Technical Field

The present invention relates to a method for distinguishing cancerous and healthy tissues on the colon tissues by means of a linear time-of-flight mass spectrometer integrated with a high power fs laser ionization mass spectrometry (fs-LIMS). Cancerous colon tissues embedded in the paraffin can be detected by means of the inventive method, wherein a reliable result can be ensured rapidly and without any human-resulting error. State of the Art

Although the incidence rate of cancer increases around the world, mortality resulting from cancer has decreased by means of advanced diagnosis and treatment methods based on the technological developments in recent years. According to World Health Organization (WHO), the incidence of colorectal cancers is app. 9,7% on both men and women, and the estimated mortality resulting from colorectal cancer is about 8,5% according to the statistics of 2012 (Organization, 2018). However, according to studies conducted in the cancer field in subsequent years after 2012, it was observed that the mortality decreased in spite of the increase of the case numbers. These rates indicate that developments in the advanced imaging technologies and researches presented in the endoscopic and colonoscopic-pathological examinations of tissues accelerate the treatment procedure (Gundogdu, Alptekin, Karabagli, Sahin, & Kilic, 2019).

The mass spectroscopy (MS) technique is one of the techniques commonly used for performing researches in tissues, wherein it provides significant information that characterizes the chemical structure of tissues. MS system is a very powerful and promising system, wherein results achieved through using MS in this implementation are saved independently from a user and thus human- resulting errors. Time-of-flight mass spectrometry (TOF-MS) system offers one of the most rapid data collection techniques ever known and in the state of the art, the tissue analysis is performed by way of using techniques such as Rapid Evaporative Ionization Mass Spectrometry (REIMS), Probe Electrospray Ionization Mass Spectrometry (PESI-MS), Desorption Electrospray Ionization (DESI), Matrix-patterned Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and the techniques are effective techniques for cancer types. In all of those techniques, since it is required to performed to sample the patient's tissue during the operation so as to analyze the tissue samples, to prepare tissues in the laboratory, and to carry out the analysis procedure, a significantly overwhelming procedure is needed, thereby causing a long time waste (Gundogdu et al., 2019). However, the current systems are insufficient to perform the detection of suspicious tissue samples in a rapid, reliable, and precise manner during the surgical operation and it is required to meet the vacancy/deficiency by means of the technology aimed to be produced through the method developed.

The use of the mass spectrometry (MS) technique dates back to the beginning of the 19th century. After the invention of lasers in the 1960s, the laser ionization mass spectrometry (LIMS) technique was started to be used commonly (Maiman, 1960). LIMS was first implemented by way of using microsecond (μs) and nanosecond (ns) pulse lasers, however ion peaks in the spectrum exhibit the extension of ion peaks with some small m/z rates by means of fragmenting efficiently the main ion. In the mass spectra achieved through using femtosecond (fs) laser pulse, extensions in the ion peaks may be minimized during the molecular decomposition/ionization and the main ion fragmentation may be also minimized (Kilic et al ., 1997). Studies on laser-mass spectrometry indicate that the pulse duration affects the ion formation mechanisms. Studies in the literature show that ion peaks achieved through the fs laser pulses are more effective compared to the pattern in mass spectra in the ns or shorter laser pulse durations as a result of the decomposition ionization procedures (Kilic et al ., 1997; Ledingham et al ., 1995). Fs laser is also used for the purpose of converting the macromolecules into gas form for the atmospheric pressure mass spectrometry. In further studies conducted in the state of the art; in the electrospray ionization-based mass spectroscopy technique combined with the fs laser for the macromolecule analysis, the main ion is dominant, because the laser beam provide a non-thermal damage procedure. However, in the same method, since the sample preparation period is required for the analysis, it constitutes a disadvantage (Gobeli & El-Sayed, 1985). MALDI-MS based studies may perform the chemical distribution of tissues, however; this technique also constitutes disadvantages such as requiring a complex preparation and being a time-consuming process. Therefore, the use of MALDI-MS is inappropriate in cases where it is required to achieve the rapid results (Karas, Bahr, & Giessmann, 1991). During the surgical procedure, surgeons would like to be sure that the cancerous tissue is completely cleaned and when a tissue area is coincided, causing hesitance, the surgeon takes a sample from the suspicious area and sends it to the pathology department, the pathological process provides an estimated result in 30-40 min. and the patient is kept under anesthesia during the period of time. The longer the duration of anesthesia, the higher risks the patient faces.

The present invention will present a significantly reasonable development and technology in the related technical field because of the deficiency of the current solutions. The present invention relates to a method for distinguishing cancerous and healthy tissues by means of using a linear time-of-flight mass spectrometry integrated with a high power fs laser ionization mass spectrometry (fs-LIMS), wherein the disadvantages are eliminated in the state of the art.

BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION

The present invention discloses a method for distinguishing cancerous tissues in analyzing the colon tissues embedded in the paraffin by means of the linear time-of-flight mass spectrometry (L-TOF-MS) employed together with the high power fs laser ionization mass spectrometry (fs-LIMS).

The inventive method allows for analysis and detection in a short period of time in a successful and reliable manner without causing any bleeding or wound, even without producing any thermal damage on the adjacent healthy tissues because of the short-term and high power application of the fs laser pulses focused on the suspicious tissue even without performing any biopsy, taking any sample or causing any bleeding and injury of suspicious tissues on organs such as colon, brain, breast, lung, liver, prostate, stomach, throat, bladder, uterus, lymphoma and skin.

Suspicious tissue area can be scanned with a micrometer precision by means of the present invention and the guidance capability is presented for the surgeon by determining the surgical margin precisely, such that it guides in few seconds where the surgeon performs the procedure.

An object of the present invention is to detect rapidly and reliably cancerous (colon, brain, breast, lung, liver, prostate, stomach, throat, bladder, uterus, lymphoma, and skin) tissues.

Another object of the present is to ensure a method without a user- independent error to distinguish cancerous (colon, brain, breast, lung, liver, prostate, stomach, throat, bladder, uterus, lymphoma, and skin) tissues. By means of the present invention, cancerous (colon, brain, breast, lung, liver, prostate, stomach, throat, bladder, uterus, lymphoma, and skin) tissues can be distinguished obviously from healthy tissues and it is aimed to reduce significantly the additional surgical duration during the surgical operation. In the inventive method, there is no sample preparation stage to detect cancerous colon tissues and the analysis can be performed directly.

DESCRIPTION OF FIGURES

FIGURE 1 (a) in the fs laser system used in the invention and (b) focusing the laser beam on the sample to be ablated through the laser pulse. The experimental assembly used to determine different colon tissues by means of implementing the PCA method,

FIGURE 2 background of the ion peaks (c) in the range of m/z 60-100 in the mass spectrum achieved from the colon cancerous tissue (a) and healthy tissue (b).

FIGURE 3 PCA statistical graph of the cancerous and healthy colon tissues achieved (a) from the block sample embedded in the paraffin and taken (b) on the glass lamella.

FIGURE 4 (a) principal component analysis (PCA) results and

(b) ion distribution graph.

FIGURE 5 (a) principal component analysis (PCA) results and

(b) ion distribution graph. DESCRIPTION OF ELEMENTS/PARTS/COMPONENTS OF THE INVENTION

Components and parts in Figures are numerated so to provide a better understanding of L-TOF_MS devices connected with the femtosecond (fs) laser system used in the invention, the equivalent of each number is provided in the following:

1.Fs Laser System

2.Beam Splitter 3.Lens

4.Sample Inlet Valve

5.Sample Ablation Chamber

6.Ionization Chamber

7.Fs Laser Beam

8.Free Flight Tube

9.Detector

10. Computer-Based Digital Storage Oscilloscope

11. Data Processing System 12. Healthy Tissue

13. Cancerous Tissue

14. Computer

Detailed Description of the Invention

The present invention relates to a method, wherein the fs-LIMS and L-TOF-MS system are employed together so as to distinguish cancerous and healthy tissues and to detect cancerous tissues. The present invention particularly discloses a method for detecting colon, brain, breast, lung, liver, prostate, stomach, throat, bladder, uterus, lymphoma, and /or skin cancerous tissues and healthy tissues.

In the present invention, the femtosecond laser fragmentation/ionization process of the spectra of the cancerous tissues is performed by means of using a femtosecond laser ionization mass spectrometry (fs-LIMS) under high vacuum conditions (under a sample pressure of ~10 ^-8 ~10 ^-6 mbar) and cancerous tissues are spectroscopically analyzed by using a linear time-of-flight mass spectrometry (L-TOF-MS) by means of the achieved mass spectra. The device used in the invention is comprised of a femtosecond (fs) laser system (1), a vacuum chamber, and data collection systems. Femtosecond laser system (1) is used as an ionization source in the wavelength range of 230-2700 nm. The mass spectrometry device used in the present invention comprises a sample ablation chamber (5), a detector for measuring ion signals with an m/z rates and saving the mass spectra, a computer-based oscilloscope (10) for carrying out the saving and analyzing process by imagining the laser signal and mass spectra, a beam splitter (2), vacuum chamber (sample ablation chamber (5)) and electrostatic lenses (3). In the sample ablation chamber (5), the interaction between cancerous and healthy tissue samples (12, 13) is ensured by means of the fs laser system (1).

In the present invention, the laser beam is focused on the tissue to different spot volumes (pm-cm) by an optic lens in a proper geometry, thereby ensuring an examination option with the desired precision. Focal volume determines the examination resolution and performs tissue analysis with micrometric precision. In the inventive method, the arriving beam is split into two by means of a beam splitter, therefore both laser beams coming from the same laser source are used in the method. The first laser beam is focussed on the tissue and a molecular sample is taken (evaporated) from the tissue, the sample beam is driven to the ionization region in the vacuum chamber by means of a venturi pump and the second laser beam is focused on the sample beam under a proper condition and geometry by means of a lens, thereby achieving the ionization of the sample.A significant amount of material is taken as neutral during the laser ablation process and they are used for the purpose of analysis, wherein a significant precise and powerful analysis is performed with few samples taken by means of ionizing the neutral tissue fragments with the second laser beam focused on the ionization chamber. These ions are focused on and accelerated towards the detector by electrostatic lenses in L-TOF-MS mass spectrometer. Ions falling on the detector are measured by the detector. All ions coming from the sample arrive at the detector, such that it depends as a function of on the time-of-flight of the ions which was converted mass and the mass spectra are saved in a single spectrum, such that it comprises all peaks. The spectrum is imagined by an oscilloscope and furthermore, transferred to computer (14) medium over LAN and saved automatically. The spectra are analyzed and imaged by oscilloscope and software programmed by our group again.

The inventive method for detecting cancerous tissues comprises the following process steps; i.Evaporating the tissue sample/samples by means of ablation through a first laser beam in the wavelength range of 230- 2700 nm sent by way of using a lens (3) with a focal distance of 3-20 cm from the femtosecond (fs) laser system (1) in the sample ablation chamber (5) with a high vacuum medium without subjecting the same to any sample preparation process, ii.Driving the evaporated sample in the ionization chamber (6) through the sample inlet valve (4), iii.performing the ionization process of the evaporated sample by means of the second laser beam in the wavelength range of 230-2700 nm, iv.Accelerating the ions occurred by flowing through the free flight tube (8) by means of electrostatic lenses and sending the same to the detector (9), v.Imaging the ions arrived at the detector (9) through the computer-based digital storage oscilloscope (10) and saving the same on the computer (14), vi.Analyzing the saved data in the data analyzing system (11) through one of the principal component analysis methods in a characteristic manner.

In the present invention, the cancerous tissues embedded in the paraffin are distinguished from the healthy tissues embedded in the paraffin by means of an ultra-fast pulse laser system and a linear time-of-flight mass spectrometry (L-TOF-MS). The experiment assembly is presented in Figure 1, which also illustrates an enlarged view of the sample in the sample ablation chamber. The linear time-of-flight mass spectrometer used in the present invention is comprised of a sample inlet unit by means of a sample inlet valve, a free flight tube of ions, a detector (microchannel Plate-MCP), electrostatic lens system, and power supplies feeding said lens and electronics. An oscilloscope is used to image and to digitize the signal collected from the detector and the Computer provides the connection between the oscilloscope and the server and is used in the data saving and data analysis. While the beam splitter feeds 10% of fs laser power on the sample in the sample ablation chamber, 90% thereof is fed to the L-TOF-MS ionization region. Thus, the ion that occurred in the sample and the sample comprised of neutral particles are used for ionizing the neutrals therein and for contributing to the ion efficiency. In the present invention, the L-TOF-MS device is used to detect the tissue samples comprising cancerous and healthy areas embedded in the paraffin. Fs-LIMS is an abbreviation of the method used, wherein L-TOF-MS is the abbreviation of the device used.

In the present invention, Resonance Laser Ablation (RLA) technique is performed by way of using laser pulses in the wavelength range of 230-2700 nm with a pulse durations in the μs-fs range used as an ionization source, particularly by way of using the resonance wavelength of the tissue and the laser wavelengths in the resonance, thus a more reliable and more powerful analysis is performed by means of employing fewer materials and less laser flow without causing any damage and injury or bleeding.

Fs laser system (1) used in the present invention is an oscilloscope laser system with a repetition frequency of 80-100 MHz, preferably 85 MHz, a 80-200 femtosecond in the wavelength of preferably 800 nm, which can generate a preferable 90 femtosecond laser pulse duration and used to pump an amplifier fs laser system (1), that can generate 80-200 femtosecond, preferably 90 femtosecond laser-pulse duration in the wavelength of 230-2700, preferably 800 nm through the repetition frequency of 1-3 kHz. Fs laser system (1) has a beam power of 3.5 W, wherein the output power of the fs laser system (1) is controlled through the annular neutral density filter and measure by means of a power meter. The fs laser system (1) is used to produce ion from the material steam obtained through the multiple photon excitation and decomposition ionization (DI) processes as an energy source for the purpose of performing the tissue ablation.

The laser beam is focused on the tissue put in the sample ablation chamber (5) in a proper geometry, evaporates the sample from the tissue surface and the evaporates samples are mainly comprised of neutrals and ionic structures. The sample cloud produced is pumped into the ionization chamber (6) of the mass spectrometry and it is ensured to ionize the neutrals therein by way of focusing a second laser beam thereon. Ionic structures of biological molecules, of which the mass spectrometer is pumped in the ionization chamber (6) are saved by the detector (9) based on the time.

In the present invention, a femtosecond linear time-of-flight mass spectrometry (Fs-L-TOF-MS) is used to detect ions and to achieve it as a function of the time-of-flight of the mass spectra. The linear time-of-flight mass spectrometry (L-TOF-MS) comprises a sample inlet valve (4), an ion source, a field-free free flight tube (8) with a length of 120 cm and a microchannel plate (MCP) detector (9) connected directly with a four-channel digital storage oscilloscope for imaging and storing the ion signal and analyzing the same in a personal computer (PC) (14) medium.

Pressure in the vacuum chamber is kept under approximately 10 ^~8 mbar prior to preferably inputting the sample and under around 10- ⁶ mbar as collecting the data by inputting the sample. All spectrum parameters are adjusted to the optimum values, through which the spectral results may be achieved.

In the fs laser system (1) used in the present invention; the beam splitter (2) splits the laser beam into two separate branches at desired rates so as to be used in two different processes. One of mentioned two beams is used to ablate the sample from the tissue, namely, to evaporate the tissue. In the second stage, the second laser beam is used to ionize the sample and afterward, the detection processes are carried out by means of the second laser beam. The first laser beam is focused on the tissue in the sample ablation chamber (5) so as to ablate or evaporate the sample and the second laser beam is focused on the sample-driven in the ionization chamber (6) in the vacuum medium. Energies of said beams are adjusted based on the sample characteristics.

In an embodiment of the present invention, a wavelength of 800 nm and 90 fs laser pulses are specifically used. The beam splitter (2) is used to split the laser beam into two branches. One of those is focused to take material from the sample in the sample ablation chamber (5) on the sample and the other one is focused on the ionization chamber (6) in the linear time-of-flight mass spectrometry, wherein it is ensured to ionize the sample by focusing on the material cloud sent in the ionization chamber (6) by taking the same from the sample ablation chamber (5) through the ablation process. In the present invention, the laser beam intensity is saved being measured prior to the lens (3) focusing the laser beam and a laser intensity values of 1 x10 ¹⁰ - 4x10 ¹⁴ W/cm ² are used. For the sample ablation, the laser power/densities of around 1x10 ¹⁰ W/cm ² are used and saved. Lenses (3) are used, which feature the focal lengths of 3-20 cm so as to focus the laser on the tissue sample embedded in the paraffin. The laser beam intensity varies as a function of the focal length. In the present invention, a lens (3) is used, which features a focal length of preferably 10 cm. In the present invention, the tissue samples are placed in the sample ablation chamber (5) with a high vacuum medium without subjecting the same to any sample preparation process and the beam coming out of the first laser branch (Beam 1) is focused on the sample by the lens (3) featuring a focal length of 3-20 cm, preferably 10 cm to cut out the materials from tissues. Beam in the second laser branch

(Beam 2) is used to ionize the sample plasma produced through Beam

1. In the result of the analysis conducted in the colon tissues by means of the inventive method, the mass spectra achieved through the colon tissues exhibit very stable m/z values. In this study, the mass spectra are statistically analyzed by way of using the ion peaks in the mass region of "m/z" 60-100 amu. In order to perform an analysis by means of the inventive method, waste colon tissue samples embedded in the paraffin (PE) and detected pathologically are used, which are representative of colon tissue samples with tumor (cancerous) and without tumor (healthy) areas. Those tissue samples are detected pathologically and sections in the thickness of ten micrometers are taken from PE blocks and every section is put on a glass lamella. Areas with tumor and without tumor are marked on the glass lamella. Tissues embedded in the paraffin are analyzed by the inventive system under vacuum conditions without conducting any preliminary study and are distinguished obviously.

The inventive method for appointing the cancerous tissues is that the biological molecules to be obtained in the gas from by evaporating the tissue through the laser beam focused on the tissue are analyzed by means of the mass spectrometry without performing any sample cuts, taking any part from the human body, such that it causes no injury or bleeding.

In the present invention, there is no need for a substantial study of any sample preparation, the result can be achieved by means of the laser ionization, decomposition, deduction, and electronic storage of the cloud in the mass spectrometry, which stems from biomolecules occurring from the direct ablation of tissue with the focused laser beam. Data saved electronically by means the detector (9) and monitored through an oscilloscope (10) are saved by transferring to the computer (14) medium and the saved mass spectrum are subjected to the PCA analysis by means of the software installed on the same computer (14) and the results are provided in a few seconds.

In the present invention, in addition to the results of the principal component analysis (PCA), a t-test may be employed and the results provided by the mass spectra are described by means of the PCA method as a statistical process. The T-test is used to observe the PCI and PC2 axes, make a decision whether the tissue samples are distinguished or not, and indicate the distinguishing degree, when necessary. In the present invention, the computer- based digital storage oscilloscope (10) is employed in imagining the laser signal and imagining and saving the spectra. Herein, the term "computer-based" defines the computer system featuring any operating system.

Furthermore, in the present invention, a linear time-of-flight mass spectrometry (L-TOF-MS) is employed to detect ions and to achieve it as a function of the time-of-flight of the mass spectra. The following equation is used to convert the mass/charge (m/z) rates per ion: m = at ² + b

While PCA, known as a dimension reduction method, maintains most of the changes in the data, it performs a dimension reduction. Statistical method order used in the present invention is as follows: fs - LIMB Raw Mass Spectral Data → Data Pre proces sing PCA Results Plotting The colon tissue embedded in the paraffin is analyzed in several steps. The mass spectra for organic compounds are detected by means of a detector (9) and the signal output by the detector (9) is input in a four-channel digital storage oscilloscope (10) triggered directly by the laser beam. The digital output from the oscilloscope (10) processes the signal presented above by means of a numerical calculation algorithm. The components obtained are selected to apply PCA as a principal component for the data processing (<m/z 100). In the mass spectrum obtained, the ion masses are selected in the region of m/z 60 - 100. The background signal does not feature any ion density and it is completely clear and no peak is observed in mass region before giving the sample to the spectrometer. Therefore, only the spectrum from the sample is observed and saved.As a result of the analyses performed within the scope of the present invention, it is obvious that all mass spectra obtained from healthy and cancerous colon tissues (12, 13) exhibit similar ion distribution in this region.

PCA results in Figure 3 illustrate the results of normal (healthy) and cancerous colon tissues obtained from the block tissues embedded in the paraffin. Said tissues further are re-analyzed by way of cutting the same onto the surface of microscope glass lamella. The tissue configurations constitute great importance in terms of interpreting the results achieved from both tissue samples. In Figure 3, it is seen that the results obtained from both tissues are compatible to each other. According to PCI axes, in the analysis of the tissues embedded in the paraffin, the tissue with tumor and the healthy tissue on the glass lamella coincide with the tissue with tumor and the healthy tissue in block state on the same side. This means that the results obtained are consistent with each other.

In the case of using a laser power of 1.8 W and laser intensity of 3.53x10 ¹⁵ W/cm ² , PCA statistical results are obtained and the experimental results are used to evaluate the statistical approaches. Two axes are used in evaluating the data obtained per group. In the analysis carried out within the scope of the present invention, each data group is regulated based on 10 different experimental results. Each point for all isomers comprises different mass components selected among the mass ion peaks in the mass spectrum, which is illustrated in Figure 4.

Fs-LIMS offers a precise, reliable, and therefore powerful novel approach in order to analyze the tissue recognition and the method both guides pathologists, surgeons, and patients during the rapid diagnosis and operation without any human error and detects the border of the cancerous region precisely. Spectrometric tissue analysis methods are capable of recognizing particularly the tissues based on the molecular structure.

In the present invention, the analyses prove that it is significantly successful in distinguishing the fresh muscle tissues from cow, sheep, and bones and cancerous and healthy tissues embedded in the paraffin. The inventive Fs-LIMS method is both sufficiently reliable and significantly rapid for analyzing the cancerous and healthy colon tissues (12, 13) embedded in the paraffin and block. The method implemented is capable of inputting the samples in the mass spectrometry and also of collecting data from biological and chemical samples and analyzing the same.

In Figure 4, (a) 17 normalized, healthy colon tissues embedded in paraffin, and a cancerous tissue embedded in paraffin are obviously differentiated from each other by using PCA statistical approach. The values herein indicate the types of 17 different healthy tissues and each point in both healthy and cancerous areas indicates the analysis of the 10 (ten) data saved successively and (b) the tissue spectra may be distinguished based on PCI and PC2 axes. The values herein indicate the mass/charge (m/z) rates which are necessary for PCA statistics and included in the study as a principal component. In Figure 5, normalized 11 cancerous tissues embedded in paraffin and the healthy tissue embedded in paraffin are distinguished from each other, (a) is PCA statistical approach, wherein the values are data of 11 different cancerous tissue types and each point in both healthy and cancerous area indicates the analysis result of the 10 data saved successively. In (b), dissemination of the tissue ion peaks can be distinguished based on PCI and PC2 axes. The values herein indicate the mass/load (m/z) rates of the tissue ions which are necessary for PCA statistics and included in the study as a principal component.

In an embodiment of the present invention, the cancerous tissue appointment can be performed in a very short period of time by way of using a laser-based MS in an intraoperative manner on the patient by means of implementing the inventive method on the tissues without paraffin.

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