KR102086716B1 - High resolution ambient mass spectrometric imaging system for analysis of living biomaterials - Google Patents

Abstract

The present invention relates to a high resolution atmospheric mass spectrometry imaging system and its use for the analysis of live biological specimens. Specifically, the present invention desorbs analyte from a sample using gold nanoparticles and a near infrared pulse laser as a desorption source, and performs mass spectrometry and imaging analysis by ionizing the desorbed analyte with a low-temperature atmospheric plasma jet or an electrospray source. A laser unit for irradiating a near-infrared laser and a mass spectrometry imaging method under atmospheric pressure-assisted gold nanoparticles comprising a step; A plasma supply unit supplying a low temperature atmospheric plasma jet to a sample; A sample stage unit on which a sample is seated and a scanning stage is mounted to enable automatic scanning; An inverted optical microscope having the sample stage mounted on an upper end thereof; An ion transfer unit transferring the desorbed and ionized analyte from the sample into a mass spectrometer; A mass spectrometer for analyzing the ionized analyte; And a monitor unit equipped with a program that collects, stores, and interprets the analysis result.

Description

High resolution ambient mass spectrometric imaging system for analysis of living biomaterials

The present invention relates to high resolution atmospheric mass spectrometry imaging systems and their use for the analysis of living biological samples.

Mass spectrometry, which is widely used in the field of analytical chemistry, is a very useful analytical method to determine the composition and structure of organic compounds by precise measurement of the atomic composition and atomic weight of organic compound ions. Molecular information of material can be obtained. However, general mass spectrometry uses a method of desorption and ionization of a sample under high vacuum conditions, and thus a biological sample containing a lot of moisture cannot be analyzed without complicated pretreatment. Therefore, secondary ion mass spectrometry (SIMS), which mainly uses liquid chromatography or electrospray ionization mass spectrometry, in which a biological sample is destroyed and then put in a solvent, or the sample is put into a vacuum chamber after pretreatment, is analyzed. ) Or matrix assisted laser desorption ionization (MALDI).

However, this method is a vacuum-based technique, which requires a number of pretreatments such as removing moisture or substituting with other substances to put a biological sample into a vacuum chamber. In this process, the sample may be damaged and an accurate analysis result may be obtained. none.

In order to overcome this problem, research has recently begun to develop atmospheric mass spectrometry techniques in the US and Europe. For example, paper spray ionization (PSI) and mid-IR lasers have been developed. Desorption ionization, direct analysis in real-time (DART), and surface acoustic wave nebulization (SAWN) using surface acoustic wave devices have been developed. In addition to contamination problems and detection limitations, new atmospheric pressure desorption ionization techniques are required because they cannot be used to implement mass spectrometry imaging by adding spatial information of samples and high resolution imaging cannot be obtained.

Therefore, the present inventors obtained various biomolecule information directly from an undamaged biological sample containing water in the study of metabolites, lipids, and proteins of biological samples to obtain cells or tissues. The present invention has been completed by chemically analyzing the composition and structure of constituent metabolites, lipids and proteins, and adding spatial information to the development of high resolution atmospheric mass spectrometry imaging systems for the analysis of living biological specimens capable of mass spectrometry imaging.

Korea Patent Registration No. 10-1748525

Accordingly, an object of the present invention is to perform mass spectrometry and imaging analysis by desorption of analytes from a sample using gold nanoparticles and near-infrared pulsed laser as desorption sources, and ionizing the desorbed analytes with a cold atmospheric plasma jet or electrospray source. It is to provide a mass spectrometry imaging method at atmospheric pressure assisted gold nanoparticles comprising the step of.

It is another object of the present invention to provide a high resolution atmospheric mass spectrometry imaging system.

It is another object of the present invention to provide a method for analyzing biomolecule information of a sample using a high resolution atmospheric mass spectrometry imaging system.

It is another object of the present invention to provide a drug screening method using a high resolution atmospheric mass spectrometry imaging system.

In order to achieve the object of the present invention as described above, the present invention desorbs the analyte from the sample by using gold nanoparticles and a near infrared pulse laser as a desorption source, the desorbed analyte to a low temperature atmospheric plasma jet or electrospray source The present invention provides a method for mass spectrometry imaging under atmospheric pressure assisted with gold nanoparticles, including ionizing to perform mass spectrometry and imaging analysis.

In one embodiment of the present invention, the gold nanoparticles may be any one selected from the group consisting of gold nanorods, gold nanospheres, gold nanowires and gold nanotubes.

In one embodiment of the present invention, the sample may be a living sample derived from an individual.

In addition, the present invention, the laser unit for irradiating the near infrared laser; A plasma supply unit supplying a low temperature atmospheric plasma jet to a sample; A sample stage unit on which a sample is seated and a scanning stage is mounted to enable automatic scanning; An inverted optical microscope having the sample stage mounted on an upper end thereof; An ion transfer unit transferring the desorbed and ionized analyte from the sample into a mass spectrometer; A mass spectrometer for analyzing the ionized analyte; And a monitor unit equipped with a program that collects, stores, and interprets the analysis results.

In one embodiment of the present invention, the ionizing material may further include a pumping unit (pumping system) inside the mass spectrometer so that the mass spectrometer may be introduced into the mass spectrometer.

In one embodiment of the present invention, when the analyte is desorbed from the sample by near-infrared laser irradiation, the sample may be treated with gold nanoparticles.

In one embodiment of the present invention, the gold nanoparticles may be any one selected from the group consisting of gold nanorods, gold nanospheres, gold nanowires and gold nanotubes.

In one embodiment of the present invention, the cold atmospheric plasma jet may be a non-thermal atmospheric helium plasma jet.

The present invention also provides a method for analyzing biomolecule information of a sample using the high resolution atmospheric mass spectrometry imaging system of the present invention.

In one embodiment of the present invention, the biomolecule may be selected from the group consisting of metabolites, lipids and proteins present in the sample in a living state.

The present invention also provides a drug screening method using the high resolution atmospheric mass spectrometry imaging system of the present invention.

In one embodiment of the present invention, the method comprises the steps of treating a candidate drug with a sample and comparing changes in the composition, content and spatial movement of metabolites, lipids or proteins in the sample with a control that has not been treated with the candidate. It may include.

The present invention relates to a high-resolution atmospheric mass spectrometry imaging system for the analysis of living biological specimens, the material analysis method using the system according to the present invention using a near-infrared pulse laser and gold nanoparticles as a desorption source, using a cold atmospheric plasma jet By using the ionization source, it is possible to analyze live samples under atmospheric pressure, obtain a large amount of biomolecule information in one analysis process, obtain high-resolution mass spectrometry imaging and optical imaging, and furthermore, It can be used for efficacy analysis and drug screening.

Figure 1a is a high-resolution atmospheric mass spectrometry imaging system according to the present invention, ① QE Orbitrap mass spectrometer, ② fs laser unit, ③ gas cylinder, ④ 5 ch. Gas flow controller, ⑤ non-thermal atmospheric plasma jet, ⑥ air-assisted ion transport, ⑦ automatic scanning (sample stage), ⑧ invert microscope, ⑨ HV driving circuit and HV probe, 조 joystick of automatic scanning, 긴 long for microscope movement Long drive scope stages, a microscopic imaging monitor, a PC for scanning software and MS software, respectively.
Figure 1b shows the overall process of sample analysis according to the present invention.
2 is a view showing the connection with the fs laser oscillator, atmospheric plasma jet and gas flow assist ion transmission equipment in the atmospheric pressure nanoPALDI mass spectrometer system according to the present invention, (a) is a photograph of the atmospheric MS system, ( b) shows a schematic of the drop-off and ionization process under atmospheric pressure on the stage, and (c) shows the different light paths between visible and near infrared light using a dichroic mirror.
In Figure 3 (a) relates to the gas flow auxiliary ion transfer equipment consisting of the ion transport tube, the chamber and the drying pump, (b) shows a customized chamber, the chamber is connected to the side hole of the chamber, the ion transport tube Air through the MS inlet. (c) is a photograph showing an analytical process during mass spectrometry on a sample stage, and (d) is a schematic diagram of an analytical process.
Figure 4a shows the results of analysis with air assisted ion transport equipment, the top graph shows the TIC (total ion flow) on the MS software before and after the pump operation, (A) is before the pump operation , (B) shows after the pump operation.
Figure 4b shows the results of optical characterization of mPEG-AuNRs, (a) is obtained by dissolving mPEG-AuNRs in distilled water to obtain UV-Vis spectrum, (b) shows the HR-TEM picture of mPEG-AuNRs. .
FIG. 5 shows mass spectrometry and optical images of tissue sections of mouse hippocampus, the upper two figures showing differential interference contrast (DIC) imaging (Before: before analysis, After: after analysis), The images represent images of ions identified at specific m / z locations of certain materials.
Figure 6 shows the results of analyzing the spatial distribution of the detected ions in the sub-region of the hippocampal tissue of the mouse by MS imaging, showing the analysis results in the CA1, CA3, DG region. (a) shows before MS analysis, (b) shows a picture after MS analysis, (c) and (d) shows the analyzed ion images compared with CA1, CA3, DG, ( e ) shows that 6 identical ROIs were assigned to both sides based on the adenine boundary of CA3 using ROI analysis function in Biomap software, (f) shows the relative distribution of analyzed ions, and (g ) Shows the results of analyzing the distribution of ionic strength of MAG (18: 1) and adenine in CA3.
Figure 7 shows the morphological changes of hippocampal tissue specimens after nanoPALDI analysis, (a) ~ (e) is a CARS (Coherent anti-stokes Raman for the vibration mode of 2750 cm ^{-1 ~} 2950cm ^{- 1} after atmospheric pressure mass spectrometry scattering image. Specifically, (a) shows a top plan view, (b) shows a side view of an orange square, (c) shows an enlarged photograph of a red square, (d) shows a CARS intensity profile, and (e) (A) shows a cross section across the horizontal dashed line in (a), (f) shows a linear pattern profile by 3D confocal imaging, and (g) shows the result of helium ion microscopy analysis.
Figure 8 shows the distortion of the original cholesterol distribution measured by vacuum-based secondary ion mass spectrometry (SIMS), (a) shows the HIM image analysis picture, (b) shows the SIMS image analysis picture.
Figure 9 shows the results of the ion image analysis according to the treatment of two different conditions for investigation of the drug effect, (a) shows the normal hippocampal tissue, (b) shows the hippocampal tissue treated with β-cyclodextrin (mβCD) It is shown.
10 is a photograph showing a laser induced desorption process in HEK cell line.
11 is a schematic diagram showing the principle of the material analysis by the high-precision atmospheric pressure mass spectrometry system according to the present invention.

The present invention is characterized in that it provides a high resolution atmospheric mass spectrometry imaging technology capable of analyzing and imaging the mass and composition of metabolites, lipids and proteins, such as metabolites, lipids and proteins in a living state without destroying cells or biological tissues. To this end, we developed atmospheric desorption and ionization sources, developed a single cellular level atmospheric pressure mass spectrometry through a gas flow ion particle delivery device, and added a scanning device to provide spatial distribution information on biomolecular information. In addition, mass spectrometry imaging of metabolites such as lipids and proteins in the sample was obtained at high resolution.

Secondary ion mass spectrometry (SIMS), a representative mass spectrometry imaging technique that has been developed and used in the past, requires a separate pretreatment process for putting a sample into a vacuum chamber and cannot analyze high molecular weights (only those under 800 Da) can be analyzed. Analysis of the sample is not possible. In addition, another technique, matrix laser desorption ionization mass spectrometry (MALDI), requires the use of an ultraviolet band laser to perform the analysis under atmospheric pressure, which is inconvenient for the system configuration because the position of the laser must be close to the sample. Is difficult to analyze. Desorption electrospray ionization (DESI) has a problem of low spatial resolution of mass spectrometry imaging, and it is impossible to analyze live samples because it is a method of wiping a sample with a solution. Meanwhile, in the present invention, a new analysis system capable of simultaneously performing mass spectrometry and imaging analysis at high resolution with respect to a living sample was developed. For this purpose, the ionization of the specimen under conditions that minimize thermal damage to the sample under atmospheric pressure And desorption process is required, so low temperature atmospheric pressure desorption / ionization source is needed, so that the analyte is desorbed from the biological sample through the optical path through the optical path using a near-infrared pulse laser to desorb the analyte from the biological sample. It was ionized using a spray source and analyzed using a high performance mass spectrometer. In addition, we have developed a new atmospheric pressure mass spectrometry imaging system by fabricating an ion delivery system that facilitates the delivery of ionized analytes to the mass spectrometer and incorporating an automatic scanning device on the stage for high resolution mass spectrometry imaging. Molecular information on various metabolites, lipids, and protein bodies was obtained and imaged.

The analysis of the metabolite in the biological sample using the high resolution atmospheric mass spectrometry imaging technology developed in the present invention will be described in more detail below.

According to the present invention, desorption of analyte from a sample using gold nanoparticles and a near-infrared pulsed laser as a desorption source, and ionizing the desorbed analyte with a low-temperature atmospheric plasma jet or an electrospray source, perform mass spectrometry and imaging analysis. Gold nanoparticles, characterized in that it provides a mass spectrometry imaging method at atmospheric pressure assisted.

In another aspect, the present invention is to provide a high-resolution atmospheric mass spectrometry imaging system, the system includes a laser unit for irradiating a near infrared laser; A plasma supply unit supplying a low temperature atmospheric plasma jet to a sample; A sample stage unit on which a sample is seated and a scanning stage is mounted to enable automatic scanning; An inverted optical microscope having the sample stage mounted on an upper end thereof; An ion transfer unit transferring the desorbed and ionized analyte from the sample into a mass spectrometer; A mass spectrometer for analyzing the ionized analyte; And a monitor unit equipped with a program for collecting, storing, and interpreting an analysis result.

The laser unit is a place for irradiating a near-infrared pulse laser to be used as a desorption source of the sample under atmospheric pressure, and the near-infrared ray is used as a source for sample desorption under atmospheric pressure because the beam is not scattered at atmospheric pressure.

The near-infrared laser focuses the beam through a high magnification objective lens to form a beam having a size of a smartimeter.

In addition, in the present invention, for more effective desorption, the nanoparticles absorbing the near infrared band were used together, and gold nanoparticles may be used as the nanoparticles.

The gold nanoparticles that can be used in the present invention is not limited thereto, but any one selected from the group consisting of gold nanorods, gold nanospheres, gold nanowires, and gold nanotubes may be used, and one embodiment of the present invention may be used. In the example, gold nanorods were prepared and used.

The plasma supply unit supplies a low temperature atmospheric plasma jet to a sample. The plasma jet is generated in a quartz tube having an inner diameter of 2 mm and an outer diameter of 3 mm.

The atmospheric plasma jet generated through the supply unit ionizes the analyte desorbed from the sample.

In addition, in one embodiment of the present invention, a non-thermal high-purity helium plasma jet was used as the plasma jet, and disposed to have an incident angle of 30 degrees with respect to the analytical sample.

The sample stage unit is a place for seating the sample to be analyzed, the scanning stage is mounted for clear and fast imaging analysis is possible automatic scanning. The coordinates of the x-y axis are set in the scanning stage, and the sample stage is positioned in a form mounted on the upper end of the inverted optical microscope.

The inverted optical microscope allows optical imaging to observe the shape of the sample through optical microscope analysis, and is also equipped with a dichroic mirror to separate the path of the near-infrared laser beam and the path of visible light to provide optical imaging and mass analysis. Imaging can be obtained simultaneously.

In addition, a dichroic beam splitter (FF720-SDi01, Semrock) was installed to introduce the fs near-infrared laser beam to the inverted optical microscope. This was possible at the same time.

The ion transfer unit serves to deliver the desorbed and ionized analyte from the sample into the mass spectrometer, and the ion transfer unit is specifically composed of a chamber, a drying pump and an ion transport tube (see FIG. 3). The chamber was mounted at the mass spectrometer inlet and the ion transport tubes used stainless steel tubes and were positioned to have a 30 degree downward bend to the sample stage. The drying pump is mounted to provide an auxiliary gas flow to enhance ion transport and is connected to a hole in the side of the chamber.

The sample-derived desorption / ionized analyte is transferred to the mass spectrometer through the ion transfer unit, and the transfer is carried out by the assist of gas flow by the pumping unit installed inside the mass spectrometer. .

In one embodiment of the present invention, the total ions of the analyte by the gas flow assisted ion transfer unit were measured. As shown in FIG. 4A, the difference in TIC before and after the pumping operation was found to be about 40 times. In other words, the gas flow assisted by the pumping operation showed that the total ions analyzed in the mass spectrometer increased about 40 times compared to the results analyzed without the pumping operation.

The mass spectrometer for analyzing the ionized analyte may use a mass spectrometer capable of measuring the mass of a conventional material. In one embodiment of the present invention, a mass spectrometer purchased from Thermo Fisher Scientific was used.

In addition, the monitor unit is equipped with a program for collecting, storing and interpreting the analysis results to derive the analysis results through a program for collecting, storing and interpreting both the results analyzed by the mass spectrometer and the inverted optical microscope analysis imaging results. This is where.

The high resolution atmospheric mass spectrometry imaging system devised in the present invention can be used to analyze biomolecule information of a sample.

In other words, it is possible to analyze and obtain information about various biomolecules such as metabolites, lipids and proteins present in the sample in a living state.

Accordingly, the present invention can provide a method for analyzing biomolecule information of a sample using a high resolution atmospheric mass spectrometry imaging system.

The biomolecules mean all the biomolecules present in the sample in the living state, but are not limited thereto, and may be metabolites, lipids, or proteins.

In one embodiment of the present invention it was confirmed that the hippocampal tissue of the mouse as a sample can analyze the content, distribution, location, etc. of the biomolecules present in the hippocampal tissue.

In addition, the present invention may provide a drug screening method using a high resolution atmospheric mass spectrometry imaging system.

Specifically, the method may comprise treating a candidate drug with a sample and comparing changes in the composition, content, and spatial movement of metabolites, lipids, or proteins in the sample with a control that has not been treated with the candidate.

In one embodiment of the present invention, cholesterol depletion in tissues treated with methyl-β-cyclodextrin and untreated tissues was analyzed to determine whether drug screening was possible using the system of the present invention.

As a result, the peak change of cholesterol in the imaging obtained by the system analysis of the present invention was clearly confirmed (see FIG. 9).

Therefore, the present inventors have found that the system of the present invention can be used both for drug efficacy analysis, screening for disease treatment agents, and the like. The effect is that the mass spectrometry and imaging results can be obtained in large quantities at one time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

<Materials and Test Methods>

① mass spectrometer

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

Positive ion mode

Mass resolution: 35000

Mass range: 1001000 m / z

-1 microscan

-100 ms maximum injection time

10 ⁶ AGC (automatic gain control)

Mass Spectrometer Temperature: 350 ° C

② Near infrared  Near infrared fs  lasers)

The fs near infrared laser oscillator was used as the desorption source in the MS (mass spectrometer) system. The fs near infrared laser is a mode-locked Ti-Sapphire oscillator (Synergy 20, Femtolasers) with 5 nJ maximum single pulse energy of less than 20 femtoseconds at 75 MHz, 802 nm. The energy of the fs laser system is controlled by a neutral density (ND) filter graded in front of the rear port of an inverted optical microscope used in the analysis phase of the sample. The fs laser beam was integrated into an inverted microscope by placing a dichroic beam splitter (FF720-SDi01, Semrock) in front of the microscope's input aperture. The final pulse energy for the sample was 2 nJ at 802 nm.

③ atmospheric pressure Plasma jet (Atmospheric pressure plasma jet)

Atmospheric pressure plasma jets used a common dual electrode array for the DBD (dielectric barrier discharge) jet. Nonthermal plasma jets were generated in quartz tubes with an inner diameter of 2 mm and an outer diameter of 3 mm. The electrode of 6 mm size was prepared by wrapping a quartz tube with copper tape, and the distance from the inner end was 7 mm. The ground electrode was faced upwards, the power supply electrode faced downwards, and a distance of 5 mm from the tube orifice. This is a dielectric barrier discharge (DBD) device with a small amount of discharge current. A sinusoidal voltage having an input voltage of 5 kV and a frequency of 27 kHz was supplied to the plasma jet apparatus. As the high purity helium gas, high purity helium gas having a flow rate of 0.5 slm was used as a discharge gas. The plasma apparatus was positioned to be in a direction having an angle of incidence of 30 with respect to the sample.

④ Gas flow  Ions for auxiliary Delivery

Ion transfer part for gas flow assistance consists of a chamber, a transfer tube and a drying pump. The chamber is a customized chamber with transfer tubing installed at the mass spectrometer inlet and pump side connected to the chamber. Stainless steel tubing (id 4.57 mm, od 6.35 mm, length 120 mm) was positioned to have a 30 degree downward bend band for the sample stage and a 60 degree curved glass tube (id 2 mm, od). 2.5 mm, length 20 mm) was connected to the transfer tube for efficient collection of molecules and ions from the sample. A drying pump (WOB-L pump 2546, Welch Vacuum) was used to provide auxiliary gas flow to improve ion transport in the feed tube. A silicon tube with 6.35 mm (1/4 inch) was connected to the side hole of the chamber and the pumping capacity of the pump was 30 l / min.

⑤ Programmable  Auto scan

Mass spectral data is obtained in the form of bundles of line scans. In the programmable motorized X-Y scanning stage (AS-MIX73-C, iNexus), stepper motors are controlled externally via joysticks and motion control programs. All elements of scanning, X-Y coordinates, scan speed, scan direction, interruption time and number of scans, were programmed using custom stage control software. The motion control program also delivered logic signals to the scanning stage and mass spectrometer simultaneously. This allows the MS data to be collected and synchronized with high accuracy during the scanning process. The sample stage is line scanned while moving in the X direction and sent in the Y direction for MS imaging. Each single line scan in the X direction was saved as a data file. These mass spectrometers can use the sequence mode of the data acquisition program to obtain hundreds of data files in one experiment. Data files of multiple scan lines from the analysis region assembled the MS image plots into one data for X and Y coordinates such that line scans were set in the X and Y directions provided.

⑥ MSI  Software for

FireFly 2.2.00 for Thermo (Prosolia) was used to extract and combine datasets in an MS imaging file format compatible with BioMAP, an image visualization and processing software program (Novartis Institutes for BioMedical Research, http: //www.maldi- msi.org). BioMAP was used to reconstruct all images and display ion distribution images in this study. Intensity maps are displayed by selecting the intensity range corresponding to the m / z value of the analyte.

⑦ mPEG-modified gold nanorods  (mPEG- AuNRs ) Produce

CTAB-AuNRs (λ _max = 800 nm) were synthesized by the following method. To this end, a seed solution was first prepared by mixing 9.75 ml of CTAB (0.1 M) containing 0.25 ml of HAuCl ₄ (0.01 M) and 0.6 ml of cold NaBH ₄ (0.01 M) solution. The mixed solution was stirred vigorously for 3 hours at a temperature of 28 ° C. for 2 minutes. Next, a growth solution was prepared, in which 3 mL of AgNO ₃ (0.01M), 20 mL of HAuCl ₄ (0.01M) and 3.2 mL of ascorbic acid (0.01M) were added to CTAB (0.1 M). It was prepared by mixing. Thereafter, 3.2 ml of the seed solution was added to prepare a reaction mixture, which was gently shaken for several seconds, and then maintained at 28 ° C. for 6 hours. When AuNRs formed, the color of the solution turned purple. AuNRs formed were observed 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 (λ max) of about 765 nm (see FIG. 4B). For the preparation of mPEG-modified AuNRs, 20 mL of CTAB-AuNRs solution was centrifuged at 15,000 rpm for 15 minutes, the supernatant was removed, and AuNRs were redispersed with 10 ml of distilled water. MPEG-SH (2.0 mg) was added to the solution followed by 10 ml of ethanol. The solution was stirred vigorously for several seconds and then gently stirred for 12 hours. Non-reacted mPEG was removed by centrifugation at 12000 rmp for 15 minutes, mPEG-modified AuNRs were suspended in water, and the optical properties of the prepared fiberized mPEG-AuNRs were analyzed.

⑧ Preparation of Hippocampal Brain Slices

Six-week-old male ICR mice were purchased from Koatech (Pyeongtaek, Korea) and bred under aseptic conditions. All experiments were performed according to the animal experiment safety education guidelines approved by Daegu Gyeongbuk Institute of Science and Technology. Brains were extracted from rats, and hippocampus was isolated and tissues were sectioned to a thickness of 200 μm using a tissue knife (McLlwain tissue chopper, Cavey Laboratory Engineering). Sections were 124 mM NaCl, 2.5 mM KCl, 1.25 mM. KH ₂ PO ₄ , 26 mM NaHCO ₃ , ₂ mMMMgSO ₄ , 2.5 mMMCaCl ₂ , 10 mM MD-glucose and oxidized sucrose artificial cerebrospinal fluid containing 4 mM D-sucrose using an Acuriumium bubbler at 32 ° C for 2 hours. Ventilated by bubbling with 95% oxygen and 5% carbon dioxide For removal of cholesterol, each section was washed with artificial cerebral spinal fluid (ACSF) without sucrose and then 25 mg / h at 32 ° C. for 6 hours. Reactions were performed with an artificial cerebrospinal fluid without sucrose treated with ml methyl β-Cyclodextrin (mβCD, Sigma-Aldrich), and the control group was treated with the non-mβCD-treated group. density 10.0 at 800 nm, 200 Each section was immersed in 5 ml of artificial cerebrospinal fluid containing 1 μl), reacted with AuNRs for 1 hour, washed hippocampal sections 10 times, and then coated with 0.1% polyethylenimine (PEI, in 25 mM borate buffer). The prepared tissue samples do not need to be completely dried when analyzed by the system of the present invention so that analysis can be started within 20 minutes.

⑨ 3D CARS Imaging

After mass spectrometry imaging analysis, samples were fixed overnight at 4% paraformaldehyde at 4 ° C. Thereafter, it was thoroughly washed with water and ethyl alcohol before being fixed to the imaging chamber. Optical images are Coherent Anti-Stokes Raman scattering scattering (CARS) microscopes made of a combination of ultra-fast pulse (~ 120fs) laser source (Insight deepsee dual, Spectra physics) and xy-direction galvanometric scanners (FV-1000, Olympus) The galvanometric metric scanner is equipped with a converted microscopic system (IX-83, Olympus) equipped with a 20 × objective (UPLSAPO20X, NA: 0.75). Lipid (fat) sensitive chemical controls were generated with a mixture of 1041 nm Stokes pulse trains (80 MHz) adjusted to a pump of 802 nm and a central vibration mode of 2860 cm ^-1 . The obtained CARS images were analyzed and processed using Olympus Fluoview 1.7a software. The images scanned in the Z-direction were stacked in an axial step size of 0.5um to produce 3D images as well as cross sections. The accumulated images were made into 3D structures for more graphical effects using Amira® 5.3.3 image analysis software (Visualization Sciences Group).

⑩ 3D Confocal  Laser scanning Imaging

After laser desorption, the tissue sections were fixed with the CARS method using 4% paraformaldehyde solution. Optical images were collected using a confocal laser scanning microscope (LSM-700, Carl Zeiss), and the confocal laser scanning microscope used in the present invention used a Zeiss AxioImager Z2m microscope equipped with a laser scanning unit. The optical train is equipped with a 100 × / 0.8 HD Zeiss EC Epiplan objective. The light was irradiated with a 5mW solid state laser which produced a monochromatic light of 405 nm. Images scanned in the Z-direction were accumulated to an axial step size of 0.7um to produce 3D images and cross-sectional views. Image data and analysis were analyzed using LSM Software (ZEN 2010M).

⑪ Helium ion microscope  image

Helium ion microscopy (HIM) analysis was performed at 35 keV and 0.05-0.5 p-A probe current using Orion NanoFab (Carl Zeiss). In particular, non-conductive coatings were applied to the samples prior to imaging analysis. Preparations for helium ion microscopy include a 55-degree slope, 0.244 pA beam current, 128 scans average, 10.92 mm working distance, 20 μm gas field ion source lifespan, 0.5 μs scan dwell time, and GFIS field of view 350 μm gas field ion source viewing angle.

< Example  1>

Atmospheric pressure nanoscale for high resolution mass spectrometry images PALDI -MS system production

 The present inventors have fabricated a high-resolution atmospheric mass spectrometry imaging system for high resolution analysis in a living state without destroying intracellular tissue. The high-precision atmospheric pressure mass spectrometry imaging system manufactured by the present invention is shown in FIG. 1, and description of each component constituting the system is as follows.

The system according to the present invention comprises: ① QE Orbitrap mass spectrometer, ② fs laser unit, ③ gas cylinder, ④ 5 ch. Gas flow controller, ⑤ non-thermal atmospheric plasma jet, ⑥ air-assisted ion transport, ⑦ automatic scanning (sample stage), ⑧ invert microscope, ⑨ HV driving circuit and HV probe, 조 joystick of automatic scanning, 긴 long for microscope movement It consists of a long drive scope stage, a microscopic imaging monitor, a PC for scanning software and MS software, the location of each component being shown in FIG.

In order to optically monitor biological samples and determine the area of analysis, a stage of inverted optical microscope (IX73, Olympus) was used as the sample stage for desorption and ionization as shown in FIG. 2A. Laser oscillators without optical amplifiers produce ultrafast laser pulses with pulse energy of 2nJ and pulse repetition rate at 802nm. Details of the laser system are described in the above method technology section, and a dichroic beam splitter (FF720-SDi01, Semrock) was installed to introduce an fs near infrared laser beam into an inverted optical microscope. This allows optical imaging monitoring and laser detachment at the same time in a biological sample without a separate objective lens as shown in FIG. 2C.

  All MS images displayed in this experiment were obtained using a 20 × objective lens (NA = 0.45; LUCPlanFLN 20X, Olympus), and the diameter of the focused laser beam can be reduced to 1.2 μm.

Especially for high quality and high spatial resolution MS imaging, the beam size of the laser for sample removal plays an important role. Focused laser beams with high magnification objectives can concentrate light energy in smaller areas, leading to better desorption effects. To confirm this, 100 x objectives (UPLSAPO100XO, NA: 1.4, Olympus) with a focused laser beam as small as 500 nm were irradiated with a small area of less than 1 μm in a single HEK cell to induce desorption. The results are shown in FIG. 10. However, due to the high magnification, shallow depths of field were not evenly scanned by the laser. Since live tissue sections sliced and aerated with tissue knives are not ideally flat or uniform, desorption by laser can only be performed in a limited area of the total analysis area, such as hundreds to thousand or two thousand square micrometers. Thus a very small laser beam focused by a high magnification lens cannot produce uniform sampling over the entire area and impairs MS imaging. In order to ensure adequate margin of field depth for uniform sampling over the entire analysis area, the present invention uses a 20x objective lens (LUCPLFLN20X, NA: 0.45, Olympus), and the resulting diameter of the focused laser beam is 1.2 micrometers. It was. In addition, high-magnification objectives for focusing the laser beam can be used on sub-micrometer spatial resolution, provided that biotissue sections of the same thickness can be obtained for the entire sample.

For accurate positioning of the region of interest at the micrometer level and samples moving at constant speed for raster scanning, a programmable motorized XY scanning stage is fixed to the inverted optical microscope.

 The configuration of the non-thermal atmospheric helium pressure plasma jet composed of a tube (tube) with a discharge gas and an electrode was such that a helium plasma medium was formed on the sample as shown in FIG. 2B. The neutral molecules desorbed by the focused laser are in strong contact with the helium plasma medium by placing two desorption / ionization sources, some of which are ionized by metastable helium atoms with an excitation energy of 19.8 eV.

As shown in Figures 2a and 2b, a pumping system for gas flow assisted ion transport was placed in the mass spectrometer for the efficient transport of molecules and ions. The gas flow auxiliary ion transfer unit is composed of a transfer tube, a chamber, and a drying pump (see FIGS. 3A and 3B), and the pump generates a gas flow from the ion transfer tube (the transfer tube) into the mass spectrometer so that the ionized analyte is mass spectrometer. Inlet (see FIGS. 3C and 3D). Using the gas flow assisted ion transfer device, the strength of the ions was increased by approximately 40 times as compared to the absence of the pump (see FIG. 4A).

In addition, the rod-shaped gold nanoparticles (AuNRs) used in the present invention were used as essential elements for efficient and stable desorption by a near infrared oscillator. Gold nanorods are thermal energy transfer materials as well as acting as photon energy storage. Most biological analytes do not absorb near-infrared (NIR) light effectively, but gold nanorods absorb near-infrared with longitudinal resonance caused by the aspect ratio and convert photon energy from the laser beam into thermal energy. Thus, contacting gold nanorods in living tissue with fs laser light can enhance the desorption effect and achieve high mass spectrum strength. But without the gold nanorods, this is not the case. In the present invention, mPEG-AuNRs were used as gold nanorods, and UV-Vis spectrum results and HR-TEM imaging images according to the use thereof are shown in FIG. 4B.

< Example  2>

Mass Spectrometry Using Live Mouse Hippocampus Tissue Imaging  analysis

Hippocampal tissue of mice was selected for imaging analysis of living tissue, which is clearly structurally separated between subregions such as cornu ammonis 1 (CA1), cornum ammonis 3 (CA3), and dentate gyrus (DG). In addition, short-term memory information plays a very important role in the integration of long-term memory and spatial search. FIG. 5 shows an optical microscope image and a mass spectrometric image of hippocampal tissue of an Institute of Cancer Research (ICR) adult mouse. FIG. The top two figures show optical micrographs before and after mass spectrometry, respectively. After the analysis was completed, desorption proceeded well in all areas of the analysis and the areas of damage due to desorption were identified. 5 is a photograph showing the spatial distribution of molecular ions in the hippocampal tissue, the ion image was generated with 433 × 300 pixels covering an area of 1800 μm × 1500 μm. As a result, the corresponding spatial resolutions in the X and Y axes were 4.2 μm and 5 μm, respectively.

Although the measurement range for mass spectrometry was set to m / z = 100 to 1,000, strong ion signals were mostly confirmed at m / z = 500 or less from mouse hippocampal tissue. Hundreds of ionic images were observed in the range m / z = 100 to 1,000, consistent with optical imaging of the analytical region, of which cholesterol, ceramides, sphingolipids and glycerophospholipids The intercept of was confirmed. Several identified ion images with sufficient ionic strength are shown in FIG. 5, where m / z of MAG (monoacylglycerol; 16: 1) at ions at 311.257, m / z at MAG 16: 0 at 313.273, MAG 18: 1 ions at m / z = 339.289, MAG 18: 0 m / z = ions at 341.305, cholesterol m / z = ions at 369.349, cholesterol m / z = ions at 385.346, adenine m / z Ions at = 136.062 and ions at ceramide 18: 0 m / z = 548.540 were observed, respectively. It was also confirmed that the CA1, CA3 and DG regions were clearly distinguished from mouse hippocampal tissue. The observed ions showed almost similar spatial distribution in hippocampal tissues except for adenine ions. Interestingly, adenine ions at m / z 136.062 were found to have distinct spatial distributions. Compared with the DIC image, it was confirmed that the spatial distribution of adenine was highly correlated with the cell body of neurons in hippocampal tissue.

In addition, analyzes were performed at higher spatial resolution for sub-regions of hippocampal tissue, the CA1, CA3 and DG regions. Atmospheric pressure nanoPALDI-MS systems are microscope-based mass spectrometry systems that offer the advantage of accurate selection of small areas. Since the sampling step, including desorption and ionization, takes place at the microscope stage, the analytical region can be established directly to the x- and y- positions of the microscope stage while viewing under the microscope. As shown in FIG. 6, it is possible to compare mass spectrometric images of specific molecular ions for selected regions. The ion image produces 433 × 100 pixels to cover an area of 600 μm × 500 μm. Therefore, the corresponding x-axis spatial resolution of 1.4 μm was analyzed at a sample movement speed of 10 μm / s, and the y-axis spatial resolution is 5 μm. In addition, because the analysis area is 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 decreasing the spacing between adjacent x-rays. However, the y-axis spatial resolution was kept at 5 μm to avoid an increase in analysis time.

Since 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 acquired, affecting the spatial resolution of MS imaging. Using the mass spectrometer's optimized parameters, the mass spectrum was obtained 433 times per minute, corresponding to 7.2 times per second.

In MS imaging obtained with atmospheric nanoPALDI method, the spatial resolution in the X-axis direction is determined by the sample movement speed in the X-axis direction (i.e., 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. Determined by the distance between. This allows the spatial resolution of the MS image to be adjusted depending on the size of the region of interest and the total analysis time required. As shown in FIG. 6, the sample moving speed was set to 30 μm / s to analyze the entire hippocampal region, but as shown in FIG. Accordingly, the spatial resolution in the X-axis direction coinciding with the MS image was improved from 4.2um in FIG. 7 to 1.4um in FIG. On the other hand, the spacing between adjacent x-axes was fixed at 5um in both experiments to maintain similar analysis time, and the spatial resolution of the y-axis was 5um in both FIGS. 6 and 7.

< Example  3>

Atmospheric pressure nano PALDI  By way Intracellular  Spatial Resolution Analysis

Mass spectrometry (MS) images with high spatial resolution require uniform and powerful desorption by a small focused laser beam and AuNR uniformly distributed within the entire tissue specimen. Therefore, after mass spectrometry, the shape of hippocampal tissue was observed. Coherent anti-stokes Raman scattering (CARS) microscopy was used to accurately measure the morphological changes of laser treated biological samples focused in a label-free manner. Near Infrared (NIR) wave pumping (802 nm and 1041 nm wavelength) CARS microscopy was used for nondestructive 3D biometric imaging. 7 shows a 3D reconstructed z-stack CARS image of a sample analyzed based on this method. 95 CARS images of CH stretched regions (2750-2960 cm ^{- 1} ) at locations scanned by 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 a z-direction with 95 layers covering a total volume of 635 x 635 x 47 μm ³ . 7A to 7C of FIG. 7 are obtained from the same target and reformatted with different viewing angles and magnifications. Since the spot size of the focused laser (about 1.2 μm) is less than 5 μm, the distance between adjacent scanning lines, it is possible to clearly visualize the track of laser scanning (see FIGS. 7B and 7C). In addition, FIG. 7D shows that one trench length by laser scanning is 5 μm, and desorption occurs in a very uniform and stable manner over the entire analysis area. As seen at the edge of the analysis region (FIG. 7E), the detached portion showed a very inclined (steep) shape, with a volume of 35 μm thick from the surface of the mouse hippocampal tissue (approximately 74% of the premature tissue layer). Desorption revealed that mass spectrometry was used. This is not the analysis of the atmospheric mass spectrometry of the present invention limited to or near the surface of the sample, but bulk analysis of analytical samples other than DESI.

In order to analyze the size of the linear crater generated by the focused laser beam in the hippocampal tissue, 3D confocal imaging of individual scan lines removed by the laser beam was carried out using a confocal laser scanning microscope (LSM-700, Carl Zeiss). ) (See FIG. 7F). The crater bottom clearly identified the plateau. The bottom and top widths of the removed linear craters were estimated to be about 3um and 5.5um at 30 points on average. When the positioning stage moved at a constant speed of 10 μm / s, the hippocampal tissue was exposed to fs laser pulses at a pulse energy of 2 nJ and a pulse repetition rate of 75 MHz. This means that hippocampal tissue was irradiated with 7.5 million laser shots per 1 μm of movement. In FIG. 7F, the sidewall width of the gradator was found to be about 1 um, indicating that laser ablation was completed within 1 um movement. Therefore, the estimated spatial resolution of 1.4um is possible by using AuNRs and ultrafast lasers under atmospheric pressure. The preceding craters observed by helium ion microscopy (HIM, Orion NanoFab, Carl Zeiss) were consistently very steep and clear as shown in FIG. 7G.

< Example  4>

Atmospheric pressure with living tissue nano Of PALDI  Analysis method

Conventional atmospheric pressure ionization methods for mass spectrometry have attempted to improve the desorption and ionization of volatile organic compounds by heating the sample area using heated gas steam or by heating the sample during sample preparation. However, the present invention uses a fs laser oscillator and a non-thermal atmospheric plasma together as a dropout / ionization source to avoid thermal sample damage. Application of AuNR to living tissue yielded effective desorption results using the fs near infrared laser oscillator. AuNR with an aspect ratio of 4.0 (λ max = 800 nm) can absorb near-infrared light and quickly convert the absorbed energy into thermal energy. By absorbing AuNR into the sample tissue membrane, AuNR acts as a hot spot within the living tissue to interact with near-infrared light, improving desorption efficiency and leading to better mass spectral intensity. In addition, the repetition rate of the fs laser oscillator was 75 MHz, which corresponds to a pulse train of 13 ns intervals, and tissue sections were continuously irradiated and detached by numerous laser saws in uniform motion. Therefore, desorption by femtosecond near-infrared laser oscillator of biological specimens was confirmed to be uniform and stable, and their profile was observed in sharp and steep form, and intracellular spatial resolution was mass spectrometrically imaged.

To ensure high quality MS imaging, the uniform distribution of AuNR in living tissue samples (samples) is critical. Therefore, we used mPEG-specific gold nanorods for uniform distribution of AuNR in tissues. Living tissues were reacted with mPEG-AuNR containing artificial cerebrospinal fluid (ACSF) for 1 hour. After the reaction, the tissues were washed only 10 times with ACSF to maintain AuNRs in the tissues. As a result, the desorption by the femtosecond near-infrared laser oscillator appeared to be uniform and stable due to the action of AuNR in the entire area of analysis (see FIGS. 7A to 7E). Despite the small beam size (1.2um diameter) of the laser, the thickness of the detached tissue was found to be 35um as shown in FIG. 7E. Because the hippocampus tissue was dried after mass spectrometry in the CARS measurement, the tissue thickness was expected to be thicker than 47 μm during the aeration process, which resulted in a particularly high aspect ratio of the laser-induced crater, which leads to intracellular spatial resolution. Done. Without the processing of AuNRs for mass spectrometry imaging, prominent mass ion peaks of biomolecules would not have been observed. Therefore, the use of AuNR is important for efficient desorption, which allows atmospheric MS imaging to produce high resolution MS images.

In addition, the atmospheric plasma jet used in the present invention used a non-thermal plasma even if it contains many kinds of charged species, including electrons, ions, and excited species, and the temperature of the non-thermal plasma was used to determine a living biological sample. Do not damage. In addition, the generated plasma jet is not in direct contact with the sample because the configuration of the sampling equipment on the sample stage is a form in which gas flow is sucked (see FIGS. 3C and 3D). This series of desorption and deionization is very effective for the analysis of live samples at atmospheric pressure. In summary, intensive ultrafast laser light using AuNR was able to effectively desorb specific volumes from biological samples, and atmospheric helium plasma was ionized with 19.8eV excited helium atoms for MS imaging of atmospheric biotissues.

With the simultaneous application of nanoparticles and isolated subsequent ionization sources, laser energy pulses were reduced compared to using the laser source alone. Lowering the pulse energy not only reduced the thermal damage of the sample, but also suppressed the scattering behavior of the desorbed material due to laser irradiation, resulting in a very clear and clear desorption pattern. This is the reason for using a cost-effective, simple structured fs laser oscillator with much lower pulse energy 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 has a feature that can analyze living tissue under atmospheric pressure analysis conditions. Therefore, MS images obtained by atmospheric nanoPALDI-MS require little sample preparation when compared to vacuum-based secondary ion mass spectrometers (SIMS) effectively used for lipid imaging with high spatial resolution (~ 1 μm). There is an advantage. Vacuum-based secondary ion mass spectrometer (SIMS) samples (samples) are prepared by freeze sectioning and drying, which often leads to distortions in lipid imaging. Distortion of the original cholesterol distribution is clearly observed in HIM images and cholesterol SIMS images (see FIG. 8). It can be seen from the HIM image that the separated cholesterol crystals formed during the SIMS sample preparation process were clearly identified (see FIG. 8A), and the SIMS image also showed a different shape from the original cholesterol distribution (see FIG. 8B). In FIGS. 5 and 6, cholesterol images derived from living tissues were found to be free from distortion due to fasting and drying of samples while maintaining the spatial resolution of SIMS. Atmospheric pressure All inputs of nanoPALDI can be transported through small tubes, such as optical fibers, plasma jet tubes, or ion transport tubes, under atmospheric pressure, so no additional technical barriers are required to develop new atmospheric mass imaging technology.

< Example  5>

Mass spectrometry With imaging  Analysis of molecular composition of specific areas of observed hippocampal tissue

Neurons in the central nervous system exhibit a variety of dendrites that are closely related to neural connectivity. In hippocampal formation, the pyramidal cells of the cornu ammonis and the granule cells of the dentate gyrus (DG) are known to be two major nerve types that exhibit distinct dendritic arbor structures. These different types of nerve types and their distinct structures allow for different molecular distributions in the CA and DG regions. As shown in Figures 5 and 6, the ion fragments of monoacylglycerol, cholesterol and ceramide showed higher signal intensity in the CA1 and CA3 regions than in the DG region. On the other hand, adenine ions at m / z = 136.062 were found to be concentrated in the DG region. Differences in the ionic image of adenine between the CA and DG regions were shown in two neuronal cell types, abstract cells and dentate gyrus granulocytes, with different distributions and different cell phenotypes in the hippocampal tissues of mice. In fact, it is known that granule cell bodies are tightly packed and small neural tubes are inserted between the cells of the tooth gyrus. Thus, the adenine ions commonly found in the nucleus were concentrated in the DG region.

Similarly, the abstract cells in the CA1 and CA3 regions structurally have two different dendrites, long thick dendrites and several basal processes, respectively, from the top and bottom of the soma. The vertices and basal projections face in opposite directions and exist in different layers, so the projection structure of the abstract cell shows the shape of a biconical. Apex and basal dendrites are known to differ in many properties, including size, geometry, electrical conductivity and responsiveness to neurotrophic factors or guidance molecules. In addition, much research has been conducted on the morphological differences between vertices and basal dendrites, and the molecular composition differs between them, as there is no suitable method for molecular composition analysis and there is no reliable molecular marker. To investigate the molecular differences between the vertices and basal dendritic spines of abstract neurons at the tissue level, sub-regions of hippocampal tissue were analyzed at high spatial resolution (see FIGS. 6C and 6D). In particular, the ionic images in CA3 were clearly divided into both sides of the adenine position, and the position of the cell bodies (ion image at m / z = 136.062) can be seen in FIGS. 6D and 6E. In CA3, the left side of the ionic image shows the dense part of the apex dendrite and the right side shows the dense part of the basal dendrites of hippocampal tissue.

The region of interest (ROI) analysis function of BioMap software was used to assign the same region of ROI to both left and right sides, and to compare the intensity of the mass spectra of monoacyl glycerol ions, cholesterol ions, and ceramide ions (FIGS. 6E, 6F). And Table 1).

The analysis showed that the MAG ions (at m / z = 311.257, m / z = 313.273, m / z = 339.289, m / z = 341.305) at the basal dendrites were 55-75% of the peak dendrites. The ceramide ions at m / z = 548.540 were observed similarly on both sides.

High-resolution MS imaging using the atmospheric nanoPALDI method revealed that MAG was 1.4-1.8 times higher in the peak dendrite-intensive region than the basal dendrite-intensive region, and at m / z = 548.540, ceramide was relatively evenly distributed in both dendrite regions. there was. In addition, the cholesterol at m / z = 385.346 in the peak dendrite-intensive region showed about 3 times higher signal intensity in the peak dendrite-intensive region than in the basal dendrite-intensive region. Specifically comparing four monoacylglycerol ions, MAGs with saturated 16: 0 and 18: 0 were found to be more unbalanced from the apex to the basal dendrite region than MAGs 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 this high resolution MS image, the present inventors confirmed the peak dendrite containing more monoacylglycerol and cholesterol compared to the basal dendrite, and ceramide is distributed evenly in both dendrite structures in CA of living hippocampal tissue. I could see that. Similar differences between the peak dendrites and the basal dendrites could also be identified in CA1 (see FIG. 6C). Detailed atmospheric nanoPALDI mass imaging (FIGS. 6C and 6D) of apical dendritic regions of CA3 was observed with mossy fiber bundles in which DG granules in the stratum lucidum extend parallel to the abstract cell layer.

In the proximal apex dendrite region of CA3, a 100 μm wide region having a low intensity of MAG was identified along the lines of the abstract cell body (see FIG. 6C). Mossy fibers in the CA3 region were of two main bundles, the major supramidal projection and intra and infra abstract projections in the clear layer were first within the proximal range of the stratum pyramid and stratum orientis, and crossed the clear layer in CA3. . CA3 pyramidal neurons are in synaptic contact with the islands of mossy fibers through an abnormally grown unusual complex of recruited spinal cole thorns. Spiny abnormal growths in distal CA3 neurons close to CA1 are mainly found in apical dendrites at a distance of 10-120 um from the soma. Considering the line profile of adenine and MAG (18: 1) from FIGS. 6C and 6D in FIG. 6G, the adenine profile is clearly seen in the abstract cell body line. The MAG (18: 1) profile shows a low intensity of 100 μm width in the apical dendritic region next to the cell body line, which is characterized by layer structure by mossy fiber projection from granular cells and spiny abnormal growth derived from CA3 neurons Matches. No similar distribution was observed for cholesterol and ceramide (18: 0) images. This observation indicates that atmospheric nano PALDI differs in molecular composition between mossy fiber projections and other peak dendrite regions. In the CA1 region no similar features were observed in the apex dendrite region as in FIG. 6C because the mossy fiber projection did not reach CA1. These results show that atmospheric nano PALDI imaging with intracellular resolution can be used to obtain multimolecular composition information at the tissue level without tagging or staining antibodies.

< Example  6>

In live hippocampus tissue methyl  beta- To cyclodextrin  Removal of cholesterol

Atmospheric pressure nano PALDI MS images of living samples can provide a great deal of spatial information about metabolites, which can be useful for drug screening at the tissue level. In order to analyze the possibility of identifying a drug that may affect hippocampal tissues using the system of the present invention, the present inventors examined atmospheric depletion of cholesterol-depleted phenomenon by methyl-β-cyclodextrin (mβCD) treatment. Analyzed. Hippocampus tissue of mice treated with mβCD under aeration conditions was dipped in ACSF solution containing mβCD (25 mg / ml) for 6 hours.

Ion images were then collected from two different hippocampal tissues, two different images of normal hippocampal tissue (see FIG. 9A) and hippocampal tissue treated with mβCD (see FIG. 9B). For hippocampal tissues treated with mβCD, ions at two cholesterol peaks, m / z 369.349 and m / z 385.346, were shown to be suppressed and disappeared throughout the assay area as shown in FIG. 9B. Interestingly, ceramide ions at m / z 548.540 were also shown to be inhibited. Other identified ions were found to be slightly reduced compared to the normal group. Therefore, the present inventors have found that the method of atmospheric pressure MS imaging analysis designed in the present invention can be useful for tissue-based drug screening, and compared with the cell-based drug screening method to which the method of killing experimental animals is applied. And it was found that the reliability is better.

 So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the appended claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (12)

delete delete delete A laser unit for irradiating a near infrared laser;
A plasma supply unit supplying a low temperature atmospheric plasma jet to a sample;
A sample stage unit on which a sample is seated and a scanning stage is mounted to enable automatic scanning;
An inverted optical microscope having the sample stage mounted on an upper end thereof;
An ion transfer unit transferring the desorbed and ionized analyte from the sample into a mass spectrometer;
A mass spectrometer for analyzing the ionized analyte; And
And a monitor unit equipped with a program for collecting, storing, and interpreting the analysis result. The method of claim 4, wherein
And a pumping system in the mass spectrometer to allow the ionized material to flow into the mass spectrometer. The method of claim 4, wherein
And when the analyte is desorbed from the sample by near-infrared laser irradiation, the sample is treated with gold nanoparticles. The method of claim 6,
The gold nanoparticles are characterized in that any one form selected from the group consisting of gold nanorods, gold nanospheres, gold nanowires and gold nanotubes, high-resolution atmospheric mass spectrometry imaging system. The method of claim 4, wherein
And the cold atmospheric plasma jet is a non-thermal atmospheric helium plasma jet. delete delete delete delete

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