System and method for identification of biological tissues

Abstract

The present invention provides for a system, method, and device for analyzing, localizing and/or identifying tissue types. The method includes analyzing, localizing and/or identifying one or more tissue samples, characterized in that the method comprises: (a) generating gaseous tissue particles from a site in the one or more tissue samples, (b) transporting the gaseous tissue particles from the site to an analyser, (c) using the analyser for generating tissue-related data based on the gaseous tissue particles, and (d) analyzing, localizing and/or identifying the one or more tissue samples based on the tissue-related data. The invention can either be used in close conjunction with a surgical procedure, when one or more surgical tools are an integrated part of ionization, or as a separate mass spectrometric probe for the analysis of one or more tissue parts.

Claims

1. A method of analyzing a sample, the method comprising: disintegrating a portion of the sample to generate gaseous particles with a disintegrating device without sample preparation, wherein the sample comprises in situ and in vivo animal tissue and wherein the disintegrating device comprises one of a visible laser, an infrared laser or an ultraviolet laser; transporting at least some of the gaseous particles toward an analyzer, wherein the disintegrating device operates by heating; and analyzing at least some of the gaseous particles using the analyzer.

2. The method of claim 1, wherein the gaseous particles comprise at least one of: individual molecules in gas phase, and clusters of molecules.

3. The method of claim 1, wherein the analyzer comprises one of: a mass spectrometer and an ion mobility spectrometer.

4. The method of claim 1, further comprising ionizing at least some of the gaseous particles away from the sample to generate gaseous 10 ions.

5. The method of claim 4, wherein said ionizing is performed by an ionization device incorporated into a fluid pump that induces a pressure gradient in a transport tube that transports the gaseous particles toward the analyzer.

6. The method of claim 4, wherein said ionizing comprises one of: corona discharge ionization and secondary electro spray ionization.

7. The method of claim 1, further comprising signaling results of the analyzing to a user in real time.

8. The method of claim 7, wherein said signaling is continuously displayed to the user.

9. The method of claim 1, wherein without sample preparation comprises the sample not being subject to chromatography.

10. A system for analyzing a sample, the system comprising: a disintegrating device for generating gaseous particles from a sample at a sample site without sample preparation, wherein the sample comprises in situ and in vivo animal tissue; a transport tube configured to transport the gaseous particles away from the sample site; an ionizing device configured to ionize at least a portion of the gaseous particles away from the sample site to generate gaseous ions; and an analyzer configured to generate data based at least on the gaseous ions, wherein the disintegrating device operates by radiative heating.

11. The system of claim 10, wherein the gaseous particles comprise at least one of: individual molecules in gas phase, and clusters of molecules.

12. The system of claim 10, wherein the analyzer comprises one of: a mass spectrometer and an ion mobility spectrometer.

13. The system of claim 10, wherein the ionizing device is incorporated into a fluid pump that induces a pressure gradient in the transport tube.

14. The system of claim 10, wherein the disintegrating device comprises an infrared laser.

15. The system of claim 10, wherein the disintegrating device comprises one of a visible laser or an ultraviolet laser.

16. The system of claim 10, wherein without sample preparation comprises the sample not being subject to chromatography.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.

(2) FIG. 1 illustrates a scheme of an in vivo mass spectrometric tissue identification system in accordance to one aspect of the present invention.

(3) FIG. 2 illustrates a scheme of an in vivo mass spectrometric tissue identification system in accordance to another aspect of the present invention.

(4) FIG. 3 illustrates the implementation of post-ionization of neutral gaseous tissue particles by means of secondary electrospray ionization.

(5) FIG. 4 illustrates the implementation of post-ionization of neutral gaseous tissue particles by means of corona discharge ionization.

(6) FIG. 5 illustrates total ion current obtained during application of device and method of the present invention in a surgical setting.

(7) FIG. 6 illustrates a full mass spectrum obtained during electrosurgical dissection of porcine liver using the setup depicted on FIG. 1.

(8) FIG. 7 illustrates three overlapping negative ion mass spectra obtained during electrosurgical dissection of porcine liver using the set up of FIG. 1.

(9) FIG. 8 illustrates mass spectra obtained from porcine liver, heart and lung in negative ion mode, during the electrosurgical dissection of corresponding organs using the setup depicted on FIG. 1.

(10) FIG. 9 illustrates mass spectra obtained from a canine melanoma, its proximal lymph node and surrounding healthy skin, in negative ion mode during electrosurgical removal of tumour and sentinel lymph node, using the setup depicted on FIG. 1.

(11) FIG. 10 illustrates a scheme of an in vivo mass spectrometric tissue identification method and device using laser means for creating gaseous tissue particles in accordance with yet another aspect of the invention.

(12) FIG. 11 illustrates a comparison between a spectrum obtained by electrosurgery (spectrum above) and a spectrum obtained by laser surgery using CO2 laser (spectrum below). Both spectra were obtained by dissecting porcine liver and analyzing the ions formed on surgical dissection without any means of post-ionization.

(13) FIG. 12 illustrates a scheme of data flow between hospitals and database development unit. Hospitals facilitate database development by providing the manufacturer with raw, histologically assigned data, while the manufacturer processes the data and stores it in a central database.

(14) FIG. 13: panel A is a picture taken during a surgical dissection of a grade III mastocytoma in a dog, panel B is a three dimensional principal component analysis of the spectra taken from the marked places in panel a), and panel C is a screenshot of real time software during surgery of panel A. 1=muscle, 2=skin, 3=subcutis, 4=mastocytoma.

DETAILED DESCRIPTION OF THE INVENTION

(15) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, unless indicated otherwise, except within the claims, the use of or includes and and vice-versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (for example including, having and comprising typically indicate including without limitation). Singular forms including in the claims such as a, an and the include the plural reference unless expressly stated otherwise.

(16) Area of Interest or Site means an area of tissue that comprises the subject of acquired tissue-related data sets. In one embodiment, an area of interest or site is suspected of containing abnormal or pathological tissue. In some embodiments, a site is believed to contain normal tissue and the data acquired is used as control or background data.

(17) In situ means the direct examination of cells or tissues. In situ includes the direct examination of tissues on a subject during a surgical procedure.

(18) Memory effect can be defined as a non-linear delay between an analysis process and acquired data. A signal corresponding to one sample (sample A) may persist even when sample A is not analyzed any more, and may interfere with the analysis of a following sample B.

(19) Subject or patient refers to an animal, including humans, in need of treatment for a condition, disorder or disease.

(20) Tumour tissue refers to any neoplastic tissue including cancerous cells. The tumour tissue may be a solid tumour or a non-solid tumour, such as those present in the circulatory system, for example leukemia.

(21) The invention will be explained in details by referring to the Figures.

(22) The applicants discovered that surgical methods employing ultrasonic or thermal disintegration (for example electrosurgery and infrared laser surgery), produce large amounts of tissue-originated gaseous tissue particles. The applicants further discovered that mass spectra of these gaseous tissue-originated particles are similar to those obtained by other mass spectrometric techniques, such as DESI, SIMS and MALDI. As such, the present invention provides for devices, systems and methods for analyzing, localizing and/or identifying tissues in real time and in situ by combining disintegrative ionization of tissues and mass spectrometry.

(23) By disintegrating tissue, charged and uncharged particles are generated in gas phase. The inventors discovered that the charged particles generated through disintegration manifest as clusters of molecules, which gradually undergo disassociation to yield molecular ions. This gradual disassociation typically starts at the point of disintegration and typically in connection with the present invention is completed to yield molecular icons in the mass spectrometer, preferably prior to mass analysis. Uncharged particles can be post-ionized, and post-ionization also produces a distribution of charged molecular clusters ranging from individual molecular ions to macroscopic droplets. These clusters also undergo gradual association to yield molecular ions. In this way, the disintegration of tissue is operable to yield charged tissue particles which in turn yield molecular ions suitable for use in mass spectrometry.

(24) As such, in one aspect, the present invention provides for a system for analyzing, localizing and/or identifying one or more tissue samples characterized in that said system comprises: (a) a disintegrating device for contacting the one or more tissue samples at a site, and for generating gaseous tissue particles from the site (b) a transport means for transporting the gaseous tissue particles from the site to an analyzer, and (c) an analyser operationally coupled to the transport means, said analyser for generating tissue-related data based on the gaseous tissue particles, wherein said tissue-related data are used for analyzing, localizing and/or identifying the one or more tissue samples.

(25) In another aspect, the present invention provides for a device for analyzing, localizing and/or identifying one or more tissue samples characterized in that the device comprises: (a) a disintegrating device for contacting the one or more tissue samples at a site, and for generating gaseous tissue particles from the site and (b) a transport means configured to be operably linked to an analyser, said transport means for transporting the gaseous tissue particles from the site to the analyzer.

(26) The novel methods, systems and devices of the present application, termed rapid evaporative ionization mass spectrometry (REIMS) involving the aerosolization of tissue to generate and identify gaseous tissue-originated ions and to localize abnormal tissue in situ may be implemented for numerous applications. According to one embodiment, REIMS techniques can be used for diagnostic purposes to screen an area of interest to identify whether tissue of a specific type or composition, or having other specific attributes, for example cancerous tissue, is present in the area of interest and, if so, to locate the cancerous tissue with a high degree of spatial resolution. These diagnostic techniques may be used for examining an area of interest that is exposed during a surgical procedure, or an area of interest exposed to an invasive or semi-invasive instrument, such as a laparoscope, endoscope, probe, fiber optic cables, or the like. In this fashion, methods, systems and devices of the present invention may be used for fast detection and diagnosis in numerous applications, including and not limited to detection of various abnormalities, including lung cancer, cancers of the digestive system organs, including esophageal cancers, colorectal cancers, and the like; skin; reproductive organs, such as prostate, ovarian, uterine and cervical cancers, breast cancer; brain cancer; cancers of the lymphatic system and bone; and the like. Other applications of the present invention will be described below.

(27) Basic setup of one aspect of the system of the present invention is shown in FIG. 1 in a surgical setting. The system shown in FIG. 1 comprises of following parts:

(28) Disintegratin Device 11. A primary function of disintegrating device is to generate an aerosol out of biological tissue via either ultrasonication or rapid boiling of water content of the tissue, which disintegrates tissue structure. Disintegration leads to formation of aerosol or gaseous particles covered with surface active molecules, i.e. membrane lipids of original structures, and contain intact and thermally degraded biomolecules. These aerosol or gaseous particles may carry a net electric charge due to uneven distribution of anionic and cationic species, and these droplets can dissociate to give individual molecular ions of membrane lipids.

(29) There are a number of different disintegration methods that can be used in the devices, systems and methods of the present invention, including ultrasonication, Joule-heating, contact heating and radiative heating, which includes electromagnetic radiation (ranging from microwave to near UV) ablation.

(30) FIG. 1 illustrates a system wherein the disintegrating device 10 performs Joule-heating by means of inducing electric current in the tissue sample of interest. The function of electrode 10 from the point of view of analysis is to bring the biological tissue into contact with remote electric power supply 70. Electric current flowing through healthy, normal tissue parts 20 and through abnormal, pathological (such as cancer) tissue parts 30 converts tissue parts 20, 30 into a gaseous mixture of charged 50 and neutral 60 species via Joule heating. Application of high-frequency (>100 kHz) alternating current (AC) can be advantageous, because direct current (DC) and low frequency AC may interfere with the electrophysiological processes of living organisms. Thus, application of DC and low frequency AC current may be dangerous or even fatal to a subject.

(31) Transformation of tissue constituents into corresponding gaseous tissue particles can be carried out by the presently used surgical tools including electrosurgery, laser surgery, water jet surgery, or ultrasonic surgery.

(32) In the case of electrosurgery, for example, current density is sufficiently high only in the proximity of sharp electrodes to cause tissue disintegration; hence tissue is evaporated where a sharp electrode is in physical contact with the body. In order to eliminate additional burns, either a large area counter electrode is used (monopolar cutting) or the two sharp electrodes are close to each other (bipolar cutting). In these cases, in one aspect of the invention, a surgical device can be equipped with a transfer tube 80 and the surgical device can be converted to a bifunctional surgical and tissue identification tool.

(33) In the case of endoscopy, standard electrosurgical, laser surgical, or ultrasonic surgical equipment can be used for sampling. Working channel of the endoscope can be used for evacuation of surgical smoke containing gaseous ions of interest. One sampling point may require the evacuation of about 1 ml of gas, hence, an applied vacuum does not result in any harmful effects.

(34) Transfer or Transport tube 80. The transport tube 80 transfers charged and/or neutral species formed on aerosolization of tissue to an analyser, such as a mass spectrometer (MS). Since an important application of the present invention is the in situ, in vivo identification, localization and/or analysis of biological tissues 20, 30 during surgery, and mass spectrometers are immensely heavy instruments compared to surgical equipment, it is important to transfer charged 50 and/or neutral species 60 formed on tissue disintegration to a remote mass spectrometer 130 instead of placing patient to close proximity of the MS instrument. As such, in aspects of the invention the MS can be placed outside the operating room. Gas flow carrying the gaseous tissue-originated particles 50, 60 through transfer tube 80 can be generated by establishing pressure a difference between two ends of tube 80. Since pressure on sampling or tissue side of the transport tube 80 is atmospheric in most of the preferred applications (e.g. surgical applications), pressure difference can be generated by decreasing pressure at the end of tube 80 which is closer to MS 130 (MS side of transport tube 80). Pressure is decreased by employing a separate a fluid pump 220, or using the vacuum system of MS 130. Transfer tube 80 can be made of any material with sufficient mechanical strength, chemical stability and sufficiently high flexibility. For example, tube 80 can be made out of various polymers, (polyethylene, polytetrafluoroethylene, polypropylene, polyvinylchloride), metals (steel, copper), glass and fused silica. Important features of material include the lack of porosity and inertness, i.e. tube wall is not supposed to retain charged 50 and neutral 60 gaseous tissue particles and also not supposed to interact with these species 50, 60 or facilitate their chemical reactions. Internal diameter of tubing 80 can be anywhere between about 0.1 mm and about 20 mm, length of tubing can be anywhere between about 0 and about 5000 mm or of enough length to interface with the MS, wall thickness of tubing can be anywhere between about 0.01 mm and about 5 mm. Transfer tube 80 can be used at ambient temperature, or at elevated temperature. Operating temperature can be set anywhere between ambient and 400 C. Elevated temperature tends to shift adsorption-desorption equilibria taking place on wall surfaces of transfer tubing 80 towards desorption, which suppresses undesired memory effects. Elevated temperature can also shift gas-phase association-dissociation equilibria towards dissociation, which decreases the recombination rate of ionic species 50 with opposite charges. Transfer tube 80 may contain minor amounts of porous or fibrous material (glass wool, fabric, etc.) to irreversibly capture large particles not producing individual gaseous ions. It is important to note, that electrically non-conductive tubing material can only be used in such cases when ion population comprising both positive and negative ions is transferred. Ion transfer efficiency in these cases can further be improved by keeping the ions off the wall of the tubing by e.g. generating a radial pseudopotential field using RF electric fields.

(35) It should be understood that transfer tube 80 may include a free portion that is flexible enough to permit a range of motion during use in conjunction with surgery, and a fixed portion that does not move during surgery, for reaching the remote analyser.

(36) Transfer tube 80 can be held next to the site where the tissue is being surgically cut such that gaseous species 50, 60 can be driven into the transfer tube 80. Alternatively, the surgical tool that serves as a disintegrating device can be co-axially connected to the transfer tube 80.

(37) Fluid Pump 220

(38) Primary function of fluid pump 220 is to generate pressure difference along transfer tube 80 and induce gas flow through transfer tube 80. Gas flow transfers charged 50 and neutral 60 species from the site of tissue disintegration towards mass spectrometer 130. Fluid pumps employing different pumping mechanisms can be employed. However, since charged 50 and neutral 60 species can be chemically aggressive, and in the case of surgical applications fluid pump device 220 need to be disinfected or disposed after each operation, the use of Venturi gas jet pumps may be desired in these cases. A Venturi pump includes a nozzle 110 and a Venturi tube 120. Venturi pumps can dilute primary gas flow and decrease concentration of charged 50 and neutral species 60 in the gas stream, however, Venturi pumps can also focus charged 50 and neutral species 60 and facilitate their electrospray or corona discharge ionization. Further advantage of Venturi pump is the lack of moving parts which decreases the chance of malfunctioning.

(39) Although fluid pump 220 can be omitted (as it is shown on FIG. 2), and the vacuum system of the mass spectrometer can be used as pumping system, this implementation may not be ideal in cases where large mass flow and linear velocity is required in transfer tube 80. Also, 220 fluid pump can become essential element of the system when ionization of neutral species 60 is performed at atmospheric pressure. It is important to note, that electrospray and corona discharge ionization methods can only performed at relatively higher pressures (p>10 torr).

(40) Post Ionization Device 320

(41) Although thermal or mechanical disintegration methods do produce charged particles 50 on the acrosolization of tissues, most of aerosolized material remains neutral in the gas phase. Furthermore, on the rapid thermal or mechanical aerosolization of tissues only certain molecules undergo ionization, which belong mainly to the group of glycerophospholipids. In order to increase ion yield, and also to increase range of molecules available for mass spectrometric analysis, ionization of neutral species 60 can be desired in certain cases. Ionization can be performed both at atmospheric pressure and in vacuum. Atmospheric pressure ionization may be preferred, since atmospheric pressure ion sources provide more stable and robust instrumental conditions and involve less serious memory effects.

(42) Post ionization methods that can be used in the systems and methods of the present invention include secondary electrospray ionization depicted on FIG. 3. Secondary electrospray ionization may be implemented by placing capillary 180 through nozzle 110 of the Venturi tube 120, pumping conductive solvent 250 through capillary 180 and applying high voltage (HV) onto conductive solvent 250 by using high voltage power supply 170. By application of HV, conductive solvent 250 is sprayed 260 from ending of capillary 180 close to mass spectrometer 130, producing charged droplets 270 of conductive solvent 250. Charged droplets 270 dissolve neutral particles 60 and those charged particles 50 which carry an opposite charge, yielding charged droplets 280 containing conductive solvent 250 and molecules originated from tissue sample 20, 30. Upon the evaporation of conductive solvent 250 from charged droplets 280, gas phase ions 50 are produced, which will be subjected to mass spectrometric analysis. Advantages of secondary electrospray ionization include that this method ionizes both volatile and non-volatile molecules. Since most serious memory effects are caused by precipitation of volatile and semi-volatile molecules, which precipitations keep a low, but constant vapor pressure for these molecules in the system, analysis of non-volatile components is important from the point of view of real-time analysis.

(43) Another post-ionization method that can be used in the system and methods of the present invention is corona discharge ionization, which is depicted on FIG. 4. Corona discharge is implemented by mounting discharge needle 200 onto Venturi tube 120 through needle mount 210, and applying high voltage onto needle 200 using high voltage power supply 170. In order to achieve optimal performance, Venturi tube 120 is equipped with a heater element 190 and heated to temperatures between ambient and 500 C. Although corona discharge ionizes predominantly volatile and semi-volatile molecules, corona discharge ionization can present as a more robust form of ionization than secondary electrospray ionization, and does not suffer from clogging and solubility effects. Similarly to corona discharge ionization, atmospheric pressure photoionization can also be implemented.

(44) Ionization can also be performed under various vacuum conditions. These methods include glow discharge ionization, chemical ionization, electron capture ionization, electron impact ionization, photoionization and any ionization method which is capable of transforming molecular clusters or individual gas phase molecules into corresponding gaseous ions.

(45) Mass Spectrometer 130

(46) Function of mass spectrometer 130 is to separate and detect ions formed either directly on tissue aerosolization, or via post-ionization of neutral particles 60. Since mass spectrometers work under high vacuum conditions, instruments capable of sampling atmospheric region may be preferred for the implementation of the present invention. Atmospheric interfaces generally consist of heated capillary 140, which serves as a primary conductance limit separating atmospheric regime from a fore vacuum regime (p1 torr) and a skimmer electrode 160 secondary conductance limit separating a fore vacuum regime from a high vacuum regime (p<10.sup.4 torr). The atmospheric interface depicted on FIGS. 1-3 consists of heated capillary 140, focusing lens 150 and skimmer electrode 160. The atmospheric interface setup illustrated in FIGS. 1-3 is advantageous since in this case central axes of heated capillary 140 and skimmer electrode 160 are substantially parallel but do not overlap. This way, ions 50 leaving heated capillary 140 can be deflected to the skimmer electrode 160 by applying appropriate DC potentials onto focusing lens 150. Since coulombic forces have no effect on neutral particles 60, charged particles 50 are effectively separated from neutral particles 60 and neutral particles 60 do not enter and contaminate high vacuum regime of mass spectrometer 130. Mass analyzers of any type can be applied for the mass analysis of gaseous ions 50, however so-called ion trap and time-of-flight instruments may be preferred. These mass analyzers collect ions for certain periods of time, then analyze the collected population of ions, which results in lower sensitivity of ion intensity ratios to signal transiency.

(47) Any suitable analyzer capable of detecting gaseous tissue-originated particles 50, 60 transported by the transport tube 80 and generating tissue-related data, including a mass spectrometer or an ion mobility spectrometer, can be used in the methods, systems and devices of the present invention.

(48) Beam of Electromagnetic Radiation 330

(49) An alternative to disintegration of tissues via Joule-heating or via ultrasound, electromagnetic radiation (ranging from microwave to near UV) disintegration of tissues can also be utilized for obtaining tissue-originated gaseous particles, including gaseous tissue-originated ions. Beam of electromagnetic radiation 330 emitted by device 340 is adsorbed by tissues 20, 30 and energy of electromagnetic radiation beam 330 is dissipated to thermal energy which converts constituents of tissue 20, 30 into ionic and neutral gaseous species 50, 60. Application of lasers with wavelength in the infrared regime may be preferred, since in these cases only the vibrational and rotational modes of molecules are excited, thus additional photochemical reactions can be avoided. A further advantage of infrared lasers includes better absorption of infrared laser beam by tissues in comparison with visible or ultraviolet lasers. Surgical laser devices work in the infrared regime exclusively, thus commercially available laser surgical equipment can be used in the systems and methods of the present invention.

(50) The surgical infrared laser can be equipped with transfer tube 80 thereby converting a surgical device into a bi-functional surgical and tissue identification tool. Disintegration of tissue occurs via rapid boiling of water content of tissues, which disintegrates tissue structure. Disintegration leads to formation of aerosol or gaseous particles covered with surface active molecules, i.e. membrane lipids of original structures, and contain intact and thermally degraded biomolecules. These aerosol or gaseous particles may carry a net electric charge due to uneven distribution of anionic and cationic species, and these droplets can dissociate to give individual molecular ions of membrane lipids, similarly to electrosurgery. Sample spectra obtained by electrosurgery and laser surgery are shown on FIG. 11.

(51) The present invention also provides for methods developed for the mass spectrometric analysis and identification of biological tissues. General implementation of invention is depicted on FIGS. 1-3. As such, in one aspect, the present invention provides for a method for analyzing, localizing and/or identifying one or more tissue samples, characterized in that the method comprises: (a) generating gaseous tissue particles from a site in the one or more tissue samples, (b) transporting the gaseous tissue particles from the site to an analyser, (c) using the analyser for generating tissue-related data based on the gaseous tissue particles, and (d) analyzing, localizing and/or identifying the one or more tissue samples based on the tissue-related data.

(52) The system depicted in FIG. 1 can be brought to stand-by position by turning on mass spectrometer 130, controller of tissue aerosolization device 70, and applying inert gas flow 100 onto fluid pump 220.

(53) Using electrodes as an example of disintegrating device 10, tissue analysis can be performed by bringing electrodes 10 (that can be incorporated into a surgical device) into close contact with tissue of interest at a site, and applying potential difference between electrodes by using electric power supply 70. Upon contact of tissue 20, 30 with electrodes 10, tissue is thermally disintegrated as a result of thermal dissipation of electric energy (Joule heating), and either the whole disintegrated tissue, or a part of it, is converted into vapor 50, 60 (meaning individual molecules in gas phase) and aerosol 50, 60 (meaning clusters of molecules in gas phase)

(54) Alternatively to electrosurgical tissue aerosolization, aerosolization of tissue parts 20, 30 by directing ultrasound, water jet or laser beam 330 onto them can also be used to generate charged 50 and neutral gaseous particles 60.

(55) Chemical composition and electrical charging of these charged 50 and neutral gaseous particles 60 depend on factors including the type of original tissue and the method used for tissue aerosolization among number of other factors. Charged 50 and neutral gaseous particles 60 enter transport tubing 80 and are transferred to either fluid pump 220 (if a fluid pump is used), or directly to the mass spectrometer 130.

(56) Heat-induced aerosolization of tissues can produce a considerable amount of charged particles 50, which allows tissue analysis without post cutting ionization (post-ionization) of neutral particles 60. In these cases, tube 80 can be directly connected to mass spectrometer 130, or fluid pump 220 can directly transfer (without post-ionization) charged particles 50 to mass spectrometer 130. When information obtained from mass spectrometric analysis of charged particles 50 (formed on tissue disintegration) is not sufficient for proper identification or detection of tissues, due to low signal intensity or lack of information content, post-ionization of neutral particles 60 can be used for enhancement of analytical information.

(57) Fluid pump 220 transfers neutral particles 60 to post ionization device, where a fraction of molecules of neutral particles 60 is converted to gaseous ions and sampled by mass spectrometer together with the gaseous ions 50 arising from the tissue. Mass spectrometer 130 can be used to separate all gaseous ions with regard to their mass-to-charge ratio and detected separately. Result of mass spectrometric analysis is mass spectrum (shown in FIGS. 6-9). Since neutral particles 60 cannot be completely separated from charged particles 50 in the atmospheric interface of mass spectrometer, charged and neutral particles 50, 60 tend to form adducts in ion optics or analyzer region of mass spectrometer. This phenomenon is undesired, since it leads to poorly resolved mass spectra (FIG. 6). Activation of ions by means of collision with inert gas molecules or inert surfaces or absorption of photons can be advantageously used to eliminate ion-molecule complexes and obtain mass spectra with appropriate resolution, as shown on FIG. 7. Since mass spectra cannot per se be directly used for identification of tissues or detection of trace amounts of certain tissues in different tissue matrix, mass spectra tissue-related data has to be processed by data analysis system 230. Mass spectra can be converted to vector format, and identified based on comparison with a database or library of mass spectra records corresponding to a plurality of tissue types. Analysis is aimed at either identification of spectra taken from pure tissues, or detection of certain type of tissue in matrix of other tissues. Alternatively, relative concentration of well defined components can also be calculated from spectra.

(58) Since mass spectrometric analysis of ions takes less than about 200 ms, and data analysis can take from about 100 to about 150 ms, information feedback according to aspects of the present invention can take less than 1 second, thereby providing real-time tissue identification.

(59) Mass spectra of tissues feature mainly membrane lipid constituents which give a tissue-specific pattern. Accordingly, in one aspect of the present invention, full spectral information can be used for the unequivocal identification of tissues. The data analysis can be based on principal component analysis (PCA), where, during the surgery, a pre-defined PCA space is used to spare analysis time. PCA space can be calculated using spectral database containing about 10,000 (ten thousands) spectra presently.

(60) Real time tissue identification can be obtained by comparing the real time tissue-related mass spectra with mass spectra of known tissue types. The real time tissue-related mass spectra can be compared to a library of mass spectra records corresponding to a plurality of known tissue types. The library of records should include spectra of all tissue types which can theoretically be sampled during a surgical intervention. In one aspect of the present invention, the library of records can include spectra converted to vectors which are noise filtered and reduced to a number of dimensions (for example from 300 dimension data to 60 dimension data) via, for example, PCA. The differentiation of tissues/organs in the library of records can be carried out with 60 dimensional linear discriminant analysis (LDA) for quantitative classification of the data. Real time classification of spectra can be performed by using the library and classifying the real time spectra. Classification can be done using, for example, Mahalanobis distances.

(61) Analysis/localization/identification of the tissues using the devices, systems and methods of the present invention can be done in at least two different ways. In the so-called alerting mode the ionic species in the surgical aerosol can be continuously analyzed and the mass spectrometric system can give continuous feedback on the nature of the tissue being dissected. Screenshot of the graphical user interface of our software taken during surgery is shown on FIG. 13c. Whenever the result of the real-time spectral identification refers to the presence of malignant proliferation or whenever the identification of tissue fails, the system can give audiovisual alerting to the user of the system (i.e. a surgeon). An alternative way of utilization of the systems or methods of the present invention can be in the microprobe mode, when the tissue features of interest are sampled actively for the purpose of identification. From the perspective of mass spectrometric tissue identification, the main difference between the two modes is the data accumulation time for individual spectrum. While in the alerting mode data are accumulated for about 0.5-1 s, in microprobe mode the data for one spectrum are accumulated as long as the disintegrating device is generating gaseous tissue particles and collecting the gaseous tissue particles. In order to demonstrate the accuracy of intraoperational tissue identification, results obtained form individual sampling points (FIG. 13a) are shown in a two dimensional PCA plot (FIG. 13b).

(62) Output information of data system can be continuously recorded and displayed on a feedback 240 device which may provide audio, visual or audiovisual information, if real-time analysis is needed. As a result, tissue parts in disintegrated volume 40 are analyzed and identified in an invasive manner, resulting in discontinuity 90 in tissue 20, 30. When discontinuity 90 is defined as a surgical cut, then the net analysis does not involve further invasiveness, compared to surgical cutting.

(63) The present invention provides, in another aspect, a method of mass spectroscopy data acquisition characterized in that said method comprises: (a) generating a yield of gaseous charged particles from an area of interest in a sample (b) transporting the gaseous sample particles from the area of interest to a mass spectrometer, and (c) using the mass spectrometer for acquiring sample-related data based on the yield of gaseous sample ions from the area of interest. In one aspect of the present invention, the sample-related data can be made available through a database, including a library of mass spectra data records, for analysis or identification of biological tissue, including by medical personnel of a hospital or clinic.

EXAMPLES

(64) The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Example 1: Analysis and Identification of Tissue Parts During Surgery

(65) By reference for example to FIG. 1, an electrosurgical unit (ICC 350, Erbe Elektromedizin GmbH) is used in combination with quadrupole ion trap mass spectrometer (LCQ Duo, ThermoFinnigan). Electrosurgical cutting electrode 10 was equipped with commercially available smoke removal unit 80 (Erbe), which was connected to fluid pump 220 (VAC 100, Veriflo) through 8 OD 2 mm ID PTFE tubing. Fluid pump 220 was mounted on LCQ instrument using heavily modified DESI ion source (OmniSpray, Prosolia) platform. Mass spectrometer 130 was operated in negative ion mode. Ions in the range of 700-800 were isolated in ion trap, and were activated using collisional activation with neutral helium atoms. Spectra were acquired in range of m/z 700-800.

(66) Canine in vivo and ex vivo data was acquired from dogs with spontaneous tumours from veterinary oncology praxis.

(67) Electrosurgical electrode 10 was used to remove malignant melanoma tumour 30 from healthy epithelial tissue 20, of the canine model. Tumour 30 was cut out together with parts of healthy skin 20 and surrounding lymph nodes carrying metastases, in order to minimize the chance for tumour recurrence. Tumour margin was determined based on mass spectrometric identification of tissue being cut. Mass spectrum of total ion current obtained during surgical intervention is shown in FIG. 5. Cutting periods during surgery are labelled as 300 and washing periods are labelled 310. It is important to point out, that mass spectrometric signals are detectable only when actual surgical cutting is performed 300 and during washing periods 310 when instrument is cleaned. Since both signal rise and fall times are extremely short compared to time of actual electrosurgical cutting, the experimental setup allows real-time analysis of tissues being cut, with minimized memory effects. Mass spectra taken from healthy epithelial tissue, melanoma and metastasis are shown in FIG. 9. Ratio of ions at m/z 746 and m/z 723 was used as tumour marker and this quantity was displayed on feedback device 240 translated to blue-red color gradient. Audio signal was also used as feedback, when frequency of beeping sound was changed a ion ratio was changed in MS spectra.

(68) Tumour 20 was successfully removed surgically, and post-surgical histological examination of removed material has proven that surgery was successful, and removed lymph node carried tumour cells.

Example 2: Determination of Drugs in Tissues for Localization of Tumour Cells

(69) An electrosurgical unit (ICC 350, Erbe Elektromedizin GmbH) is used in combination with quadrupole ion trap mass spectrometer (LCQ Duo, ThermoFinnigan). Electrosurgical cutting electrode 10 was equipped with commercially available smoke removal unit 80 (Erbe), which was connected to fluid-pump 229 (VAC 100, Veriflo) through 8 OD 2 mm ID PTFE tubing. Fluid pump 220 was mounted on LCQ instrument using heavily modified DESI ion source (OmniSpray, Prosolia) platform. Fluid pump 220 was equipped with secondary electrospray post-ionization unit, comprising capillary 180 and high voltage power supply 170. Electrospray 260 and mass spectrometer 130 were operated in positive ion mode. Ions at m/z 447 and 449 were monitored with m/z 446 as background signal.

(70) Nude mice carrying NCI-H460 human non-small cell lung cancer xenograft were house in a temperature- and light-controlled room, feed and water were supplied ad libitum. At age of 8 weeks, the mice were dosed with 220 mg/bw kg gefitinib. Following 3 days of drug treatment, tumour xenografts were sampled in vivo, under phenobarbital anesthesia.

(71) Electrosurgical electrode 10 was used to remove 30 non-small cell lung cancer tumour from 20 healthy lung tissue of the murine model. Animals were subjected to pre-operational chemotherapy using Gefitinib. Gefitinib (molecular weight is 446) selectively binds to epithelial growth factor receptor (EGFR), which is overexpressed by NSCLC tumour cells. Thus, gefitinib can be used for the chemical labeling of these tumours. Molecular ions of gefitinib were monitored to localize infiltrating tumours.

(72) Tumour 20 was cut out together with parts of 20 healthy lung tissue. Tumour margin was determined based on mass spectrometric identification of tissue being cut. Ratio of ions at m/z 447 and m/z 446 was used as tumour marker and this quantity was displayed on feedback device 240 translated to blue-red color gradient. Audio signal was also used as feedback, when frequency of beeping sound was changed as ion ratio was changed in MS spectra.

(73) Tumour 20 was successfully removed surgically, and post-surgical histological examination of removed material has proven that surgery was successful.

Example 3: Localization and Identification of Bacterial Infections on Mucous Membranes

(74) Home built thermal tissue disintegration device comprising DC power supply 70 and metal electrodes 10 is used in combination with quadrupole ion trap mass spectrometer (LCQ Duo, ThermoFinnigan). Metal electrodes 10 were connected to fluid pump 220 (VAC 100, Veriflo) through 8 OD 2 mm ID PTFE tubing. Fluid pump 220 was mounted on LCQ instrument using heavily modified DESI ion source (OmniSpray, Prosolia) platform. Mass spectrometer 130 was operated in negative ion mode. Ions in the range of 640-840 were isolated in ion trap, and were activated using collisional activation with neutral helium atoms. Spectra were acquired in range of m/z 640-840.

(75) Electrodes 10 were used to sample upper epithelial layer of mucous membranes infected by various bacteria. Since present application of method and device is aimed at minimally invasive analysis of mucous membranes, about 0.1-0.4 mg of total material comprising epithelial cells, bacteria, and mucus was disintegrated for recording one fully interpretable mass spectrum. Tissue part 20 (laryngeal mucous membrane) in contact with electrodes 10 was heated up to 850 C. Full mass spectra were compared to database comprising of mass spectra of 122 bacterial strains. Spectral similarity was defined as cosinus of 200 dimensional mass spectral data vectors. Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphilococcus aureus, and Streptococcus pneumoniae were successfully identified, with the appropriate data entry at the first position of database search hit list. In most of the cases, the first three hits also belonged to the same genus.

Example 4: Business Model

(76) The present invention includes four main identified application areas: robotized surgery, general oncosurgery, pathology and microbial diagnostics. Since price range of instruments used in everyday practice is considerably lower than market price of mass spectrometric systems, the mass spectrometer part of the present invention can be sold to clinics, pathology labs, outpatient offices etc. at net cost of manufacturing. Actual profit can be realized by making parts 10, 80, 220, and post-ionization devices as single-use consumable parts of the system of the invention. This can be a desirable feature because otherwise these parts 10, 80, 220 need to be thoroughly cleaned and disinfected after each surgical or diagnostic intervention. Further possible source of profit can be the software used for interpretation of data and identification of individual mass spectra. Both search engine and database can be continuously developed and sold to users for a low, but recurrent fee. All sold systems can be linked to an internet-based network, which continuously provides the development team 380 with raw data 390, and facilitates the development of central tissue spectrum database 370 as it is depicted on FIG. 12. Hospitals 360 can receive fully uniformized and reliable data 400 via internet.

(77) The above disclosure generally describes the present invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. Other variations and modifications of the invention are possible. As such modifications or variations are believed to be within the sphere and scope of the invention as defined by the claims appended hereto.