Determination of plume arrival time in laser ablation

Abstract

A method and system for calculating the arrival time window of a plume at an analyzer is presented. The plume is generated from a tissue or geological sample by laser ablation. In some embodiments, external sensors measure ambient parameters such as temperature and pressure to more accurately calculate the arrival time window. In other embodiments, direct measurements of the plume's arrival time or non-contact measurements of the plume's flow velocity in a pipe are used to determine the arrival time window of the plume at the analyzer.

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 428,912, filed November 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical Field

[0002] This disclosure relates to laser ablation and the generation and analysis of plumes of biological material. [Background technology]

[0003] background In existing Image Mass Cytometry™ (IMC™) or Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICPMS) systems, each pixel of an image corresponding to a sample is generated by analyzing one or more plumes created by ablating the portion of the sample corresponding to that pixel. A laser pulse can be used to convert a solid sample into a plume of nanoparticles.

[0004] To accurately correlate imaging mass cytometry (IMC) and other laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) imaging techniques, some method is needed to correlate the production of the material analyzed for a given pixel in the image with the corresponding mass spectrum (or spectra) produced from that material. In this way, the acquisition system generates a total "count" per pixel.

[0005] In a typical IMC or LA-ICPMS system, a solid sample is converted into a plume of nanoparticles by at least one laser pulse. Pulse trains can also be used, depending on sample, laser, and pixel size considerations. The aerosol plume expands into an ablation gas (usually helium). The ablation gas flows over the sample surface, carrying the plume material to an ionization source, usually through a transfer tube for downstream analysis.

[0006]

[0006] To achieve accurate correlation between the mass spectrum (or mass spectra) of one or more plumes and a particular pixel in an image, knowledge of the exact travel time of the plume from the ablation location to the downstream analysis stage is required. Accurate correlation between one or more mass spectra and one or more ablation locations on the sample allows the acquisition system to generate the correct total "count" per pixel. In other words, this allows the acquisition system to correctly tally data from one or more plumes corresponding to the same pixel. As pixel acquisition rates increase, the requirement for good timing fidelity also increases, as the time width of the acquisition window decreases as the pixel acquisition rate increases. Therefore, the higher the acquisition rate, the larger the percentage of the window width that fixed timing jitter or drift will be. Summary of the Invention [Means for solving the problem]

[0007] overview

[0007] In one aspect, a method for use in imaging mass spectrometry is disclosed, the method comprising generating a plume from sample tissue using laser ablation, causing the plume to flow in a carrier gas along a plume transport path to an analyzer, measuring a set of parameters of the carrier gas at one or more locations along the plume transport path, the set of parameters including at least one of temperature and pressure, and utilizing the set of parameters in combination with a flow rate of the carrier gas to determine the arrival time of the plume at the analyzer.

[0008] The plume arrival time can be determined by applying one or more corrections based on the amount of variation of a set of parameters relative to a reference set of parameters. In some embodiments, a mass flow controller (MFC) can be used to measure the flow rate of the plume, and the MFC flow rate can be adjusted based on the set of parameters to correct for the arrival time of the plume at the analyzer. In some embodiments, the set of parameters can be updated based on a predetermined schedule.

[0009]

[0009] In a related aspect, a method for use in imaging mass spectrometry is disclosed, the method comprising generating a plume from sample tissue using laser ablation, causing the plume to flow to an analyzer, measuring the plume arrival time at the analyzer for a plurality of plumes, and determining the timing of a plume acquisition window based on the measurements, wherein determining the plume acquisition window is based on an external standard contained on the same substrate as the tissue sample.

[0010]

[0010] By way of example, the external standard may be at least one of a fused bead and a tuning tape.

[0011]

[0011] In a related aspect, a method for use in imaging mass spectrometry is disclosed, the method including generating a plume from sample tissue using laser ablation, causing the plume to flow through a transfer tube to an analyzer, measuring the flow velocity of the plume in the transfer tube with a non-contact sensor, and calculating a plume acquisition window based on the measurement.

[0012]

[0012] By way of example, the non-contact sensor may be an ultrasonic transducer.

[0013]

[0013] Some embodiments provide an imaging mass spectrometry system that includes a laser configured to generate radiation suitable for causing ablation of the sample to generate an ablation plume; an analyzer configured to receive the ablation plume and generate a mass imaging analysis of the plume; a transfer tube positioned to receive the plume and provide a flow path for delivering the plume to the analyzer; a carrier gas source configured to provide a flow of carrier gas over the sample to promote flow of the ablation plume through the transfer tube; at least one temperature sensor and pressure sensor positioned relative to the transfer tube to measure at least one of temperature and pressure at one or more positions along the flow path; and a controller in communication with the temperature and pressure sensors, the controller configured to receive any of the temperature and pressure data generated by the respective sensors and process the data to correct a reference travel time of the plume from the ablation site to the analyzer.

[0014] In some embodiments, the imaging mass spectrometry system can further include a mass flow controller (MFC) configured to measure a flow rate of the plume. In some embodiments, the controller can be further configured to send a control signal to the MFC to adjust the MFC flow rate based on the corrected transit time.

[0015]

[0015] Various aspects of the embodiments can be better understood by reference to the following detailed description in conjunction with the associated drawings, which are briefly described below.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS The drawings are not necessarily to scale or exhaustive. Instead, emphasis is generally placed on illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments consistent with the present disclosure. Together with the description, the drawings serve to explain the principles of the present disclosure. [Brief explanation of the drawings]

[0017]

[0017] [Figure 1]

[0018] 3 is a flow chart illustrating the various steps in an embodiment of a method according to the invention for correcting the arrival time of a plume. [Figure 2]

[0019] 1 illustrates a schematic diagram of a system for correcting the arrival time of a plume according to one embodiment. [Figure 3]

[0020] An example of a plume associated with a TAG and potential crosstalk is shown, showing the observed change in the optimal delay in plume position relative to the laser TAG trigger (also known as Plume Start Delay) over time. [Figure 4]

[0021] Experimental determination of plume onset values ​​is shown. [Figure 5]

[0022] Experimental measurements of temperature and pressure are shown. [Figure 6]

[0023] Experimental correction results for plume initiation error are presented. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description

[0024] The present embodiments relate to the analysis of samples, such as geological or biological samples (e.g., tissues and cells), and / or individual microparticles or nanoparticles, for analysis such as mass spectrometry and mass cytometry using inductively coupled plasma and similar elemental ionization techniques.

[0019]

[0025] As described above for a typical IMC or LA-ICPMS system, a solid sample is converted into an aerosol plume of nanoparticles by at least one laser pulse. Pulse trains can also be used, depending on sample, laser, and pixel size considerations. The aerosol plume expands into an ablation gas (usually helium). The ablation gas flows over the sample surface, typically through a transfer tube, carrying the plume material downstream to the ionization source of the mass analyzer.

[0020]

[0026] Due to engineering constraints related to gas handling, RF shielding, and thermal management, there is typically a minimum distance that the plume must travel to be transported between the sample and the downstream ionization source.

[0021]

[0027] In some conventional systems (e.g., some conventional LA-ICPMS systems), such distance may be 40-60 cm, allowing dual mode use of the mass analyzer, including pause mode and imaging mode.

[0022]

[0028] This distance results in delay and is an important consideration for establishing a correlation between the generation of a plume at the ablation site, e.g., generated by laser ablation, associated with a pixel generated by an image analyzer, and the detection of the plume by a mass analyzer. Establishing such a correlation between plume generation and detection by a mass analyzer requires consideration of the transit time of the plume from the ablation site to the mass analyzer, e.g., through the ionized plasma, ion optics, and detector. In many cases, the time the plume spends traveling between the ablation site and the ionized plasma of the downstream mass analyzer, e.g., through a transfer tube, accounts for a significant portion of this time, e.g., about 10-40 ms, compared to a typical transit time of about 30 μs within the ion optics.

[0023]

[0029] Imaging Mass Cytometry™ (IMC) requires good time fidelity of the arrival of the ablation plume into the plasma in order to construct an ion image from the sequence of detected plumes. In this context, “time fidelity” refers to how well the arrival time of the plume into the plasma correlates with the time of the corresponding laser shot, and how narrowly distributed the arrival times of the individual particles that make up the plume are. These two effects may be described as “arrival time jitter” between individual plumes and as “temporal spreading” of individual plumes during their passage. Without sufficient timing fidelity, plumes from successive laser ablation events may merge and blur, resulting in undesirable “smearing” of the final tissue image (reconstructed from the ion signal) or other serious imaging artifacts.

[0024]

[0030] Conventional approaches for calculating the correlation between pixel plume generation and plume detection assume that the time it takes for the plume to travel through the transfer tube is constant for different plumes arriving at different times. Therefore, in such conventional approaches, the plume transit time (also referred to herein as the plume transit time interval) is measured as a function of one or more parameters of the carrier gas flow used to facilitate the transport of the plume through the transfer tube to the downstream mass analyzer. The delay time between the trigger signal for generating the laser ablation pulse and the start of the mass analyzer detector acquisition window is set based on this plume transit time interval. However, assuming a constant delay time is only a valid assumption if both the ambient temperature and pressure remain constant. In reality, the ambient temperature and / or pressure may change during the data acquisition interval, thus affecting the plume transit time from the ablation site to the mass analyzer. For example, fluctuations in ambient temperature and / or pressure may affect the operation of a mass flow controller utilized to establish the desired flow rate of the carrier gas, thereby introducing errors into conventional techniques for determining plume transit time.

[0025]

[0031] By way of example and to facilitate discussion of various embodiments of the present invention, a brief description of the operation of a mass flow controller will be provided. A mass flow controller (MFC) is a device that uses a thermal sensor and a proportional valve to set a desired gas flow rate through it. It works by assuming that the heat transfer capacity of a gas depends only on the number of gas molecules flowing over the sensor unit, although some MFCs may also compensate for thermal variations in the heat capacity of the gas. By adding heat to the gas and measuring the increase in gas temperature at a point downstream, the flow rate through the sensing element can be inferred. A valve within the MFC is then used to stabilize the flow (in molecules / second) at the desired setpoint selected by the user.

[0026]

[0032] The flow rate through an MFC is given by the units (SLPM) used to specify the mass flow rate through the MFC. The units are "standard liters per minute," where "standard" refers to standard temperature and pressure conditions, i.e., 0°C and 100kPa. It is only at standard temperature and pressure (STP) conditions that the mass flow rate (SLPM) from an MFC equals the volumetric flow rate (L / min). Deviations from STP conditions will affect the volumetric flow output.

[0027]

[0033] At typical conditions in a gas conduit, the behavior of MFC gases is well modeled by the ideal gas law, which models the volume of a gas as linearly proportional to its temperature and inversely proportional to its pressure.

number

[0028]

[0034] In Equation 1, V represents the volume of the gas under consideration. The number n represents the number of moles of gas in the volume. R represents the gas constant, 8.314 J / mol.K, which is related to the Boltzmann constant through Avogadro's number multiplied by the Boltzmann constant. T and P represent the temperature and pressure of the gas in volume V, respectively.

[0029]

[0035] It can be seen that if the flux of gas molecules is constant (i.e., a constant n), as would typically be expected in a gas conduit at a pressure of about 1 atm and room temperature, then V will be a constant only if both T and P are constant.

[0030]

[0036] In gas conduits, the assumption that the plume transit time from the ablation site to the ionized plasma is constant is accurate only if the gas flow velocity remains constant during data acquisition. However, the gas flow velocity is determined by the volumetric flow rate and the dimensions of the inner channel of the transfer tube. The gas volumetric flow rate can depend on the ambient temperature and pressure. As a result, the gas flow velocity can also depend on the ambient pressure and temperature. Drift in the gas flow velocity leads to a drift in the optimal plume onset. In IMC or other laser ablation regions with high pixel rates, it was unclear whether a drift in plume onset would affect the measurement of the ion signal at a particular pixel.

[0031]

[0037] In experiments measuring the change in the optimal delay, also known as the plume onset delay, a large shift (approximately 20 TOF pushes) in the plume's position relative to the laser TAG trigger was observed over time, as shown in Figure 6. As shown in Figure 3, each plume is associated with a "tag" input. This is a channel in the acquisition system that identifies the arrival of the laser pulse and allows the system to identify the range of the TOF (time-of-flight) push corresponding to that laser shot. For example, the portion of the signal from one laser shot that falls inside another's window is the "crosstalk" (CT) of that plume. Typically, several plumes are summed together to create a cleaner, more representative "accumulation" transient, which is used to measure crosstalk. TOF pushes are used in the software as the unit of timing for the CyTOF®. In the CyTOF XT™, the time between two consecutive pushes is 13 μs.

[0032]

[0038] The observed shift was found to correlate with the recorded temperature and pressure changes during data acquisition. In this particular experiment, which involved operation at a pulse ablation rate of approximately 800 Hz, the acquisition window was set to a width of 96 pushes (corresponding to the duration between successive laser shots, measured in TOF push intervals). Thus, in such a case, a 20-push drift in the plume position accounts for a non-negligible fraction of a typical plume width. This shift manifests itself as an additional source of pixel-to-pixel crosstalk, i.e., the spread of the signal from the drifting plume across multiple pixels.

[0033]

[0039] Without being limited to any particular theory, it is believed that such variations are due to variations in the velocity of gas flow through the transfer tube. Therefore, shortening the transfer tube may reduce such variations. For example, a given change in flow rate will result in a shift that is 10 times larger in a 40 cm long tube than in a 4 cm long tube. However, shortening the transfer tube is not always easy, or even possible. Additionally, it is desirable to minimize such variations, whatever the length of the transfer tube.

[0034]

[0040] FIG. 1 is a flowchart illustrating various steps in one embodiment of a method for use in imaging mass spectrometry. The method includes triggering a laser to generate an ablative radiation pulse directed at a sample, causing ablation of a portion of the sample to generate a plume, and using a carrier gas to transport the plume downstream to a mass imaging analyzer via a flow path through a transfer tube. Correlation between the laser trigger and detection of the plume by the mass analyzer can be established using a reference time corresponding to the time it takes the plume to travel from the ablation site to the mass analyzer. The temperature and / or pressure of the carrier gas at one or more locations along the flow path can be monitored, for example, according to a predetermined schedule or continuously, and such temperature / pressure measurements can be used to apply a correction factor to the reference time to substantially compensate for any inaccuracies in the reference time due to fluctuations in either temperature and / or pressure. Such automatic correction allows for improved automation of the correlation of the time spent by the plume traveling between the ablation site and the plasma, improving the assignment of ion signals to corresponding pixels.

[0035]

[0041] According to some embodiments, compensation for such variations in gas velocity due to changes in ambient temperature and / or pressure can be achieved by measuring multiple operating parameters during initial tuning of the instrument. More specifically, in some embodiments, the tuning parameters can include T, P, and P, i.e., initial temperature (T), initial pressure (P), and optimal plume onset (P). Often, all tuning parameters are recorded simultaneously. As noted above, plume onset (P) refers to the time delay utilized by systems according to various embodiments of the present teachings, corresponding to the time interval between the time when the ablation laser pulse associated with a pixel is triggered and the beginning of the time window for integrating the signal of that particular pixel. In some embodiments, P can be measured in units of TOF push.

[0036]

[0042] In some embodiments, the temperature and pressure can be monitored (measured) multiple times during data acquisition, e.g., periodically. The temperature and pressure data can also be used to correct the plume transit time through the transfer tubing. Such correction of the plume transit time can be achieved, for example, by adjusting the plume start time (PS) using the acquired temperature and pressure data.

[0037]

[0043] For example, in some embodiments, periodically during data acquisition, the current value of PS may be corrected using the following relationship:

number

[0038]

[0044] During the ceremony, P.S. n represents the nth correction applied to the PS0 plume onset value, and T n and P n are the measured temperature and pressure, respectively, for the nth correction.

[0039]

[0045] The above parameters a and b can be used to adjust the respective contributions of the temperature and pressure terms. In some embodiments, the values ​​of the a and b parameters can be determined theoretically and / or empirically. By way of example and not limitation, the following default values ​​can be used: a=-1 and b=1. These default values ​​reflect that the hotter the gas, the larger the volume it occupies. Thus, increasing the gas temperature reduces the plume arrival time due to the increased gas velocity traveling through the transfer pipe. The opposite is true for pressure.

[0040]

[0046] According to various embodiments, different equations can be used to compensate for temperature and pressure variations. For example, in some embodiments, the parameters a and b can be utilized to adjust the magnitude of the respective corrections. Furthermore, the above relationships for correcting pulse start time can be used over a wide range of temperatures and / or pressures. Given typical temperature and pressure variations, the amount of correction is often small enough that linearization is appropriate.

[0041]

[0047] In experiments conducted according to such an embodiment, the data showed that the method provided appropriate correction of plume onset values ​​based on ambient T and P measurements. As shown in Figure 4, laser microdissection (LMD) was recorded hourly from 4 × 100 ROIs on the tuning film during an overnight batch run. Plume onset values ​​were extracted by convolving an 800 Hz acquisition window with the raw plume data and finding peaks in the resulting waveform. Transient calibration used the same steps as the initial plume onset estimation. Ambient temperature and pressure values ​​were simultaneously recorded using a Dracal USB-PTH200 sensor unit, as shown in Figure 5. The correction results are shown in Figure 6. The standard deviation of plume onset values ​​on the tuning film is approximately 5 pushes. A correction error of less than 5 pushes would be undetectable. Temperature- and pressure-based correction showed improvement in plume onset error, suggesting that further improvement is possible by optimizing the a and b parameters for linearization.

[0042]

[0048] In some embodiments, plume onset can be corrected by actively recording plume arrival times throughout the data acquisition period and using the arrival times as feedback. These embodiments have the advantage of not requiring the assumption that plume arrival time drift depends on T and P. Rather, plume arrival time drift is directly measured and corrected. For example, changes in arrival time may be identified and used to correct the plume arrival time. For example, CyTOFs utilize tuning films (thin plastic films on microscope slides doped with controlled amounts of detectable elements) to automatically adjust various operating parameters in IMC mode. One of the parameters measured by the analyzer (which can be implemented as software (SW)) is plume onset. Measurements are made by firing laser pulses at a low repetition rate and observing the signal transients corresponding to each laser shot. By analyzing the shape and delay of the transients, SW derives an optimal value for plume onset from these transients. If the system needs to correct for plume onset drift, one possible solution is to periodically pause tissue imaging, return to the tuning film, and measure a new value for optimal plume onset. This method introduces confusion into the imaging process and complicates the SW logic. As used herein, the term "transient" refers to several plumes associated with a pixel, all grouped together. A laser shot generates a plume of particles from the sample. The plume travels into the plasma and through the plasma to the mass spectrometer. Ion signals from the ablated material generate a transient signal, often simply referred to as a transient.

[0043]

[0049] In yet another embodiment, the sample of interest contains sufficient elements that can be used to measure transients with sufficient fidelity to derive an optimal plume onset. Correction for plume onset drift can therefore be performed simultaneously with sample ablation. This approach has the disadvantage of requiring the addition of a special, relatively bright elemental stain to every sample, which adds complexity and extra work that the user must perform.

[0044]

[0050] In some embodiments, plume arrival times can be measured at intervals during data acquisition (e.g., during acquisition of imaging mass data from a tissue sample) using external standards contained on the same substrate as the sample, e.g., the same slide. Some examples of such external standards include, but are not limited to, fused beads or tuning tapes (WO2020055743A1, WO2019210233A1, which are incorporated by reference in their entirety).

[0045]

[0051] In some embodiments, plume arrival time can be measured by applying some internal standard to a sample (e.g., a tissue sample). By way of example, such an internal standard can exhibit uniform ablation characteristics and metal content and not affect the quality of the sample (e.g., tissue) image. In these embodiments, ablating the sample generates both data of interest and "real-time tuning" data that can be used to measure and correct instrument drift related to plume arrival time on the fly. In other words, in some embodiments, the optimal plume start value (PS) can be measured while imaging the tissue sample. In such a case, the optimal value of (PS) can be corrected in real time by switching to a correction value. This is in contrast to a workflow in which tissue imaging is stopped, the system switches to a tuning protocol, and then the system returns to imaging the sample after the tuning protocol.

[0046]

[0052] In some embodiments, a gas flow rate controller different from the MFC gas flow controller can be utilized to measure the gas flow rate. For example, in some such embodiments, the velocity of the gas flow through the transfer tube can be measured. In these embodiments, the gas flow measurement is preferably performed without disturbing the plume during measurement. By way of example, in some embodiments, a non-contact sensing device can be used to measure the velocity of the gas flow through the transfer tube. An example of such a non-contact sensing device is, but is not limited to, an ultrasonic transducer.

[0047]

[0053] In some embodiments, the MFC flow rates for both the makeup gas and / or the chamber gas can be adjusted based on the measured temperature and pressure to stabilize the plume arrival time. In such embodiments, it is important to note that variations in the mass flow rate of gas through the injectors of the MFC flow controllers can affect the composition of the plasma and potentially have an effect on sensitivity or oxide formation. Therefore, in these embodiments, users can compensate for this effect by adjusting the RFG power to maintain stable sensitivity / oxide formation.

[0048]

[0054] In some embodiments, optimal plume initiation can be maintained constant by varying the gas flow. The next parameter to control is plume broadening. Again, two parameters (makeup gas and chamber gas) can be used to keep plume initiation and plume broadening relatively stable. However, changes in gas flow into the plasma can also affect the temperature in the ionization zone, which can affect ionization efficiency and oxide production. Therefore, in some cases, two gases alone may not be sufficient to stabilize the three parameters of plume initiation, plume broadening, and plasma temperature in the ionization region. In some embodiments, RF power can be used to control the temperature in the ionization region.

[0049]

[0055] The present teachings advantageously enable the time width of the data acquisition window associated with each pixel to be reduced, thereby increasing the pixel acquisition rate. As discussed above, a narrower data acquisition window can make measurements more sensitive to plume arrival time drift due to variations in ambient conditions. In various embodiments, correction for plume arrival time drift can be used to acquire data at a high pixel rate while maintaining image quality.

[0050]

[0056] FIG. 2 shows a schematic diagram of an imaging mass spectrometry system 100 according to one embodiment, including a laser 101 that generates a beam of laser radiation suitable for ablation of a sample 104. The laser 101 operates under the control of a controller 102 that provides a trigger for the laser to emit an ablation pulse. Interaction of the laser ablation pulse with the sample causes ablation of a portion of the sample exposed to the laser beam, generating a plume, e.g., a nanoparticle plume. The plume spreads in a carrier gas (also referred to herein as an ablation gas), which flows over the sample surface and carries the plume material downstream to a mass imaging analyzer 106 via a transfer tube. A temperature / pressure measurement device 107, operating under the control of the controller 102, can be coupled to the transfer tube 105 to measure the temperature and pressure of the carrier gas and the entrained plume at one or more locations along the path provided by the transfer tube. The controller can receive these temperature / pressure measurements and utilize the measurements in the manner disclosed herein to generate correction factors to apply to a reference transit time that the controller can calculate based on the carrier gas flow rate and the distance between the ablation location and the downstream imaging mass analyzer (e.g., the ionization chamber of the downstream imaging mass analyzer).

[0051]

[0057] As mentioned above, in some embodiments, instead of measuring temperature and / or pressure, other systems can be utilized to: (1) Actively record the arrival time of the plume with the analyzer 106 throughout the data acquisition period and use it as feedback using the controller 102. (2) Plume arrival times can be measured at intervals during data acquisition (e.g., during acquisition of imaging mass data from a tissue sample) using external standards contained on the same substrate as the sample, e.g., the same slide. (3) In some embodiments, the plume arrival time can be measured by application of some internal standard to the sample (e.g., tissue sample).

[0052] Conclusion and General Terms

[0058] The above detailed description makes reference to the accompanying drawings. The same or similar reference numbers may be used in the drawings or description to refer to the same or similar parts. Also, similarly named elements may perform similar functions and may be similarly designed, unless otherwise specified. Details have been described to provide an understanding of the exemplary embodiments. Embodiments, for example, alternative embodiments, may be practiced without some of these details. In other instances, well-known techniques, procedures, and components have not been described in detail to avoid obscuring the described embodiments.

[0053]

[0059] The foregoing description of the embodiments has been presented for illustrative purposes only. It is not exhaustive and does not limit the embodiments to the precise form disclosed. While several exemplary embodiments and features have been described, modifications, adaptations, and other implementations may be possible without departing from the spirit and scope of the embodiments. Thus, unless expressly stated otherwise, the description may relate to one or more embodiments and should not be construed as limiting the embodiments as a whole. This is true regardless of whether the disclosure states that a feature relates to "a," "the," "one," "one or more," "some," or "various" embodiments. As used herein, the singular forms "a," "an," and "the" may include the plural unless the context clearly dictates otherwise. Furthermore, the term "coupled" does not exclude the presence of intermediate elements between the coupled items. Also, a statement that a feature may be present indicates that the feature may be present in more than one embodiment.

[0054]

[0060] In this disclosure, the terms "include," "comprise," "contains," and "have," when used after a collection or system, refer to an open inclusion and do not exclude other unlisted elements from being added to the collection or system. Furthermore, unless otherwise stated or inferred from the context, the conjunction "or," when used, is inclusive, meaning instead and / or, rather than exclusive. Furthermore, when these terms are used, a subset of a set may include one or more (including all) elements of the set.

[0055]

[0061] Furthermore, as used in this disclosure, unless stated or deduced otherwise, a first variable is an increasing function of a second variable if the first variable does not decrease but instead generally increases when the second variable increases. Conversely, a first variable is a decreasing function of a second variable if the first variable does not increase but instead generally decreases when the second variable increases. In some embodiments, a first variable can be an increasing or decreasing function of a second variable if the first variable is directly or inversely proportional to the second variable, respectively.

[0056]

[0062] The disclosed systems, methods, and apparatus are not limited to any particular aspect or feature or combination thereof, and the disclosed systems, methods, and apparatus do not require that any one or more particular advantages be present or problems be solved. While any theory of operation is for ease of explanation, the disclosed systems, methods, and apparatus are not limited to such theory of operation.

[0057]

[0063] Modifications and variations are possible in light of the above teachings or may result from practicing the embodiments. For example, the described steps need not be performed in the same order or to the same extent as discussed. Likewise, various steps may be omitted, repeated, combined, or performed in parallel as necessary to achieve the same or similar purpose. Likewise, the described systems need not necessarily include all components described in the embodiments, and may include other components not described in the embodiments. Accordingly, the embodiments are not limited to the details set forth above, but instead are defined by the appended claims in light of their full scope of equivalents. Furthermore, the present disclosure is directed to entirely novel and unobvious features and aspects of the various disclosed embodiments, both alone and in various combinations and subcombinations with one another.

[0058]

[0064] While the present disclosure has been particularly described in conjunction with specific embodiments, many alternatives, modifications, and variations will become apparent in light of the foregoing description. It is therefore intended by the appended claims to embrace any such alternatives, modifications, and variations that fall within the true spirit and scope of the present disclosure.

Claims

1. 1. A method for use in imaging mass spectrometry, comprising: generating a plume from the sample tissue using laser ablation; causing the plume to flow in a carrier gas along a plume transport path to an analyzer; measuring a set of parameters of the carrier gas at one or more locations along the plume transport path, the set of parameters including at least one of temperature and pressure; utilizing the set of parameters in combination with the carrier gas flow rate to determine the arrival time of the plume at the analyzer; A method comprising:

2. The method of claim 1 , wherein determining the arrival time comprises applying one or more corrections based on an amount of variation of the set of parameters relative to a reference set of parameters.

3. 10. The method of claim 1, further comprising measuring a flow rate of the plume using a mass flow controller (MFC), and adjusting the MFC flow rate based on the set of parameters to correct for plume arrival time at the analyzer.

4. The method of claim 1 , further comprising updating the set of parameters according to a predetermined schedule.

5. 1. A method for use in imaging mass spectrometry, comprising: generating a plume from the sample tissue using laser ablation; causing the plume to flow to an analyzer; measuring plume arrival times at the analyzer for a plurality of plumes; determining plume acquisition window timing based on said measurements; and Including, The method, wherein said determining said plume acquisition window is based on an external standard contained on the same substrate as the tissue sample.

6. The method of claim 5 , wherein the external standard is one of a fused bead and a tuning tape.

7. 1. A method for use in imaging mass spectrometry, comprising: generating a plume from the sample tissue using laser ablation; causing the plume to flow through a transfer tube to an analyzer; measuring the flow velocity of the plume in the transfer pipe with a non-contact sensor; calculating a plume acquisition window based on said measurements; A method comprising:

8. The method of claim 7 , wherein the non-contact sensor comprises an ultrasonic transducer.

9. 1. An imaging mass spectrometry system, comprising: a laser configured to generate radiation suitable for causing ablation of the specimen to generate an ablation plume; an analyzer configured to receive the ablation plume and produce a mass imaging analysis of the plume; a transfer tube positioned to receive the plume and provide a flow path for delivering the plume to the analyzer; a carrier gas source configured to provide a flow of carrier gas over the specimen to facilitate flow of the ablation plume through the transfer tube; at least one of a temperature sensor and a pressure sensor positioned relative to the transfer tube to measure at least one of a temperature and a pressure at one or more locations along the flow path; a controller in communication with the temperature sensor and the pressure sensor, the controller configured to receive any of the temperature and pressure data generated by the respective sensors and process the data to correct a reference travel time of the plume from the ablation site to the analyzer; Including, the system.

10. The system of claim 9 , further comprising a mass flow controller (MFC) configured to measure a flow rate of the plume.

11. 11. The system of claim 10, wherein the controller is further configured to send a control signal to the MFC to adjust the MFC flow rate based on the corrected transit time.

Images