Source-detector synchronization in multiplexed secondary ion mass spectrometry

Abstract

This disclosure primarily provides a method and system comprising the following steps: directing an ion beam to a region of a sample to release charged particles from the region of the sample, wherein the directed ion beam is pulsed at a first repetition rate; deflecting a first subset of the released charged particles from a first path to a second path different from the first path in response to a gate signal synchronized with the repetition rate of the pulsed ion beam; and detecting the first subset of the released charged particles in a time-of-flight (TOF) mass spectrometer to determine information about the sample, wherein the gate signal is a common reference time set for the TOF mass spectrometer for the first subset of charged particles released by each pulse of the ion beam.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Patent Application No. 62 / 636,220, filed February 28, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to scanning biological samples using ion beams and to determining mass spectrometry information of said samples based on ion beam exposure. Background of the Invention

[0005] Immunohistochemistry has been used to visualize protein expression in biological samples, such as tumor tissue biopsies. Such methods typically involve exposing the sample to an antibody that binds to a fluorescent group or an enzyme reporter that generates a colored pigment. Analysis of the spectral images of the tagged sample yields information that can be used to assess protein expression levels and co-expression events. Summary of the Invention

[0006] This disclosure features a multiplexed ion beam imaging method for analyzing protein expression and other biological events and structures in tissue samples. The sample is labeled with an antibody conjugated to a mass tag, such as a lanthanide element, and then exposed to a primary ion beam. The primary ions are incident on the sample, and secondary ions are generated based on the mass tag. Spatial-resolution and mass-resolution analysis of the secondary ions from the sample can provide information about protein expression and other biological events at specific sample locations.

[0007] The mass resolution of secondary ions can be achieved by measuring the time-of-flight of these ions. In one configuration for such measurements, the secondary ions traveling from the sample are redirected to a new, typically orthogonal direction, thus resetting the time-of-flight of the ions in that direction. This type of orthogonal configuration allows the process of generating secondary ions by the ion beam source to be separated from the measurement of the time-of-flight of these ions. Therefore, compared to a linear instrument configuration that directly measures the time-of-flight of secondary ions generated by the ion beam, the ion beam can generate more secondary ions in a shorter time. Measuring a large number of secondary ions leads to increased sensitivity of the device. Furthermore, more data about the sample can be collected in a shorter time.

[0008] The orthogonal configuration can be further improved by synchronizing the pulses of the ion beam with the duty cycle of the measuring device. Synchronization allows for the collection and measurement of a larger portion of the secondary ions generated from the sample.

[0009] Additional methods for targeting a specific range of quality labels are disclosed, wherein the additional methods utilize the orthogonal instrument configuration for measurement. Eliminating unwanted quality labels based on the measurements results in an increased signal-to-noise ratio, thereby further improving the sensitivity of measurements involving labels of interest.

[0010] Typically, in a first aspect, this disclosure is characterized by a method comprising the steps of: directing an ion beam to a region of a sample to release charged particles from the region of the sample, wherein the directed ion beam is pulsed at a first repetition rate; deflecting a first subset of the released charged particles from a first path to a second path different from the first path in response to a gate signal synchronized with the repetition rate of the pulsed ion beam; and detecting the first subset of the released charged particles in a time-of-flight (TOF) mass spectrometer to determine information about the sample, wherein the gate signal sets a common reference time for the TOF mass spectrometer for the first subset of charged particles released by each pulse of the ion beam.

[0011] Implementations of the method may include any one or more of the following features.

[0012] Based on the difference in the detection time of the first subset of charged particles relative to the common reference time, the TOF mass spectrometer can distinguish mass-to-charge ratio differences among the charged particles in the first subset. The gate signal can be synchronized with the repetition rate of the pulsed ion beam so that the first subset includes a substantial portion of at least one type of released charged particles propagating along the first path. The substantial portion may include greater than 50%, or preferably greater than 70%.

[0013] The pulse width of the directional ion beam can be between 5 nanoseconds and 100 microseconds (e.g., between 100 nanoseconds and 50 microseconds, or between 1 microsecond and 50 microseconds). The first repetition rate can be between 1 kHz and 200 kHz (e.g., between 10 kHz and 100 kHz). The deflection from the first path to the second path can be between 60 degrees and 120 degrees. The gate signal can include a voltage pulse applied to the ion optical electrode to cause the deflection. The pulse width of the voltage pulse can be between 500 nanoseconds and 50 microseconds.

[0014] The released charged particles can travel along the first path at different speeds depending on their mass, and the time delay between the voltage pulse and the pulsed ion beam can be set to select a range of charged particle masses from the released charged particles that will be in the first subset. The time delay between the voltage pulse and the pulsed ion beam can be set to select heavier masses among the charged particles in the first subset.

[0015] The method may include blocking a second subset of the released charged particles propagating along the first path from entering the chamber containing the TOF mass spectrometer. The blocking may include using adjustable mechanical stops and / or using adjustable electromagnetic fields generated by ion optical electrodes. The blocking of the second subset may occur at a time period different from the deflection and may also be synchronized with the repetition rate of the pulsed ion beam.

[0016] The released charged particles may include secondary element atomic ions derived from a mass tag associated with the sample. The pulsed ion beam can release the charged particles by ionizing the mass tag. The sample may be a flat sample comprising biological material on a conductive substrate.

[0017] The method may include scanning the pulsed ion beam relative to the sample to irradiate additional regions of the sample, and further includes performing the deflection and the detection on each of the additional regions of the sample.

[0018] Unless otherwise expressly stated, implementations of the method may also include any other features disclosed herein, including features disclosed in any combination of different implementations.

[0019] In another aspect, this disclosure is characterized by a mass spectrometry system comprising: a pulsed ion source configured to direct an ion beam to a region of a sample to release charged particles from the region of the sample, wherein the pulsed ion source generates pulses of the ion beam at a first repetition rate; an ion optics device controllable to adjustably deflect a first subset of the released charged particles from a first path to a second path different from the first path; an ion optics controller coupled to the pulsed ion source and configured to generate a gate signal synchronized with the repetition rate of the pulsed ion beam to cause the ion optics device to deflect the first subset from the first path to the second path; and a time-of-flight (TOF) mass spectrometer positioned to detect the first subset of the released charged particles to determine information about the sample, wherein the gate signal sets a common reference time for the TOF mass spectrometer for the first subset of charged particles released by each pulse of the ion beam.

[0020] The implementation of the system may include any one or more of the following features.

[0021] The TOF mass spectrometer can be configured to distinguish mass-to-charge ratio differences among the first subset of charged particles based on the difference between the detection time for the flight time of the first subset of charged particles and the common reference time.

[0022] The gate signal can be synchronized with the repetition rate of the pulsed ion beam so that the first subset includes a substantial portion of at least one type of the released charged particles propagating along the first path. The substantial portion may include greater than 50%, or preferably greater than 70%.

[0023] The pulse width of the directional ion beam can be between 5 nanoseconds and 100 microseconds (e.g., between 100 nanoseconds and 50 microseconds, or between 1 microsecond and 50 microseconds). The first repetition rate can be between 1 kHz and 200 kHz (e.g., between 10 kHz and 100 kHz). The deflection from the first path to the second path can be between 60 degrees and 120 degrees.

[0024] The gate signal may include a voltage pulse, and the ion optics controller may be configured to apply the gate signal to the ion optics electrode to induce the deflection. The pulse width of the voltage pulse may be between 500 nanoseconds and 50 microseconds.

[0025] The released charged particles can travel along the first path at different speeds depending on their mass, and the ion optical controller can be configured to set a time delay between the voltage pulse and the pulsed ion beam to select a range of charged particle masses from the released charged particles that will be within the first subset. For example, the ion optical controller can be configured to set the delay between the voltage pulse and the pulsed ion beam to select heavier masses among the charged particles in the first subset.

[0026] The system may include a blocking member positioned to prevent a second subset of the released charged particles propagating along the first path from entering the chamber containing the TOF mass spectrometer. The blocking member may include an adjustable mechanical stop. The ion optics controller may be configured to generate one or more electromagnetic fields using ion optical electrodes to directionally direct the second subset of the released charged particles toward the blocking member. The blocking of the second subset may occur at a time period different from the deflection and may also be synchronized with the repetition rate of the pulsed ion beam.

[0027] The released charged particles may include secondary element atomic ions derived from a mass tag associated with the sample. The pulsed ion beam can release the charged particles by ionizing the mass tag. The sample may be a flat sample comprising biological material on a conductive substrate.

[0028] The ion optics controller can be configured to generate one or more control signals that cause the pulsed ion source to scan the pulsed ion beam relative to the sample to irradiate additional regions of the sample, and to generate one or more control signals that cause the ion optics device to adjustably deflect a first subset of released charged particles corresponding to each additional region from a first path to a second path different from the first path. The TOF mass spectrometer can be configured to detect the first subset of released charged particles corresponding to each additional region.

[0029] Unless otherwise expressly stated, implementations of the system may also include any other features disclosed herein, including features disclosed in any combination of different implementations.

[0030] Details of one or more embodiments of the subject matter of this specification are set forth in the following figures and description. Other features, aspects, and advantages of the subject matter will become more apparent from the description, figures, and claims. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating an example system for multiplexed ion beam imaging.

[0032] Figure 2 This is a schematic diagram of the controller and auxiliary devices.

[0033] Figures 3A to 3E This is a schematic diagram showing various exposure patterns of a single ion beam on a sample.

[0034] Figure 4 This is a schematic diagram showing an example of a portion of an ion beam optics device. Figure 5 This is a schematic cross-sectional view of a sample marked with a quality label.

[0035] Figure 6 It is a schematic cross-sectional view of a tissue section sample placed on a substrate.

[0036] Figure 7 It is a schematic cross-sectional view of a sample having multiple cells placed on a substrate.

[0037] Figure 8 This is a schematic diagram showing an example of a portion of an ion optics device.

[0038] Figure 9 This is a schematic diagram showing an example of an ion trap.

[0039] Figure 10 This is a schematic diagram showing another example of an ion trap.

[0040] Figure 11 This is a schematic diagram illustrating an example of a linear time-of-flight detector.

[0041] Figure 12 This is a schematic diagram showing an example timing diagram for a linear time-of-flight detector.

[0042] Figure 13 This is a schematic diagram illustrating an example of an orthogonal time-of-flight detector.

[0043] Figure 14 This is a schematic diagram showing an example timing diagram for an orthogonal time-of-flight detector.

[0044] Figure 15 This is a schematic diagram showing an example of an electrostatic tap valve.

[0045] In each of the figures, the same reference numerals and names indicate the same elements. Detailed Implementation

[0046] A. General Overview – Multiplexed Ion Beam Imaging System

[0047] Multipath visualization of protein expression, along with other biochemical groups and structures, allows researchers to identify important associations between biological functional events. As part of diagnostic testing, visualization of protein expression can be used to assess malignancies in resected tissue samples and, in particular, to provide crucial information about the development of signaling pathways and related structures within tumor tissue.

[0048] This disclosure features a system and method for multi-channel visualization of antigens and other biochemical structures and groups in biological samples using secondary ion mass spectrometry. Structure-specific antibodies are conjugated to specific mass tags, typically in the form of metallic elements (e.g., lanthanides). When a sample is exposed to the conjugated antibody-mass tag, the tag binds to the corresponding antigen. Exposing the labeled sample to a primary ion beam releases secondary ions corresponding to the conjugated mass tag from the labeled sample. Performing spatially resolved detection on the secondary ions generated from the sample allows direct visualization of the localization of specific antigens in the sample and extraction of quantitative information (e.g., antigen concentration) as a function of spatial location. This information can be combined with other structural information (e.g., information about tumor margins, cell type / morphology) to develop a detailed assessment of tumor viability and progression in the sample.

[0049] Figure 1 This is a schematic diagram illustrating an example system 100 for multiplexed ion beam imaging. System 100 includes an ion beam source 102, ion beam optics 104, a stage 106, a voltage source 108, an ion collecting optics 110, and a detection device 112. Each of these components is connected to a controller 114 via signal lines 120a to 120h. During operation of system 100, controller 114 can adjust the operating parameters of each of the following: ion beam source 102, ion beam optics 104, stage 106, voltage source 108, ion collecting optics 110, pressure regulation system 130, and detection device 112. Furthermore, controller 114 can exchange information with each of the aforementioned components of system 100 via signal lines 120a to 120h.

[0050] During operation, ion beam source 102 generates an ion beam 116 comprising a plurality of primary ions 116a. The ion beam 116 is incident on a sample 150 located on a stage 106 inside a sample chamber 126. Optionally, in some embodiments, voltage source 108 applies a potential to a substrate 152 supporting the sample 150. The primary ions 116a in the ion beam 116 interact with the sample 150, thereby generating secondary ions 118a as a secondary ion beam 118. The secondary ions 118a are attracted by extraction electrode 132 and focused into an ion optics device 110. The secondary ion beam 118 is collected by ion collecting optics 110 and directed to a detection device 112. The detection device 112 measures one or more ion counts corresponding to the secondary ions 118a in the secondary ion beam 118 and generates an electrical signal corresponding to the measured ion count. The controller 114 receives the measured electrical signal from the detection device 112 and analyzes the electrical signal to determine information about the secondary ion 118a and the sample 150.

[0051] The controller 114 can adjust a variety of different operating parameters of the various components of the system 100, and can transmit information (e.g., control signals) and receive information (e.g., electrical signals corresponding to measurement and / or status information) from the components of the system 100. For example, in some embodiments, the controller 114 can activate the ion beam source 102 and can adjust the operating parameters of the ion beam source 102, such as the ion current of the ion beam 116, the waist of the ion beam 116, and the propagation direction of the ion beam 116 relative to the central axis 122 of the ion beam source 102. Typically, the controller 114 adjusts the operating parameters of the ion beam source 102 by transmitting appropriate control signals to the ion beam source 102 via signal line 120a. Furthermore, the controller 114 can receive information from the ion beam source 102 via signal line 120a (including information about the ion current of the ion beam 116, the waist of the ion beam 116, the propagation direction of the ion beam 116, and various potentials applied to the components of the ion beam source 102).

[0052] Various primary ion beams 116 generated by ion source 102 can be used to expose sample 150. For example, in some embodiments, the primary ion beam 116 consists of multiple oxygen ions. In some embodiments, the primary ion beam 116 includes one or more of the following: gallium ions, helium ions, cesium ions, neon ions, krypton ions, xenon ions, and / or argon ions.

[0053] For example, in some embodiments, ion source 102 may be implemented as an oxygen dual-plasma tube source that generates a primary ion beam 116 (e.g., a Direct Extraction Negative Ion Duoplasmatron available from National Electrostatics Corp., Middleton, Wisconsin). Alternatively or additionally, ion source 102 may be implemented as a Cs liquid metal ion gun, as described, for example, by Umemura et al. in Rev. Sci. Instrum. 65, 2276 (1994), available at https: / / doi.org / 10.1063 / 1.1144676, the entire contents of which are incorporated herein by reference.

[0054] Ion beam optics 104 typically includes various elements that use electric and / or magnetic fields to control the properties of the ion beam 116. For example, in some embodiments, ion beam optics 104 includes one or more beam focusing elements that adjust the spot size of the ion beam 116 at an incident position 124 on the sample 150. In some embodiments, ion beam optics 104 includes one or more beam deflection elements that deflect the ion beam 116 relative to an axis 122, thereby adjusting the incident position 124 of the ion beam 116 on the sample. Ion beam optics 104 may also include various other elements, including one or more apertures, extraction electrodes, beam blocking elements, and other elements that assist in guiding the ion beam 116 onto the sample 150.

[0055] The controller 114 can typically adjust the characteristics of any of the aforementioned elements via a suitable control signal transmitted via signal line 120b. For example, the controller 114 can adjust the focusing characteristics of one or more beam focusing elements of the ion beam optics 104 by adjusting the potential applied to the beam focusing element via signal line 120b. Similarly, the controller 114 can adjust the propagation direction of the ion beam 116 (and the incident position 124 of the ion beam 116 on the sample 150) by adjusting the potential applied to the beam deflection element via signal line 120b. Furthermore, via a suitable control signal transmitted on signal line 120b, the controller 114 can adjust the position of one or more apertures and / or beam blocking elements in the ion beam optics 104, and adjust the potential applied to the extraction electrodes in the ion beam optics 104. In addition to adjusting the characteristics of the ion beam optics 104, the controller 114 can also receive information from various components of the ion beam optics 104, including information about the potential applied to the components of the ion beam optics 104 and / or information about the position of the components of the ion beam optics 104.

[0056] Stage 106 includes a surface for supporting sample 150 (and substrate 152). Typically, stage 106 can be translated in each of the x, y, and z coordinate directions. Controller 114 can translate stage 106 in one of these directions by transmitting a control signal on signal line 120d. To achieve translation of the incident position 124 of the ion beam 116 on sample 150, controller 114 can perform the following operations: adjusting one or more potentials applied to the deflection element of ion beam optics 104 (e.g., to deflect the ion beam 116 relative to axis 122), adjusting the position of stage 106 via the control signal transmitted on signal line 120d, and / or adjusting both the deflection element of ion beam optics 104 and the position of stage 106. Furthermore, controller 114 receives information about the position of stage 106 transmitted along signal line 120d.

[0057] In some embodiments, system 100 includes a voltage source 108 connected to substrate 152 via electrodes 108a and 108b. When activated by controller 114 (via an appropriate control signal transmitted on signal line 120c), voltage source 108 applies a potential to substrate 152. The applied potential assists in capturing a secondary ion beam 118 from sample 150 because the potential repels secondary ions 118a, causing the secondary ions to exit sample 150 along the direction of ion collecting optics 110.

[0058] like Figure 1 As shown, sample 150 is typically a relatively flat sample extending in the x-coordinate direction and / or y-coordinate direction and having a measured thickness in the z-coordinate direction. The support surface of stage 106 also extends in the x-coordinate direction and y-coordinate direction.

[0059] In some embodiments, system 100 includes an extraction electrode 132. A controller 114 can activate the extraction electrode 132 by applying a suitable potential (e.g., via signal line 120g). The applied potential (which represents a potential difference relative to the potential applied to substrate 152) generates an electric field that accelerates secondary ions 118a away from the sample and toward the extraction electrode 132.

[0060] Via signal line 120e, controller 114 applies a potential to one or more elements of ion-collecting optics 110 to create a potential difference between extraction electrode 132 and the elements of ion-collecting optics 110. This potential difference accelerates secondary ions 118a collected by extraction electrode 132 to a predetermined energy, thereby efficiently transporting secondary ions 118a from extraction electrode 132 to ion-collecting optics 110. As a result, a secondary ion beam 118 composed of multiple secondary ions 118a is captured by ion-collecting optics 110.

[0061] Typically, the ion collecting optics 110 may include various electric field generating elements and magnetic field generating elements for deflecting and focusing the secondary ion beam 118. Furthermore, the ion collecting optics 110 may include one or more apertures, beam blocking elements, and electrodes. As discussed above in conjunction with the ion beam optics 104, the controller 114 can adjust the potential applied to each component of the ion collecting optics 110 via a suitable control signal transmitted on signal line 120e. By transmitting the control signal on signal line 120e, the controller 114 can also adjust the position of the apertures, beam blocking elements, and other movable components of the ion collecting optics 110. Furthermore, the controller 114 can receive information on signal line 120e regarding operating parameters (e.g., voltage, position) of various components of the ion collecting optics 110.

[0062] Ion collecting optics 110 guides a secondary ion beam 118 into a detection device 112. The detection device 112 measures the number or current of ions corresponding to various types of secondary ions 118a in the secondary ion beam 118 and generates an output signal containing information about the measured number or current of ions. By transmitting appropriate control signals on signal line 120f, a controller 114 can adjust various operating parameters of the detection device 112, including a maximum and minimum ion count detection threshold, signal integration time, the range of mass-charge (m / z) values ​​on which ion counts are measured, the dynamic range of the counting technique on which it is measured, and the potential energy applied to various components of the detection device 112.

[0063] The controller 114 receives an output signal from the detection device on signal line 120f, the output signal including information about the measured ion count or current. Furthermore, the controller 114 also receives information via signal line 120f about the operating parameters of the various components of the detection device 112, including the various operating parameter values ​​discussed above.

[0064] The detection device 112 may include various components for measuring the ion count / current corresponding to the secondary ion beam 118. For example, in some embodiments, the detection device 112 may correspond to a time-of-flight (TOF) detector. In some embodiments, the detection device 112 may include one or more ion detectors, such as a Faraday cup, which generate electrical signals when ions are incident on their active surface. In some embodiments, the detection device 112 may be implemented as a multiplier detector, in which incident ions enter an electron multiplier in which they generate corresponding electron bursts. The electron bursts may be detected directly as electrical signals, or they may be incident on a transducer that generates photons (i.e., optical signals) in response to the incident electrons. The photons are detected using a photodetector that generates an output electrical signal.

[0065] In some implementations, a pressure regulation system 130 is used to maintain the sample chamber 126, the ion optics 110, and the detection device 112 under reduced pressure. For example, the pressure regulation system 130 may maintain 1.0 x 10⁻⁶ pressure in one or more components of the system. -4 Torr or less (e.g., 1.0 x 10⁻⁶) -5 Toe or smaller, 1.0 x 10 -6 Toe or smaller, 1.0 x 10 -7 Toe or smaller, 1.0 x 10 -8 Toe or smaller, 1.0x10 -10 (or smaller), the one or more components include an ion source 102, an ion beam optics 104, a sample chamber 126, an ion optics 110, and a detection device 112.

[0066] The pressure regulation system 130 can maintain equal pressure in two or more different components, or even all components, of the system. Alternatively, the pressure in some components can be different. For example, in some embodiments, the pressure regulation system 130 can individually regulate the pressure of each of the following: ion source 102, ion beam optics 104, sample chamber 126, ion optics 110, and detection device 112, such that the gas pressure in each component can independently fall within any of the above ranges. Depending on the nature of the sample, the signal being measured, and the different operating conditions within the system, the gas pressure in any of these components can be different or the same.

[0067] The pressure regulation system 130 transmits the pressure of these components to the controller 114 via line 120h. Based on this information, the controller 114 can adjust the operating parameters of the system 130 via line 120h to achieve and maintain reduced pressure in these separate components.

[0068] Operating under reduced pressure can help reduce contamination and / or signal noise in system 100. For example, reducing pressure can reduce the number of unwanted particles detected in the system. Detecting such particles can lead to increased background noise and potential saturation of the detector.

[0069] The decompression operation can also reduce the number of unwanted particles in the system, which can react or collide with secondary ions generated from sample 150 traveling to detection device 112. In addition to modifying the energy distribution of the ions of interest, collisions between secondary ions and such unwanted particles can cause the secondary ions to prematurely disintegrate via unwanted disintegration pathways, resulting in a decrease in the peak signal intensity of the secondary ion molecular ions.

[0070] Furthermore, in some embodiments, maintaining a reduced pressure in the system during operation can facilitate ion transport within the system. For example, by maintaining different portions of the system at different reduced pressures during operation, a pressure gradient can be established between the different portions of the system, and this pressure gradient can be used to facilitate the transport of secondary ions between the different portions of the system.

[0071] As discussed above, controller 114 is capable of: adjusting various operating parameters of system 100, receiving and monitoring the values ​​of said operating parameters, and receiving electrical signals containing information about secondary ions 118a (and other types) generated from sample 150. Controller 114 analyzes said electrical signals to extract information about secondary ions 118a and other types. Based on the extracted information, controller 114 can adjust the operating parameters of system 100 to improve system performance (e.g., m / z resolution, detection sensitivity) and to improve the accuracy and reproducibility of data measured by system 100 (e.g., ion counts). Controller 114 can also perform the following operations: display operations to provide the system user with an image of sample 150 showing the distribution of various mass tags within sample 150; and storage operations to store information related to said distribution in a non-volatile storage medium.

[0072] As discussed above, any steps and functions described herein can be performed by controller 114. Typically, controller 114 may include a single electronic processor, multiple electronic processors, one or more integrated circuits (e.g., application-specific integrated circuits), and any combination of the foregoing. Controller 114 executes software-based instructions and / or hardware-based instructions to perform the steps and functions discussed herein. Figure 2As shown, controller 114 may include processor 210 and data storage system (including memory 220 and / or storage elements (e.g., storage device 230)) interconnected via system bus 250. The controller may be connected to at least one input device and at least one output device such as display 240. Each set of software-based instructions, embodied as a software program stored on a tangible, non-transitory storage medium (e.g., optical storage medium such as CD-ROM or DVD, magnetic storage medium such as hard disk, or persistent solid-state storage medium) or device, may be implemented using a high-level programming language, an object-oriented programming language, assembly language, or machine language.

[0073] B. Sample exposure and secondary ion imaging

[0074] Multiplexed ion beam imaging (MIBI) is a surface-sensitive technique that allows the detection and localization of various chemical components on the surface of biological samples. For example, MIBI methods can be used to resolve single molecular targets (e.g., single RNA molecules, DNA molecules, proteins, or protein complexes) or to determine the amount of certain proteins, nucleic acids, or molecules in biological samples containing cells. The "multiplexing" in MIBI refers to the use of multiple markers to simultaneously or sequentially detect and measure multiple analytes in a single biological sample.

[0075] In visualizing protein expression, the MIBI method offers many advantages over conventional multiplexed immunohistochemical techniques. For example, conventional techniques rely on optical detection of fluorescence emission from samples labeled with fluorophores conjugated to multiple antibodies. These conjugated fluorophores specifically bind to the corresponding antigens in the sample, and imaging of the fluorescence emission from the sample is used to assess the spatial distribution of the fluorophores. For samples where the antigen concentration is relatively low, signal amplification (e.g., using multivalent enzyme-linked secondary antibodies) can be used to aid visualization. However, the use of signal amplification techniques can impair quantitative information (e.g., antigen concentration information) that might otherwise be extracted from the sample images.

[0076] Other constraints are also encountered in conventional multiplexed immunohistochemical visualization techniques. Optically detecting and separating the spectral features of multiple fluorophores is a complex problem, especially when the fluorescence spectra of these fluorophores show significant overlap. Without robust discrimination between the spectral features of the fluorophores, important expression-related information may not be detected. Furthermore, such techniques often rely on primary antibodies generated from different host species. These factors limit the applicability of conventional multiplexed immunohistochemical visualization techniques in predictive biomarker development and clinical diagnosis.

[0077] In contrast, the MIBI method can be used to simultaneously resolve the spatial distribution of a relatively large number of mass tags applied to a sample, while achieving image resolution comparable to high-magnification optical microscopy. Furthermore, images acquired via MIBI technology do not suffer from signal overlap issues and allow for highly precise quantification of antigen concentrations. They are unaffected by antibody incompatibility caused by host-target mismatch.

[0078] The method of labeling sample 150 with a suitable quality tag (e.g., an antibody-conjugated quality tag) will be discussed in subsequent chapters. After sample 150 has been labeled, in order to perform multiplexed ion beam imaging, a primary ion beam 116 is directed to multiple different incident positions 124 on sample 150. At each position 124, the primary ion beam 116 generates secondary ions 118a by ionizing molecular species from the sample surface. In particular, the secondary ion 118a corresponds to the portion of the quality tag at that position that is conjugated and bound to the antibody of sample 150.

[0079] Secondary ions 118a forming the secondary ion beam 118 are measured and analyzed to determine spatially resolved information about the biochemical structure of sample 150. For example, secondary ions 118a are transferred to a detection device 112, such as a mass spectrometer, where they are mass-analyzed and quantified using standard mass analysis techniques (e.g., time-of-flight, magnetic sector, quadrupole, ion trap, and combinations thereof). Using time-of-flight (“TOF”) technology, the secondary ions 118a can be separated by the time it takes for them to travel through the detection device 112, the time corresponding to the mass of the ion. In other words, measuring the time between the generation and detection of the secondary ions (time of flight) enables mass analysis of the generated secondary ions. Suitable methods for detecting and quantifying the second ion 118a will be discussed more extensively in later sections.

[0080] To obtain spatially resolved information from sample 150, a primary ion beam 116 is translated to multiple different incident positions 124 on sample 150. These multiple different incident positions form a two-dimensional exposure pattern of the primary ion beam 116 within the plane of sample 150 (i.e., within a plane parallel to the xy plane). Mass spectra collected from the sample at each location along the sample surface can be used to generate a two-dimensional (2D) spatially resolved image of the scanned portion of the sample. At each location, mass spectrometric information relating to secondary ions 118a detected at that location is determined. Accordingly, at each pixel in the resulting image, mass spectrometric information corresponding to different mass tags bound to the sample at the location corresponding to that pixel is obtained.

[0081] Typically, a wide variety of different exposure patterns can be used. For example, in some embodiments, the exposure pattern corresponds to a square or rectangular array of incident positions 124 on the sample 150 of the primary ion beam 116. Figure 3A This is a schematic diagram showing a square array of incident positions 124 of the primary ion beam 116 on the sample 150, thereby forming a square exposure pattern 300 on the sample 150. Each row of the exposure pattern 300 includes 10 different incident positions 124 of the primary ion beam 116 on the sample 150 spaced apart along the x-coordinate direction. Each column of the exposure pattern 300 includes 10 different incident positions 124 of the primary ion beam 116 on the sample 150 spaced apart along the y-coordinate direction. In total, the exposure pattern 300 includes 100 different incident positions 124 of the primary ion beam 116.

[0082] Typically, each row and each column of the exposure pattern 300 may include any number of different incident positions 124 of the primary ion beam 116 on the sample 150. For example, in some embodiments, each row and / or each column of the exposure pattern 300 includes 10 or more (e.g., 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more) different incident positions 124 of the primary ion beam 116.

[0083] To expose sample 150 to primary ion beam 116 according to the exposure pattern, the different incident positions 124 constituting the exposure pattern 300 can typically be accessed by primary ion beam 116 in any order. However, in some embodiments, the different incident positions 124 are accessed in a specific order. For example, the square exposure pattern 300 in FIG3 can be implemented such that the ion beam 116 is scanned once sequentially along each row of the exposure pattern. After each incident position 124 in a single row is accessed sequentially (e.g., by translating the primary ion beam 116 parallel to the x-coordinate direction), the primary ion beam 116 is translated parallel to the y-coordinate direction to the next row in the exposure pattern 300, and then each incident position 124 in the next row is accessed sequentially.

[0084] This example exposure sequence corresponds to a single grating scan pattern of ion beam 116 on sample 150. (As in...) Figure 3A As shown, the ion beam 116 visits positions 302a to 302j in sequence, then visits positions 304a to 304j in sequence, and so on, until the last row of positions 320a to 320j is visited in sequence.

[0085] Exposure pattern 300 includes a total of 100 different incident positions of primary ion beam 116 on sample 150. However, more generally, exposure pattern 300 may include any number of different incident positions of primary ion beam 116. For example, in some embodiments, exposure pattern 300 includes 25 or more (e.g., 50 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 5000 or more, 10000 or more, 20000 or more, 30000 or more, 50000 or more, 100000 or more, 200000 or more, 500000 or more) different incident positions of primary ion beam 116 on sample 150.

[0086] The maximum dimension of the exposure pattern 300 measured in the direction parallel to the x-coordinate is Lx, and the maximum dimension of the exposure pattern 300 measured in the direction parallel to the y-coordinate is Ly. Typically, Lx and Ly are selected as desired based on the spatial dimensions of the portion of the sample 150 to be analyzed. For example, in some embodiments, Lx and Ly may each be independently 25 micrometers or larger (e.g., 50 micrometers or larger, 100 micrometers or larger, 200 micrometers or larger, 300 micrometers or larger, 400 micrometers or larger, 500 micrometers or larger, 700 micrometers or larger, 1.0 mm or larger, 1.5 mm or larger, 2.0 mm or larger, 2.5 mm or larger, 3.0 mm or larger, 5.0 mm or larger).

[0087] Figure 3A The exposure pattern 300 in the image is a square pattern. However, more generally, the exposure pattern formed by the set of incident positions 124 of the primary ion beam 116 on the sample 150 does not have to be square or rectangular. Two-dimensional exposure patterns with various different shapes and intervals between the incident positions of the primary ion beam 116 can be implemented. For example, the array can be hexagonal or have an irregular (e.g., random or spatially varied) shape. Figure 3B This is a schematic diagram showing an exposure pattern 300 in which rows of an exposure pattern are spatially offset in the y-direction to form an offset array. Figure 3C This is a schematic diagram showing a radial exposure pattern 300 in which the various incident positions of one ion beam 116 are sequentially exposed along radial lines 322a to 322h.

[0088] Figure 3D It is shown in Figure 3A The diagram shows a helical exposure pattern 300 on a square array. The dark squares indicate the incident positions where the primary ion beam 116 is exposed sequentially along a helix starting at position 312e and ending at position 304d.

[0089] Figure 3E This is a schematic diagram showing a square exposure pattern 300 in which incident positions 302a to 310e overlap (e.g., at the darker shaded areas 330a and 330b). The degree of overlap between incident positions can typically be selected as desired, depending on the specific sample and measurement application.

[0090] Return to Figure 3A When a sample 150 is exposed to a primary ion beam 116 according to an exposure pattern 300, the exposure can be performed based on a single execution of the exposure pattern 300 or multiple executions of the exposure pattern 300. In other words, in some embodiments, the sample 150 is exposed to the primary ion beam 116 by directing the primary ion beam 116 to visit each position in the exposure pattern 300 once. In some embodiments, the sample 150 is exposed to the primary ion beam 116 by directing the primary ion beam 116 to visit each position in the exposure pattern 300 multiple times. Typically, for example, after the primary ion beam 116 has visited each position in the exposure pattern 300 once, the primary ion beam 116 follows a second exposure sequence in which the beam visits the position in the exposure pattern 300 a second time. Subsequent exposure sequences can be implemented in which the primary ion beam 116 repeats the exposure sequence defined by the exposure pattern 300 as desired multiple times.

[0091] In some implementations, multiple exposures of the primary ion beam 116 may occur at each location in the exposure pattern 300 before the primary ion beam is moved to a new location within the exposure pattern 300. That is, at each location in the pattern, the sample may be exposed to the primary ion beam 116 two or more times (e.g., three or more, four or more, five or more, seven or more) before the primary ion beam 116 moves to the next location in the pattern. Exposing the sample to the primary ion beam 116 in this manner can have certain advantages. For example, in cases where it is desirable to expose the sample to the primary ion beam 116 multiple times, exposing the sample in this manner can reduce the total time required for sample scanning by reducing the number of times the primary ion beam 116 is moved.

[0092] Typically, the accuracy and reproducibility of the ion count / ion current measured by the detection device 112 depend on the number of secondary ions 118a generated by the interaction between the primary ion beam 116 and the sample 150. The number of secondary ions generated at each incident position 124 of the primary ion beam 116 is, in turn, a function of the total primary ion dose at each position. As the primary ion dose increases, all other factors remain constant, and the number of secondary ions generated also increases. As discussed above, the total primary ion dose at each incident position 124 can be delivered either via a single exposure of the primary ion beam 116 at each position or via multiple exposures of the primary ion beam 116 at each position (i.e., by repeating the exposure pattern 300).

[0093] In summary, as used in this article, the term "exposure pattern" (with examples from...) Figures 3A to 3E The exposure pattern 300 (illustrated in the diagram) refers to the set of incident spatial locations of the primary ion beam 116 on the sample 150, and the set of residence time (also known as exposure time), ion dose, ion beam current, and other exposure parameters associated with each spatial incident location of the primary ion beam 116 on the sample 150. In some embodiments, the controller 114 retains information corresponding to the exposure pattern in volatile and / or non-volatile memory units. During operation of the system 100, in response to the ion count / ion current measured by the detection device 112, and / or in response to adjusting performance-related metrics of the system 100 (such as signal resolution, signal-to-noise ratio, and data repeatability and / or accuracy), the controller 114 can change the exposure pattern by modifying the set of incident locations of the primary ion beam 116 associated with the exposure pattern, and / or by modifying any exposure parameters associated with the set of spatial locations.

[0094] The above discussion regarding the "exposure pattern" can also consider the cross-sectional shape of the ion beam at incident position 124, which corresponds to the spatial cross-sectional distribution of ions within the ion beam at incident position 124. Typically, many different cross-sectional shapes of the ion beam can be used, depending on the nature of the ion source 102 and the ion beam optics 104. For example, in some embodiments, the cross-sectional shape can be circular or elliptical, such that the ion distribution within the cross-sectional region of the beam is approximately uniform. The cross-sectional shape of the ion beam 116 on the sample surface can depend on the propagation direction of the ion beam relative to the sample surface. For example, in some embodiments, when the ion beam 116 is incident on the surface at a non-orthogonal angle, the cross-sectional shape of the ion beam 116 on the sample surface can be elliptical.

[0095] In some embodiments, the cross-sectional shape of the ion beam 116 on the sample surface can reflect the spatial variation distribution of ions within the ion beam. For example, the cross-sectional shape of the ion beam 116 can be Gaussian or more generally reflect another spatial ion distribution within the ion beam, in which the ion density is maximum at the beam center and decreases towards the edges of the beam. More complex shapes are also possible, where the ion density reaches local maxima at multiple locations within the cross-sectional region of the ion beam 116, thereby effectively forming a "multipolar" cross-sectional shape. Dipole, tetrapole, hexapole, and octole cross-sectional shapes can be formed via appropriate configuration of the ion source 102 and the ion beam optics 104.

[0096] In some embodiments, to control the incident position 124 of the primary ion beam 116 on the sample 150, the controller 114 translates the stage 106 in the x and y coordinate directions via control signals transmitted on the signal line 120d. When the primary ion beam 116 is guided to a static position, the movement of the stage 106 in the x and y coordinate directions affects the translation of the sample 150 relative to the primary ion beam 116 in the x and y coordinate directions, thereby moving the incident position 124 of the primary ion beam 116.

[0097] Alternatively or additionally, in some embodiments, controller 114 adjusts one or more elements of ion beam optics 104 to translate the position of primary ion beam 116 on sample 150. Figure 4 This is a schematic diagram showing an example of a portion of an ion beam optics device 104. The ion beam optics device 104 includes a housing 402 that surrounds various components, including focusing elements 404 and 406 (implemented as annular electrostatic lenses), a first pair of deflection electrodes (due to perspective in the figure, ...), and so on. Figure 4 Only one of the first pair of deflecting electrodes (electrode 408a) and the second pair of deflecting electrodes 410a and 410b are shown. The ion beam optics 104 also includes a beam blocking element 412.

[0098] Controller 114 is electrically connected via signal line 120b to focusing elements 404 and 406, and electrically connected to the first pair of deflection electrodes (in Figure 4 (As shown via the connection to electrode 408a) and electrically connected to the second pair of deflection electrodes 410a and 410b. By transmitting appropriate signals on signal line 120b, controller 114 regulates the potential applied to each element to which the controller is connected.

[0099] During operation of system 100, a primary ion beam 116 passes through a hole in housing 502 (in... Figure 4(Not shown) The beam enters the ion beam optics 104 and nominally propagates along the central axis 122 of the ion beam optics 104. By applying a suitable potential to the annular focusing elements 404 and 406, the controller 114 adjusts the focal position of the primary ion beam 116 along the axis 122.

[0100] By adjusting the potential applied to the first pair of deflection electrodes and the second pair of deflection electrodes via a control signal transmitted along signal line 120b, controller 114 can adjust the incident position 124 of the primary ion beam 116 on the sample 150. For example, by adjusting the potential applied to the first pair of deflection electrodes (electrode 408a and...) Figure 4 The primary ion beam 116 is deflected in a direction parallel to the x-coordinate direction by the potential of the second electrode (not shown). Therefore, in order to scan the primary ion beam 116 in a direction parallel to the x-coordinate direction in the exposure pattern, the controller 114 adjusts the potential applied to the first pair of deflecting electrodes.

[0101] Similarly, by adjusting the potential applied to the second pair of deflection electrodes 410a and 410b, the deflection component generated by the primary ion beam 116 is parallel to the y-coordinate direction. Therefore, in order to scan the primary ion beam 116 in the exposure pattern in a direction parallel to the y-coordinate direction, the controller 114 adjusts the potential applied to the second pair of deflection electrodes.

[0102] To prevent the primary ion beam 116 from incident on the sample 150, the controller 114 can adjust the potential applied to one or both pairs of deflection electrodes so that the primary ion beam 116 is intercepted by a beam-blocking element. For example, by applying a suitable potential to electrodes 410a and 410b, the primary ion beam 116 can be deflected such that the beam is blocked by the beam-blocking element 512 in the ion beam optics 104. The beam-blocking element can also be located outside the ion beam optics 104, and the potential applied to the deflection electrodes can be adjusted to redirect the primary ion beam 116 to incident on the external beam-blocking element.

[0103] After mass spectrometry information about the sample has been acquired through the following operations, the mass spectrometry information is used by controller 114 to form one or more images of the sample by scanning an ion beam 116 across the sample according to an exposure pattern and measuring secondary ions 118a generated from the sample. The images formed in this manner can be analyzed to identify the boundaries of individual cells and / or subcellular features within individual cells. Various methods, such as image segmentation, can be used to identify cell boundaries, as generally described, for example, in Ko et al., J. Digital Imaging 22:259-74 (2009), and Ong, Comput. Biol. Med. 26:269-79 (1996), the entire contents of which are incorporated herein by reference. Examples of computational techniques that can be used for image segmentation include, but are not limited to, thresholding techniques (as described, for example, by Korde et al. in Anal. Quant. Cytol. Histol. 31:83-89 (2009) and Tuominen et al. in Breast Cancer Res. 12, R56 (2010)), adaptive attention windows (as described, for example, by Ko et al. cited above), and gradient flow tracking (as described, for example, by Li et al. in J. Microscopy 231:47-58 (2008)). The entire contents of each of the foregoing references are incorporated herein by reference.

[0104] Once individual cells are identified, the measured secondary ion signals corresponding to each individual cell (or its subcellular features) can be further processed to determine information about the sample on a per-cell basis. For example, signals corresponding to individual cells can be integrated to generate quantitative information about each mass tag of interest within each cell. In this way, the abundance of different antigens, nucleic acids, and other biological entities of interest bound to the mass tags can be quantitatively determined within each cell.

[0105] Determining the number of each quality tag associated with each cell allows for the classification of cells in the sample. That is, individual cells and / or individual subcellular features can be assigned to one or more categories based on the type and / or number of quality tags associated with the cell / subcellular feature. Controller 114 can use this information to generate one or more output images, in which the categories of various cells and / or various subcellular features are displayed to a system user, for example, via output display 240.

[0106] For example, controller 114 can generate a pseudo-color image in which cellular and / or subcellular features are color-coded according to the type and number of quality tags bound to the cells. The color intensity of each pixel in the image can be correlated with the signal intensity obtained at the corresponding location on the sample. In other words, the color intensity in any single pixel of the cell image can be correlated with the amount of one or more specific quality tags bound to the sample at the corresponding sample location.

[0107] In some embodiments, MIBI technology can be used to selectively obtain information about the relatively thin outer layer of a sample, since secondary ions 118a are generated from the sample primarily from said thin outer layer. For example, in some embodiments, secondary ion generation (and therefore information determination) occurs at a sample thickness of 1.0 micrometer or thinner (e.g., 500 nm or thinner, 200 nm or thinner, 100 nm or thinner, 50 nm or thinner, 20 nm or thinner, 10 nm or thinner, 5 nm or thinner, 2 nm or thinner, 1 nm or thinner).

[0108] Furthermore, typically, the dose of primary ions exposed to the sample is relatively small, allowing organic compounds present in the sample to be ionized while maintaining their chemical structure, thus enabling the analysis of organic compounds (e.g., identification from mass spectrometry information) without undergoing significant degradation. Reducing sample degradation can reduce background noise in the resulting spectral data, which would otherwise exist due to stray signals from degradation fragments of organic compounds.

[0109] C. Sample preparation and quality labeling

[0110] MIBI relies on the release of secondary ions from a sample using a focused ion beam, the sample being specifically labeled with one or more different types of mass tags. “Labeling” refers to the process of attaching a detectable group (“tag”) to a structure of interest within a sample. Such structures are collectively referred to herein as “analytes,” and can include a variety of cellular components (e.g., cell wall, cytoplasm, nucleus, nuclear membrane, mitochondria) as well as biological structures and biochemical entities in biological samples (e.g., antigens, antibodies, proteins, peptides, nucleic acids, enzymes, enzyme receptors). By attaching tags to analytes, the presence of analytes in the sample can be determined and quantified by detecting the corresponding binding tags.

[0111] Detectable groups or labels typically include a quality tag and a binding agent. "Specific tagging" or "specific binding" refers to a binding agent that firmly, relatively exclusively, and preferentially binds to the analyte, compared to nonspecific binding where the binding agent attaches to many different parts of the sample (cellular components and / or biochemical structures and / or biochemical entities). For example, antigen-antibody binding is an example of specific binding. When the antigen (analyte) is present in the sample, the binding agent, including a complementary antibody, preferentially binds to the analyte antigen. Therefore, it is also preferable to attach a quality tag that binds to the antibody to the analyte antigen.

[0112] Specific binding can refer to an interaction in a sample that distinguishes between a desired (targeted) analyte and an undesired analyte (e.g., antigen 502), and in some embodiments, the interaction is greater than about 10 to 100 times or more (e.g., greater than about 1000 times or 10,000 times). In some embodiments, when the binding agent and the analyte specifically bind in the capture agent / analyte complex, the affinity between the binding agent and the analyte is characterized by: less than 10... -6 M of K D (dissociation constant), less than 10 -7 M of K D Less than 10 -8 M of K D Less than 10 -9 M of K D Less than 10 -11 M of K D Or less than about 10 -12 M or below K D .

[0113] Antigens and complementary, specifically binding antibodies will be discussed by example in this section. However, it should be understood that the systems and methods disclosed herein are not limited to attaching quality tags via antigen-antibody binding. Various other specific binding agents may also be used, including aptamers (that specifically bind to nucleic acids), chromogenic stains, and / or other chemical agents.

[0114] For example, in some embodiments, the staining agent may be one or more of the following: phalloidin, gadodiamine, acridine orange, Bismarck brown, carmine, Coomassie blue, tar violet, crystal violet, DAPI, hematoxylin, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hearst stain, iodine, malachite green, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetraoxide, rhodamine, safranin, phosphotungstic acid, osmium tetroxide, ruthenium tetroxide, ammonium molybdate, cadmium iodide, carbazide, ferric chloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid, potassium ferrocyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate, thallium nitrate, thioaminourea, uranyl acetate, uranyl nitrate, vanadium oxysulfate, or any derivative thereof. Stains can be specific to any feature of interest, such as proteins or protein classes, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, organelles (e.g., cell membrane, mitochondria, endoplasmic reticulum, pectin, nuclear envelope, etc.), and cell compartments (e.g., cytosol, nuclear fraction, etc.). Stains can also be used to enhance the contrast or imaging of intracellular or extracellular structures.

[0115] In some implementations, the staining agent may be applicable to a live subject. The staining agent can be administered to the subject by any suitable means such as uptake, injection (e.g., into the bloodstream), or local application (e.g., during surgery). This staining agent may be specific to the tissue, biological structure (e.g., blood vessels, lesions), or cell type of interest. The staining agent can be bound to the cells of a subject involved in cellular processes such as glucose uptake. Examples of such staining agents include, but are not limited to, gadolinium, cisplatin, halogenated carbohydrates (e.g., fluorinated, chlorided, brominated, iodinated carbohydrates), etc. Other injectable staining agents used in imaging techniques (such as MRI, PET scans, CT scans, etc.) may be conjugated to the quality label and administered to a live subject if not inherently associated with the quality label. A sample can be obtained from the subject after administration for use in the methods described herein.

[0116] As discussed above, the mass tag binds to a binding reagent, and when the binding reagent binds to the analyte, the mass tag is attached to the analyte. As used herein, a "mass tag" is a detectable group that can be identified by its atomic mass and / or mass spectrometric profile.

[0117] Figure 5This is a schematic cross-sectional view of sample 150 on substrate 152. Sample 150 contains proteins expressing multiple different antigens 502. Antibodies 504 are labeled with mass tags 506 specifically for each antibody type. The tagged antibodies 504 specifically bind to the corresponding antigen 502 on the surface of sample 150. When the sample is exposed to a focused ion beam 116, the mass tags 506 are released from sample 150 as secondary ions. The tags 506 are collected and analyzed to identify and quantify the presence of antigens 502 corresponding to the released mass tags 506 in the scanned portion of sample 150.

[0118] Examples of targetable antigens include, but are not limited to, carcinoembryonic antigen (for recognizing adenocarcinoma), cytokeratin (for recognizing cancer, but also expressed in some sarcomas), CD15 and CD30 (for Hodgkin's disease), alpha-fetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD117 (for gastrointestinal stromal tumors), CD10 (for renal cell carcinoma and acute lymphoblastic leukemia), prostate-specific antigen (for prostate cancer), estrogen and progesterone (for tumor recognition), CD20 (for recognizing β-cell lymphoma), and CD3 (for recognizing T-cell lymphoma).

[0119] Examples of antibody 504 include, but are not limited to, κ and λ light chains and equivalents of α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ heavy chains, or other types. A full-length immunoglobulin “light chain” (approximately 25 kDa or approximately 214 amino acids) contains a variable region of approximately 110 amino acids at the NH2 terminus and a constant region of κ or λ at the COOR terminus. A full-length immunoglobulin “heavy chain” (approximately 50 kDa or approximately 446 amino acids) similarly contains a variable region (approximately 116 amino acids) and one of the aforementioned heavy chain constant regions, such as γ (approximately 330 amino acids). Antibody 504 may also typically include any isotype of antibody or immunoglobulin, antibody fragments that retain specific binding to antigens (including, but not limited to, Fab, Fv, scFv, and Fd fragments), chimeric antibodies, humanized antibodies, microantibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. The term "antibody" also encompasses Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to antigens and monoclonal antibodies. Antibodies can exist in a variety of other forms and as single chains, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional (i.e., bispecific) hybrid antibodies.

[0120] The mass of mass tag 506 can range from 21 atomic mass units to 238 atomic mass units. More than 100 non-biological stable isotopes of elements between 21 and 238 AMU can be simultaneously measured via MIBI without significant overlap. Examples of stable isotopes used in the mass tag include isotopes of transition metals, post-transition metals, halides, noble metals, or lanthanides, or any other element not commonly found in the analyzed sample. These can include, but are not limited to, high molecular weight members of: transition metals (e.g., Rh, Ir, Cd, Au), post-transition metals (e.g., Al, Ga, In, Tl), metalloids (e.g., Te, Bi), alkali metals, halogens, and actinides. Mass tag 506 can also consist of lower molecular weight transition elements (e.g., Al, W, and Hg) not commonly found in biological matrices. In some embodiments, the tag isotopes may contain non-lanthanides that can form stable metal chelating agent tags for the applications described herein.

[0121] As noted above, in some embodiments, mass label 506 may include one or more lanthanide elements. Lanthanide elements with atomic numbers between 58 and 71 are sometimes referred to as "rare earth metals". Examples of lanthanide elements that may be used in mass label 506 include lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0122] Lanthanides are particularly useful as mass markers for several reasons. These elements typically have very low natural abundances in living tissues, and therefore, the detection signal induced by ions of such elements can often be considered as a specific marker of the tissue, rather than as a result of the naturally occurring concentration of such elements. Furthermore, lanthanides have well-defined masses, and the measurement signal induced by ions of such elements can be easily identified compared to background noise contributions. Further, lanthanides do not undergo fragmentation, degradation, or other physical or chemical processes that would otherwise generate multiple ion signals and / or reduce the amplitude of the molecular ion peak. Still further, due to the absence of such degradation pathways, ions derived from lanthanides can be "collected" after generation, effectively increasing the integration time or signal averaging time during ion current measurements without signal loss due to collisional fragmentation and / or spontaneous fragmentation.

[0123] As discussed above, in some embodiments, the mass label 506 may include one or more noble metal atoms. Suitable examples of noble metals include, but are not limited to, palladium, silver, iridium, platinum, and gold.

[0124] In some embodiments, mass tag 506 may include a chelating ligand (e.g., a chelating polymer composed of repeating units of a metal chelating agent) that chelates one or more atoms of a single abiotic isotope (e.g., a lanthanide), such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The chelating ligand can be used (e.g., by binding a specific lanthanide) to selectively alter the mass of certain mass tags, thereby modulating the detection window (i.e., the range of mass-to-charge ratio (m / z) values ​​of said mass tags that can be detected). In this way, mass tags can be selectively detected within a detection window that is less “crowded” in the presence of contributions from other background species. Alternatively or additionally, signals from certain mass tags can be “compressed” into a specific detection window, resulting in a smaller range of scanned m / z ratios and consequently reducing the overall measurement time.

[0125] In some implementations, optical staining methods can be combined with mass tags to enable multiplexed measurements of features of interest within the sample. This combination of techniques allows for the differentiation of some regions of the sample via optical methods and other regions via mass spectrometry information. For example, in some implementations, a certain type of mass tag may be located in multiple different cells and / or cell compartments. Consequently, it is difficult to explicitly assign the secondary ion signals induced by the mass tag to a specific cell / compartment.

[0126] However, if the sample is also stained with one or more chromogenic and / or fluorescent staining agents that differentially label the sample, secondary ion signals can be assigned to specific cells / compartments based on the localization of the staining agents in the sample (as determined by optical measurements of the sample). In this way, signals induced by common types of secondary ions (e.g., common types of mass tags) can be localized to different regions of the sample. For example, consider a sample that has been mass-tagged and also stained with Her2 membrane staining and ER nuclear staining. Based on the co-localization of secondary ion signals induced by mass tags (e.g., lanthanide-based tags) and optical absorption / emission signals induced by Her2 and ER staining agents, the secondary ion signals are assigned to different cell compartments within the individual sample cells.

[0127] Alternatively, in some embodiments, spatially resolved secondary ion signals attributed to different mass tags can be used to distinguish regions of a sample co-localized with two or more chromogenic and / or fluorescent staining agents. For example, consider a sample stained with ER nuclear staining agent and either dsDNA or histone H3 staining agent (both co-localized with said ER staining agent in the sample). If the different regions of the sample where these co-localized staining agents are located are also distinctly labeled with mass tags, the secondary ion signals induced by said mass tags can be used to assign ER signals and dsDNA / histone H3 signals to said different regions.

[0128] In some implementations, additional structural groups can exist as intermediate structures between the mass tag and the specific binding agent. Such structural groups can serve as linking members that facilitate the attachment of the mass tag to the specific binding agent. Specifically, these structural groups can allow the use of general preparation protocols rather than specialized synthetic methods for each different type of adduct to prepare a wide range of different mass tag-specific binding agent adducts.

[0129] Typically, the mass tag 506 (MT) can bind or conjugate to an active group (R), which in turn binds or conjugates to a specific binding reagent (SBR) such as an antibody. The structure of this adduct can be represented as MT-R-SBR, where the mass tag and the active group form the intermediate compound RT. Further, in some embodiments, a spacer group (S) can be present between the active group and the mass tag, such that the structure of the adduct can be represented as MT-SR-SBR.

[0130] For example, in some embodiments, R can be a thiol-active maleimide or a halogen-containing, amine-active N-hydroxysuccinimide (NHS)-carbonate and / or a hydroxyl-active N,N-diisopropyl-2-cyanoethylphosphamide. Such active groups can react with other groups on the specific binding reagent (e.g., cysteine ​​or other residues of the antibody or the thiol group of the oligonucleotide).

[0131] In some embodiments, the MT may be, for example, a polymer of 10 to 500 units, wherein each unit of the polymer comprises a coordinating transition metal atom. Suitable examples of the active group R and quality-labeled MTs including DOTA- and DTPA-based polychetants containing a coordinating group are described in Biochim. Biophys. Acta 883:430-467 (1986) by Manabe et al. and in U.S. Patent Nos. 6,203,775 and 5,364,314, the entire contents of which are incorporated herein by reference.

[0132] Additional examples of quality labels MT and active groups R are described in U.S. Patent Application Publication No. 2008 / 0003316 and U.S. Patent Nos. 6,203,775, 7,267,994, 6,274,713, and 5,364,313, the entire contents of each of which are incorporated herein by reference. A method for manufacturing polymer-based quality labels is also described in Zhang et al., Agnew. Chem. Int. Ed. 46:3111-3114 (2007), the entire contents of which are incorporated herein by reference.

[0133] Various chelating agents / groups can also be used to coordinate and bind metal ions in mass tags. Examples of such chelating agents / groups include, but are not limited to, EDTA, EGTA, and heme. Typically, chelating agents / groups can bind metal ions carrying single, double, triple, and tetracharges. Methods for attaching such agents / groups to specific binding reagents are known in the art.

[0134] Examples of samples 150 that can be analyzed using MIBI include bulk tissue (e.g., tumor tissue removed during a biopsy or another type of tissue sample repaired via another invasive or non-invasive surgical procedure), cell populations (e.g., blood cells), or organelles within single cells extracted from human or animal patients. For example, in some embodiments, sample 150 corresponds to a formalin-fixed, paraffin-embedded tissue sample. Such samples can be prepared from cancerous tumors and other anatomical structures during histological examination of the biopsy tissue.

[0135] Biological structures and components that can be examined include cell walls, nuclei, cytoplasm, membranes, keratin, muscle fibers, collagen, bone, proteins, nucleic acids, and other types of cellular macromolecules (e.g., carbohydrates, lipids). MIBI can be used in a variety of ways, from highlighting muscle fibers or connective tissue in tissue samples to classifying different blood cells in cell samples.

[0136] Figure 6 This is a schematic cross-sectional view of sample 150 on substrate 152. Figure 6 An optional coating 154 is positioned between the substrate 152 and the sample 150. The coating 154 prevents the sample from becoming charged, which can lead to damage to the primary ion beam and, in some environments, disrupt the process of generating secondary ions from the sample. When present, the coating 154 can be electrically connected to a voltage source 108 via electrodes 108a and 108b, such as... Figure 1 As shown in the figure.

[0137] The substrate 152 can be implemented as a microscope slide or another flat support structure, and can be formed from a variety of materials including various types of glass, plastics, silicon, and metals. In one example, it is cut to 18mm. 2 A block of silicon wafers (available from Silicon Valley Microelectronics in Santa Clara, California) was used as substrate 152. The silicon wafers were rinsed twice with methanol and polished with a cotton swab applicator. The cleaned wafer substrates were then immersed in a 2% poly-1-lysine solution (available from Sigma-Aldrich in St. Louis, Missouri) for 10 minutes and baked at 30°C for 1 hour.

[0138] If present, coating 154 may be formed of one or more metallic elements and / or one or more non-metallic compounds having relatively high electrical conductivity. Examples of metallic elements used to form coating 154 include, but are not limited to, gold, tantalum, titanium, chromium, tin, and indium. In some embodiments, coating 154 may be implemented as a plurality of different claddings, each of which may be formed as a single layer of metallic element or a single layer of non-metallic compound having relatively high electrical conductivity.

[0139] As in Figure 6 As shown, sample 150 is generally flat and extends in the x-coordinate direction and / or y-coordinate direction, and has a measured thickness d in the z-coordinate direction. Depending on the method of preparing sample 150, the sample may have a generally constant thickness d over the entire plane of the sample parallel to the xy-coordinate plane. Alternatively, multiple real samples corresponding to the removed tissue may have a non-constant thickness d over the entire plane of the sample parallel to the xy-coordinate plane. Figure 6 In the figure, sample 150 (shown in cross-section) has a non-constant thickness d measured along the z-coordinate direction.

[0140] Typically, the thickness d of sample 150 depends on the method by which sample 150 is obtained and processed before being mounted onto substrate 152. For example, some samples are sliced ​​from a larger tissue block using a microtome and can have a relatively constant thickness. As another example, some samples are obtained directly via excision and can have a variable thickness. The thickness d of sample 150 can range from 500 nm to 500 μm (e.g., from 1 μm to 300 μm, from 5 μm to 200 μm, from 10 μm to 150 μm, from 25 μm to 100 μm).

[0141] In some embodiments, substrate 152 may also include one or more additional coating materials to promote adhesion between sample 150 and substrate 152. In the absence of coating 154, the one or more additional coating materials may be applied directly to substrate 150, forming a layer between sample 150 and substrate 152. In the presence of coating 154, for example, the one or more additional coating materials may be applied on top of coating 154, forming a layer between coating 154 and sample 150. Suitable additional coating materials for promoting adhesion of sample 150 include, but are not limited to, poly-1-lysine.

[0142] In some embodiments, sample 150 corresponds to an array of single cells on a substrate. The array may be naturally occurring and correspond to a regular, ordered arrangement of cells in a tissue sample. Alternatively, the cell array may be a product of sample preparation. That is, the sample can be prepared by manually or automatically placing individual cells onto substrate 152 (e.g., in a series of recesses or depressions formed in substrate 152) to form the cell array.

[0143] Figure 7 A schematic cross-sectional view of a cell sample 150 located on a substrate 152 is shown. The substrate 152 optionally includes one or more conformal cladding layers 154 as discussed above. Furthermore, the substrate 152 includes an array of recesses 156 corresponding to depressions formed in the surface of the substrate 152. Each recess 156 contains a portion 150a to 150c of the sample 150. Typically, although in Figure 7 The substrate 152 includes three recesses 156 containing three separate portions 150a to 150c of the sample 150, but more generally, the substrate 152 may include any number of recesses 156, and the sample 150 may be disposed in any one or more recesses 156.

[0144] The recesses 156 (and the portions of the sample 150 distributed within the recesses 156) can typically be arranged in various patterns in the substrate 152. For example, the recesses 156 can be formed in a linear (i.e., one-dimensional) array in the substrate 152. Alternatively, the recesses 156 can be distributed along one dimension of the plane of the substrate 152, with irregular spacing between some or all of the recesses.

[0145] As another example, the recesses 156 may be formed in a two-dimensional array in the substrate 152, with a regular spacing between adjacent recesses in both the x-coordinate direction and the y-coordinate direction in a plane parallel to the substrate 152. Alternatively, at least some of the recesses 156 may be irregularly spaced in one or both of the x-coordinate direction and the y-coordinate direction in a plane parallel to the substrate 152.

[0146] When the recesses 156 form a two-dimensional array in the substrate 152, the array can take various forms. In some embodiments, the array of recesses 156 can be a square array or a rectangular array. In some embodiments, the array can be a hexagonal array, a polar array with radial symmetry, or another type of array with geometric symmetry in the plane of the substrate 152.

[0147] As discussed above, each of the portions 150a to 150c of sample 150 may include one or more cells. During sample preparation, each portion 150a to 150c may be dispensed into or disposed in a corresponding recess 156 of substrate 152 to form sample 150. For example, each portion 150a to 150c of sample 150, which is a cell suspension in liquid culture medium, may be dispensed into a corresponding recess 156, and the liquid culture medium may then be removed (e.g., by washing or heating) to leave the cells in each recess 156.

[0148] Typically, for any sample type 150, to facilitate various biochemical structural analyses of the sample 150 (such as protein expression), the sample 150 can be tagged with one or more different types of mass tags. When the sample 150 is exposed to a primary ion beam 116, the mass tag is ionized and released from the sample 150. The ionized mass tag corresponds to secondary ions 118a and forms a secondary ion beam 118 emanating from the sample 150. Based on the incident position 124 of the ion beam 116 on the sample 150, the controller 114 analyzes the secondary ions 118a present in the secondary ion beam 118, which can generate rich information about the biochemical structure of the sample 150 at each incident position 124.

[0149] To apply quality tag 506 to sample 150, each quality tag can be conjugated to a specific antibody 504 that selectively binds to antigen receptor 502 in sample 150. For example, a solution of each antibody-conjugated quality tag can be prepared, and the sample 150 can then be labeled by exposing it to each quality tag solution. In some embodiments, sample 150 is exposed sequentially and / or in parallel to multiple quality tag solutions so that sample 150 can be labeled with multiple different quality tags.

[0150] Various methods can be used to prepare suitable mass-tagged solutions. For example, in some embodiments, the solution is prepared via a sequence of three steps: loading a polymer linker with a mass-tagged element (e.g., a lanthanide metal), antibody reduction, and conjugating the metal-loaded polymer to the antibody. For example, in the first step, a metal-chelated polymer is loaded with a specific metal element. To perform this step, the polymer can be suspended in buffer 1 (available from IONpath, Menlo Park, California), and the metal element of interest can be added. The mixture can then be incubated at 37°C for 45 minutes and subsequently transferred to a filter (e.g., a 3 kDa molecular weight cutoff (MWCO) available from MilliporeSigma, Burlington, Massachusetts). A rotary filter is used to retain the polymer and to remove unbound metal tags by washing the polymer twice with buffer 1.

[0151] A second step, which can be performed in parallel with the first step, is the preparation of an antibody to receive the polymer. The antibody can be transferred to a filter (e.g., a 50 kDa MWCO filter) that retains the antibody. The antibody is first filtered using a rotary filter and then washed twice with buffer 2 (available from IONpath in Menlo Park, California). Next, the antibody can be incubated at 37°C for 30 minutes with a reducing agent (such as 4 mM tris(2-carboxyethyl)phosphine (TCEP)) to partially reduce the antibody and expose thiol residues for conjugation to the maleimide-containing polymer. After incubation, the antibody can be washed twice with buffer 3 (available from IONpath in Menlo Park, California) using the same 50 kDa MWCO filter to remove TCEP and prevent further reduction of the antibody.

[0152] In the third step, the metal-tagged polymer and the reduced antibody can be conjugated and incubated at 37°C for 60 to 90 minutes to allow the polymer to conjugate with the antibody. After this incubation, the mixture can be washed three times with buffer 4 (available from IONpath, Menlo Park, California) using the same 50 kDa MWCO filter, which preserves the conjugated antibody and allows removal of unbound polymer. The concentration of the metal-tagged antibody can be determined by measuring the absorbance of the solution of the metal-tagged antibody at 280 nm in buffer 5 (available from IONpath, Menlo Park, California) in a ThermoFisher Scientific NanoDrop spectrophotometer (available from ThermoFisher Scientific, Waltham, Massachusetts), diluted to a concentration between 200 Tg / mL and 500 Tg / mL, and stored at 4°C.

[0153] In some implementations, in order to prepare a sample consisting of a cell array, such as Figure 7 As shown, cells in a suspension can be enhanced with surface marker antibodies, and the cells are incubated at room temperature for approximately 30 minutes. After incubation, the cells are washed twice with a mass tagging solution to label the cells. The cells can then be diluted in PBS to produce the desired cell concentration per unit volume (e.g., approximately 10⁻⁶). 7 Aliquots of labeled cells (cells / mL) were placed in recess 156 and allowed to adhere for approximately 20 minutes. The adhered cells were then gently rinsed with PBS, fixed in PBS containing 2% glutaraldehyde for approximately 5 minutes, and then rinsed twice with deionized water. After rinsing, the sample could be dehydrated by fractionating ethanol, air-drying at room temperature, and storing in a vacuum desiccator for at least 24 hours prior to analysis.

[0154] In some implementations, in order to prepare intact tissue samples (such as samples obtained from biopsies), such as Figure 6As shown, tissue samples can be mounted on substrate 152. After fixation, the samples can be baked at approximately 65°C for 15 minutes, dewaxed in xylene (if the samples are obtained from FFPE tissue blocks), and rehydrated via a series of graded ethanol solutions. The samples can then be immersed in epitope repair buffer (10 mM sodium citrate, pH 6) and placed in a pressure cooker (available from Electron Microscopy Sciences, Hatfield, Pennsylvania) for approximately 30 minutes. Subsequently, the samples can be rinsed twice with deionized water and once with washing buffer (TBS, 0.1% Tween, pH 7.2). Residual buffer solution can be removed by gently touching the samples with lint-free cotton paper. In some embodiments, the samples can then be incubated for approximately 30 minutes with blocking buffer (TBS, 0.1% Tween, 3% BSA, 10% donkey serum, pH 7.2).

[0155] In some embodiments, the blocking buffer can then be removed, and the sample can be labeled overnight in a humidified chamber at 4°C with a mass labeling solution. After labeling, the sample can then be rinsed twice in washing buffer (PBS, 2% glutaraldehyde), fixed for approximately 5 minutes, rinsed in deionized water, and stained with Harris hematoxylin for 10 seconds. In some embodiments, after rinsing, the sample is dehydrated by a fractionated ethanol series, air-dried at room temperature, and stored in a vacuum desiccator for at least 24 hours prior to analysis.

[0156] For example, breast tumor tissue sections for MIBI imaging can be prepared as follows: Tissue sections (e.g., 4 μm thick) can be cut from formalin-fixed paraffin-embedded (“FFPE”) tissue blocks of human breast tumors using a microtome, and the tissue sections are mounted on a poly-L-lysine-coated silicon substrate for MIBI analysis. In some embodiments, the sections mounted on the silicon substrate can then be baked at 65°C for 15 minutes, dewaxed in xylene, and rehydrated via a series of graded ethanol fractions. The sections can then be immersed in epitope repair buffer (10 mM sodium citrate, pH 6) and placed in a pressure cooker (e.g., Electron Microscopy Sciences, Hatfield, Pennsylvania) for up to 30 minutes. In some embodiments, after pressure cooking, the sections can be washed twice with dH2O and once with a washing buffer (e.g., a buffer containing Tris-buffered saline (“TBS”), 0.1% Tween, pH 7.2). For example, residual buffer can be removed by gently touching the surface with lint-free cotton paper. The sections can then be incubated for 30 minutes with blocking buffer (TBS, 0.1% Tween, 3% BSA, 10% donkey serum, pH 7.2). The sections can be rinsed twice in washing buffer (PBS, 2% glutaraldehyde), fixed for 5 minutes, and then rinsed in dH2O. Finally, the sections can be dehydrated by fractionating ethanol, air-drying at room temperature, and then storing in a desiccator in a vacuum desiccator for at least 24 hours before imaging.

[0157] Antigen retrieval can be performed using a decloaking chamber (e.g., Concord, CA, Biocare Medical) with citrate buffer at pH 6.0, 125°C, and 15 psi. The total incubation time for sections is up to 45 minutes. Primary antibody incubation can be performed overnight in a humidified chamber at room temperature. Normal goat serum can be used for blocking. Biotinylated goat anti-rabbit (1:1000) can be used as a secondary antibody with the Vectastain ABC Kit Elite. The peroxidase substrate kit DAB (e.g., Vector Labs, Burlingame, CA) can be used separately for signal amplification and visualization. Tissue containing each assessed antigen is known to be used as a positive control.

[0158] It should be understood that the above preparation steps are provided only as examples of methods for preparing samples, and modifying the order of the above steps also produces samples that are appropriately labeled with quality tags and ready for MIBI analysis. In particular, the above preparation step sequence can be modified based on the properties of the sample (e.g., the type of tissue to which the sample corresponds).

[0159] D. Ion Optical Devices

[0160] Return to Figure 1 The system 100 includes an ion optics device 110 for guiding secondary ions 118a from the sample chamber 126 into the detection device 112. Typically, the ion optics device 110 can include a variety of different ion optical elements for the purpose of guiding secondary ions 118a. Figure 8 This is a schematic diagram illustrating an embodiment of the ion optical device 110. Figure 8 The ion optics 110 includes a housing 810 characterized by an inlet aperture 812 and an outlet aperture 814. During operation, secondary ions 118a enter the ion optics 110 from the sample chamber 126 through the inlet aperture 812 and propagate in direction 818 along the central axis 816 toward the outlet aperture 814. The secondary ions 118a exit through the aperture 814 and enter the detection device 112.

[0161] The ion optics device 110 may include various different ion optical elements for guiding secondary ions 118 and modulating the properties of said ions. In some embodiments, such as in Figure 8 As shown, the ion optics 110 includes one or more focusing elements 802, 804, and 806 connected to the controller 114 via signal line 120e. The ion optics 110 also includes a beam deflection element 808 connected to the controller 114 via signal line 120e. During operation of the system 100, secondary ions 118a generated in chamber 126 enter the ion optics 110 through aperture 812 and generally propagate along direction 818. The controller 114 adjusts the potentials applied to the focusing elements 802, 804, and 806 and to the beam deflection element 808 to ensure that direction 818 is substantially parallel to axis 816. Furthermore, in addition to adjusting the potentials applied to the focusing elements and the beam deflection element, the controller 114 also adjusts the diameter of the beam of secondary ions 118a and the trajectory of the secondary ions 118a as they exit the ion optics 110 through aperture 814.

[0162] In some implementation schemes, in addition to Figure 8 Other than the elements shown or as Figure 8 As an alternative to the elements shown, ion optics 110 may include one or more ion traps. Ion traps may be used to temporarily “collect” or concentrate the secondary ions 118a generated in chamber 126 before delivering them to detection device 112.

[0163] A wide variety of different types of ion traps can be used in the ion optics 110, and the choice of ion trap can depend on various factors such as the yield of secondary ions 118a. For example, in some embodiments, the ion optics 110 may include a ring-shaped ion trap. Figure 9 This is a schematic diagram of an annular ion trap 900 including electrodes 902 and 904 connected to controller 114 via signal line 120e. During operation of the ion trap 900, secondary ions 118a enter the ion trap through orifice 906. Controller 114 adjusts the potential applied to electrodes 902 and 904 such that the electrodes apply annular trapping force to the secondary ions 118a, causing the secondary ions to undergo precession within the gap 910 between electrodes 902 and 904. The potential applied to electrodes 902 and 904 can be swept so that after the secondary ions 118a are trapped within the annular gap 910, secondary ions 118a with a specific m / z ratio are selectively ejected from the trap 900 through orifice 908. In this way, according to their m / z ratios, the trapped ions can be directed to detection device 112, allowing detection device 112 to detect secondary ions at high resolution. For example, other features and aspects of the toroidal ion trap are disclosed in Lammert et al., “Miniature Toroidal Radio Frequency Ion Trap Mass Analyzer,” J. Am. Soc. Mass Spectrom. 17:916-922 (2006) and Austin et al., “Halo Ion Trap Mass Spectrometer,” Anal. Chem. 79:2927-2932 (2007), the entire contents of each of which are incorporated herein by reference.

[0164] As another example, in some embodiments, the ion optics 110 may include a radial ion trap. Figure 10A schematic diagram of a radial ion trap 1000 is shown, including an inlet orifice 1002, an outlet orifice 1006, and one or more trap electrodes 1004 connected to a controller 114 via signal lines 120e. During operation, secondary ions 118a enter the trap 1000 through the orifice 1002. The controller 114 applies a potential to the one or more trap electrodes 1004, thereby generating a radial trap field within the ion trap 1000, which causes the secondary ions 118a to undergo precession within a trap region 1008. The controller 114 changes the potential such that only secondary ions 118a within a selected range of m / z values ​​are retained and trapped within region 1008. Secondary ions 118a falling outside this range of m / z values ​​are ejected through the orifice 1006. By changing the potential, secondary ions with different m / z ratios can be selectively “sweeped” out of the ion trap 1000 and into the detection device 112.

[0165] In ion trap 1000, the shape of trap region 1008 determines the nature of the trap field generated by controller 114. For example, in some embodiments, trap region 1008 corresponds to an elliptical aperture formed in electrode 1004, and ion trap 1000 is an elliptical ion trap with an elliptical trap field. In some embodiments, trap region 1008 corresponds to a circular aperture formed in electrode 1004, and ion trap 1000 is a circular ion trap with a circular trap field. Other aspects and features of radial ion traps are discussed in, for example, Patterson et al., “Miniature Cylindrical Ion Traps Mass Spectrometer,” Anal. Chem. 74:6145-6153 (2002), Blain et al., “Towards the Hand-Held Mass Spectrometer: Design Considerations, Simulation, and Fabrication of Micrometer-Scaled Cylindrical Ion Traps,” Int. J. Mass Spectrom. 236:91-104 (2004), and Riter et al., “Analytical Performance of a Miniature Cylindrical Ion Trap Mass Spectrometer.” The full contents of each of the references are incorporated herein by reference in "Spectrometer (Analytical Performance of Small Cylindrical Ion Trap Mass Spectrometer)," Anal. Chem. 74:6154-6162 (2002), and the entire contents of each of the references are incorporated herein by reference.

[0166] The foregoing discussion provides various examples of different elements that may be included in the ion optical device 110; however, it should be understood that the ion optical device 110 may also include other elements. Furthermore, in some embodiments, the ion optical device 110 may not include any elements. That is, secondary ions 118a can be delivered directly from the chamber 126 to the detection device 112 without penetrating or being manipulated by any element corresponding to the ion optical device 110.

[0167] E. Detection device

[0168] After passing through the ion optics 110, secondary ions 118a are detected by the detection device 112. The detection device 112 may include a variety of different elements. Typically, the detection device 112 is used to generate an electrical signal (e.g., voltage, current) that qualitatively and / or quantitatively represents the secondary ion cluster entering the detection device 112.

[0169] For example, in some embodiments, the detection device 112 may include one or more Faraday cup detectors connected to the controller 114 via signal line 120f. When secondary ions 118a are incident on a Faraday cup detector, the detector generates an electrical signal that is transmitted to the controller 114 via signal line 120f. When a suitable ion trap for m / z resolution is used in the ion optics 110 as discussed above, the signal generated by the Faraday cup detector can be measured by the controller 114 according to m / z, thereby providing quantitative information about the secondary ion 118a group.

[0170] In some implementations, the detection device 112 may include a linear time-of-flight (lin-TOF) detector. Figure 11 This is a schematic diagram illustrating an embodiment of a lin-TOF detector 1100. The detector 1100 optionally includes one or more electrodes 1104 and a detection element 1106 (such as a Faraday detector) connected to a controller 114 via signal line 120f. During operation, secondary ions 118a enter the detector 1100 through an aperture 1102. By applying a suitable potential to the electrodes 1104, the controller 114 can optionally adjust (e.g., deflect and / or focus the secondary ions 118a) the characteristics of the ions. The secondary ions 118a then generally propagate toward the detection element 1106 along direction 1110, traversing the drift region 1108.

[0171] Secondary ions 118a of different masses propagate at different speeds. Therefore, the transit time of each secondary ion 118a across the drift region 1108 is related to the ion's m / z ratio. The controller 114 measures the electrical signal generated by the detection element 1106 based on time, where the time associated with each signal represents a specific m / z ratio. The controller 114 uses calibration information to convert the arrival time of ions at the detection element 1106 into a specific m / z ratio, thereby enabling the quantitative determination of the ion cluster based on the m / z ratio.

[0172] In time-of-flight detectors, the ion detection cycle begins at time zero. Since the m / z ratio associated with each signal is a direct function of time relative to time zero when the signal is measured, it is important to accurately and repeatably create time zero during each detection cycle. For lin-TOF detectors, time zero can be created in different ways depending on how the secondary ion 118a is generated.

[0173] In some embodiments, ion source 102 generates a continuous primary ion beam 116, thereby continuously generating secondary ions 118a from sample 150. In this case, time zero at detector 1100 is defined by controller 114. Before executing an ion detection cycle, controller 114 adjusts the potential applied to electrode 1104 so that secondary ions 118a entering orifice 1102 are deflected and blocked by orifice 1112 and do not reach detection element 1106. To initiate the ion detection cycle, controller 114 adjusts the potential applied to electrode 1104 so that secondary ions 118a can propagate through detector 1100 and reach detection element 1106. The time at which controller 114 initiates the detection cycle by adjusting the potential applied to electrode 1104 corresponds to time zero (t = 0) in the detection cycle, and the measurement time of the ion signal generated by detection element 1106 is referenced to this time zero.

[0174] Typically, controller 114 initiates an ion detection cycle by adjusting the potential applied to electrode 1104 for a relatively short period of time, allowing pulses or bursts of secondary ions 118a to propagate to and be detected by detection element 1106. However, after allowing the pulses of secondary ions 118a, controller 114 readjusts the potential applied to electrode 1104 such that, while detecting the allowed secondary ions 118a, it blocks any additional secondary ions 118a reaching aperture 1112.

[0175] Figure 12This is a schematic timing diagram illustrating the relationship between the permission window 1202 and the measured electrical signal 1204 corresponding to the secondary ion 118a, the permission window corresponding to the adjustment of the potential applied to the electrode 1104 by the controller 114. The rising edge of the permission window 1202 corresponds to time zero (t=0) of the detection period, and the electrical signal 1204 is measured at times t1, t2, and t3 relative to t=0.

[0176] Alternatively, in some embodiments, the ion source 102 is operated in a pulsed mode by the controller 114. That is, the controller 114 transmits a suitable control signal to the ion source 102 via signal line 120a, causing the ion source 102 to generate a pulsed primary ion beam 116. In this operating mode, the control signal transmitted to the ion source 102 to initiate a primary ion pulse can be used as a clock signal for the ion detection cycle. That is, refer to... Figure 12 For the purpose of detecting secondary ions 118a, the control signal transmitted to the ion source 102 to initiate the pulsed primary ion also defines t=0. In some embodiments, when the ion source 102 operates in pulsed mode, the secondary ions 118a are not selected by the electrodes 1104 and orifice 1112 in the detector 1100. The secondary ion signal measured by the detection element 1106 is at each reference time t=0, which is defined by the primary ion pulse control signal transmitted by the controller 114. The continuous control signals transmitted to the ion source 102 are sufficiently separated in time such that the ion detection sequence is completed after each primary ion pulse before the next primary ion pulse is generated.

[0177] In some implementations, the detection device 112 includes an orthogonal time-of-flight (TOF) detector. Figure 13 This is a schematic diagram of an ortho-TOF detector 1300. During operation, secondary ions 118a enter the detector 1300 from the ion optics 110 and travel along path 1326 to the detection element 1324.

[0178] Secondary ions 118a have a velocity in the z-direction when they enter detector 1300. Controller 114 applies a "push out" voltage pulse (i.e., a "gate signal") to push-out electrode 1302 via signal line 120f, and controller 114 also applies the "push out" voltage pulse to grids 1306 and 1310 via signal line 120f to accelerate a large number of secondary ions 118a in the y-direction, thereby deflecting the path of secondary ions 118a relative to the z-direction by a deflection angle θ. The deflection angle θ, corresponding to the angle by which secondary ions 118a deviate from their initial propagation direction, can typically be between 60 degrees and 120 degrees (e.g., between 60 degrees and 110 degrees, between 70 degrees and 90 degrees, or between 80 degrees and 90 degrees).

[0179] Electrode 1308 is connected to controller 114 via signal line 120f. Controller 114 applies a potential to electrode 1308 to accelerate deflected secondary ions 118a toward ion reflector 1314 in the y-direction.

[0180] After passing through grid 1310, the deflected secondary ions 118a propagate in the field-free space 1312 until they reach the ion reflector 1314. The ion reflector 1314 includes grids 1316 and 1318 and a ring electrode 1320, each of which is connected to a controller 114 via a signal line 120f. The controller 114 applies a suitable potential to grids 1316 and 1318 and to the ring electrode 1320, causing the ion reflector to act as an "ion mirror," thereby altering the propagation direction of the secondary ions 118a in the y-direction and effectively "reflecting" the ions back through the field-free space region 1312.

[0181] Secondary ions 118a penetrate the grid 1322 and are incident on the detection element 1324. The detection element 1324 typically has a width 1326, measured in the z-direction, between 0.5 cm and 5 cm (e.g., between 1 cm and 3 cm). To detect all ejected ions 118a, the width 1304 of the ejection plate 1302 in the z-direction is typically within the same range as, and can be similar to, the width of the detection element 1324.

[0182] In the ortho-TOF detector 1300, the flight time of the secondary ion 118a is measured in the y-direction, while the initial propagation direction of the secondary ion 118a when it enters the detector 1300 from the ion optics 110 is in the z-direction. Thus, the ortho-TOF detector 1300 effectively resets the time to zero for the secondary ion 118a before measuring its flight time in the detector. In the lin-TOF detector described above, time zero is defined, for example, by a pulse control signal transmitted to the ion source 102, while in the ortho-TOF detector 1300, time zero is effectively defined by an ejection signal applied to the electrode 1302.

[0183] When measuring secondary ions, ortho-TOF detectors offer significant advantages over lin-TOF detectors. For example, in a lin-TOF detector used with a pulsed ion source 102, the time zero referenced to the measured secondary ion signal corresponds to the control signal transmitted to the ion source to generate a primary ion pulse. Therefore, the time pulse width of the primary ion beam 116 is designed to be relatively short (e.g., approximately 10 ns) to precisely measure only the ions generated from the sample in response to this primary ion pulse. Furthermore, since the control signal transmitted to the ion source creates the time zero for the entire ion detection cycle, the control signal is typically much shorter than the ion detection cycle to ensure that the uncertainty in mass resolution (dependent on the time zero) is sufficiently low. That is, to make the uncertainty in mass resolution acceptable, the time width of the control signal is significantly shorter than the ion detection cycle.

[0184] Conversely, since the pulse width of the primary ion beam 116 is not zero at the ortho-TOF detector settling time, the pulse width of the primary ion beam can be significantly larger (e.g., 3 μs). As a result, a larger amount of secondary ions 118a are generated from the sample, which in turn leads to a larger measured ion signal.

[0185] Furthermore, while the lin-TOF detector can measure almost all secondary ions 118a generated from the sample (effectively without loss due to the duty cycle), the time taken to complete a detection cycle is considerably longer than the duration of the primary ion pulse that generates the secondary ion 118a. Therefore, for example, a typical detection cycle can be completed in approximately 20 microseconds for secondary electrons generated in response to a 10 ns primary ion pulse. As a result, for every 10 ns of sample ionization, 20 microseconds are consumed in detecting the secondary ion signal. In other words, for every second of analysis time, secondary ions are generated in only approximately 500 microseconds. This can severely limit the system's throughput and may be a significant problem when dealing with large samples and / or large scan areas.

[0186] As described above in conjunction with the lin-TOF detector, an ortho-TOF detector can be used in system 100, wherein ion source 102 continuously generates a primary ion beam 116. While the primary ions are being generated, secondary ions 118a are also being generated continuously from sample 150 and enter ortho-TOF detector 1300 near ejection electrode 1302.

[0187] However, the ortho-TOF detector 1300 operates in a pulsed mode, where the controller 114 applies an ejection signal to the ejection electrode 1302 at a specific repetition rate. For example, in some embodiments, while the repetition rate is approximately 100 kHz, the detector 1300 can typically operate at a wide variety of different repetition rates. Generally, because secondary ions 118a are allowed to enter the detector 1300, the repetition rate of the ejection signal is selected to allow sufficient time for signal measurement.

[0188] Furthermore, the duration of the ejection signal applied to the ejection electrode 1302 by the controller 114 is selected to ensure that the ejection region of the detector 1300 is filled with secondary ions 118a. However, after the ejection region of the detector 1300 is filled, space charge effects and ion scattering events can adversely affect the accurate establishment of time zero. For example, in some embodiments, although the duration of the ejection signal applied to the ejection electrode 1302 by the controller 114 is approximately 3 microseconds, a wide variety of different ejection signal widths can typically be used.

[0189] In summary, both the repetition rate and the time pulse width of the ejection signal applied to the ejection electrode 1302 by the controller 114 are selected to ensure that the detector accurately and reproducibly measures the secondary ion signal. However, when the primary ion beam 116 continuously generates secondary ions 118a by the ion source 102, the ortho-TOF detector detects a relatively modest proportion of the total amount of secondary ions 118a generated. For example, an ortho-TOF with a 3-microsecond ejection signal operating at a repetition rate of 100 kHz can only measure approximately 30% of the total amount of secondary ions 118a generated from the sample.

[0190] Measuring a relatively low proportion of secondary ions 118a can have some undesirable consequences. First, by measuring only a relatively small proportion of the total amount of generated secondary ions 118a, the measured secondary ion signal is significantly weaker than the signal when detecting a larger proportion of the total amount of secondary ions. As a result, the measurement sensitivity of system 100 is lower than that when detecting a larger proportion of secondary ions. For example, by detecting only about 30% of the generated secondary ions, the measurement sensitivity of system 100 can be reduced by 3 to 4 times.

[0191] Secondary ions 118a that are not deflected along path 1326 within detector 1300 are simply scattered within the detector. Therefore, for example, if the duty cycle period of the detector is 10 microseconds, and secondary ions 118a are deflected along path 1326 for only 3 microseconds of the duty cycle period, then for the remaining 7 microseconds, the secondary ions 118a entering detector 1300 simply disperse within the detector. However, some of these dispersed ions can still reach detection element 1324 and generate a false background signal. This background signal effectively serves as noise within detector 1300, making the discrimination of TOF-based secondary ion signals more difficult. Therefore, in addition to reducing the sensitivity of system 100 by selectively detecting a relatively small proportion of secondary ions 118a, the dispersion of undetected secondary ions can also lead to increased baseline noise, which is the case when measuring the secondary ion signal.

[0192] Furthermore, a significant portion of the sample's signal generation is "wasted" by generating a relatively large proportion of undetected secondary ions 118a. As the primary ion beam 116 scans the sample and generates secondary ions 118a, the sample is ablated by the primary ion beam 116. Thus, only a limited number of secondary ions 118a are generated per sample before it is consumed. By discarding a significant proportion of the generated secondary ions 118a, a large portion of the sample is effectively wasted during measurement.

[0193] To improve the matching between the amount of secondary ions 118a generated from the sample and the capacity of the ejection region within the detector 1300, and to better utilize the sample to generate the actual detected secondary ions 118a, the ion source 102 can be operated in pulsed mode. The controller 114 transmits a control signal via signal line 120a to cause the ion source 102 to generate a pulsed primary ion beam 116. Typically, the control signal is periodic and includes a series of trigger pulses or gate pulses with a selected time width and a selected repetition rate. Each trigger pulse or gate pulse causes the ion source 102 to generate a primary ion pulse. Therefore, the repetition rate of the pulsed primary ion beam 116 is the same as the repetition rate of the control signal transmitted by the controller 114.

[0194] Operating the ion source 102 in pulsed mode can also be advantageous because the average primary ion current of the ion source 102 can be maintained at the same level as when operating in continuous mode. However, since the source is pulsed, the primary ion current increases instantaneously per pulse. Therefore, sample erosion due to exposure to the primary ion beam 116 occurs at the same rate, but the measurement sensitivity of the system 100 is increased because a larger proportion of secondary ions released from the sample is measured.

[0195] Typically, controller 114 can adjust the repetition rate and time pulse width of the control signal transmitted to ion source 102. Specifically, each of these parameters can be adjusted to increase the proportion of secondary ions generated as measured by detector 1300.

[0196] For example, in some implementations, the time pulse width of the control signal transmitted to the ion source 102 can be adjusted based on the ion capacity of the ejection region of the detector 1300. The ejection region of the detector 1300 corresponds to the spatial region between the ejection electrode 1302 and the grid 1306. Due to space charge effects, collision events, and geometric constraints, the ejection region has a limited storage capacity N for secondary ions 118a. 充满 Based on the average ion transport rate v within system 100 离子 Starting from the time when secondary ions 118a are first generated by the primary ion beam 116, during the time period t 充满 The inner region (initially not containing secondary ions) is filled with N. 充满 One ion, which roughly corresponds to t 充满 =N 充满 / v 离子 To increase the proportion of secondary ions measured by detector 1300, controller 114 can adjust the pulse duration in the control signal transmitted to ion source 102 to match the time period t required to fill the ejection region of detector 1300 with secondary ions. 充满 For example, in some embodiments, the pulse width of the control signal transmitted to the ion source 102 is the same as the time period t required to fill the ejection region of the detector 1300 with secondary ions. 充满 The difference between them is the time period t. 充满 10% or less (e.g., 8% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less).

[0197] After the ejection region of detector 1300 is filled with secondary ions 118a, controller 114 applies an ejection signal to ejection electrode 1302 to deflect the secondary ions 118a along path 1326 in detector 1300. As discussed above, the ejection signal is a periodic signal consisting of repetitive pulses with amplitude and time width.

[0198] The pulse duration in the control signal transmitted to ion source 102 effectively defines the duration of the primary ion pulse in the primary ion beam 116. Furthermore, since each primary ion pulse generates a large number of secondary ions 118a, the pulse duration in the control signal transmitted to ion source 102 also effectively defines the pulse duration in response to the secondary ions 118a generated from the sample by the pulsed primary ion beam.

[0199] The pulse width in the control signal transmitted to the ion source 102 is compared with the time interval t during which the ejection region of the detector 1300 is filled with secondary ions 118a. 充满 Matching ensures that the ion capacity of detector 1300 is more fully utilized, and therefore, the amplitude of the secondary ion signal measured by detection element 1324 is greater than that when the ejection region of detector 1300 is filled with fewer secondary ions.

[0200] In some implementations, controller 114 can adjust the repetition rate of the control signal transmitted to ion source 102 (i.e., the repetition rate of pulsed primary ion beam 116) based on the repetition rate of the ejection signal applied to ejection electrode 1302 by controller 114. Specifically, by ensuring that the repetition rate of the control signal transmitted to ion source 102 matches the repetition rate of the ejection signal applied to ejection electrode 1302 by controller 114, a significantly larger proportion of secondary ions 118a generated from the sample can be deflected and measured within detector 1300. Because controller 114 selects the repetition rate of the ejection signal based on the analysis of the time required to complete the ion detection cycle, matching the repetition rate ensures that each pulse or group of secondary ions 118a arriving at detector 1300 is deflected and analyzed, and that another pulse or group of secondary ions 118a arrives for analysis precisely at the completion of the ion detection cycle.

[0201] In this way, the matching of the repetition rate of the control signal applied to ion source 102 and the repetition rate of the ejection signal applied to ejection electrode 1302 ensures that a higher proportion of the generated secondary ions are measured, leading to several advantages. Specifically, as discussed above, a significantly higher proportion of the sample is consumed in the generation of the secondary ions 118a actually measured, rather than secondary ions discarded only during the ion detection cycle. Furthermore, background noise in detector 1300 is reduced due to a significant reduction in the contribution from discarded secondary ions 118a. In some embodiments, the repetition rate of the pulses in the control signal transmitted to ion source 102 differs from the repetition rate of the pulses in the ejection signal applied to ejection electrode 1302 by 10% or less (e.g., 8% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less).

[0202] The repetition rate of the pulses in the control signal transmitted to ion source 102 effectively defines the repetition rate of the primary ion pulses in the primary ion beam 116 and the repetition rate of the pulses of secondary ions 118a released from the sample in response to the primary ion pulses. Accordingly, in some embodiments, the repetition rate of the primary ion pulses in the primary ion beam 116 differs from the repetition rate of the pulses in the ejection signal applied to ejection electrode 1302 by 10% or less (e.g., 8% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less). Further, the repetition rate of the pulses of secondary ions released from the sample differs from the repetition rate of the pulses in the ejection signal applied to ejection electrode 1302 by 10% or less (e.g., 8% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less).

[0203] The process of generating a primary ion pulse and transmitting it to the sample to generate secondary ions 118a occurs over a non-zero time period. Further, as generated from the sample, secondary ions 118a propagate from chamber 126 through ion optics 110 to detection device 112 over an additional non-zero time period. To further increase the proportion of secondary ions 118a deflected and measured within detector 1300, controller 114 can account for the average delay time between transmitting the control signal to ion source 102 and applying the ejection signal to ejection electrode 1302 by introducing a time offset (i.e., phase offset) between the transmission of the control signal to ion source 102 and the arrival of the secondary ion pulse or group at detector 1300. The magnitude of the time offset is chosen to ensure that the ejection signal is applied to ejection electrode 1302 precisely when the ejection region of detector 1300 is filled with secondary ions 118a. This ensures that as many secondary ions as possible are deflected into detector 1300 for analysis.

[0204] In practice, by generating pulses or groups of secondary ions 118 from the test sample, the controller 114 can select an appropriate time offset and iteratively adjust the time offset between the two signals until the detector 1300 measures a secondary ion signal of sufficient amplitude. As the magnitude of the time offset becomes closer to the delay time between transmitting the control signal to the ion source 102 and the filling time of the ejection region in the detector 1300, the measured amplitude of the secondary ion signal is expected to increase.

[0205] The aforementioned operation of matching the repetition rate of the control signal transmitted to ion source 102 with the ejection signal applied to ejection electrode 1302 and optionally adjusting the time offset between the signals generates a control signal transmitted to ion source 102 (which generates a pulsed primary ion beam 116 with a repetition rate), said control signal being synchronized with the ejection signal applied to ejection electrode 1302. Although synchronization and pulse width adjustment are used in the methods and systems discussed herein to significantly improve the sensitivity of system 100 and reduce baseline noise, such steps are generally not implemented in routine mass spectrometry analysis of biological samples, partly because conventional methods typically lack precise control over the timing of ion generation in the sample.

[0206] For example, methods such as electrospray ionization are used to analyze many biological samples. In such methods, accurately establishing zero time is difficult, or even impossible. Electrospray ionization is typically performed continuously, rather than in a pulsed manner. Furthermore, even when performed in a pulsed manner, the ionization process depends on the analyte flow rate and other solution transport parameters. Therefore, in many cases, it is not possible to generate packets or pulses of ions with a precise repetition rate. As a result, as discussed above, the adjustment of pulse width and the repetition rate (and time offset) of the control signal is often not used in conjunction with the analysis of biological samples.

[0207] As discussed above, synchronization of the ejection signal applied to the ejection electrode 1302 with the control signal transmitted to the ion source 102 (and the repetition rate generated by the pulsed primary ion beam 116) can increase the proportion of secondary ions 118a generated from the sample 150 and detected by the detector 1300. Synchronization can result in a substantial proportion of the generated secondary ions 118a measured by the detector 1300 (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or even more).

[0208] Figure 14 This is a schematic timing diagram illustrating an example of a control signal transmitted by controller 114 and a measurement signal obtained by detector 1300. Controller 114 transmits a control signal consisting of a periodic sequence 1402 of voltage pulses to ion source 102, causing ion source 102 to generate a primary ion beam 116 consisting of a sequence of primary ion pulses. The primary ion pulses interact with sample 150, thereby generating pulses or groups of secondary ions 118a that propagate to detection device 112 (e.g., detector 1300).

[0209] The controller 114 applies an ejection signal consisting of a periodic sequence of voltage pulses 1404 to the ejection electrode 1302, thereby deflecting and accelerating secondary ions 118a in the ejection region of the detector 1300 along the y-direction. When the secondary ions reach the detection element 1324, the detection element measures the arrival time of the secondary ions relative to time zero (t0), which is established by each corresponding pulse in the ejection signal, as discussed above. Since the time of flight of the secondary ions 118a in the detector 1300 (i.e., along path 1326) is related to the mass-to-charge ratio of the ions, the measured secondary ion signal 1406 corresponds to an ion abundance measurement based on the m / z of the secondary ions 118a.

[0210] Each pulse 1402 in the control signal transmitted to the ion source 102 has an amplitude 1402a and a time interval (or “width”) 1402b. The pulse sequence 1402 has a time period 1402c that defines the frequency of the pulse sequence. Each pulse 1404 in the ejection signal applied to the ejection electrode 1302 has an amplitude 1404a, a time interval (or “width”) 1404b, and a time period 1404c that defines the frequency of the pulses in the sequence.

[0211] As discussed above, the time width 1402b of pulse 1402 can be synchronized with the filling time t of the ejection region of detector 1300. 充满 The approximate matching ensures that the amount of secondary ions 118a generated from the sample according to each incident primary ion pulse is approximately sufficient to just fill the exit region of the detector 1300. For example, in some embodiments, the time width 1402 may be between 5 nanoseconds and 100 microseconds (e.g., between 50 nanoseconds and 80 microseconds, between 100 nanoseconds and 50 microseconds, between 500 nanoseconds and 50 microseconds, between 1 microsecond and 50 microseconds, between 1 microsecond and 30 microseconds, between 1 microsecond and 20 microseconds, between 1 microsecond and 10 microseconds, between 2 microseconds and 10 microseconds, between 2 microseconds and 8 microseconds, between 3 microseconds and 5 microseconds).

[0212] Because the time width 1402b of pulse 1402 effectively determines the time width of a single ion pulse in a single ion beam 116, the time pulse width of a single ion beam 116 can be between 5 nanoseconds and 100 microseconds (e.g., between 50 nanoseconds and 80 microseconds, between 100 nanoseconds and 50 microseconds, between 500 nanoseconds and 50 microseconds, between 1 microsecond and 50 microseconds, between 1 microsecond and 30 microseconds, between 1 microsecond and 20 microseconds, between 1 microsecond and 10 microseconds, between 2 microseconds and 10 microseconds, between 2 microseconds and 8 microseconds, between 3 microseconds and 5 microseconds).

[0213] The time width 1404b of the pulse 1404 can be selected as desired to ensure that the secondary ions 118a are deflected and accelerated within the detector 1300. For example, in some embodiments, the time width 1404b can be between 5 nanoseconds and 100 microseconds (e.g., between 50 nanoseconds and 100 microseconds, between 100 nanoseconds and 100 microseconds, between 500 nanoseconds and 100 microseconds, between 700 nanoseconds and 100 microseconds, between 1 microsecond and 80 microseconds, between 1 microsecond and 50 microseconds, between 2 microseconds and 50 microseconds, between 10 microseconds and 50 microseconds, between 3 microseconds and 30 microseconds, between 3 microseconds and 20 microseconds).

[0214] Typically, the amplitudes 1402a of pulse 1402 and 1404a of pulse 1404 can be selected as desired to achieve suitable control functions. For example, in some embodiments, the pulse amplitude 1402a can be between 1000V and 5000V. In some embodiments, the pulse amplitude 1404a can be between 100V and 3000V.

[0215] As described above, the time periods 1402c of the pulse 1402 sequence and 1404c of the pulse 1404 sequence are approximately matched to ensure that each pulse or group of secondary electrons 118a generated from the sample arrives at the detection device 112 (e.g., detector 1300) after the previous ion detection cycle has ended. As a result, all secondary ions in each arriving secondary ion pulse or group can be deflected into and measured by the detector 1300; a small number (if any) of secondary ions are dispersed or otherwise repelled.

[0216] The durations of time periods 1402c and 1404c are selected by controller 114 based on the elapsed time during each ion detection cycle. The elapsed time depends on the length of the flight path in detector 1300, the voltage suitable for accelerating secondary ions, the m / z of the secondary ions, and the speeds of various hardware and software components of detector 1300. For example, in some embodiments, time periods 1402c and 1404c are each between 5 microseconds and 50 microseconds (e.g., between 10 microseconds and 40 microseconds, or between 15 microseconds and 30 microseconds).

[0217] In some embodiments, the frequency (or repetition rate) of the control signal (i.e., pulse 1402) transmitted to the ion source 102 and the ejection signal (i.e., pulse 1404) applied to the ejection electrode 1302 are each between 1 kHz and 200 kHz (e.g., between 10 kHz and 100 kHz, or between 80 kHz and 100 kHz). Because each pulse 1402 transmitted to the ion source 102 generates a primary ion pulse forming a primary ion beam 116, the repetition rate of the pulsed primary ion beam 116 can also be between 1 kHz and 200 kHz (e.g., between 10 kHz and 100 kHz, or between 80 kHz and 100 kHz).

[0218] As discussed above, the time offset (or phase offset) 1408 between pulses 1402 and 1404 reflects the non-zero delay time between the controller 114 transmitting the control signal to the ion source 102 and the arrival of the corresponding pulses or packets of secondary ions 118a at the detection device 112. The controller 114 is typically configured to adjust the time offset value 1408 to control the amplitude s of the measured secondary ion signal. The magnitude of the delay time and therefore the magnitude of the time offset 1408 depends on a number of factors, including: the length of the path 1326; the m / z of the measured secondary ions; and the various accelerating, decelerating, focusing, and deflecting fields through which the primary and secondary ions pass. For example, in some implementations, the size of time offset 1408 may be 80% or less of time period 1402c and time period 1404c (e.g., 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or even less).

[0219] It should be noted that as the pulses or groups of secondary ions 118a propagate from the sample 150 to the detection device 112, the spatial distribution of secondary ions within the pulse widens due to the different m / z values ​​between the ions. By adjusting the time width 1402b of the pulse 1402 in the control signal transmitted to the ion source 102, the time t required to fill the ejection region of the detector 1300 can be determined. 充满 The changes.

[0220] In some embodiments, not all secondary ions 118a arrive at the ejection region of detector 1300 within the time width 1404b of the ejection signal pulse due to the spatiotemporal widening of the pulse as it propagates through system 100. In this case, time offset 1408 can be adjusted to utilize this spatiotemporal widening to selectively deflect and detect a specific subset of secondary ions with m / z values ​​within detector 1300. Because the arrival time of secondary ions within the ejection region will depend on their m / z values, by adjusting time offset 1408 such that an ejection signal pulse is applied to ejection electrode 1302 when a specific subset of secondary ions with m / z values ​​is present within the ejection region, the measurement and analysis of secondary ions by detector 1300 can be effectively limited to that subset of m / z values. For example, in some embodiments, by adjusting time offset 1408, secondary ions 118a corresponding to relatively heavy masses (e.g., secondary ions derived from lanthanide-based mass tags) can be selectively measured by detector 1300. In some embodiments, by adjusting the time offset 1408 to different values, secondary ions 118a corresponding to lighter masses (e.g., secondary ions originating from atoms such as sodium and potassium) can be selectively measured by the detector 1300. By preferentially selecting the relatively heavier masses for measurement, the average mass of the unmeasured secondary ions is less than the average mass of the measured secondary ions. Conversely, by preferentially selecting the relatively lighter masses for measurement, the average mass of the unmeasured secondary ions is greater than the average mass of the measured secondary ions.

[0221] Other mechanisms and steps can also be used to effectively filter secondary ions 118a so that the detection device 112 measures only ions with m / z values ​​within a specific range. In some embodiments, the ion optics 110 may include a "snap cap" valve that preferentially directs secondary ions to the detection device 112 (e.g., detector 1300). Figure 15 This is a schematic diagram illustrating an embodiment of the flap valve 1500. The valve 1500 includes a housing 1516, which includes an inlet port 1506 and two ports 1510 and 1512. A blocking element 1514 is located at the end of port 1510, and an outlet port 1508 is located at the end of port 1512. Deflection electrodes 1502 and 1504 are located within the housing 1516 and are connected to a controller 114 via a signal line 120e.

[0222] During operation, pulses or groups of secondary ions 118a enter valve 1500 through inlet orifice 1506. As discussed above, the spatiotemporal expansion of the secondary ion pulses during propagation through system 100 causes spatial dispersion of secondary ions within the pulse, with smaller m / z ions leading larger m / z ions. By applying a suitable potential to electrodes 1502 and / or 1504, valve 1500 can be used to preferentially guide a subset of secondary ions corresponding to a selected range of m / z values ​​out of orifice 1508. When no potential is applied to electrodes 1502 and / or 1504, secondary ions 118a pass through the electrodes undeflected and are intercepted by blocking member 1514, preventing them from reaching detection device 112. However, when a suitable potential is applied to electrodes 1502 and / or 1504, secondary ions 118a are deflected along path 1518, thus exiting orifice 1508 and entering detection device 112. By applying a suitable potential to electrodes 1502 and / or 1504 during a specific time window when secondary ions 118a pass through valve 1500, controller 114 can selectively direct a subset of secondary ions corresponding to a specific range of m / z values ​​to detection device 112 for analysis.

[0223] In addition to valve 1500, other mechanisms and devices may be used to selectively direct a subset of the released secondary ions 118a, corresponding to a specific range of m / z values, into detection device 112 for measurement. Such devices include, but are not limited to, various adjustable mechanical blocking members and electrode assemblies. Where such mechanisms (such as valve 1500) include an electrode to which a controller 114 delivers a control signal to selectively deflect the secondary ions 118a, this control signal may be synchronized (with an appropriate time offset) with a control signal transmitted to ion source 102 and with a discharge signal applied to discharge electrode 1302 to ensure that approximately the same subset of m / z ratios is selected from each pulse of secondary ions 118a for analysis. Furthermore, the time width of the control signal delivered to such mechanisms (e.g., a potential applied to electrode 1502 and / or electrode 1504) can be adjusted to select the width of the distribution of m / z values ​​to be analyzed.

[0224] Valve 1500 is particularly useful for selectively analyzing secondary ions, providing certain types of information of different kinds. For example, detection device 112 can detect many relatively bright secondary ions (such as sodium and potassium ions) to provide an image of the overall structure of the sample (e.g., tissue structure). Since these ions are generally widely distributed throughout the sample, they produce relatively strong measurement signals and thus provide a bright, detailed, and non-selective image of the sample.

[0225] Conversely, secondary ions derived from mass tags typically provide information about the local distribution of a particular sample component, such as a protein. Therefore, depending on the distribution of a particular sample component across different sample regions, the intensity of images derived from these types of secondary ions may be relatively low in some corresponding regions of the sample.

[0226] By selectively allowing secondary ions with a certain range of m / z values ​​into the detection device 112 for analysis, two types of information can be obtained. For example, in some embodiments, valve 1500 (or another mechanism) can be operated by controller 114 to first acquire one or more images of sample 150 by detecting only relatively bright secondary ions of the element (such as sodium and / or potassium). These images provide an “overview” or profile of the sample. Controller 114 can then operate valve 1500 (or another mechanism) to acquire one or more images of sample 150 by detecting secondary ions derived from the mass label. These images provide structure-specific information about the location, distribution, and quantity of various sample components.

[0227] In some embodiments, controller 114 is configured to sequentially acquire two types of images by selectively detecting different types of secondary ions using valve 1500 or another mechanism. For example, controller 114 may be configured to operate valve 1500 such that for each set (n-1) images of sample 150 acquired by detecting secondary ions derived from a mass tag, a study image of sample 150 is acquired by selectively detecting relatively bright secondary ions (e.g., Na, K) from sample 150. The study image may be acquired before or after (n-1) structure-specific images and may be used, for example, to select exposure parameters and exposure patterns for the sample.

[0228] Mechanisms such as valve 1500 can be used in conjunction with other methods and system components discussed above to further improve the precision and accuracy of measurements performed using system 100. Specifically, by filtering out and preventing uninteresting secondary ions 118a from reaching detection device 112, baseline signal noise can be reduced, thereby allowing the weaker signals from the secondary ions of interest to be distinguished from the baseline signal.

[0229] Other implementation plans

[0230] While this specification contains numerous details of specific implementations, these should not be construed as limiting the scope of this disclosure or the scope of what may be claimed, but rather as descriptions of features specific to particular embodiments. Unless otherwise expressly stated, features described herein in the context of individual embodiments may also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented individually or in any suitable sub-combination in multiple embodiments. Furthermore, although features may be described above as functioning in certain combinations, and even initially stated so, one or more features of a claimed combination may be removed from the combination, and the claimed combination may involve sub-combinations or variations thereof.

[0231] Similarly, while operations may be depicted in a specific order in the accompanying drawings, this should not be construed as requiring such operations to be performed in the specific order shown or in an ordered sequence, or that all illustrated operations can be performed to achieve the desired result. In some cases, multitasking and parallel processing may be advantageous. Furthermore, the separation of various system patterns and components in the embodiments described above should not be construed as requiring such separation in all embodiments, and it should be understood that the described program components and systems can generally be integrated together in a single software product or packaged into multiple software products.

[0232] Specific embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims.

Claims

1. A method for determining information about a sample, comprising: An ion beam is directed to a region of a sample to release charged particles from that region of the sample, wherein the directed ion beam is pulsed at a first repetition rate. In response to a gate signal synchronized with the repetition rate of the pulsed ion beam, a first subset of the released charged particles is deflected from a first path to a second path different from the first path; and The first subset of the released charged particles is detected in a time-of-flight (TOF) mass spectrometer to determine information about the sample, wherein the gate signal sets a common reference time for the TOF mass spectrometer for the first subset of charged particles released by each pulse of the ion beam.

2. The method according to claim 1, wherein, Based on the difference between the detection time of the time of flight of charged particles in the first subset and the common reference time, the TOF mass spectrometer distinguishes the mass-to-charge ratio differences among charged particles in the first subset.

3. The method according to claim 1 or claim 2, wherein, The gate signal is synchronized with the repetition rate of the pulsed ion beam so that the first subset includes a substantial portion of at least one type of the released charged particles propagating along the first path.

4. The method according to claim 3, wherein, The aforementioned substantial portion includes more than 50%.

5. The method according to claim 3, wherein, The aforementioned substantial portion includes more than 70%.

6. The method according to any one of the preceding claims, wherein, The pulse width of the guided ion beam is between 5 nanoseconds and 100 microseconds.

7. The method according to claim 6, wherein, The pulse width of the guided ion beam is between 100 nanoseconds and 50 microseconds.

8. The method according to claim 6, wherein, The pulse width of the guided ion beam is between 1 microsecond and 50 microseconds.

9. The method according to any one of the preceding claims, wherein, The first repetition rate is between 1 kHz and 200 kHz.

10. The method according to claim 9, wherein, The first repetition rate is between 10 kHz and 100 kHz.

11. The method according to any one of the preceding claims, wherein, The deflection from the first path to the second path is between 60 and 120 degrees.

12. The method according to any one of the preceding claims, wherein, The gate signal includes a voltage pulse applied to the ion optical electrode to cause the deflection.

13. The method according to claim 12, wherein, The pulse width of the voltage pulse is between 500 nanoseconds and 50 microseconds.

14. The method according to claim 12, wherein, Depending on the mass of the released charged particles, the released charged particles travel along the first path at different speeds, and wherein a time delay between the voltage pulse and the pulsed ion beam is set to select from the released charged particles a range of masses that will be within the first subset.

15. The method according to claim 14, wherein, The time delay between the voltage pulse and the pulsed ion beam is set such that the heavier masses of the released charged particles are selected into the first subset of charged particles.

16. The method according to any one of the preceding claims, further comprising blocking a second subset of the released charged particles propagating along the first path from entering the chamber containing the TOF mass spectrometer.

17. The method according to claim 16, wherein, The obstruction includes the use of an adjustable mechanical stop.

18. The method according to claim 16, wherein, The barrier includes the use of an adjustable electromagnetic field generated by an ion optical electrode.

19. The method of claim 16, wherein, The blocking of the second subset occurs during a time period different from the deflection and is also synchronized with the repetition rate of the pulsed ion beam.

20. The method according to any one of the preceding claims, wherein, The released charged particles include secondary element atomic ions derived from mass tags associated with the sample.

21. The method according to claim 20, wherein, The pulsed ion beam releases the charged particles by ionizing the mass tag.

22. The method according to claim 20, wherein, The sample is a flat sample containing biological material on a conductive substrate.

23. The method of claim 1, further comprising: The pulsed ion beam is scanned relative to the sample to irradiate additional regions of the sample, and the deflection and detection are further performed on each of the additional regions of the sample.

24. A mass spectrometry system, comprising: A pulsed ion source configured to direct an ion beam to a region of a sample to release charged particles from the region of the sample, wherein the pulsed ion source generates pulses of the ion beam at a first repetition rate; An ion optical device, wherein the ion optical device is controllable to adjustably deflect a first subset of released charged particles from a first path to a second path different from the first path; An ion optics controller, coupled to the pulsed ion source and configured to generate a gate signal to deflect the first subset from the first path to the second path, the gate signal being synchronized with the repetition rate of the pulsed ion beam; and A time-of-flight (TOF) mass spectrometer is configured to detect a first subset of the released charged particles to determine information about the sample, wherein the gate signal sets a common reference time for the TOF mass spectrometer for the first subset of charged particles released by each pulse of the ion beam.

25. The system according to claim 24, wherein, The TOF mass spectrometer is configured to distinguish mass-to-charge ratio differences among charged particles in the first subset based on the difference between the detection time of the flight time of charged particles in the first subset and the common reference time.

26. The system according to claim 24 or claim 25, wherein, The gate signal is synchronized with the repetition rate of the pulsed ion beam so that the first subset includes a substantial portion of at least one type of the released charged particles propagating along the first path.

27. The system according to claim 26, wherein, The aforementioned substantial portion includes more than 50%.

28. The system according to claim 26, wherein, The aforementioned substantial portion includes more than 70%.

29. The system according to any one of claims 24 to 28, wherein, The pulse width of the guided ion beam is between 5 nanoseconds and 100 microseconds.

30. The system according to claim 29, wherein, The pulse width of the guided ion beam is between 100 nanoseconds and 50 microseconds.

31. The system according to claim 29, wherein, The pulse width of the guided ion beam is between 1 microsecond and 50 microseconds.

32. The system according to any one of claims 24 to 31, wherein, The first repetition rate is between 1 kHz and 200 kHz.

33. The system according to claim 32, wherein, The first repetition rate is between 10 kHz and 100 kHz.

34. The system according to any one of claims 24 to 33, wherein, The deflection from the first path to the second path is between 60 and 120 degrees.

35. The system according to any one of claims 24 to 34, wherein, The gate signal includes a voltage pulse, and the ion optics controller is configured to apply the gate signal to the ion optics electrode to induce the deflection.

36. The system according to claim 35, wherein, The pulse width of the voltage pulse is between 500 nanoseconds and 50 microseconds.

37. The system according to claim 35, wherein, Depending on the mass of the released charged particles, the released charged particles travel along the first path at different speeds, and wherein the ion optical controller is configured to set a time delay between the voltage pulse and the pulsed ion beam to select from the released charged particles a range of masses that will be within the first subset.

38. The system according to claim 37, wherein, The ion optical controller is configured to set the time delay between the voltage pulse and the pulsed ion beam such that the heavier mass of the released charged particles is selected into the first subset of charged particles.

39. The system according to any one of claims 24 to 38, further comprising a blocking member arranged to block a second subset of the released charged particles propagating along the first path from entering the chamber containing the TOF mass spectrometer.

40. The system according to claim 39, wherein, The blocking component includes an adjustable mechanical stop.

41. The system according to claim 39, wherein, The ion optical controller is configured to generate one or more electromagnetic fields using ion optical electrodes to guide the second subset of the released charged particles onto the blocking member.

42. The system according to claim 39, wherein, The blocking of the second subset occurs during a time period different from the deflection and is also synchronized with the repetition rate of the pulsed ion beam.

43. The system according to any one of claims 24 to 42, wherein, The released charged particles include secondary element atomic ions derived from mass tags associated with the sample.

44. The system according to claim 43, wherein, The pulsed ion beam releases the charged particles by ionizing the mass tag.

45. The system according to claim 43, wherein, The sample is a flat sample containing biological material on a conductive substrate.

46. ​​The system according to claim 24, wherein: The ion optical controller is configured to: One or more control signals are generated, which cause the pulsed ion source to scan the pulsed ion beam relative to the sample to irradiate additional regions of the sample; as well as One or more control signals are generated, which cause the ion optics to adjustably deflect a first subset of released charged particles corresponding to each additional region from a first path to a second path different from the first path; as well as The TOF mass spectrometer is configured to detect the first subset of the released charged particles corresponding to each additional region.

Images