WO2019013464A1

WO2019013464A1 - Method for treating biological tissue, laser treatment device and atmospheric pressure mass spectrometry imaging system

Info

Publication number: WO2019013464A1
Application number: PCT/KR2018/007051
Authority: WO
Inventors: 김재영; 문대원
Original assignee: 재단법인대구경북과학기술원
Priority date: 2017-07-10
Filing date: 2018-06-22
Publication date: 2019-01-17

Abstract

The present invention relates to a method for treating biological tissue, a laser treatment device and an atmospheric pressure mass spectrometry imaging system. The method comprises the steps of: a) preparing a pretreatment material solution for absorbing energy of near-infrared and visible band laser light; b) applying the pretreatment material solution on biological tissue to be treated and thus allowing the pretreatment material to be absorbed into the membrane of the biological tissue; c) focusing and irradiating near-infrared and visible band laser light to the biological tissue which has absorbed the pretreatment material and thus treating the biological tissue.

Description

A method for processing biological tissue, a laser processing apparatus and an atmospheric pressure mass spectrometry imaging system

TECHNICAL FIELD The present invention relates to a method for processing biological tissue, a laser processing apparatus, and an atmospheric pressure mass spectrometry imaging system, and more particularly to a femtosecond laser oscillator or continuous oscillation laser for processing biological tissue under atmospheric pressure, A laser processing apparatus, and an atmospheric pressure mass spectrometry imaging system.

In general, mass spectrometry, which is widely used in the field of analytical chemistry, is a very useful analytical method to grasp the composition and structure of organic substances through precise measurement of atomic composition and atomic weight of organic compound ions. The molecular information of the substance can be obtained. However, since general mass spectrometry uses a method of desorbing and ionizing a sample under high vacuum conditions, a biological sample containing a large amount of moisture can not be analyzed without complicated pretreatment. Therefore, the secondary ion mass spectrometry (SIMS (mass spectrometry)) method is mainly used in liquid chromatography or electrospray ionization mass spectrometry, in which a biological sample is destroyed and analyzed in a solvent, or the sample is subjected to a pre- ) Or matrix assisted laser desorption ionization (MALDI).

However, this method is a vacuum-based technique. Therefore, in order to put a biological sample into a vacuum chamber, various pretreatments such as elimination of moisture or substitution with other substances are required. In this process, samples are damaged and accurate analysis results are obtained none.

In order to overcome these problems, researches have been started to develop atmospheric pressure mass spectrometry technology mainly in USA and Europe. For example, paper spray ionization (PSI) and mid-IR lasers (DART), surface acoustic wave nebulization (SAWN), etc. have been developed. However, these methods are still used for the analysis of biological samples There is a problem and a detection limit. In addition, since it can not be used at all to implement mass spectrometry imaging combined with spatial information of a sample, high resolution imaging can not be obtained, and a new atmospheric pressure desorption ionization technology is required.

In addition, the development of treatment methods of living tissue using laser has been used in various fields in the medical field, such as resection of living tissues using laser. However, continuous oscillation laser can be focused, but there is no hurricane, The laser light of the upper broad band and the near infrared ray band has little influence on the living tissue, the laser light of the middle infrared band is invisible because it is invisible, and the focusing lens is very expensive.

On the other hand, a microwave pulse laser including a femtosecond laser has a very high attachment power in a very short time, and after that it has a rest period, which can reduce the thermal damage caused by the laser. However, There was a problem of leaving a large ablation mark more than a meter.

Also, Y.-K. Kim, H.-K. Na, S.-J. Kwack, S.-R. Ryoo, Y. Lee, S. Hong, S. Hong, Y. Jeong, and D.-H. Min, "Synergistic effect of graphene oxide / MWCNT films on laser desorption / ionization mass spectrometry of small molecules and tissue imaging" ACS Nano, 5 (6), 45504561 (2011) When a sample is placed on a double layered chip and the laser is irradiated, the energy of the laser is absorbed in the double-layered chip, and heat energy is transferred to the sample using the photothermal effect. Thus, the material is desorbed and ionized And mass spectrometry is carried out.

However, since the laser is located above the sample, the laser must pass through the sample to reach the double-layer chip. Therefore, the sample is thick or heterogeneous, which absorbs the energy of the laser in some areas, and partial absorption of energy causes distortion in the analysis of the whole sample.

Therefore, in the conventional method, it is possible to analyze a thin intercept structure of 15 탆 or less, and there is a problem that the range of application is very limited when it is used for mass analysis of a biological sample.

SUMMARY OF THE INVENTION The present invention provides a method and a laser processing apparatus for processing a biological tissue in an atmospheric pressure environment, the method being capable of minimizing the traces of treatment while preventing thermal damage from occurring.

Another object of the present invention is to provide a method and a laser processing apparatus for processing a living tissue capable of performing a cut-off process at a depth of 100 mu m or less within a width of 5 mu m using a laser of a relatively low output .

It is another object of the present invention to provide an atmospheric pressure mass spectrometry imaging system capable of desorbing an analyte from a biological tissue or a biological sample and imaging the desorbed analyte at a high resolution.

Another object of the present invention is to provide a high-resolution atmospheric pressure mass spectrometry for analyzing live biological samples capable of performing mass spectrometry by desorbing substances at atmospheric pressure using a laser of low output irrespective of the thickness or homogeneity of the sample And an analysis imaging system.

According to an aspect of the present invention, there is provided a method for processing biological tissue, comprising the steps of: a) preparing a pretreatment material solution that absorbs energy of laser light in a near-infrared light and a visible light band; b) Applying a substance solution to a biological tissue to be treated so that a pretreatment substance is absorbed into the biological tissue; and c) irradiating a living tissue absorbing the pretreatment substance with near-infrared light and visible light, And processing the tissue.

According to another aspect of the present invention, there is provided a laser processing apparatus for processing biological tissues, comprising: a light source for emitting laser light in a near-infrared light and a visible light band; a light source for converging the laser light of the light source to absorb energy of laser light in the near- And an objective lens for converging and irradiating the pretreatment material for converting into thermal energy to the biological tissue absorbed in the film.

According to another aspect of the present invention, there is provided an atmospheric pressure mass spectrometry imaging system including a laser processing apparatus for emitting laser light in a near-infrared light and a visible light band, a laser processing apparatus for converging laser light of the laser processing apparatus, An ionizing unit for ionizing the analyte desorbed from the living tissue, and a mass analyzer for collecting the ionized analyte and analyzing the mass of the analyte, .

According to another aspect of the present invention, there is provided an atmospheric pressure mass spectrometric imaging system comprising: a light source substrate for supporting living tissue and absorbing energy of laser light to generate heat; a laser light source for irradiating laser light in a near- An ionization unit for ionizing an analyte desorbed from the living tissue, and a mass spectrometer for collecting the ionized analyte and analyzing the mass of the analyte.

A method and a laser processing apparatus for treating a living body tissue according to the present invention are capable of processing a biological tissue at atmospheric pressure to increase the absorption rate of laser light energy and processing using a relatively low power laser in the processing, It is possible to prevent heat damage to the tissue.

In addition, biological tissues can be processed using near-infrared and visible laser beams, and focusing can be facilitated while using a low-cost focusing lens, thereby achieving an effect that the biological tissue can be finely processed.

The present invention can perform mass spectrometry with high reliability irrespective of the thickness and homogeneity of a sample by allowing a substance to be desorbed from a living tissue by utilizing a photothermal effect without transmitting a biological sample, It is effective.

In addition, the present invention increases the absorption rate of laser light energy absorbed in living tissue at atmospheric pressure, and enables processing using a relatively low power laser in the process, thereby obtaining a large amount of biomolecule information through one analysis process And it is possible to obtain a high-resolution mass barge imaging.

1 is a flowchart of a method for processing a living tissue according to a preferred embodiment of the present invention.

FIG. 2 is a spectrum of the optical characteristics of the gold nano-rod.

3A to 3D are helium ion micrographs of mouse hippocampal tissue specimens.

4 is a helium ion micrograph of a living tissue treated by the method for treating a living tissue of the present invention.

FIG. 5 is a photograph of a hippocampal tissue pretreated with gold nanospheres treated with a 532 nm continuous oscillation laser.

6 is a configuration diagram of an atmospheric pressure mass spectrometric imaging system according to an embodiment of the present invention.

7 is a configuration diagram of an atmospheric pressure mass spectrometric imaging system according to another embodiment of the present invention.

8 is a photomicrograph of a mouse hippocampal tissue before and after desorption.

9 is a mass spectrometric image of hippocampal tissue of an ICR (Institute of Cancer Research) adult mouse.

FIGS. 10A to 10G are results of analysis of the spatial distribution of detected ions for the subregion of the hippocampal tissue of the mouse by MS imaging.

11 is a morphological comparison of hippocampal tissue specimens after nanoPALDI analysis.

12A and 12B are images showing distortion of the original cholesterol distribution measured by a vacuum based secondary ion mass spectrometer (SIMS).

13A and 13B are images showing the result of ion image analysis according to two different condition treatments for drug effect investigation.

FIG. 14 is a block diagram of a high-resolution atmospheric pressure mass spectrometry imaging system for analyzing live biological samples according to another embodiment of the present invention.

Fig. 15 is a configuration diagram of the light modulation substrate in Fig. 14. Fig.

16 is a spectrum obtained by analyzing the optical properties of gold nano-rods.

17 is a block diagram of a high resolution atmospheric pressure mass spectrometry imaging system for analyzing live biological samples according to another embodiment of the present invention.

18A to 18D are helium ion micrographs of mouse hippocampal tissue specimens.

- Explanation of symbols -

100: laser processing device 200: light source

300: ionization processing unit 310: supply pipe

400: mass spectrometer 410: induction tube

420: chamber 500: vacuum pump

600: image pickup unit 610: objective lens

700: computer 800: light-emitting substrate

810: glass plate 820: light heat layer

Hereinafter, a method for processing biological tissue of the present invention, a laser processing apparatus, and an atmospheric pressure mass spectrometry imaging system will be described in detail with reference to the accompanying drawings.

The embodiments of the present invention are provided to explain the present invention more fully to those skilled in the art, and the embodiments described below can be modified into various other forms, The scope of the present invention is not limited to the following embodiments. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an," and "the" include plural forms unless the context clearly dictates otherwise. Also, " comprise " and / or " comprising " when used herein should be interpreted as specifying the presence of stated shapes, numbers, steps, operations, elements, elements, and / And does not preclude the presence or addition of one or more other features, integers, operations, elements, elements, and / or groups. As used herein, the term " and / or " includes any and all combinations of one or more of the listed items.

Although the terms first, second, etc. are used herein to describe various elements, regions and / or regions, it is to be understood that these elements, parts, regions, layers and / . These terms do not imply any particular order, top, bottom, or top row, and are used only to distinguish one member, region, or region from another member, region, or region. Thus, the first member, region or region described below may refer to a second member, region or region without departing from the teachings of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing embodiments of the present invention. In the figures, for example, variations in the shape shown may be expected, depending on manufacturing techniques and / or tolerances. Accordingly, embodiments of the present invention should not be construed as limited to any particular shape of the regions illustrated herein, including, for example, variations in shape resulting from manufacturing.

FIG. 1 is a flow chart of a method for processing a living tissue according to a preferred embodiment of the present invention. As shown in FIG. 1, in the atmospheric pressure environment, a treatment for increasing the energy absorption rate of laser light in the near- And a main processing step S200 of irradiating and processing the pre-processed biological tissue by focusing the laser light of the near-infrared light and the visible light band.

The preprocessing step S100 may include preparing a pretreatment material including nanoparticles (particles having an average particle diameter of 10 to 100 nm) of a material having a high absorption rate of laser light in the near-infrared light and visible light band (S110) (S120) of applying the pretreatment substance to the surface of the living tissue to be treated, and a step (S130) of waiting for the pretreatment substance to be sufficiently absorbed by the living tissue.

The pretreatment material prepared in the step S110 may be a solution containing a gold nanorod, a gold nanosphere, a graphene oxide, a reduced graphene graphene, and the energy of the laser light in the near infrared ray and visible light band may be more Nanoparticles of absorbable material can be used.

What should be considered here is that the nanoparticles are absorbed into the tissues of the organism, so they choose a metal or carbon compound known to be harmless.

Various methods known in the art can be used for the preparation of the pretreatment material. In the present invention, the following gold nano-rods are prepared.

First, 9.75 ml of CTAB (0.1 M) containing 0.25 ml of HAuCl4 (0.01 M) and 0.6 ml of a solution of NaBH4 (0.01 M) were mixed to prepare a seed solution and incubated at a temperature of 28 캜 for 3 hours And stirred vigorously.

Then, 3 ml of AgNO3 (0.01 M), 20 ml of HAuCl4 (0.01 M) and 3.2 ml of ascorbic acid (0.01 M) were added to CTAB (0.1 M) and mixed to prepare a growth solution. Thereafter, 3.2 ml of the seed solution was added to the growth solution to prepare a reaction mixture, which was gently shaken for several seconds and then held at 28 ° C for 6 hours to allow the gold nanorod to form.

When AuNRs were formed, the color of the solution turned purple. The AuNRs formed were found to have an average length of 41.9 (± 4.4) nm, a width of 12.2 (± 1.3) nm and a surface plasmon resonance peak (λmax) of about 765 nm.

For the preparation of AuNRs, 20 ml of the CTAB-AuNRs solution was centrifuged at 15,000 rpm for 15 minutes, and after removing the supernatant, the AuNRs were redispersed with 10 ml of distilled water. To this solution was added mPEG-SH (2.0 mg) and then 10 ml of ethanol was added. The solution was vigorously stirred for several seconds and then gently stirred for 12 hours.

The non-reacted mPEG was removed by centrifugation at 12000 rpm for 15 minutes, and the mPEG-modified AuNRs were prepared by suspending in water.

Through this process, CTAB-AuNRs (λmax = 800 nm) can be synthesized.

Next, in step S120, the pretreatment material is applied to the surface of the living tissue to be treated. The gold nano-rods, gold nanospheres, graphene grains, and reduced graphene grains included in the pretreatment material are absorbed into the biotissue by the action of the biotissue.

In step S130, the gold nano-rods, gold nanospheres (grains), oxidized grains or reduced graphene grains are allowed to be sufficiently absorbed into the biotissues. After 30 to 60 minutes, the substances for the pretreatment are sufficiently absorbed into the membrane of the biotissue.

In the present invention, the living tissue is a live living tissue, and the term 'treatment' of the living tissue includes micro-excision and micro-perforation, and it should be understood as a concept including desorption of a living tissue material for analysis of living tissue. Micro-cutting and micro-perforation may be widely used in the medical field. As will be described later in detail, the present invention can prevent the occurrence of thermal damage in the treatment of living tissue using a laser, thereby enabling elimination of painlessness and bloodlessness.

Next, in the main processing step (S200), the biological tissue processed through the preprocessing step (S100) is processed by using a laser processing apparatus.

The laser processing apparatus for emitting the laser light includes a light source section for emitting a near-infrared laser beam and a visible light laser beam having a wavelength of 532 to 802 nm, and an objective lens for converging a laser beam emitted from the light source section.

The laser processing apparatus may further include a moving unit capable of moving the light source unit and the focusing lens on a plane or in a space. In the case where the living tissue is a living body specimen, the living body specimen may include moving means capable of relatively moving the living body specimen in a plane or in a space.

More preferably, the waveguide can freely adjust the optical path of the laser beam emitted from the light source unit, and can be inserted into the body.

As described above, gold nano-rods absorbed in living tissue can absorb energy of near-infrared laser light and can obtain effective treatment results.

Gold nanorods with an aspect ratio of 4.0 (λmax = 800 nm) can absorb near-infrared light and quickly convert absorbed energy into heat energy.

When a gold nanorod is inserted into a biotissue through a biotissue membrane, the gold nanorod acts as a hotspot interacting with the near-infrared laser light in the living tissue and improves the efficiency of processing such as ablation and desorption, The spectral intensity can be derived.

FIG. 2 is a spectrum of the optical characteristics of the gold nano-rod. As shown in the figure, the absorption coefficient is maximized at a wavelength of 800 nm, and it is possible to absorb good laser light energy in the near-infrared and visible wavelength bands.

3A to 3D are helium ion micrographs of mouse hippocampal tissue specimens.

In Fig. 3A, the hippocampal tissue of the A specimen is a specimen which has not undergone any treatment, and it can be seen that the hippocampus is not resected by the focused laser beam of the laser treatment apparatus.

The above experiment was carried out with the energy of 300, 200, 100, 75, 50 mW in the energy range indicated by the laser beam of 532 to 802 nm wavelength, and it was found that the sample A was not accurately excised even with the energy of 300 mW have. In the drawing, the portion indicated by the dotted line is a virtual representation of the portion to be cut out.

3B is a pretreated specimen using a gold nanospherite solution as a pretreatment material, FIG. 3C is a grafted oxide specimen, and FIG. 3D specimen D is a specimen treated with reduced oxidized graphene.

The B specimens and the D specimens were subjected to very precise excision at an energy range of 200 to 300 mW, and in the case of the C specimens, the excision was performed at an energy of 100 to 300 mW.

As a result of the test, it can be confirmed that the specimens treated with the pretreatment material absorb the energy of the laser light and are excised by the laser light of relatively low energy.

As described above, the present invention increases the energy absorption rate of the laser light by pretreatment of the living tissue, so that the living tissue can be treated with laser light having a lower energy than the conventional one, so that heat damage to the living tissue can be prevented.

Further, laser light having a visible light range can be used, which facilitates the processing and facilitates focusing.

4 is a helium ion micrograph of a living tissue treated by the method for treating a living tissue of the present invention. In FIG. 4, the living tissue is a hippocampal tissue pretreated with gold nanorods and resected by a femtosecond laser having an 802 nm wavelength.

At this time, the width of the resection portion is in the range of 4.7 to 5.7 mu m, and it is possible to perform fine resection, and the biopsy around the resection portion is not damaged.

FIG. 5 is a photograph of the hippocampal tissue pretreated with gold nanospheres treated with a 532 nm continuous oscillation laser, and it can be confirmed that heat damage did not occur.

As described above, the portion treated by the method for biotissue treatment of the present invention may have the meaning of the resection or perforation formation. Further, the biological tissue can be optically monitored and analyzed during the processing.

That is, by using the present invention, the mass and composition of lipids, proteins, and other intracellular metabolites can be analyzed and visualized in a living state without destroying cells or living tissues.

As described above, according to the present invention, the biological tissue can be pretreated and treated with relatively low energy, and the treatment includes desorption.

The desorption can be performed by mass spectrometry and imaging analysis by desorbing the analyte from the biological tissue or the biological sample and ionizing the desorbed analyte.

6, an atmospheric pressure mass spectrometry imaging system according to an embodiment of the present invention includes a laser processing apparatus 100 for irradiating a pre-processed biological tissue (or sample 1) with laser light having a wavelength of 532 to 802 nm, An ionization processing unit 300 for ionizing the analyte desorbed from the biological tissue 1 as the light is irradiated, and an analyzing unit 300 for collecting and analyzing the ionized analyte using the suction force generated in the vacuum pump 500 A mass spectrometer (400), a light source unit (200) for providing illumination to the living tissue (1), an imaging unit (600) for capturing an image of the living tissue (1) through an objective lens (610) And a computer 700 for displaying a photographing result of the photographing unit 600.

As shown in FIG. 7, the supply pipe 310 of the ionization processing unit 300 may be connected to the induction pipe 410 of the mass spectrometer 400 to ionize the analyte.

The ionization processing unit 300 may use a plasma generator or an electric spray source.

Reference numeral 130 denotes a beam splitter which processes the laser light so that the image pickup unit 600 and the laser processing apparatus 100 can share the objective lens 610. [

Hereinafter, the structure and operation of the atmospheric pressure mass spectrometry imaging system according to an embodiment of the present invention will be described in more detail.

First, the laser processing apparatus 100 can be focused through the objective lens 610 by providing a laser beam having a wavelength of 523 to 802 nm in a near-infrared or visible light band.

The objective lens 610 may be a high magnification objective lens of a microscope, and the objective lens 610 may be shared with the imaging unit 600.

The imaging unit 600 may be an optical microscope and may obtain an image of the processing result of the living tissue 1.

The objective lens 610 is located on the lower side of the living tissue 1. [ The biological tissue 1 is placed on a substrate made of a transparent material such as glass. The objective lens 610 is located on the bottom side and the light source unit 200 is located on the upper side of the biological sample 1.

The light source unit 200 provides illumination enabling the biological sample 1 to be photographed using the imaging unit 600.

The laser light having a wavelength of 532 to 802 nm provided from the laser processing apparatus 100 is reflected through the first reflector 110 to be irradiated on the bottom surface of the biological sample 1 and analyzed by the pretreated biological sample 1 The material is desorbed.

As described in detail above, the pretreatment biological tissue is processed by the laser light of the laser processing apparatus 100, and not only fine resection or perforation can be formed, but also analyte such as protein is desorbed during the formation of the resection or perforation .

The thus-desorbed analyte is ionized by the plasma generated in the ionization treatment unit 300. The ionization of the analyte is for separating the analyte easily and can be supplied to the mass spectrometer 400 through the ionization post-induction tube 410 as in the example shown in FIG.

The ionization processing unit 300 may use an atmospheric pressure plasma apparatus capable of generating plasma at atmospheric pressure. The ionization processing unit 300 includes a supply pipe 310 for supplying a plasma to a position close to the biological tissue 1 in order to increase the ionization rate of the desorbed analyte. The supply pipe 310 may use a quartz tube.

The living tissue 1 may be placed on a stage capable of moving in a plane, and the living tissue 1 may be moved and processed on a plane.

7 and the supply pipe 310 are connected to the induction pipe 410 so that the analytical material moving through the induction pipe 410 can be ionized by the action of the vacuum pump 500. [

The induction pipe 410 may be made of stainless steel and the vacuum pump 500 forms an air flow so that the analyte moves through the induction pipe 410 and is supplied to the mass spectrometer 400.

The mass spectrometer 400 measures the mass of the analyte, and can measure the molecular mass of protein, metabolite, and lipid.

The light of the light source 200 is provided on the upper surface of the living tissue 1 and the laser beam L is emitted from the laser processing unit 100 to the imaging unit 600 The enlarged image of the living tissue 1 can be taken. At this time, the light incident on the image sensing unit 600 may be the light reflected through the second reflection unit 120 provided on the lower side of the objective lens 610.

As described above, the present invention can reduce the size of the apparatus and simplify the apparatus by making the optical path of the laser light of the laser processing apparatus 100 and the light incident to the image sensing unit 600 the same.

In addition, since the surface on which the laser light is directly processed can be imaged at the same position, an accurate image can be obtained after the laser processing.

The reason why the relatively inexpensive objective lens 610 can be used for focusing the laser light depends on the range of the wavelength of the laser, and the range of the laser wavelength is due to the pretreatment of the biological tissue 1.

The computer 700 is provided with a mass analysis program, and can transmit, analyze, and display analysis results of the mass spectrometer 400.

FIG. 8 is a micrograph of the hippocampal tissue of an adult mouse of the Institute of Cancer Research (ICR) before desorption and after desorption.

Desorption is well progressed in all laser irradiated areas and the area of damage due to desorption can be identified.

Hereinafter, the present invention will be described in more detail with reference to specific examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

The mass spectrometer 400 was a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer purchased from Thermo Fisher Scientific. For mass analysis, the operating requirements of the analyzer were to be performed under the following conditions.

- positive ion mode

- Mass resolution: 35000

- mass range: 1001000 m / z

- 1 micro scan

- Maximum injection time of 100 ms

- 106 Automatic gain control (AGC)

- Mass analyzer temperature: 350 ° C

The laser processing apparatus 100 used a mode-locked Ti-Sapphire oscillator (Synergy 20, Femtolasers) with a maximum single pulse energy of 5 nJ at 75 MHz and less than 20 femtoseconds at 802 nm.

The laser light was integrated into an inverted microscope by placing a dichroic beam splitter (FF720-SDi01, Semrock Co.) in front of the input aperture of the objective lens of the microscope. The final pulse energy for the sample was 2 nJ at 802 nm.

The ionization processor 300 uses a conventional dual electrode arrangement for a dielectric barrier discharge (DBD) jet. A nonthermal plasma jet was generated in a quartz tube having an inner diameter of 2 mm and an outer diameter of 3 mm. A 6 mm electrode was fabricated by wrapping a quartz tube with copper tape and spacing 7 mm from the inner end. The ground electrode was facing upward, the power supply electrode was facing downward, and the distance was 5 mm from the pipe orifice.

This is a dielectric barrier discharge (DBD) device with a small discharge current. A sinusoidal voltage having an input voltage of 5 kV and a frequency of 27 kHz was supplied to the ionization processing unit 300. The high purity helium gas was a high purity helium gas with a flow rate of 0.5 slm as the discharge gas. The ionization treatment unit 300 was positioned so as to have an angle of incidence of 30 degrees with respect to the living tissue.

The induction tube 410 was placed with a stainless steel tubing (id 4.57 mm, od 6.35 mm, length 120 mm), with a downward bending band of 30 degrees relative to the sample stage, A curved glass tube (id 2 mm, od 2.5 mm, length 20 mm) connects to the transfer tube for efficient collection of molecules and ions from the sample. A vacuum pump (WOB-L pump 2546, Welch Vacuum, 500) is used to create a flow of air to induction tube 410, A silicon tube having a 1/4 inch (6.35 mm) diameter was connected to the side opening of the chamber 420 and the pumping capacity of the pump was 30 l / min.

FireFly 2.2.00 for Thermo (Prosolia) was used to extract and combine data sets in MS imaging file format compatible with BioMAP, an image visualization and processing software program (Novartis Institutes for BioMedical Research, msi.org). BioMAP reconstructed all images and was used to display ion distribution images in this study. Intensity maps were chosen to indicate the intensity range corresponding to the m / z value of the analyte.

&Lt; Example 1 >

Mass spectrometry imaging analysis using living mouse hippocampal tissue

The mouse hippocampal tissue was selected for imaging analysis of living tissue, the hippocampal tissue was clearly structurally spaced between sub-regions such as cornu ammonis 1 (CA1), cornum ammonis 3 (CA3) and dentategyrus (DG) Short-term memory information plays a very important role in the integration of long-term memory and spatial search.

Fig. 9 shows a mass spectrometry image of hippocampal tissue of an ICR (Institute of Cancer Research) adult mouse showing the spatial distribution of molecular ions in hippocampal tissues.

The ion images were generated with 433 × 300 pixels covering the area of 1800 μm × 1500 μm. As a result, the spatial resolutions of the X and Y axes were 4.2 μm and 5 μm, respectively.

The measurement range for mass spectrometry was set at m / z = 100 to 1,000, but most of the strong ion signals were found at m / z = 500 or less from the hippocampal tissue of the mouse. Hundreds of ion images consistent with the optical imaging of the analysis area were observed in the range of m / z = 100 to 1,000, including cholesterol, ceramides, sphingolipids, and glycerophospholipids. Were identified. Several confirmed ion images with sufficient ionic strength were analyzed with an ion at m / z = 311.257 of MAG (monoacylglycerol; 16: 1), an ion at m / z = 313.273 of MAG 16: 0, m ion at m / z = 339.289, ion at MAG 18: 0 m / z = 341.305, ion at cholesterol m / z = 369.349, ion at cholesterol m / z = 385.346, And ceramide 18: 0 m / z = 548.540, respectively.

It was also confirmed that the CA1, CA3 and DG regions were clearly distinguished from the mouse hippocampus tissue. Observed ions showed almost similar spatial distribution in hippocampal tissue except for adenine ion. Interestingly, the adenine ion at m / z 136.062 was found to have a distinct spatial distribution. Compared with the DIC image, the spatial distribution of adenine was highly correlated with the soma of neurons in hippocampal tissue.

Additionally, analysis was performed at higher spatial resolutions for CA1, CA3, and DG regions, the sub-regions of hippocampal tissue. The atmospheric pressure nanoPALDI-MS system is a microscope-based mass spectrometry system that has the advantage of accurately selecting small areas. Since the sampling step involving desorption and ionization takes place in the microscope stage, the analysis area can be established directly at the x- and y- positions of the microscope stage while viewing under the microscope.

As shown in FIGS. 10A-10G, mass spectrometry images of specific molecular ions for selected regions can be compared. The ion image produces 433 × 100 pixels to cover the area of 600 μm × 500 μm. Therefore, the corresponding x-axis spatial resolution of 1.4 μm is analyzed at a sample migration rate of 10 μm / s and the y-axis spatial resolution is 5 μm. In addition, the area of analysis can be reduced, the x-axis spatial resolution of the ion image can be increased to the limit, and higher resolution MS imaging can be obtained. The y-axis spatial resolution could be increased by reducing the spacing between adjacent x-rays.

However, the spatial resolution in the y-axis direction was kept at 5 μm to avoid an increase in analysis time.

Because desorption and ionization continue at atmospheric pressure, the mass spectrometer's internal analysis rate (scan rate) in sequence mode can significantly affect the speed and number of mass spectra obtained, affecting the spatial resolution of MS imaging.

The optimized parameters of the mass spectrometer were set and used. The mass spectrum was obtained 433 times per minute, which corresponds to 7.2 times per second.

The spatial resolution in the X axis direction in the MS imaging obtained by the atmospheric pressure nanoPALDI method is determined by the sample movement velocity in the X axis direction (that is, the movement speed of the sample stage) and the spatial resolution in the Y axis direction is determined by two adjacent X axis laser beam paths Lt; / RTI > This allows the spatial resolution of the MS image to be adjusted according to the size of the region of interest and the total analysis time required.

As shown in Figs. 10A to 10G, the moving speed of the living tissue was set to 30 mu m / s in order to analyze the entire hippocampal region. However, in order to analyze the lower region of the hippocampus tissue, . Therefore, the spatial resolution in the X-axis direction in the MS image was improved from 4.2 m in Fig. 11 to 1.4 m in Figs. 10a-10g.

On the other hand, the spacing between adjacent x-axes was fixed at 5 占 퐉 in both experiments to maintain a similar analysis time, and the spatial resolution in the y-axis was found to be 5 占 퐉 in both Figs. 10a-10g and Fig.

&Lt; Example 3 >

Analysis of intracellular spatial resolution by atmospheric pressure nano PALDI method

To obtain a mass spectrometry (MS) image with a high spatial resolution, a uniformly strong desorption action by a small-sized focused laser beam and uniformly distributed AuNR in the entire tissue sample is required. After mass analysis, the morphology of hippocampal tissue was observed. A coherent anti-stokes Raman scattering (CARS) microscope was used to accurately measure the morphological changes of the laser-treated biological specimens focused in the unlabeled manner.

Near IR (NIR) wave pumping (802 nm and 1041 nm wavelength) CARS microscopy was used for nondestructive 3D biomedical imaging. Figure 11 shows a 3D reconstructed z-stack CARS image of a sample analyzed based on this method.

95 CARS images of the CH stretched region (2750-2960 cm ^-1 ) at the location scanned with randomly selected mass spectrometry were obtained with a z-axis step spacing of 0.5 μm and then merged into one feature using 3D visualization software . More specifically, 2D CARS images composed of 512 x 512 pixels were collected in z direction in 95 layers covering a total volume of 635 × 635 × 47 μm ³ . 11a to 11c were obtained from the same target and were reformatted at different viewing angles and magnifications. Because the spot size of the focused laser (about 1.2 μm) is less than 5 μm, the spacing between adjacent scanning lines, the track of the laser scanning can be clearly visualized (see FIGS.

Also, Figure 11d shows that the length of one trench by laser scanning was 5 占 퐉, and the desorption occurred in a very uniform and stable manner over the entire analysis area. As shown at the edge of the analysis area (Fig. 11e), mass volume of 35 um thickness was actually desorbed and mass analysis was used. This is a bulk analysis of analytical samples different from DESI, rather than an atmospheric pressure mass spectrometry of the present invention localized to the surface or near the surface of the sample.

To analyze the size of the linear craters generated by the focused laser beam in the hippocampal tissue, the 3D confocal imaging of the individual scan lines removed by the laser beam was performed using a confocal laser scanning microscope (LSM-700, Carl Zeiss ) (See Fig. 11, f).

The crater floor clearly identified the plateau. The widths of the bottom and top of the removed linear craters were estimated to be about 3 μm and 5.5 μm, respectively, from an average of 30 points. When the positioning stage moved at a constant speed of 10 m / s, the hippocampal tissue was exposed to fs laser pulses at a pulse repetition rate of 75 MHz with a pulse energy of 2 nJ. This means that the hippocampus tissue was scanned with 7.5 million laser shots per μm movement. In Fig. 11F, the width of the side wall of the crater is about 1 um, which means that laser ablation is completed within 1 um of movement. Therefore, the estimated spatial resolution of 1.4 um is possible by using AuNRs and ultrafast laser at atmospheric pressure. The leading crayers observed with a helium ion microscope (HIM, Orion NanoFab, Carl Zeiss) were consistently very steep and clean as shown in Figure 7, g.

Analysis method of atmospheric pressure nano PALDI using living tissue

Conventional atmospheric pressure ionization methods for mass spectrometry have attempted to improve desorption and ionization of volatile organic compounds by heating the target sample region with heated gas steam or heating the sample during sample preparation. However, the present invention uses fs laser oscillator and non-thermal atmospheric plasma as a dropout / ionization source together to avoid thermal damage to the sample. The application of gold nanorods to living tissues resulted in effective desorption results using fs near-infrared laser oscillators. Gold nanorods with an aspect ratio of 4.0 (λmax = 800 nm) can absorb near-infrared light and quickly convert absorbed energy into heat energy. Through the absorption of gold nanorods (AuNRs) into the biotissue membrane, within the living tissue, AuNR acts as a hotspot interacting with near-infrared light, improving the desorption efficiency and obtaining better mass spectral strength. In addition, the repetition rate of the fs laser oscillator was 75 MHz, corresponding to a pulse train of 13 ns intervals, and tissue sections were continuously irradiated and desorbed by a number of laser beams in a uniform motion. Therefore, the desorption by the femtosecond near infrared laser oscillator of the biological specimen was confirmed to be uniform and stable, and their profiles were observed in a sharp and steep shape, and the intracellular spatial resolution was mass-analyzed by imaging.

In order to obtain high-quality MS imaging, uniform distribution of AuNR in living tissue is a very important factor.

Thus, mPEG-specific gold nanorods were used for uniform distribution of AuNR in tissue.

Live living tissue was reacted with mPEG-AuNR containing artificial cerebral spinal fluid (ACSF) for 1 hour. After the reaction, the tissues were washed 10 times with ACSF to maintain AuNRs in the tissue. As a result, the desorption by the femtosecond-near-infrared laser oscillator appeared uniform and stable due to the action of AuNR in the region before analysis.

Despite the small beam size (1.2 m diameter) of the laser, the thickness of the desorbed tissue was found to be 35 um, as shown in Fig. Because the hippocampal tissue was dried after mass analysis in the CARS measurement, the thickness of the tissue in the aeration process was expected to be thicker than 47 um, which resulted in a specific increase in the aspect ratio of the laser-induced citer, . Without the treatment of AuNRs for mass spectrometric imaging, the prominent mass ion peak of the biomolecule would not have been observed. Therefore, the use of AuNR is important for efficient desorption, and its use enables atmospheric pressure MS imaging to deliver high resolution MS images.

In addition, as an example of the ionization treatment unit used in the present invention, the atmospheric plasma jet uses a non-thermal plasma even though it contains many kinds of charged species including electrons, ions and excited species, Do not damage live biological samples.

The generated plasma jet is not in direct contact with the sample because the configuration of the sampling equipment on the sample stage is such that the gas flow is inhaled. This series of desorption and deionization is very effective at analyzing living samples at atmospheric pressure conditions. In summary, intensive ultrafast laser light using AuNR was able to effectively remove a definite volume from a biological sample, and atmospheric helium plasma ionized to 19.8 eV of excited helium atom for MS imaging of atmospheric biomedical tissue.

With the simultaneous application of nanoparticles and a separate subsequent ionization source, the laser energy pulse was reduced compared to using a laser source alone. Lowering the pulse energy not only reduces the heat damage of the sample but also suppresses scattering of the desorbed material due to the laser irradiation, so that a very clear and clear desorption pattern can be obtained. This is why we used a cost-effective, simple-structure fs laser oscillator with much lower pulse energies than using an fs laser amplifier (several nanojoules vs. several hundred microjoules, for instance, 24 nJ vs. 150800 μJ) as the desorption source.

In addition, the atmospheric pressure nanoPALDI method according to the present invention is characterized in that living tissue can be analyzed under atmospheric pressure analysis conditions. Thus, the MS image obtained by atmospheric pressure nanoPALDI-MS has little to prepare for the sample compared to the vacuum-based secondary ion mass spectrometer (SIMS), which is effectively used for geological imaging with high spatial resolution (~ 1 μm) There are advantages. Vacuum-based secondary ion mass spectrometry (SIMS) samples (specimens) are prepared by frozen sectioning and drying, which can often lead to distortions in lipid imaging. The distortion of the original cholesterol distribution is clearly observed in HIM and cholesterol SIMS images (see FIGS. 12A and 12B). It can be seen that the separated cholesterol crystals formed in the SIMS sample preparation in the HIM image are clearly confirmed (see Fig. 12A), and the SIMS image can confirm the original cholesterol distribution and other shapes (see Fig. 12B). 9 and 10A to 10G, it was found that the cholesterol image derived from living tissues was not distorted due to the fasting and drying of the sample while the spatial resolution remained almost the same as SIMS. Atmospheric pressure No additional technical barriers are required to develop new atmospheric pressure mass imaging techniques because all of the nanoPALDI input can be transported through a small tube such as an optical fiber, a plasma jet tube, or an ion transport tube at atmospheric pressure.

&Lt; Example 5 >

Analysis of molecular composition of specific regions of hippocampal tissue observed by mass spectrometry imaging

Neurons in the central nervous system exhibit various dendritic structures closely related to neuronal connectivity. In hippocampal formation, granular cells of CA (cornu ammonis) pyramidal cells and DG (dentate gyrus) are known to be two major neuronal types that represent distinct dendritic arbor structures. These different types of neuronal types and their distinct structures allow different molecular distributions in the CA and DG regions. As shown in Figs. 8 and 10A to 10G, ionic fragments of monoacylglycerol, cholesterol, and ceramide showed higher signal intensities in the CA1 and CA3 regions than in the DG region. In contrast, ions at adenine m / z = 136.062 were concentrated in the DG region. Differences in the ion image of adenine between the CA and DG regions appeared as two neuronal cell types, an abstract cell and a dendritic granule cell, with different distribution and different cell phenotypes in the hippocampal tissue of the mouse. In fact, the granule cell body is packed tightly and a small neural tube is inserted between the cells of the tooth. Therefore, the adenine ions generally observed in the nucleus were concentrated in the DG region.

In a similar fashion, the abstract cells in the CA1 and CA3 regions structurally have two different dendrites, long and thick dendrites and several basal projections from the top and bottom of the soma, respectively. The apex and base protrusions are opposite to each other and exist in different layers, and the protrusion structure of the abstract cell shows the shape of biconical. Vertex and basal dendrites are known to differ in many properties, including size, geometry, electrical conduction, and responsiveness to neurotrophic factors or guidance molecules. In addition, much research has been done on morphological differences between vertex and basal dendrites, and the different molecular composition between them has not yet been elucidated, as there is no suitable method for molecular composition analysis and no reliable molecular markers. To investigate the molecular differences between the apex of abstract neurons and the basolateral dendrites at the tissue level, subregions of hippocampal tissue were analyzed at high spatial resolution (see FIGS. 10c and 10d). In particular, in CA3 ion images were clearly divided into both adenine sites, and the cell body locations (ion image at m / z = 136.062) could be seen in FIGS. 10d and 10e. In CA3, the left side of the ion image shows the dense part of the apex dendrites, and the right side shows the dense part of the basal dendrites of the hippocampus.

Using the BioMap software's region of interest (ROI) analysis function, the same region of the ROI was assigned to both left and right, and the intensity of the mass spectra of monoacylglycerol ion, cholesterol ion, and ceramide ion was compared (Figures 10e, 10f And Table 1).

As a result of analysis, the MS intensity of MAG ion ((at m / z = 311.257, m / z = 313.273, m / z = 339.289, m / z = 341.305) The ceramide ions at m / z = 548.540 were observed similarly in both cases.

High-resolution MS imaging using the atmospheric-pressure nanoPALDI method showed that the MAGs were 1.4 to 1.8 times higher than the basal dendritic-intensive regions and 1.4 to 1.8 times higher than the basal dendritic-intensive regions, and the ceramides were relatively evenly distributed in both dendritic regions at m / z = 548.540 there was. In addition, the cholesterol at m / z = 385.346 in the peak dendritic intense region showed about 3 times higher signal intensity in the peak dendritic intensity region than the base dendritic intensity region. A comparison of four monoacylglycerol ions specifically showed that MAGs with saturation of 16: 0 and 18: 0 showed an imbalance toward the basal dendritic region from the peak to MAG with unsaturated 16: 1 and 18: 1 fatty acids (1.8: 1 and 1.5: 1 vs. 1.4: 1 and 1.4: 1). With such a high resolution MS image, the present inventors confirmed apical dendrites containing monoacylglycerol and cholesterol more than the basophilic dendrites, and the ceramides were uniformly distributed in both dendritic structures in the CA of living hippocampal tissues . Similar differences between the apical dendritic region and the basal dendritic region were also confirmed in CA1 (see Fig. 10C).

Detailed atmospheric pressure nanoPALDI mass imaging (Fig. 10c and 10d) of the apical dendritic region of CA3 was observed as a mossy fiber bundle in which the DG granule cells in the stratum lucidum expanded parallel to the abstract cell layer.

In the proximal apex dendrite region of CA3, a region of 100 mu m width with low intensity of MAG could be confirmed along the line of the abstract cell body (see Fig. 10C). In the CA3 region, the moss fibers were in two major bundles, the main suprapyramidal projections in the clear layer and the intra and infra abstract projections, first within the proximal extent of the stratum pyramids and stratum orientations, and crossing the transparent strata in CA3 . CA3 pyramidal neurons are in synaptic contact with the islets of the moss fiber via the overgrown specific complexes of the recruited spinal callus. In the distal CA3 neurons near CA1, aberrant abnormal growth is mainly found in apical dendrites at a distance of 10 to 120 μm from the soma. Considering the line profiles of adenine and MAG (18: 1) from Figures 10g to 10c and 10d, the adenine profile is evident in the abstract cell line. The MAG (18: 1) profile exhibited a low intensity of 100 μm width in the apical dendrites adjacent to the cell line, resulting in a layer structure due to mossy fiber projection and visible abnormal growth from CA3 neurons Match. No similar distribution of cholesterol and ceramide (18: 0) images was observed. This observation indicates that the atmospheric nano PALDI differs in molecular composition between moss fiber projections and other apex dendritic regions. Similar features in the apical dendritic region as shown in Figure 10C in the CA1 region were not observed because the mossy fiber projection did not reach CA1. These results indicate that atmospheric nano PALDI imaging with intracellular resolution can be used to obtain information on the molecular composition at the tissue level without tagging or staining of the antibody.

&Lt; Example 6 >

Removal of Cholesterol by Methyl Beta-Cyclodextrin in Living Hippocampal Tissues

Atmospheric pressure on living samples The nano PALDI MS image can provide a lot of spatial information about the metabolites, which can be useful for drug screening at tissue level. Therefore, the inventors of the present invention found that the cholesterol depletion phenomenon by methyl- beta -cyclodextrin (m beta CD) treatment was analyzed by atmospheric pressure nano-PALDI MS imaging Respectively. The hippocampal tissues of mice treated with mβCD in an aeration condition were immersed in an ACSF solution containing mβCD (25 mg / ml) for 6 hours.

Thereafter, ion images were collected from two different hippocampal tissues, the two different images being normal hippocampal tissue (see FIG. 13A) and hippocampal tissue treated with mβCD (see FIG. 13B), respectively. For hippocampal tissue treated with m [beta] CD, the two cholesterol peaks at m / z 369.349 and m / z 385.346 were found to be suppressed and disappeared in the entire assay region as shown in Fig. 13b. Interestingly, the ceramide ion at m / z 548.540 was also inhibited. Other identified ions were slightly decreased compared to the normal group. Therefore, the present inventors have found that the atmospheric pressure MS imaging analysis method designed in the present invention can be used for screening of tissue-based drugs, and it is more accurate than the cell-based drug screening method of killing laboratory animals And reliability.

As described above, the atmospheric pressure mass spectrometry imaging system of the present invention is capable of drug screening.

Specifically, the candidate drug is treated with a biotissue, and the analyte is desorbed from the biotissue, so that the action of the drug can be directly confirmed by comparing the components, the content, and the spatial shift of metabolites, lipids or proteins with those of the control without the drug treatment .

FIG. 14 is a block diagram of a high resolution atmospheric pressure mass spectrometry imaging system for analyzing live biological samples according to an embodiment of the present invention.

14, a high-resolution atmospheric pressure mass spectrometry imaging system for analyzing a live biological sample according to an embodiment of the present invention includes a photo-thermal substrate 800 on which a biological tissue (or a sample) 1 is mounted, 800 for irradiating laser light having a wavelength of 532 to 802 nm and an ionization processing unit 300 for ionizing analytes desorbed from the living tissue 1 as the laser light is irradiated, A mass spectrometer 400 for collecting and analyzing ionized analytes using a suction force generated by a vacuum pump 500, a light source 200 for providing illumination to the living tissue 1, An imaging unit 600 for imaging an image of the living tissue 1 through the imaging unit 610 and a computer 700 for displaying an imaging result of the imaging unit 600.

Hereinafter, the structure and operation of a high-resolution atmospheric pressure mass spectrometry imaging system for analyzing a live biological sample according to a preferred embodiment of the present invention will be described in more detail.

First, the photo-thermal substrate 800 is assumed to be mounted on a stage that moves substantially in a plane.

Fig. 15 is a configuration diagram of the light modulation substrate 800. Fig.

Referring to FIG. 15, the photo-thermal substrate 800 has a structure in which a glass plate 810 and a photo-thermal layer 820 are formed on the glass plate 810.

When the laser light is irradiated to the photothermal layer 820, the photothermal layer 820 absorbs the energy of the laser to emit heat to form the biotissue 1 ). &Lt; / RTI >

As described above, the laser is preferably irradiated directly to the photothermal layer 820 without passing through the living tissue 1. [ To this end, the present invention is configured to form a photo-thermal layer 820 on a glass substrate 810 through which laser is transmitted, and to irradiate the laser light from the bottom of the glass substrate 810 to the photo-thermal layer 820.

That is, the laser of the laser processing apparatus 100 is reflected through the reflecting portion 110 and irradiated to the photo-thermal layer 820 through the glass substrate 810. [

Therefore, the energy of the laser is not absorbed in a part of the living tissue 1, and distortion in mass analysis can be prevented from occurring.

The laser processing apparatus 100 provides a laser beam having a wavelength of 523 to 802 nm in a near infrared ray or visible light band and the irradiation direction is switched by the reflecting unit 110 in the vertical direction of the optical thermal substrate 800, 610). &Lt; / RTI >

The objective lens 610 may be a high magnification objective lens of a microscope. As described above, the objective lens 610 performs focusing of the laser light, identifies the biological tissue 1 processed through the imaging unit 600 And serves to provide the light from the light source unit 200 to the image sensing unit 600 so that an image can be picked up.

The material is desorbed from the sample of the biological tissue (1) by the photothermal action of the photothermal layer (820) together with the irradiation of the laser. The light absorption layer 820 absorbs a part of the light energy of the laser to generate heat, and the laser which is not absorbed by the light modulation layer 820 is irradiated to the biological tissue 1 to desorb the material. At this time, the material is more easily detached by heat generated in the photothermal layer 820.

As a specific example of the photothermal layer 820, there may be used graphene grains coated on the upper surface of the glass substrate 810, reduced graphene grains, nano-rods of nano-particles of metal having high absorptance of near- Layer.

The metal having a high light energy absorption rate of the near-infrared light and visible light broadband laser may be the same.

16 is a spectrum obtained by analyzing the optical properties of gold nano-rods. As shown in the figure, the absorption coefficient is maximized at a wavelength of 800 nm, and it is possible to absorb good laser light energy in the near-infrared and visible wavelength bands.

In order to stabilize the graphene oxide, the reduced graphene oxide, the gold nanorod, or the gold nanoparticle layer, it may be coated with a coating layer having high heat resistance and high light transmittance.

This is to prevent foreign matter from being generated when the laser light is irradiated to the photothermal layer 820. Examples of the coating layer include PEI (Ultem).

The desorbed material is ionized by the plasma generated in the ionization processing unit 300. The ionization of the material is for separating the analyte easily, and can be supplied to the mass spectrometer 400 through the ionization post-induction tube 410 as in the example shown in FIG.

The ionized analyte may be supplied to the mass spectrometer 400 through the induction tube 410.

17, the other configuration is the same as that of the atmospheric pressure mass spectrometry imaging system described in detail with reference to FIG. 14, except that the supply pipe 310 of the ionization processing unit 300 is connected to the induction pipe 410, Can be treated and ionized as it passes through the membrane 410.

The ionized analyte is introduced into the chamber 420 and supplied to the mass spectrometer 400 for analysis.

18A to 18D are helium ion micrographs of mouse hippocampal tissue specimens.

As shown in FIGS. 18A to 18D, when the hippocampal tissue specimens having various thicknesses were subjected to CW laser light output of 300, 200, 100, 75 and 50 mW, respectively, the material was detached, One can confirm that the analyte is desorbed uniformly in the output range of 300 to 100 mW.

That is, since the present invention irradiates the laser light below the photothermal layer 820, the analyte can be desorbed from a living sample irrespective of the thickness of the sample.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention will be.

INDUSTRIAL APPLICABILITY The present invention is industrially applicable as an analyte can be desorbed from a biological sample using a laser having a relatively low output by increasing the energy absorption rate of laser light.

Claims

A light source for emitting a laser beam having a wavelength of 532 to 802 nm; And

The laser light of the light source is focused and the pretreatment substance which absorbs the energy of the laser light in the wavelength band of 532 to 802 nm is focused and irradiated onto the internal tissue of the membrane or the laser light of the wavelength band of 532 to 802 nm And an objective lens for irradiating the living body tissue through a light-heat substrate which generates heat by absorbing the laser light.
The method according to claim 1,

Wherein the pretreatment material absorbed in the membrane of the biotissue comprises:

A gold nanoparticle, a gold nanoparticle, a gold nanoparticle, a gold nanoparticle, a gold oxide nanoparticle, a gold nanoparticle, a gold nanoparticle, a graphene oxide graphene nanoparticle, or a reduced graphene graphene nanoparticle.
The method according to claim 1,

Wherein the light-

Glass substrate,

And a light heating layer which is positioned on the upper surface of the glass substrate and on which the living tissue is deposited and absorbs energy of laser light in a wavelength band of 532 to 802 nm to generate heat.
The method of claim 3,

The light-

Gold nanorod layer, gold nanoparticle layer, oxidized graphene nanoparticle layer, or reduced oxidized graphene nanoparticle layer.
The method according to claim 2 or 3,

And the energy of the laser beam is 100 to 300 mW.
A laser processing apparatus for emitting laser light in a 532 to 802 nm wavelength band;

A pretreatment material for absorbing the energy of the laser light and converting the laser light into heat energy is focused on the living tissue absorbed in the film by being positioned on the lower side of the living tissue and focusing the laser light of the laser processing device, An objective lens for focusing and irradiating the biological tissue located on the object;

An ionization unit for ionizing an analyte desorbed from the living tissue; And

And a mass analyzer for collecting and analyzing the mass of the ionized analyte.
The method according to claim 6,

An imaging unit having the same optical path as that of the laser processing apparatus and imaging the living tissue through the objective lens; And

Further comprising a computer for receiving and displaying an imaging result of the imaging unit and analysis results of the mass spectrometer.
8. The method of claim 7,

The energy of the laser light is 100 to 300 mW,

The pretreatment material absorbed in the membrane of the biotissue may be one or more of gold nano-rods, gold nanoparticles, oxidized graphene nanoparticles, and reduced oxidized graphene nanoparticles to absorb energy and convert to heat energy Pressure mass spectrometry imaging system.
The method according to claim 6,

Wherein the light-

A glass substrate,

And a light heating layer positioned on an upper surface of the glass substrate to receive the living tissue and generate heat by absorbing energy of the laser light.
10. The method of claim 9,

Wherein the light-

Further comprising a coating layer coated on the surface of the photothermographic layer.
11. The method according to claim 9 or 10,

The light-

A gold nanoparticle layer, a gold nano-rod layer, a gold nanoparticle layer, a graphene oxide graphene nanoparticle layer, or a reduced graphene oxide graphene nanoparticle layer.
11. The method of claim 10,

Wherein the coating layer comprises:

PIE. &Lt; / RTI >
a) preparing a pretreatment material solution that absorbs energy of laser light in the near-infrared light and visible light bands;

b) applying the pretreatment material solution to the biological tissue to be treated so that the pretreatment material is absorbed into the biological tissue; And

c) a step of irradiating the living tissue absorbing the pretreatment substance with laser light in the near-infrared light and visible light band and irradiating the same to treat the living tissue.
14. The method of claim 13,

The pre-

Wherein the gold nanoparticles are one or more of gold nanoparticles, gold nanoparticles, gold nanoparticles, gold nanoparticles, oxidized graphene nanoparticles, and reduced oxidized graphene nanoparticles.
15. The method of claim 14,

The step b)

After the pretreatment material solution is applied, it is allowed to stand for 30 to 60 minutes,

Characterized in that one or two or more of the gold nanorod, gold nanoparticle, oxidized graphene nanoparticle, and reduced oxidized graphene nanoparticle is absorbed into the membrane of the biotissue .
16. The method of claim 15,

In the step c)

The wavelength of the laser light in the near-infrared and visible light bands is 532 to 802 nm,

One or two or more of the gold nanorods, gold nanoparticles, oxidized graphene nanoparticles, and reduced oxidized graphene nanoparticles absorbed in the membrane of the biotissue absorbs energy of the laser light and is converted into thermal energy To the living body tissue.
14. The method of claim 13,

The process of step c)

A method for treating a living tissue, characterized by being resection, perforation or desorption of living tissue.
18. The method of claim 17,

Wherein the energy of the laser light irradiated in the step (c) is 100 to 300 mW.