample stage and into the sample, the laser focus detection unit further comprising at least one photodetector configured to generate at least one detection signal by detecting the laser light from the at least one reference laser, the at least one detection signal being indicative of one or more properties of the sample when at least a portion of the sample is ablated, and / or being indicative of a distance from the objective lens to the sample stage, and / or being indicative of a distance from the objective lens to a surface of the sample; wherein the laser focus detection unit is further configured to transmit the at least one detection signal to a controller configured to dynamically control one or more parameters of the ablation laser and / or a position of the objective lens and / or a position of the receiver based on the at least one detection signal.

2. The apparatus of claim 1, wherein the laser focus detection unit is configured to detect the laser light from the at least one reference laser with a temporal resolution of 1-10 nanoseconds or less and transmit the at least one detection signal to the controller.

3. The apparatus of claim 1 or 2, wherein the one or more properties of the sample comprise at least one of a formation of a cavity in the sample caused by the laser light from the ablation laser, a formation of an ablation plume resulting from ablated material ejected from the sample, and a change in thickness of the sample.

4. The apparatus of claim 1 or 2, wherein the controller is configured to control one or more parameters of the ablation laser, the one or more parameters comprising a laser pulse energy and / or a laser pulse frequency.

5. The apparatus of claim 1 or 2, wherein the controller is configured to continuously adjust a position of the objective lens and / or a position of the receiver based on the at least one detection signal.

6. The apparatus of claim 1 or 2, wherein the at least one photodetector comprises a fast photodetector configured to detect laser light from the at least one reference laser that has reflected and / or transmitted through at least one surface of the sample stage and / or sample.

7. The apparatus of claim 6, wherein at least one fast photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that has reflected off a surface of the sample stage and is directed back through the objective lens.

8. The apparatus of claim 6, wherein the at least one photodetector comprises a photodetector configured to detect laser light from the at least one reference laser that has reflected off a surface of the sample facing the receiver and is directed back through the objective lens.

9. The apparatus of claim 7, wherein the photodetector comprises an array detector for peak detection.

10. The apparatus of claim 6, wherein the at least one photodetector comprises a photodetector disposed on the first side of the sample and configured to detect laser light from the at least one reference laser that has transmitted through the sample.

11. The apparatus of claim 1 or 2, wherein at least one photodetector is configured to resolve the at least one detection signal with sub-nanosecond temporal resolution.

12. The apparatus of claim 1 or 2, wherein the at least one reference laser comprises at least a first reference laser configured to direct first laser light to the objective lens along an axis that is offset from an optical axis of the objective lens, and the at least one photodetector is configured to detect the first laser light reflected back through the objective lens along the axis that is offset from the optical axis of the objective lens.

13. The apparatus of claim 1 or 2, wherein the at least one reference laser comprises at least a second reference laser configured to direct second laser light to the objective lens along an optical axis of the objective lens, and the at least one photodetector is configured to detect the second laser light reflected back through the objective lens along the optical axis of the objective lens.

14. The apparatus of claim 13, wherein the at least one photodetector configured to detect the second laser light reflected back along the optical axis of the objective lens forms part of an interferometer.

15. The apparatus of claim 14, wherein the second laser light reflected back along the optical axis of the objective lens simultaneously provides an interference signal from a cavity formed in the sample by the laser light from the ablation laser.

16. The apparatus of claim 1 or 2, further comprising a light source disposed on the first side of the sample, the light source configured for transillumination of the sample.

17. The device of claim 1 or 2, further comprising a fluorescence excitation source configured for epi-fluorescence imaging and / or analysis of the sample, preferably disposed at the second side of the sample.

18. The device of claim 17, further comprising a spectrometer configured for spectroscopic and / or time-resolved fluorescence or Raman emission from the sample, preferably disposed at the second side of the sample.

19. The device of claim 1 or 2, wherein the ablation laser is a femtosecond laser.

20. The device of claim 19, wherein the ablation laser is a near-infrared laser.

21. The device of claim 1 or 2, wherein the at least one reference laser is a diode laser.

22. The device of claim 21, wherein the at least one reference laser is in the form of a photonic integrated circuit.

23. The device of claim 1 or 2, wherein the optical assembly is configured to be able to ablate a target region having a diameter of 50 pm + 10% or less.

24. A system for ablating and analyzing a target region of a sample, the system comprising: an imaging and ablating device according to any one of claims 1 to 23; and an analyzer configured to receive and analyze at least a portion of the ablated material received by the receiver.

25. The system of claim 24, wherein the analyzer comprises one or more of a PCR machine, a sequencing machine, a spectrometer, and a mass spectrometer.

26. The system of claim 25, wherein the mass spectrometer is configured as a time-of-flight (TOF) mass spectrometer, an Orbitrap mass spectrometer, a linear ion trap mass spectrometer, a quadrupole mass spectrometer, a quadrupole ion trap mass spectrometer, a sector magnetic mass spectrometer, or a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.

27. The system of any one of claims 24 to 26, further comprising a liquid chromatography column associated with the analyzer, the liquid chromatography column configured to receive the ablated material prior to delivery of the ablated material to the analyzer.

28. A method of imaging and ablating a sample to enable analysis of an ablated portion of the sample, the method comprising: providing a system according to any one of claims 24 to 27; acquiring an image of the sample; selecting a region of interest within the sample; delivering laser light from the ablation laser to the region of interest to ablate at least a portion of the region of interest; and capturing at least a portion of the ablated material on the receiver; optionally transferring at least a portion of the ablated material from the receiver to the analyzer; and dynamically adjusting one or more parameters of the ablation laser and / or a position of the objective lens and / or a position of the receiver based on at least one detection signal from the laser focus detection unit.

29. The method of claim 28, wherein a plurality of laser pulses are applied to the sample from the ablation laser, and wherein one or more of a laser pulse frequency, a laser pulse energy level, or a laser pulse depth is dynamically varied across the plurality of laser pulses.

30. The method of either one of claims 28 or 29, wherein the image of the sample is acquired with a spatial resolution value, and wherein the laser is focused at a depth of no more than R times the spatial resolution value, measured from an upper surface of the sample, where R is a value of about 5 to about 30.

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