Lock mass library for internal correction

Abstract

A method of calibrating or optimising an analytical instrument is disclosed that comprises analysing analyte from a sample using an analytical instrument, determining a sample type of the sample based on analysis of analyte from the sample, identifying one or more species of the analyte that are known to be endogenous to the determined sample type, and calibrating or optimising the analytical instrument using the one or more identified endogenous species.

Claims

1. A method of calibrating or optimising an analytical instrument comprising: analysing analyte from a sample using an analytical instrument by measuring one or more physico-chemical properties of said analyte; determining a sample type of said sample based on one or more measured physico-chemical properties of analyte from said sample, wherein: said sample type is one or more of: (a) a phenotypic characteristic, (b) a genotypic characteristic, and/or (c) a disease state of said sample, or said sample is a microbial sample and said sample type is one or more of: (A) information about the genus, (B) information about the species, and/or (C) information about a strain of a microbe present in said sample; identifying one or more species of said analyte that are known to be endogenous to said determined sample type; and calibrating or optimising said analytical instrument using one or more measured physico-chemical properties of said one or more identified endogenous species; wherein said step of identifying one or more species of said analyte that are known to be endogenous to said determined sample type comprises determining whether one or more species of said analyte correspond to one or more species for said determined sample type that are present in a predetermined list or library, wherein said predetermined list or library includes one or more selected species that are endogenous to each of a plurality of known sample types.

2. A method as claimed in claim 1, wherein said sample comprises: (i) a living or non-living tissue sample; (ii) a histopathology sample; or (iii) a microbe culture.

3. A method as claimed in claim 1, further comprising ionising said analyte and/or said sample using: (i) Rapid Evaporative Ionisation Mass Spectrometry (REIMS); and/or (ii) Desorption ElectroSpray Ionisation (DESI) so as to produce a plurality of ions.

4. A method as claimed in claim 1, wherein said step of analysing said analyte from said sample comprises measuring one or more physico-chemical properties of said analyte and/or said plurality of ions, wherein said one or more physico-chemical properties comprise: (i) mass or mass to charge ratio; (ii) mass or mass to charge ratio peak shape or width; (iii) ion mobility, collision cross section or interaction cross section; and/or (iv) ion mobility, collision cross section or interaction cross section peak shape or width.

5. A method as claimed in claim 1, wherein said step of determining said sample type of said sample comprises determining said sample type of said sample based on said analysis of said analyte and/or on prior analysis of analyte from said sample.

6. A method as claimed in claim 1, wherein said step of determining said sample type comprises determining said sample type from a plurality of known sample types.

7. A method as claimed in claim 1, wherein said sample type comprises: (i) a diseased or non-diseased type of living or non-living tissue; (ii) a diseased or non-diseased type of histopathology sample; or (iii) a diseased or non-diseased type of microbe culture.

8. A method as claimed in claim 1, wherein said step of identifying one or more species of said analyte that are known to be endogenous to said determined sample type comprises identifying one or more species of said analyte that are known to be endogenous to said determined sample type based on said analysis of said analyte and/or on prior analysis of analyte from said sample.

9. A method as claimed in claim 1, wherein said one or more endogenous species comprise one or more lipids.

10. A method as claimed in claim 1, further comprising using said calibrated or optimised analytical instrument for subsequent analysis of analyte from said sample.

11. A method as claimed in claim 1, wherein said step of calibrating or optimising said analytical instrument comprises: generating a calibration for said analytical instrument; and/or updating, modifying and/or correcting an existing calibration for said analytical instrument; and/or optimising one or more operational parameters of said analytical instrument.

12. A method as claimed in claim 1, wherein said step of identifying one or more species of said analyte that are known to be endogenous to said determined sample type comprises identifying one or more species of said analyte that are known to be endogenous to said determined sample type and that are sufficiently stable, consistent, abundant, clear and/or isolated.

13. A method as claimed in claim 1, further comprising postponing said calibration or optimisation of said analytical instrument when one or more of said known endogenous species cannot be identified or accurately identified.

14. A method as claimed in claim 13, further comprising recording when one or more of said known endogenous species cannot be identified or accurately identified and/or when said calibration or optimisation is postponed.

15. A method as claimed in claim 13, further comprising reducing a confidence or weight assigned to data acquired when one or more of said known endogenous species cannot be identified or accurately identified and/or when said calibration or optimisation is postponed.

16. A method as claimed in claim 1, comprising while analysing analyte from said sample, repeatedly performing said steps of: determining said sample type of said sample; identifying one or more species in said analyte that are known to be endogenous to said determined sample type; and calibrating or optimising said analytical instrument using said one or more identified endogenous species.

17. An analytical instrument comprising: an analyser arranged and adapted to analyse analyte from a sample by measuring one or more physico-chemical properties of said analyte; and a control system arranged and adapted: (i) to determine a sample type of said sample based on one or more measured physico-chemical properties of analyte from said sample, wherein: said sample type is one or more of: (a) a phenotypic characteristic, (b) a genotypic characteristic, and/or (c) a disease state of said sample; or wherein said sample is a microbial sample and said sample type is one or more of: (A) information about the genus, (B) information about the species, and/or (C) information about a strain of a microbe present in said sample; (ii) to identify one or more species in said analyte that are known to be endogenous to said determined sample type by determining whether one or more species of said analyte correspond to one or more species for said determined sample type that are present in a predetermined list or library, wherein said predetermined list or library includes one or more selected species that are endogenous to each of a plurality of known sample types; and (iii) to calibrate or optimise said analytical instrument using one or more measured physico-chemical properties of said one or more identified endogenous species.

18. A method comprising: identifying one or more species endogenous to each of one or more sample types; determining one or more values of one or more physico-chemical properties for each of said one or more species; and storing said one or more determined values for each of said one or more species together with an indication of the corresponding sample type.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 illustrates schematically an analytical instrument in accordance with various embodiments;

(3) FIG. 2 illustrates schematically the Rapid Evaporative Ionisation Mass Spectrometry (REIMS) technique according to various embodiments; and

(4) FIG. 3 illustrates schematically the Desorption ElectroSpray Ionisation (DESI) technique according to various embodiments.

DETAILED DESCRIPTION

(5) Various embodiments relating to methods for calibrating or optimising an analytical instrument, such as a mass and/or ion mobility spectrometer, will now be described.

(6) FIG. 1 illustrates an analytical instrument in accordance with various embodiments. As shown in FIG. 1, the analytical instrument may comprise an ion source 1 and an analyser 2 for analysing ions generated by the ion source 1.

(7) The ion source 1 may comprise any suitable ion source, such as a Rapid Evaporative Ionisation Mass Spectrometry (REIMS) ion source, or a Desorption ElectroSpray Ionisation (DESI) ion source. Ions generated by the ion source 1 are transferred to the analyser 2 for analysis.

(8) The analyser 2 may comprise any suitable device(s) or stage(s) for analysing analyte ions, e.g. in terms of their mass to charge ratio and/or ion mobility, such as one or more devices for separating ions according to their mass to charge ratio and/or ion mobility, one or more devices for filtering ions according to their mass to charge ratio and/or ion mobility, one or more ion detectors, etc.

(9) The analytical instrument may also comprise a control system 3 that is configured to control the operation of the ion source 1 and the analyser 2, e.g. in the manner of the various embodiments described herein. The control system 3 may comprise suitable control circuitry that is operable to cause the ion source 1 and/or the analyser 2 to operate in the manner of the various embodiments described herein. The control system may also comprise suitable processing circuitry operable to perform any one or more or all of the necessary processing and/or post-processing operations in respect of the various embodiments described herein.

(10) According to various embodiments, endogenous species from a sample being analysed by the analytical instrument are used to correct the instrument calibration. According to various embodiments, the instrument is calibrated or optimised using knowledge of the possible sample types, together with knowledge of species that will be present in the possible sample types, and post-processing steps.

(11) According to various embodiments, a list or library of species that are endogenous to each of a set of known sample types is generated, e.g. prior to analysis of a sample and/or offline. The set of known sample types may include sample types that are expected based on the particular sample being or to be analysed.

(12) For example, the sample may be a living tissue, a histopathology sample, a microbe culture, etc., and the known sample types may include diseased or non-diseased types of living or non-living tissue (e.g. tissue from different organs, etc.), diseased or non-diseased types of histopathology sample, or diseased or non-diseased types of microbe culture, etc. The endogenous species may comprise, for example, one or more lipids.

(13) The library may be generated by identifying one or more species endogenous to each of one or more sample types, determining one or more values of one or more physico-chemical properties for each of the one or more species, and storing the one or more determined values for each of the one or more species together with an indication of the corresponding sample type, e.g. in a suitable memory device or storage medium.

(14) For example, in various embodiments, the theoretical mass to charge ratio (m/z) of one or more selected molecular species endogenous to various types of sample are identified and/or calculated, and stored in a library that may be indexed by sample type.

(15) According to various embodiments, one or more endogenous species are selected for each of the known sample types for inclusion in the library. This may done, for example, on the basis of the physico-chemical properties (e.g. mass to charge ratio and/or ion mobility) of the species or ions derived from the species. Various criteria for selecting the endogenous molecular species to be used may be considered and used.

(16) For example, species that give rise to ion peaks that are always or very commonly present (e.g. for the particular form of ionisation being used) and that appear at values of the physico-chemical properties that are sufficiently separated or isolated from other peaks (i.e. so as to avoid interferences) and/or that are particularly intense, etc., may be selected and used in the library.

(17) According to various embodiments, when it is desired to analyse a sample, the analytical instrument (e.g. mass and/or ion mobility spectrometer) may optionally be calibrated, e.g. using a standard calibration mixture (e.g. lock mass), prior to commencement of each experiment and a null calibration modification (or base calibration) may be initialized.

(18) According to various embodiments, during an acquisition or analysis of a sample, the following steps may be iterated: (i) the current sample type is updated based on analysis of recent data; (ii) the measured mass to charge ratio (m/z) values, peak shapes and/or metadata are substantially continuously monitored, and endogenous (molecular) species corresponding to the current sample type are identified; (iii) if possible, the calibration modification (or calibration) is modified or updated using some or all of the species identified in recently acquired data; and (iv) the current calibration modification is applied to the current data.

(19) Thus, according to various embodiments, analyte from a sample, such as a living or non-living tissue sample, a histopathology sample, or a microbe culture, is analysed.

(20) The analyte may comprise an aerosol that may have been generated, e.g., by subjecting the sample to alternating electric current at radiofrequency by, for example, using a surgical diathermy device. This analyte may be transported to the analytical instrument for analysis.

(21) Thus, according to various embodiments, the analytical instrument (e.g. mass and/or ion mobility spectrometer) may comprise or may be coupled to another device, such as a surgical diathermy device. According to various embodiments, the method may comprise the analytical instrument and/or the analyser 2 receiving analyte, e.g. from the other device.

(22) According to various embodiments, the sample, analyte or aerosol may be ionised, e.g. using known Rapid Evaporative Ionisation Mass Spectrometry (REIMS) techniques.

(23) FIG. 2 illustrates the Rapid Evaporative Ionisation Mass Spectrometry (REIMS) technique according to various embodiments.

(24) FIG. 2 illustrates a method of rapid evaporative ionisation mass spectrometry (REIMS) wherein bipolar forceps 4 may be brought into contact with in vivo tissue 5 of a patient 6. Other arrangements would be possible, such as the use of a surgical diathermy device in place of the bipolar forceps 4.

(25) An RF voltage from an RF voltage generator 7 may be applied to the bipolar forceps (electrodes) 4 which causes localised Joule or diathermy heating of the tissue 5 or sample. As a result, an aerosol or surgical plume 8 is generated. The aerosol or surgical plume 8 may then be captured or otherwise aspirated through an irrigation port of the bipolar forceps 4. The irrigation port of the bipolar forceps 4 may therefore be reutilised as an aspiration port. The aerosol or surgical plume 8 may then be passed from the irrigation (aspiration) port of the bipolar forceps 4 to tubing 9. The tubing 9 is arranged to transfer the aerosol or surgical plume 8 to an atmospheric pressure interface of a mass and/or ion mobility spectrometer 2.

(26) According to various embodiments a matrix comprising an organic solvent such as isopropanol may be added to the aerosol or surgical plume 8 at the atmospheric pressure interface. The mixture of aerosol and organic solvent may then be arranged to impact upon a collision surface within a vacuum chamber of the mass and/or ion mobility spectrometer 2. The collision surface may be heated. The aerosol may be caused to ionise upon impacting the collision surface resulting in the generation of analyte ions. The ionisation efficiency of generating the analyte ions may be improved by the addition of the organic solvent. However, the addition of an organic solvent is not essential.

(27) Analyte ions which are generated by causing the aerosol, smoke or vapour 8 to impact upon the collision surface may then be passed through subsequent stages of the mass and/or ion mobility spectrometer 2 and subjected to analysis such as mass analysis and/or ion mobility analysis in a mass analyser or filter and/or ion mobility analyser.

(28) According to various other embodiments, the sample or analyte may be ionised using Desorption ElectroSpray Ionisation (DESI).

(29) FIG. 3 illustrates the Desorption ElectroSpray Ionisation (DESI) technique according to various embodiments.

(30) As shown in FIG. 3, the desorption electrospray ionisation (DESI) technique is an ambient ionisation method that involves directing a spray of (primary) electrically charged droplets 11 onto a surface 12 with analyte 13 present on the surface 12 and/or directly onto a surface of a sample 14. The electrospray mist is pneumatically directed at the sample by a sprayer 10 where subsequent ejected (e.g. splashed) (secondary) droplets 15 carry desorbed ionised analytes (e.g. desorbed lipid ions).

(31) The sprayer 10 may be supplied with a solvent 16, nebulising gas 17 such as nitrogen, and voltage from a high voltage (HV) source 18. The solvent 16 may be supplied to a central capillary of the sprayer 10, and the nebulising gas 17 may be supplied to a second capillary that may (at least partially) coaxially surround the central capillary. The arrangement of the capillaries, the flow rate of the solvent 16 and/or the flow rate of the gas 17 may be configured such that solvent droplets are ejected from the sprayer 10. The high voltage may be applied to the central capillary, e.g. such that the ejected solvent droplets 11 are charged.

(32) The charged droplets 11 may be directed at the sample such that subsequent ejected (secondary) droplets 15 carry desorbed analyte ions. The ions travel through air into an atmospheric pressure interface 19 of a mass and/or ion mobility spectrometer or analyser (not shown), e.g. via a transfer capillary 20.

(33) The desorption electrospray ionisation (DESI) technique allows for ambient ionisation of a trace sample at atmospheric pressure with little sample preparation. The desorption electrospray ionisation (DESI) technique allows, for example, direct analysis of biological compounds such as lipids, metabolites and peptides in their native state without requiring any advance sample preparation.

(34) It would also be possible to use other ionisation techniques. For example, the ion source may comprise (i) a rapid evaporative ionisation mass spectrometry (REIMS) ion source; (ii) a desorption electrospray ionisation (DESI) ion source; (iii) a laser desorption ionisation (LDI) ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption (LDTD) ion source; (vi) a desorption electro-flow focusing (DEFFI) ion source; (vii) a dielectric barrier discharge (DBD) plasma ion source; (viii) an Atmospheric Solids Analysis Probe (ASAP) ion source; (ix) an ultrasonic assisted spray ionisation ion source; (x) an easy ambient sonic-spray ionisation (EASI) ion source; (xi) a desorption atmospheric pressure photoionisation (DAPPI) ion source; (xii) a paperspray (PS) ion source; (xiii) a jet desorption ionisation (JeDI) ion source; (xiv) a touch spray (TS) ion source; (xv) a nano-DESI ion source; (xvi) a laser ablation electrospray (LAESI) ion source; (xvii) a direct analysis in real time (DART) ion source; (xviii) a probe electrospray ionisation (PESI) ion source; (xix) a solid-probe assisted electrospray ionisation (SPA-ESI) ion source; (xx) a cavitron ultrasonic surgical aspirator (CUSA) device; (xxi) a focussed or unfocussed ultrasonic ablation device; (xxii) a microwave resonance device; or (xxiii) a pulsed plasma RF dissection device.

(35) According to various embodiments, one or more physico-chemical properties of the analyte or ions derived from the analyte, such as mass or mass to charge ratio, mass or mass to charge ratio peak shape or width, ion mobility, collision cross section or interaction cross section, and/or ion mobility, collision cross section or interaction cross section peak shape or width, are measured (and in various embodiments continuously monitored) by the analytical instrument.

(36) According to various embodiments, the sample type of the sample being analysed is determined e.g. using known tissue-typing methods. According to various embodiments this is done based on recent analysis of the sample being analysed, e.g. based on the analysis of the analyte and/or on prior analysis of analyte from the (same) sample (e.g. by the analytical instrument during the same experimental run, set of experimental runs or surgical procedure), i.e. based on the measured physico-chemical properties of the analyte or ions derived from the analyte.

(37) The sample type of the sample may be the identity and/or any phenotypic and/or genotypic characteristic of the sample. For example, the sample type of a human or animal tissue sample may be the type of the tissue, e.g., liver, kidney, or lung. Alternatively or in addition, it may be the disease state of the sample, e.g., healthy or cancerous. The sample type of a microbial sample may, e.g. be information about the genus, species, and/or strain of a microbe present in the sample.

(38) The determination of the sample type may involve using a device to generate aerosol, smoke or vapour from the sample, mass and/or ion mobility analysing said aerosol, smoke, or vapour, or ions derived therefrom so as to obtain spectrometric data, and analysing said spectrometric data. The method may comprise analysing analyte ions derived from the aerosol, smoke or vapour. Analysing the spectrometric data may comprise analysing one or more sample spectra so as to classify an aerosol, smoke or vapour sample. This may comprise developing a classification model or library using one or more reference sample spectra, or may comprise using an existing library. For example, an identification of the sample type may be made if the spectrometric data corresponds more closely to one library entry than any other library entry. Analysing the one or more sample spectra so as to classify the aerosol, smoke or vapour sample may comprise unsupervised analysis of the one or more sample spectra (e.g., for dimensionality reduction) and/or supervised analysis of the one or more sample spectra (e.g., for classification). An exemplary method for tissue-typing using spectrometric analysis is disclosed in Balog et al. Science Translational Medicine 17 Jul. 2013, vol 5, issue 194, 194ra93.

(39) One or more known endogenous species for the determined sample type are then identified, e.g. using the list or library. That is, one or more species of the analyte that are known to be endogenous to the determined sample type are identified, e.g. based on the analysis of the analyte and/or on prior analysis of analyte from the sample.

(40) This may be done by determining whether one or more species of the analyte correspond to one or more species for the determined sample type that are present in the predetermined list or library. An appropriate window or error may be used in this determination, in order to account for instrument drifts.

(41) According to various embodiments, where possible, the instrument is then calibrated or optimised using the identified endogenous species, i.e. using the measured physico-chemical properties of the identified endogenous species.

(42) A new calibration may be generated for the analytical instrument, and/or an existing or current calibration (e.g. the initial calibration or a subsequent calibration) may be updated, modified and/or corrected.

(43) The calibration type may include a polynomial, spline or probabilistic calibration.

(44) According to various embodiments, the step of calibrating the instrument or modifying a or the calibration may comprise: (i) modifying one or more calibration parameters (e.g. polynomial coefficients, gain, etc.); (ii) modifying an underlying base or initial calibration; and/or (iii) applying an extra calibration (which may be subject to some constraints, e.g. polynomial order) after the main or initial calibration.

(45) The calibration may be an absolute calibration or a relative calibration, e.g. relative to an initial calibration made at the beginning of an experiment.

(46) Additionally or alternatively, one or more operational parameters of the analytical instrument may be optimised using the identified endogenous species, i.e. using the measured physico-chemical properties of the identified endogenous species. According to various embodiments, in a feedback mode of operation, the data corresponding to the identified molecular species may be used to guide modification of one or more instrument parameters to improve data quality.

(47) The parameter(s) that are optimised may include, for example, one or more voltages (e.g. detector voltage), one or more temperatures, one or more gas pressures, one or more flow rates, etc., of the instrument. The parameter(s) that are optimised may include one or more parameters of the ion source 1 and/or one or more parameters of the analyser 2.

(48) For example, where the ion source 1 comprises a Rapid Evaporative Ionisation Mass Spectrometry (REIMS) ion source, the parameter(s) that are optimised may include, for example, the amplitude and/or frequency of the RF voltage applied to the electrodes 4, the composition, temperature and/or flow rate of the solvent, the temperature of the heated collision surface, the position and/or orientation of the electrodes 4, etc.

(49) Where the ion source 1 comprises a Desorption ElectroSpray Ionisation (DESI) ion source, the parameter(s) that are optimised may include, for example, the composition, flow rate and/or temperature of the solvent 16, the composition, flow rate and/or temperature of the nebulising gas 17, the magnitude of the high voltage, the position and/or orientation of the sprayer 10 and/or the capillary 20, etc.

(50) The calibrated or optimised analytical instrument is in various embodiments then used for subsequent analysis of analyte from the sample and/or the calibration is applied to the current data.

(51) According to various embodiments, the steps for calibrating or optimising the instrument (i.e. determining the sample type and identifying known endogenous species, etc.) may be iterated, e.g. periodically, at predetermined time intervals, or after a predetermined number of experiments. According to various embodiments, as the composition of the sample (potentially) changes, e.g. between different sample types, then the determined sample type and corresponding known endogenous species used for the calibration can also change. This ensures that an optimum calibration is maintained as the sample type changes.

(52) For example, where the ion source 1 is scanned (e.g. in a raster pattern) across the surface of the target or sample (and/or where the sample is scanned relative to the ion source 1), then as the composition of the sample changes between different positions on the sample, e.g. from sample type to different sample type, then the determined sample type and the corresponding known endogenous species that are selected and used for the calibration may change.

(53) Additionally or alternatively, where the composition of the sample changes as the sample is consumed due to the ionisation process or otherwise, then the determined sample type and corresponding known endogenous species that are selected and used for the calibration may change. For example, as a sample is consumed when using the REIMS technique, e.g. during a surgical procedure, the sample type may change e.g. from a diseased tissue to a non-diseased tissue, and so the determined sample type and corresponding known endogenous species that are selected and used for the calibration may also change in order to ensure that an optimum calibration is maintained.

(54) According to various embodiments, the calibration or optimisation of the analytical instrument may be postponed when one or more of the known endogenous species, i.e. present in the list or library, cannot be identified or accurately identified.

(55) According to various embodiments, the system may be configured such that the calibration modification is updated only once a sufficient number of ions have been measured or acquired, i.e. such that adequate statistics may be produced for the calibration.

(56) For example, a number of recently acquired spectra may be summed, e.g. over a time period shorter than the characteristic timescale of the expected calibration drift for this purpose. According to various embodiments, the minimum number of spectra necessary for adequate statistics may be summed for this purpose, so as to reduce any problems associated with instrument drifts.

(57) According to various embodiments, the calibration or optimisation may be postponed where the one or more identified species are not sufficiently stable, consistent, abundant, clear and/or isolated in the measurement. Additionally or alternatively, species that are not sufficiently stable, consistent, abundant, clear and/or isolated in the measurement may be (temporarily) removed from consideration for the calibration (and other species may be relied on where present).

(58) For example, if for one or more given species, unexpected rapid changes in the measured mass to charge ratio (m/z) and/or changes in the peak shape are observed, which, e.g., may be due to interference from other species present in the sample, then these one or more species may temporarily be removed from consideration.

(59) According to various embodiments, the calibration or optimisation may be postponed where metadata, such as information regarding detector saturation and/or instrument warning states, indicates that the acquired data is not sufficiently reliable for the calibration.

(60) According to various embodiments, in any such cases where acceptable reference measurements are unavailable and/or the calibration is postponed, the most recent good calibration modification (or calibration) or optimisation may be retained and used, e.g. until a new calibration optimisation is produced.

(61) When the calibration is postponed, a record may be made, and the confidence or weight assigned to data acquired during this time can be reduced. For example, if some predetermined maximum time has elapsed since the last good modification (or calibration) was obtained, a mass accuracy warning flag may be set. Inferences regarding the composition of the current sample may be modulated in light of this information.

(62) According to various embodiments, diagnostic information obtained from the calibration procedure, e.g. evidence (marginal likelihood), curvature or residuals, may be used to enable automatic selection of a high quality subset of data for use at any particular time during the analysis.

(63) Although the above embodiments have been described primarily in terms of mass to charge ratio (m/z) calibration, according to various other embodiments, the same techniques may be used in ion mobility, collision cross section (CCS) or interaction cross section calibration i.e. in internal lock CCS. Ion mobility or collisional cross section (CCS) calibrations may be updated in real-time based on measurement of endogenous species.

(64) Aspects of the above described embodiments may also be applied to ion imaging techniques, such as Desorption Electrospray Ionisation (DESI) or Matrix-Assisted Laser Desorption/lonisation (MALDI) imaging techniques. It should be understood that as used herein, the terms image, imaging or similar relate to any type of spatial profiling of a sample surface, i.e. where spatially resolved data is acquired for a sample surface (and that, for example, in these embodiments, an image need not be displayed or otherwise formed).

(65) According to a known imaging technique, a lock mass sample is provided on or together with the two-dimensional sample to be imaged. For example, a lock mass patch may be provided in one corner of a tissue section sample. While imaging the sample by raster scanning across the sample, a periodic lock mass calibration may be acquired by periodically returning to and analysing the lock mass patch.

(66) According to various embodiments, when imaging a sample (e.g. a two dimensional sample such as a tissue section sample), the analytical instrument (e.g. mass and/or ion mobility spectrometer) may be calibrated or optimised using a portion of the sample that has been determined to be particularly useful for the calibration or optimisation, e.g. for which one or more of the known endogenous species or particularly useful known endogenous species (e.g. as described above) are present. The calibration may be performed by (e.g. repeatedly and/or periodically) returning to and analysing the identified particular portion of the sample. This then means that no lock mass patch is required (and according to various embodiments, no lock mass patch is provided).

(67) Thus, according to various embodiments, the method comprises imaging a sample, identifying a part of the sample that comprises one or more species that are known to be endogenous to the sample type of the sample, and calibrating or optimising the analytical instrument using the identified part of the sample.

(68) According to various embodiments, imaging the sample comprises analysing the sample, optionally by ionising the sample, optionally by (raster) scanning across the sample.

(69) According to various embodiments, identifying a part of the sample that comprises one or more species that are known to be endogenous to the sample type of the sample may comprise identifying a part of the sample that comprises one or more species that are known to be endogenous to the sample type of the sample and that are particularly useful for the calibration or optimisation.

(70) According to various embodiments, a portion of the sample may be determined to be particularly useful for calibration where one or more known endogenous species (e.g. as described above) are present and/or where one or more selected endogenous species are present, such as one or more known endogenous species that are sufficiently or particularly stable, consistent, abundant, intense, clear and/or isolated (e.g. as described above).

(71) According to various embodiments, calibrating or optimising the analytical instrument using the identified portion of the sample may comprise calibrating or optimising the analytical instrument using the known endogenous species present in the identified portion of the sample (e.g. as described above).

(72) According to various embodiments, the sample type of the sample may be determined (e.g. as described above) during the imaging experiment.

(73) According to various embodiments, the particular portion of the sample that is used for the calibration may be changed or updated e.g. when an improved portion is discovered during the imaging experiment.

(74) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.