Methods, apparatuses, and systems for cryogenic sample processing by plasma focused ion beam

Abstract

This invention relates to a method, apparatus, and system for processing frozen biological samples using a focused plasma ion beam (FIB). The samples can then be used for mass spectrometry (MS); genomics, such as gene sequencing analysis or next-generation sequencing (NGS) analysis; and proteomics. The invention particularly relates to a method for processing at least one biological sample. This method is particularly suitable for high-performance microscopy; proteomics analysis; sequencing, such as NGS, etc. According to the invention, the method includes the step of providing at least one biological sample in frozen form. Milling is performed by including O... + and / orXe + At least a portion of the sample is treated with a plasma ion beam of at least one of the plasmas.

Description

[0001] This application is a divisional application of the invention patent application with application number 201811514187.1, application date December 11, 2018, entitled "Method, Apparatus and System for Processing Biological Cryogenic Samples by Plasma Focused Ion Beam".

[0002] This application claims the benefit and priority of U.S. Patent Application No. 62 / 611153, filed December 28, 2017, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a method, apparatus, and system for processing frozen biological samples using focused ion beams (FIB). The samples can then be used for mass spectrometry (MS); genomics, such as gene sequencing analysis or next-generation sequencing (NGS) analysis; and proteomics. Background Technology

[0004] The development of electron and scanning probe microscopy in the latter half of the 20th century has enabled stunning images of the internal structure and composition of matter at nanoscale, molecular, and atomic resolution. This progress was largely achieved through computer-aided microscopy manipulation, data acquisition, and analysis methods. Advances in imaging technology in the early 21st century have paved the way for the availability of highly accurate structural and functional information. From a hardware perspective, high-resolution imaging methods now typically resolve atomic positions with sub-angstrom precision, providing in-depth insights into the atomic structure and dynamics of materials.

[0005] Various microscopy techniques may be associated with this invention, such as electron microscopy, charged particle microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning transmission electron microscopy (STEM), as well as various sub-types, such as so-called "dual-beam" tools (e.g., FIB-SEM), which additionally employ a focused ion beam (FIB) to allow supporting behaviors such as ion beam milling or ion beam-induced deposition (IBID) or ion-type imaging. This is a non-exclusive list of high-performance microscopy methods.

[0006] More specifically, in SEM, irradiating a sample with a scanning electron beam induces the emission of “auxiliary” radiation from the sample in the form of, for example, secondary electrons, backscattered electrons, X-rays, and photoluminescence (infrared, visible, and / or ultraviolet photons); then, one or more components of this emitted radiation flux are detected and used for image accumulation purposes.

[0007] In TEM, the electron beam used to irradiate the sample is selected to have sufficiently high energy to penetrate the sample (for this purpose, the sample will typically be thinner than in the case of a SEM sample); the transmitted electrons emitted from the sample can then be used to create an image. When such a TEM operates in scanning mode (and thus becomes STEM), the image in question is accumulated during the scanning action of the electron beam.

[0008] Instead of using electrons as the irradiation beam, other types of charged particles can be used to perform charged particle microscopy. In this regard, the phrase "charged particles" should be understood broadly to encompass, for example, electrons, positive ions (e.g., Ga or He ions), negative ions, protons, and positrons.

[0009] Regarding ion microscopy, some additional information can be gathered, for example, from the following sources: WHEscovitz, TRFox and R. Levi-Setti, Scanning Transmission Ion Microscope with a Field Ion Source, Proceedings of the National Academy of Sciences of the United States of America 72(5), pp. 1826-1828 (1975).

[0010] It should be noted that in addition to imaging, charged particle microscopes (CPMs) may also have other functions, such as performing spectroscopy, examining diffraction patterns, and performing (targeted) surface modifications (e.g., milling, etching, deposition).

[0011] While various forms of scanning microscopes have been known for decades, they share a common drawback that has begun to manifest as a bottleneck in many scientific and technological fields. This drawback relates to the fact that scanning imaging is often a relatively slow and cumbersome process, and has therefore traditionally been limited to studying very small (partial) samples, such as tens of nanometers typically in CPM and tens of micrometers in confocal microscopy. However, in many fields to which humanity is dedicated, there is a growing need for these techniques to expand their imaging area by several orders of magnitude while maintaining the resolution they provide. For example, in cell biology, there is a need to extend cellular-level imaging to the tissue level, for instance, to study more clearly the interactions between (different types of) cells, particularly in brain tissue, muscle tissue, tumors (cancer tissue), etc. In mineralogy / petrology, there is a need to extend molecular-level analysis to the grain and grain boundary levels, for example, to more clearly study gas trapping behavior in matrix materials during follow-up studies; precious metal encapsulation in quartz formations; and undesirable inclusions in synthetic gemstones and other crystals. In the semiconductor industry, for example, when conducting quality control on critical dimensions, coverage, contamination, etc., there is a desire to extend research from the individual node level to the broader scope of the entire device and device array.

[0012] However, extending current scanning microscopy techniques to such a large imaging scale would require a significantly increased image accumulation time, making such an extension practically unsustainable. Therefore, despite the immense demand and need, existing techniques are so impractical that they are excluded from real-world applicability in this respect.

[0013] Another problem with current scanning microscopy techniques becomes apparent when imaging radiation-sensitive samples, such as (live) biological samples, cryogenic samples, etc. Irradiating these samples with high-energy beams (especially charged particles) tends to cause damage (such as molecular rearrangement / mutation, thawing, drying, etc.) at or near the impact footprint of the irradiation beam. To mitigate this effect, reducing the intensity and / or increasing the scanning speed of the irradiation beam can be considered, but these measures usually result in an undesirable decrease in the signal-to-noise ratio (SNR).

[0014] These techniques are further described in EP 2 963 673 A1 and EP 2 958 131 A1, which were granted to the assignee of this invention. In recent years, the assignee has also extensively developed this method.

[0015] Another approach in microscopy involves targeted analysis, deposition, and material ablation using focused ion beams (FIB). This technique is particularly useful in the semiconductor industry, materials science, and increasingly in the biological field. An FIB setup is a scientific instrument similar to a scanning electron microscope (SEM). However, while SEM uses a focused electron beam to image samples in a compartment, an FIB setup instead uses a focused ion beam. FIB can also be incorporated into systems with both electron and ion beam columns, allowing the use of either beam to study the same characteristics.

[0016] The most common instruments use liquid metal ion sources (LMIS), especially gallium ion sources. Ion sources based on elements gold and iridium can also be used. In gallium LMIS, gallium metal comes into contact with a tungsten needle. The heated gallium wets the tungsten and flows towards the needle tip, where opposing forces of surface tension and electric field cause the gallium to form a sharp tip called a Taylor cone. The radius of this cone tip is extremely small (about 2 nm). The huge electric field at this small tip (greater than 1 × 10⁸ volts per centimeter) causes the ionization of gallium atoms and field emission.

[0017] The source ions are then typically accelerated to energies of 1–50 keV (kiloelectron volts) and focused onto the sample through an electrostatic lens. LMIS generates a high-current-density ion beam with very small energy diffusion. Modern FIBs can deliver tens of nanoamperes of current to the sample, or image the sample with spot sizes on the order of a few nanometers.

[0018] Focused ion beam (FIB) systems have been commercially available for about two decades, primarily for use by large semiconductor manufacturers. FIB systems operate similarly to scanning electron microscopes (SEMs), but as the name suggests, they use a precisely focused ion beam (typically gallium) instead of an electron beam. This beam can be used for imaging at low beam currents or for point sputtering or milling at high beam currents.

[0019] Gallium (Ga + The primary ion beam bombards the sample surface and sputters a small amount of material, which serves as secondary ions (i). + or i - ) or neutral atom (n 0 The primary beam also produces secondary electrons (electrons) that leave the surface. - When the primary beam creates a grating on the sample surface, signals from sputtered ions or secondary electrons are collected to form an image.

[0020] At low primary beam currents, very little material is sputtered, and modern FIB systems can easily achieve an imaging resolution of 5 nm (the imaging resolution of Ga ions is limited to about 5 nm by sputtering and detector efficiency). At higher primary currents, large amounts of material can be removed by sputtering, allowing samples to be precisely milled to the submicron or even nanometer scale.

[0021] If the sample is non-conductive, a low-energy electron correction gun can be used to provide charge neutralization. In this way, by using a primary positive ion beam to image with secondary positive ions, even highly insulating samples can be imaged and milled without a conductive surface coating, as required in SEM.

[0022] Until recently, FIB (Film Injection Biology) has been widely used in the semiconductor industry. Applications such as defect analysis, circuit modification, photomask repair, and point-to-point transmission electron microscopy (TEM) sample preparation on integrated circuits have become common procedures. Modern FIB systems possess high-resolution imaging capabilities; in many cases, this capability, combined with in-situ sectioning, eliminates the need to examine FIB section samples in a separate SEM instrument. Achieving the highest resolution imaging while preventing damage to sensitive samples still requires SEM imaging. However, combining SEM and FIB columns in the same compartment allows the utilization of the benefits of both.

[0023] Unlike electron microscopy, FIB is inherently destructive to the sample. When high-energy gallium ions bombard the sample, they sputter atoms from the surface. These gallium atoms are also implanted a few nanometers into the upper layer of the surface, making the surface amorphous.

[0024] Due to its sputtering capabilities, FIB can be used as a micro- and nano-machining tool for modifying or processing micro- and nano-scale materials. FIB micro-machining itself has become a widely used field, but nano-machining combined with FIB is an area still under development. Typically, the minimum beam size for imaging is 2.5–6 nm. The minimum milling feature size is slightly larger (10–15 nm) because it depends on the total beam size and the interaction with the sample being milled.

[0025] FIB tools are designed for etching or machining surfaces. Ideally, an FIB could remove an atomic layer without damaging the atoms in the next layer or leaving any damage on the surface. However, currently, due to sputtering, machining typically roughens the surface at the submicron scale. FIBs can also be used for depositing materials via ion beam-induced deposition. When gases, such as tungsten hexacarbonyl, are used... （ W(CO) 6）When introduced into a vacuum chamber and chemisorbed onto the sample, FIB-assisted chemical vapor deposition occurs. By scanning the region with the beam, the precursor gas is broken down into volatile and non-volatile components; the non-volatile components, such as tungsten, remain as deposits on the surface. This is useful because the deposited metal can act as a sacrificial layer to protect the sample below from destructive sputtering by the beam. With lengths ranging from nanometers to hundreds of micrometers, tungsten metal deposition allows for the placement of metal wires where needed. Other materials, such as platinum, cobalt, carbon, and gold, can also be deposited locally.

[0026] A drawback of FIB sample preparation is the surface damage and implantation mentioned above, which can be significantly affected when using techniques such as high-resolution "lattice imaging" TEM or electron energy loss spectroscopy. This damaged layer can be minimized by using FIB milling with lower beam voltages or lower ion energies, or by further milling with a low-pressure argon ion beam after the FIB process.

[0027] FIB preparation can be used with cryogenically frozen samples in appropriately equipped instruments, allowing for cross-sectional analysis of samples containing liquids or fats, such as biological samples, pharmaceuticals, foams, inks, and food.

[0028] FIB is also used for secondary ion mass spectrometry (SIMS). After sputtering the sample surface with a primary focused ion beam, the ejected secondary ions are collected and analyzed.

[0029] At lower beam currents, FIB imaging resolution begins to rival that of the more familiar scanning electron microscope (SEM) in terms of imaging morphology. However, FIB's two imaging modes, which use secondary electrons and secondary ions generated by the primary ion beam, offer many advantages over SEM.

[0030] FIB secondary electron imaging reveals strong contrasts in particle orientation. As a result, particle morphology can be easily imaged without the aid of chemical etching. Particle boundary contrast can also be enhanced by carefully selecting imaging parameters. FIB secondary ion imaging also reveals chemical differences and is particularly useful in corrosion studies, as the secondary ion yield of metals can increase by three orders of magnitude in the presence of oxygen, thus clearly revealing the presence of corrosion.

[0031] Another advantage of FFIB secondary electron imaging is that the ion beam does not alter the signal of the fluorescent probe used in protein labeling, thus creating the opportunity to correlate FIB secondary electron images with images obtained by fluorescence microscopy.

[0032] Another potential application area is mass spectrometry (MS). This is an analytical technique that ionizes chemical species and classifies ions based on their mass-to-charge ratio. Simply put, mass spectrometry measures the mass in a sample. Mass spectrometry is used in many different fields and can be applied to both pure samples and complex mixtures.

[0033] Mass spectrometry is a graph of ion signal intensity as a function of mass-to-charge ratio. These spectra are used to determine the elemental or isotopic characteristics of a sample, the mass of particles and molecules, and to elucidate the chemical structure of molecules such as peptides and other compounds.

[0034] In a typical MS procedure, a sample, which can be solid, liquid, or gas, is ionized, for example, by bombarding it with electrons. This may cause some sample molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and placing them in an electric or magnetic field: ions with the same mass-to-charge ratio will experience the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. The result is displayed as a spectrum of the relative abundance of the detected ions, as a function of mass-to-charge ratio. Atoms or molecules in the sample can be identified by correlating known masses with identified masses or by characteristic fragmentation patterns.

[0035] Mass spectrometry is a crucial method, particularly for protein characterization and sequencing. Two main methods for ionizing whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). To maintain the performance and mass range of available mass spectrometers, two methods are available for protein characterization. In the first method, the whole protein is ionized using one of the two techniques mentioned above and then introduced into the mass analyzer. This method is known as a "top-down" strategy for protein analysis. However, the top-down approach is primarily limited to low-throughput single-protein studies. In the second method, after electrophoretic separation, the protein is enzymatically digested into smaller peptides, solutions, or gels using proteases such as trypsin or pepsin. Other proteolytic agents are also used. This batch of peptide products is then introduced into the mass analyzer. When proteins are identified using characteristic patterns of peptides, this method is called peptide mass fingerprinting (PMF), and if identification is performed using sequence data determined in tandem MS analysis, it is called de novo peptide sequencing. These protein analysis procedures are also known as “bottom-up” methods. However, a third approach, an intermediate “middle-down” approach, is being introduced, which involves analyzing proteolytic peptides that are larger than typical trypsin peptides.

[0036] Another area of ​​interest is massively parallel sequencing (or massively parallel sequencing), which refers to any of the high-throughput DNA sequencing methods that utilize the concept of massively parallel processing; it is also known as next-generation sequencing (NGS) or second-generation sequencing. Some of these technologies emerged between 1994 and 1998 and have been commercially available since 2005. These technologies use miniaturized and parallel platforms, sequencing 1 million to 43 billion short reads (50-400 bases each) per instrument run.

[0037] Many NGS platforms differ in their engineering configuration and sequencing chemistry. They are examples of massively parallel sequencing technologies that share DNA templates from clonal amplification or single DNA molecules via spatial separation in flowing cells. This design is very different from Sanger sequencing (also known as capillary sequencing or first-generation sequencing), which is based on the electrophoretic separation of chain termination products generated in individual sequencing reactions.

[0038] Various techniques have been used, but each has its limitations. TEM can reveal structural details of proteins in their native environment, but the electron beam is very harmful to biological tissues, and the dose must be kept very low, which limits signal and resolution. Omics studies (such as genomics, proteomics, and metabolomics) are not spatially or temporally resolved. ChIP-Seq and CLIP-Seq procedures require cumbersome biochemical separation steps. In situ sequencing techniques, such as FISSEQ, cannot resolve dense regions, such as nucleoli or stress granules. Mass spectrometry proteomics can provide comprehensive quantitative analysis of intracellular protein composition. However, MS does not provide any inherent spatial localization and is limited by the specificity of the extraction methods used, such as fractionation or immunoprecipitation.

[0039] FIB milling of low-temperature samples is widely used, but Ga + The FIB is limited by a maximum beam current of typically 65 nA. Given the typical size of biological samples, Ga FIB milling is generally impractical. Furthermore, Ga contamination can negatively impact subsequent analyses. + The advantages of FIB milling are high compatibility (milling artifacts, such as curtains, are reduced and there is no Ga contamination) and high material removal rate for biological tissues, which enables new separation and purification techniques, such as partial biological deletion and / or organelle removal.

[0040] All previously cited patent and non-patent literature, as well as any citations contained therein, are incorporated herein by reference. Summary of the Invention

[0041] In view of the above, the objective of the present invention is to provide an improved and / or alternative method, apparatus and system for processing, in particular, biological samples in the form of freezing or cryopreservation.

[0042] The objective is achieved by means of the method, apparatus and system according to the invention.

[0043] This invention relates particularly to a method for processing at least one biological sample. This method is especially suitable for high-performance microscopy; proteomics analysis; sequencing, such as NGS, etc. According to the invention, the method includes the step of providing at least one biological sample in a frozen form. This can be in a vitreous or any other form suitable for milling and further analysis. Milling is performed by including O... + and / orXe + At least a portion of the sample is treated with a plasma ion beam containing at least one of the following plasmas. Alternatively, a plasma containing Ar can also be used. + N + Kr + Ne + He + and / or H + The ion beam.

[0044] This specifically includes any mixture of these plasmas or any other plasmas. When further referred to as O... + Or O + plasma or O + When using plasma beams, O is covered + O2 + and other species and containing O + O2 + Plasma or plasma beams of other O-type related species.

[0045] It also includes dual plasmas, such as PFIB.

[0046] When using Xe + When using a plasma beam or plasma jet, O2 gas can be added. This has been shown to be particularly advantageous in protecting samples, especially around the beam, to avoid any damage to biological samples.

[0047] Milling can be performed by including at least 10% O + Plasma ions, preferably at least 25%, more preferably at least 50%, more preferably at least 66%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, and more preferably almost or exactly 100%. + Plasma ion beams are used. These plasma ions can also provide advantageous sample protection when probes or samples are sputtered and thus subjected to physical and chemical stresses in other ways.

[0048] The method according to the invention may also include milling biological samples that are preferably cryopreserved or in a cryogenic form or in any relevant state.

[0049] It also enables a further step of milling at least a portion of a sample using at least one plasma ion beam with an effective beam current of at least 6 nA, preferably at least 10 nA, preferably at least 50 nA, more preferably at least 100 nA, more preferably at least 200 nA, even more preferably at least 300 nA, even more preferably at least 400 nA, even more preferably at least 500 nA, even more preferably at least 600 nA, even more preferably at least 650 nA, even more preferably at least 700 nA, even more preferably at least 800 nA, even more preferably at least 900 nA, even more preferably at least 1000 nA. This allows for considerably faster sample handling and milling than before. In any case, the sample is not damaged or suffers minimal damage.

[0050] Further steps of milling at least a portion of the sample can be achieved by a mass removal rate of at least 100 micrometers. 3 / second, preferably at least 200 micrometers 3 / second, more preferably at least 300 micrometers 3 / second, more preferably at least 500 micrometers 3 / second and more preferably at least 1000 micrometers 3 At least one plasma ion beam is applied per second. This is several times, or even ten times, or even tens of times faster than existing technologies, while still preserving biological samples. For example, 500 micrometers... 3 A milling rate of / second can be achieved with a beam current of 1 μA and a crushing efficiency of 0.5 micrometers. 3 / nanoku (μm) 3 The target material ( / nC) is used to obtain it. If a larger beam current of 2 μA is used, 1000 micrometers can be obtained. 3 Removal rate per second.

[0051] The present invention may include milling a sample to perform at least one of the following steps: removing unwanted portions of the sample; and extracting the target. If necessary, the milling may be controlled accordingly by several erosion steps or cyclic point-by-point or face-by-face erosion.

[0052] It also enables further steps to accumulate multiple targets in a cumulative sample. This can be applied to many different tasks such as image generation, proteomics, and genomics.

[0053] The processing can be submicron processing, and the method can further include the step of generating at least one microscopic image for visualizing and controlling one or both of the milling operations.

[0054] SEM and / or TEM microscopy can generate microscopic images. Other microscopic methods or combinations thereof can also be used.

[0055] Further steps in analyzing or accumulating samples can also be performed using mass spectrometry, preferably via a so-called orbitrap fusion mass spectrometer, which has shown particular suitability for analyzing these samples, especially for proteomics purposes. Therefore, the present invention can provide further steps for analyzing and / or determining proteins in samples.

[0056] Alternatively or additionally, further steps can be performed to sequence the target using next-generation sequencing (NGS).

[0057] Preferably, when analyzing proteins contained in a sample, the present invention can also be used to track the spatial information of the target and further for target analysis.

[0058] The present invention also relates to an apparatus for processing at least one biological sample, particularly for carrying out the aforementioned method, comprising at least one support for at least one biological sample in frozen form; and at least one plasma ion beam generator configured to generate at least one component containing O + or Xe + A plasma ion beam containing at least one of the following plasmas. Alternatively, a plasma ion beam containing Ar may also be used. + N + Kr + Ne + He + and / or H + The ion beam. Any other features described above and below and claimed in connection with the method are also included in the apparatus category of this invention.

[0059] The present invention also includes a dual plasma ion generator.

[0060] The plasma ion beam generator of the device can be configured to generate a plasma ion beam containing at least 10% O. + Plasma ions, preferably at least 25%, more preferably at least 50%, more preferably at least 66%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, and more preferably almost or exactly 100%. + Plasma ion beams.

[0061] The apparatus can also be configured to achieve the effective beam current and / or mass removal rate mentioned above and claimed in the context of the method according to the invention.

[0062] The apparatus may also include a microscope, preferably a SEM or TEM microscope, for generating microscopic images of at least one of the milling operations for visualization and control.

[0063] The present invention also relates to a system or component comprising the apparatus described above and below, and at least one of the following: a microscope, preferably a SEM or TEM microscope; a mass spectrometer, preferably an orbital trap fusion mass spectrometer; and a sequencing station, preferably an NGS station. Such a system enables new and advantageous workflows for proteomics analysis and sequencing.

[0064] The present invention also covers the use of the methods described above and claimed below, which are used for at least one of: proteomics analysis; genomics, such as gene sequencing; and high-performance microscopy, such as SEM or TEM.

[0065] The exact properties and quantity of the plasma components depend on many factors, including the RF power. In some cases, if there is evidence that a particular species is more effective when milling a sample, it is preferable to adjust the RF power to emphasize that species (preferably the one produced) rather than another. Higher RF power tends to produce more O. + Lower RF power tends to generate more O2. + Therefore, adjustments can be made accordingly.

[0066] This invention preferably uses fibrillation-based cellular microscopy (FIB) milling of cryopreserved cells to isolate the target or region of interest (ROI) for localization of proteomics data, thereby obtaining a more comprehensive picture of cellular protein composition. In contrast, using standard techniques, proteins are not localized and cannot be so easily located at specific sites within the cell.

[0067] Laser dissection methods exist for isolating certain regions, but the samples must remain either viable (discarding time-related factors) or chemically fixed, which can affect proteomics results and reduce certain signals. The present invention preferably avoids this situation.

[0068] This invention enables sensitive protein analysis in biological samples with high temporal and spatial resolution. Currently, various techniques are used for protein analysis in developmental biology research, but each technique has limitations in certain aspects. This invention can use Xe... + Or even more so O + Plasma FIB (and other ion species, such as Ar) + N + Kr + Ne + He + and / or H +It provides extremely high removal rates for cryopreserved biological samples and sensitive protein detection with the help of the assignee's mass spectrometer.

[0069] This invention also includes methods or workflows for separating and concentrating cryogenic biological samples for mass spectrometry protein analysis and gene sequencing analysis.

[0070] Cryogenic preparation is crucial because the catalytic cooling process immediately halts cellular activity, thus preserving the organism's state at a specific moment. In addition to this temporal resolution, high spatial resolution within the sample can be achieved using FIB milling techniques. Specifically, the assignee's recent work demonstrates that O + Plasma-based FIB is highly effective in removing large amounts of material from organic substrates, including cryogenic biological tissues.

[0071] One of the two possible embodiments uniquely utilizes O in PFIB + High milling speed: First, O + Milling can be used to remove specific regions of a sample, resulting in subsequent protein analysis of the remaining regions not being contaminated or diluted by the overall protein signature of the sample. Protein distribution can vary between different regions within a cell or organism, and this method of removing unwanted regions helps to focus on the true signal of interest, which is important for studies of organism development. Secondly, traditional segmentation and lifting techniques can be used to remove target regions within a sample. The process can be repeated on multiple identical samples as needed to obtain the necessary amount of material suitable for the detection limits of subsequent analytical tools.

[0072] Currently, the mass spectrometry instrument with the best sensitivity for proteomics (large-scale protein extraction and analysis) is the recipient's fusion mass spectrometer, therefore, combined with O + The workflow of plasma-enhanced immunosorbent assay (FIB) technology combined with the recipient's mass spectrometry technology will result in a uniquely powerful tool for biological sample analysis. The low-temperature preparation and in-situ specifications offered by FIB enable users to capture regions of interest with high spatial and temporal resolution, while the sensitivity of the mass spectrometer allows for the detection of subtle protein changes. Therefore, this invention could be a powerful new tool for biological research.

[0073] According to the present invention, frozen samples are subjected to freezing treatment, affected or scab formation, and may also be in the form of cryopreservation or low-temperature preservation.

[0074] O + Plasma beams enable practical sample preparation on these larger biological samples, providing value for future switchable ion species PFIBs.

[0075] This technique will now be discussed with reference to the accompanying drawings. Attached Figure Description

[0076] Figure 1 An embodiment of a workflow based on the prior art is described.

[0077] Figure 2 The schematic layout of the FIB milling station is shown.

[0078] Figure 3 The milling results according to the present invention are depicted.

[0079] Figure 4 The preparation of the meshes for the anterior and posterior parts of the embryo is shown respectively. Detailed Implementation

[0080] Figure 1 An embodiment of a typical workflow practiced in the art is schematically depicted. The biological sample 1 shown is intended for further analysis, the biological sample comprising one or more organisms, cells, structures, proteins, DNA, RNA, etc. To analyze the cells, cellular fractions, proteins, RNA, and / or DNA contained in the biological sample, sample 1 is exposed to one or more stages by one or more digestants or enzymes (represented by respective containers 2) to undergo their selected degradation and disintegration, etc. Fractions, molecules, proteins, etc., can be further separated in a high-performance liquid chromatography (HPLC) instrument 3, thereby allowing for better isolation of fractions of particular interest. They can then be transferred to a mass spectrometer 4 for further isolation and specification of fractions, molecules, proteins, etc., of interest. One drawback is that chemical and physical separations are rather ambiguous and do not localize these proteins, etc.

[0081] Furthermore, laser dissection is known to isolate the region of interest. However, samples should be kept viable, and time-related factors should be eliminated, or chemical fixation should be performed; otherwise, proteomics results may be affected and signal quality reduced.

[0082] Figure 2 This demonstrates an exemplary example of FIB sample preparation. For example... Figure 2 As shown, the GA + Gallium (Ga) generated by ion generator 5 +） A primary ion beam bombards the sample surface and sputters a small amount of material, which serves as secondary ions (i). + or I - ) or neutral atom (n 0 The primary beam also produces secondary electrons (electrons) that leave the surface. − When the primary beam creates a grating on the sample surface, signals from sputtered ions or secondary electrons are collected to form an image.

[0083] The primary ion beam according to the present invention now comprises O + and / or xenon+ As described above and claimed below, it may also contain Ar. + N + Kr + Ne + He + and / or H + .

[0084] At low primary beam currents, very little material is sputtered, and modern FIB systems can easily achieve an imaging resolution of 5 nm (the imaging resolution of Ga ions is limited to about 5 nm by sputtering and detector efficiency). At higher primary currents, large amounts of material can be removed by sputtering, allowing samples to be precisely milled to the submicron or even nanometer scale.

[0085] Gas-assisted etching and / or optional gas gun 6 can achieve the selected deposition. This can be applied according to the invention, especially when Xe + When used as a primary ion beam, O2 is advantageously added via gas gun 6.

[0086] If the sample is non-conductive, a low-energy electron correction gun 7 can be used to provide charge neutralization. In this way, by using a primary positive ion beam to image with secondary positive ions, even highly insulating samples can be imaged and milled without a conductive surface coating, as required in SEM.

[0087] according to Figure 3 According to the present invention, the biological sample 1 can be processed by selecting a target 11 and / or removing unwanted portions 12. As shown, this can be accomplished by milling the target 11 out of the unwanted portions 12 or by milling and sputtering away the unwanted portions 12 from the target 11. The latter involves the removal of a large amount of unwanted portions or volume.

[0088] Figure 4 Two graphs are displayed. The left graph illustrates the preparation of meshes for two different regions of the embryo (anterior (y-axis) and posterior (x-axis)) and demonstrates a preferred advantage of the invention: the additional use of spatial information. More specifically, Figure 4 This illustrates the spatial selection or distribution of targets in biological samples according to the present invention. In the present case, a grid has been developed for two distinct regions of the embryo (Drosophila embryo), the anterior and posterior. Further regions can also be tracked.

[0089] The samples were run on the assignee's orbital trap fusion mass spectrometer to identify a variety of proteins. As mentioned above, those can also be spatially distributed.

[0090] exist Figure 4The right side shows the distribution of protein expression targets relative to RNA expression targets.

[0091] When relative terms such as “about,” “substantially,” or “approximately” are used in this specification, such terms should also be interpreted as including exact terms. That is, for example, “substantially straight” should be interpreted as including “(perfectly) straight.”

[0092] Whenever steps are described in the foregoing or appended claims, it should be noted that the order in which the steps are described herein may be a preferred order, but may not necessarily be performed in the order described. That is, unless otherwise stated or unless it is clear to a person skilled in the art, the order in which the steps are described may not be mandatory. That is, when this document states, for example, that a method comprises steps (A) and (B), it does not necessarily mean that step (A) precedes step (B), but may also mean that steps (A) and (B) are performed simultaneously (at least partially) or that step (B) precedes step (A). Furthermore, when it is said that step (X) precedes another step (Z), this does not mean that there are no steps between steps (X) and (Z). That is, step (X) preceding step (Z) includes the case where step (X) is performed directly before step (Z), and the case where (X) is performed before one or more steps (Y1) up to step (Z). The corresponding considerations apply when terms such as "after" or "before" are used.

[0093] Comparison Examples Ga has already been used + Xe + and O + The testing involved selectively removing most of the cryopreserved fruit flies (fruit fly embryos) during sample preparation. The following has been observed.

[0094] Using Ga + When using a plasma ion beam, the available current is approximately 50 nA. The milling rate decreases as the dose accumulates on the sample. The milling time becomes unfavorable for the desired sample volume.

[0095] Using Xe + At that time, the increased current increased the milling rate by 10 times. However, even using O2 gas can increase the milling rate.

[0096] For the aforementioned application, use O + It showed the best results as a primary ion beam, achieving up to 4500 micrometers. 3 Milling rate per second (compared to Ga) +(45-fold enhancement). Another advantage is that the samples do not exhibit the ion beam damage that would occur with such high milling rates (potentially due to localized heating). This is even more pronounced with smaller samples or for samples that are unsupported / immersed in ice.

[0097] O + The plasma ion beam, with a voltage of 30 keV at a setting of 1 μA, measured an effective current of 680 nA. Compared to Ga... + The ion beam setup uses a current more than 13 times greater.

[0098] In the above use of O + In the example of the plasma ion beam, the beam underwent a CCS mode with a z-depth of 15 μm, a dwell time of 1 μs, 65% x-overlap, and 85% y-overlap. Therefore, half of the fruit fly embryos could be removed in approximately 7 minutes.

[0099] Therefore, according to the O of the present invention + Plasma ion beams can create workflows that allow for the preparation of multiple samples in a single period. Ultimately, this may require approximately 100 cells per sample, thus saving significant time compared to standard methods.

Claims

1. A method for processing at least one biological sample, comprising the following steps: Provide at least one biological sample in frozen form; and By including O + The plasma ion beam mills at least a portion of the sample.

2. The method according to claim 1, the method further comprising the step of milling at least a portion of the sample with at least one plasma ion beam having an effective beam current of at least 500 nA.

3. The method according to claim 1, the method having the further step of milling at least a portion of the sample with at least one plasma ion beam having an effective beam current of at least 1000 nA.

4. The method according to any one of the preceding claims, wherein the method has a mass removal rate of at least 100 micrometers. 3 / second, more preferably at least 500 micrometers 3 / second and more preferably at least 1,000 micrometers 3 A further step of milling at least a portion of the sample with at least one plasma ion beam per second.

5. The method according to any one of the preceding claims, the method comprising the step of milling the sample to perform at least one of the following: removing unwanted portions of the sample; and extracting a target.

6. The method according to the preceding claim, wherein the method has a further step of accumulating a plurality of said targets in an accumulated sample.

7. The method according to any one of the preceding claims, wherein the processing is a submicron process, and the method further comprises the step of generating at least one microscopic image for visualizing and controlling one of the milling operations, particularly wherein the microscopic image is generated by at least one of SEM and TEM microscopy.

8. The method according to any one of the preceding claims, the method having the further step of analyzing the sample or the accumulated sample, particularly proteins in the sample, by mass spectrometry, preferably by orbital trap fusion mass spectrometry.

9. The method according to any one of claims 5 to 8, wherein the method has a further step of sequencing one or more of the targets by next-generation sequencing (NGS).

10. The method according to any one of claims 5 to 9, wherein preferably, when analyzing the proteins contained in the sample, the spatial information of the target can also be tracked and the information can be further used in its analysis.

11. An apparatus for processing at least one biological sample, particularly for carrying out the method according to any one of the preceding claims, comprising: At least one support for at least one biological sample in frozen form; At least one plasma ion beam generator, the plasma ion beam generator being configured to generate at least one containing O + Plasma ion beam; and An air gun used to add O2.

12. The apparatus of claim 11, wherein the apparatus has a microscope for generating a microscopic image for at least one of the milling operations, preferably a SEM or TEM microscope.

13. A system comprising the apparatus of claim 11 or 12 and at least one of the following: a microscope, preferably a SEM or TEM microscope; a mass spectrometer, preferably an orbital trap fusion mass spectrometer; and a sequencing station, preferably an NGS station.

14. Use of the method according to any one of claims 1 to 10, wherein it is used for at least one of: proteomics analysis; genomics, such as gene sequencing; and high-performance microscopy, such as SEM or TEM.

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