JP2014126439A - Observation method for scanning type probe microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an observation method for a scanning type probe microscope, whereby a cell can be satisfactorily observed in a solution by scanning a probe.SOLUTION: An observation method comprises: a cell holding step S1 in which a cell that becomes hard when exposed to light is held in a solution; a light exposure step S2 in which the hardness of the cell is adjusted by exposing the cell, held in the solution, to light; and an observation step S3 in which the cell whose hardness is adjusted is observed in the solution by scanning a probe.

Description

  The present invention relates to an observation method of a scanning probe microscope.

  It is important to study the individual cells that make up living organisms in order to elucidate life phenomena.

  A cell is composed of a cytoplasm, an organelle and a nucleus covered with a cell membrane.

  Rafts that are membrane proteins, sugar chains, or a complex of these are present on the cell membrane that separates cells from the outside world and forms receptors that exchange information with the outside world. In addition, a cytoskeleton that supports the shape of cells and the arrangement of functional molecules exists inside the cell membrane. Furthermore, an extracellular matrix exists outside the cell membrane. These tissues cooperate to control exchanges with the outside world and maintain them so that they can act as living bodies.

  Therefore, it is important to know the shape, arrangement, and dynamics of biomolecules on the cell membrane during cell research.

  For this reason, in order to know the arrangement and shape of biomolecules, cells are solidified chemically or with liquid nitrogen, and the shape of the surface is observed with an electron microscope.

  In addition, in order to know the dynamics of biomolecules, the molecules that make up the cells are fluorescently modified, or the target protein molecules are fluorescent by modifying them so that the fluorescent protein is expressed in the gene that is the design drawing of the protein molecule. The movement and distribution of molecules are also observed with a fluorescence microscope.

  In addition, since the size is several nanometers to several tens of nanometers, biomolecules are also observed with a scanning probe microscope. For example, an isolated and purified membrane protein fused with a lipid bilayer can be developed on a hard substrate such as mica or glass, or a self-assembled membrane can be formed on the same substrate, Observation of changes in shape and structure in an aqueous solution such as a saline solution, a buffer solution, or a culture solution with an atomic force microscope is also performed.

  Attempts to observe biomolecules on living cells using an atomic force microscope are continuing because of their high resolution and ability to handle measurements in solution.

  However, since the electron microscope is used in a vacuum, the physiological activity of the cells is completely lost.

  Moreover, it cannot be said that the fluorescence microscope reflects the movement of the original molecule itself on the premise of the fluorescence modification, and cannot exceed the optical resolution.

  In Non-Patent Document 1 (Cytoskeleton, February 2012 69: 101-112; Forced Extension of Delipidated Red Blood Cell Cytoskeleton with Little Indication of Spectrin Unfolding), molecules that interact with membrane proteins on cells using atomic force microscopy The interaction force is measured. In order to measure attractive force, page 103 of Non-Patent Document 1 discloses a method of aminating a glass substrate in order to fix red blood cells on glass. Since the cytoplasm inside the erythrocyte is a fluid, it easily deforms when the tip of the cantilever comes into contact with it, making imaging based on repulsive force difficult, and even in Non-Patent Document 1, the atomic force microscope is Only the analysis based on the molecules adsorbed when the tip comes into contact with the observation object and the attractive force acting on the tip of the cantilever is performed, and details of the atomic force microscope image are not disclosed.

  The present invention has been made in view of such a situation, and an object of the present invention is to provide an observation method capable of observing cells in an aqueous solution satisfactorily by scanning with a probe.

  The observation method according to the present invention includes a cell holding step for holding cells that are hardened by irradiation with light in a solution, and a light irradiation step for adjusting the hardness of the cells by irradiating the cells held in the solution with light. And an observation step of observing the cells whose hardness has been adjusted in a solution by scanning with a probe.

  ADVANTAGE OF THE INVENTION According to this invention, the observation method which can observe a cell favorably in aqueous solution by the scanning of a probe is provided.

The flowchart of the observation method by embodiment is shown. The apparatus for performing the light irradiation process shown by FIG. 1 is shown. It is the optical microscope image of the slide glass after the light irradiation process shown in FIG. 1 obtained through the 40 times objective lens. It is the optical microscope image of the slide glass after the light irradiation process shown by FIG. 1 obtained through the 4 times objective lens. Is. 2 shows an apparatus for performing the light irradiation step and the observation step shown in FIG. 1. It is an atomic force microscope image of the slide glass after the light irradiation process shown by FIG. 1 shows an atomic force microscope disclosed in Japanese Patent Application Laid-Open No. 2004-69428. The structure of red blood cells is schematically shown. It shows a situation when erythrocytes are observed with an atomic force microscope according to conventional techniques.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  In the present embodiment, cells to be observed are cells that are irradiated with light and whose cytoplasm becomes harder than before receiving light irradiation. The case of erythrocytes is shown as a typical example of cells.

  As shown in FIG. 8, erythrocytes have a form in which a cytoplasm 41 containing a large amount of hemoglobin is covered with a cell membrane 42 (lipid bilayer membrane) in the same manner as normal cells. On the cell membrane 42, membrane protein 43 (for example, band 3 protein) is embedded in a lipid bilayer membrane, and these enter and exit substances inside and outside and transmit information.

  Until now, the arrangement and shape of the membrane protein 43 on the cell membrane surface of erythrocytes could not be satisfactorily observed with an atomic force microscope in substantially the same aqueous solution as in the living body. As shown in FIG. 9, when the cell membrane 42 of red blood cells is very soft, when the probe 45 at the tip of the cantilever 44 contacts the membrane protein 43 and the membrane protein 43 receives a force from the probe 45, This is because the liquid cytoplasm 41 is deformed and the cell membrane 42 moves.

  As the inventors proceeded with research on observation of cells using such an atomic force microscope, when the cells were irradiated with light under appropriate conditions, the cells became hard and difficult to deform, and living bodies on the cell membrane We obtained a new finding that the molecules can be observed well. The reason why the cells become hard is not clear, but is thought to involve photodenaturation of molecules contained in the cytoplasm. For example, it is estimated as follows.

  By irradiation with light, the tertiary structure of the biomolecule contained in the cytoplasm is broken and denatured from a particulate form to a linear molecular structure. As a result, the entanglement between the molecules advances and the cytoplasm gels. Thereby, the elastic modulus of the cytoplasm increases, the movement of the cytoskeleton is attenuated, and the cell membrane can be supported. As a result, movement of membrane proteins and extracellular matrix existing on the cell membrane is reduced.

  If the cytoplasm contains cells that denature upon irradiation with light, it is expected that the hardness of the cells themselves can be adjusted by irradiating the cells with light.

  The observation method of the present embodiment is based on such knowledge, and as shown in FIG. 1, a cell holding step S1 for holding cells that are hardened by irradiation with light in the solution, and in the solution. A light irradiation step S2 for adjusting the hardness of the cells by irradiating the held cells with light, and an observation step S3 for observing the cells with adjusted hardness in the solution by scanning with a probe.

  The cell contains, for example, hemoglobin. More specifically, the cell may be a red blood cell. The cell holding step S1 includes, for example, preliminarily adsorbing a substance for enhancing the adsorptivity of cells onto the surface of a transparent member such as a glass substrate. As a substance for enhancing the adsorptivity of cells, for example, an amino group appears on the surface to be adsorbed by the cells.

  The light to irradiate is light having a wavelength shorter than 505 nm, for example. More specifically, the irradiated light may be light in a wavelength band of 360 nm to 370 nm or light in a wavelength band of 400 to 440 nm. In the light irradiation step S2, for example, light irradiation is performed using an optical microscope. In the light irradiation step S2, a light irradiation region is defined by the illumination field of view of the optical microscope. Moreover, light irradiation process S2 prescribes | regulates the light irradiation time with the illumination time of an optical microscope. The optical microscope may be, for example, a laser microscope. In this case, in the light irradiation step S2, the light irradiation region is defined by the laser light scanning range of the laser microscope. In the light irradiation step S2, the light irradiation time is defined by the scanning speed and the number of scans of the laser light of the laser microscope.

  In the observation step S3, the cells are scanned with a probe from the opposite side of the cell holding portion held in the cell holding step S1.

  According to this observation method, the cells are hardened by irradiating the cells with light, so that even if the probe of the scanning probe microscope contacts the probe, the cells are not easily deformed. It becomes possible to observe well in an aqueous solution. For example, it becomes possible to satisfactorily observe the shape and arrangement of biomolecules on the cell membrane that could not be measured so far.

  FIG. 2 shows an apparatus for performing the light irradiation step S2. This apparatus includes an inverted optical microscope 10.

  The optical microscope 10 includes a mercury lamp 11, a collimating lens 13, an attenuation filter 14, a dichroic mirror 15, an objective lens 17, and an observation stage 18. The objective lens 17 can be switched in magnification. That is, one of a plurality of objective lenses having different magnifications is selectively arranged on the optical path.

  The optical microscope 10 also acquires an optical image by using a halogen lamp 12 that is used in place of the mercury lamp 11 and a half mirror 16 that is used in place of the dichroic mirror 15 in order to acquire an optical microscope image. A camera 19 is provided.

  On the observation stage 18, a slide glass 21 holding red blood cells 22 in a buffer solution 23 is placed. Such a slide glass 21 is produced as follows. First, the erythrocytes 22 diluted to an appropriate concentration with the buffer solution 23 are developed on the aminated slide glass 21. Red blood cells 22 are adsorbed on the aminated slide glass 21 at a certain ratio. Next, the buffer solution 23 is replaced. As a result, red blood cells that have not been adsorbed on the aminated slide glass 21 are removed.

  The substrate for holding the red blood cells 22 is not limited to the aminated slide glass 21, and any substrate may be used as long as it does not interfere with the physical adsorption of the red blood cells 22.

  When the light irradiation step S <b> 2 is performed, the mercury lamp 11, the attenuation filter 14, and the dichroic mirror 15 are disposed on the optical path of the optical microscope 10. The light emitted from the mercury lamp 11 is collimated by the collimating lens 13, attenuated by the attenuation filter 14, reflected by the dichroic mirror 15, focused by the objective lens 17, and is applied to a partial region on the slide glass 21. Irradiated. For example, the output of the mercury lamp 11 is 100 W, the transmittance of the attenuation filter 14 is 6%, the magnification of the objective lens 17 is 60 times, and the irradiation time is 30 minutes. The dichroic mirror 15 has a characteristic of reflecting light having a wavelength of 505 nm or less. Therefore, the red blood cells 22 are irradiated with light having a wavelength of 505 nm or less among the light generated from the mercury lamp 11.

  After completion of the light irradiation step S2, the mercury lamp 11 is switched to a halogen lamp, the attenuation filter 14 is removed, and the dichroic mirror 15 is switched to the half mirror 16 as necessary. Thereby, the state of the red blood cells 22 can be observed as an optical microscope image by the camera 19.

  The slide glass 21 after completion of the light irradiation step S2 is then subjected to observation with a scanning probe microscope such as an atomic force microscope.

  The peak of light absorption of the hemoprotein constituting hemoglobin is from 400 nm to 450 nm. By receiving light having a wavelength of 505 nm or less, hemoglobin, which is the main component of the cytoplasm of red blood cells, absorbs the energy of the irradiation light, and the tertiary The structure is broken and the particle structure is changed to a linear molecular structure. As a result, the entanglement between the molecules advances and the cytoplasm gels.

  On the other hand, if the red blood cells that have not been irradiated with light are replaced with sterilized water, the shape of the erythrocytes cannot be maintained due to the water entering the cells having a high salt concentration through the cell membrane, and eventually ruptures.

  FIG. 3 is an optical microscopic image obtained by observing erythrocytes using a 40 × objective lens after replacing the buffer solution of erythrocytes irradiated with light having a wavelength of 505 nm or less using a 60 × objective lens with sterile water. The optical microscope image of FIG. 3 moves the observation stage 18 on which the slide glass 21 is placed and moves the observation area 18 through the objective lens 60 times and the boundary between the area where the light is not irradiated and the center of the visual field. The images were aligned and observed through a 40 × objective lens. On the left side of the broken line in FIG. 3, erythrocytes whose cytoplasm is gelled by light irradiation can be seen. On the other hand, on the right side of the broken line in FIG. 3, traces of red blood cells in which red blood cells not irradiated with light have been ruptured by replacement of buffer solution and sterilized water can be seen.

  The optical microscope image in FIG. 4 is an image observed through a 4 × objective lens with the center of the region irradiated with light through the 60 × objective lens aligned with the center of the field of view. In the region surrounded by the one-dot chain line circle, red blood cells that have gelled and maintained the cytoplasmic shape can be seen.

  As described above, since the light irradiation step S2 is performed using the optical microscope 10, it is possible to select light with a high degree of freedom.

  The wavelength of the light for promoting photodegeneration of the red blood cells 22 can be changed by installing a color selection filter after the mercury lamp 11 and further replacing the color selection filter with one having different optical characteristics. .

  The amount of light for promoting photodenaturation of the red blood cells 22 can be changed by replacing the attenuation filter in the subsequent stage of the mercury lamp 11 with one having a different transmittance.

  The location of light irradiation for promoting photodegeneration of the red blood cells 22 can be changed by moving the slide glass 21 by the observation stage 18.

  The light irradiation region for promoting the photodegeneration of the red blood cells 22 is defined by the illumination field of the optical microscope 10. The illumination field of view of the optical microscope 10 can be changed by installing an illumination stop on the illumination optical path and changing the size and shape of the illumination stop. It can also be changed by switching the objective lens 17 to one having a different magnification.

  In addition, the optical microscope may be equipped with various light sources for performing various observations, and any light source that emits light that promotes photodenaturation of the red blood cells 22 may be used as the light source. It may be applied to light irradiation for promoting photo-denaturation of water. For example, a light source that emits ultraviolet light may be applied to light irradiation for promoting photodenaturation of red blood cells 22.

  FIG. 5 shows an apparatus for performing the light irradiation step S2 and the observation step S3. This apparatus has a configuration in which an optical microscope 10 and an atomic force microscope 30 are combined. The optical microscope 10 is an inverted optical microscope and is the same as that shown in FIG. In other words, this apparatus has a configuration in which an atomic force microscope 30 is added to the optical microscope 10 of FIG.

  The atomic force microscope 30 includes a cantilever 31 disposed above the observation stage 18, a scanning mechanism 32 that scans the cantilever 31, a displacement detection unit 33 that optically detects the displacement of the cantilever 31, and a scanning mechanism 32. The controller 34 is configured to drive and to form an atomic force microscope image based on information detected by the displacement detector 33.

  With such a configuration, coexistence of atomic force microscope observation from above, light irradiation from below and optical microscope observation is realized.

  That is, in this apparatus, the glass slide 21 holding the red blood cells 22 together with the buffer solution 23 is irradiated with light that promotes photodegeneration of the red blood cells 22 from below using the mercury lamp 11, the attenuation filter 14, and the dichroic mirror 15. If necessary, optical microscope observation can be performed from below using the halogen lamp 12 and the half mirror 16, and further, atomic force microscope observation can be performed from above. The optical microscope observation and the atomic force microscope observation may be performed separately or simultaneously.

  Since the cytoplasm 41 of the erythrocyte 22 is gelated by light irradiation, the cell membrane 42 is supported by the cytoplasm gel and is not easily deformed. Therefore, the membrane protein 43 present on the cell membrane 42 of the erythrocyte 22 is removed by an atomic force microscope. 30 enables good observation in the solution.

  FIG. 6 is an atomic force microscope image of erythrocytes whose surface was observed as a result of gelation. The observation environment is in a buffer solution. The distance of each observed white grain is about 100 nm, and an interval equivalent to the interval of band 3 protein observed with an electron microscope or the like is observed.

  In addition to the apparatus shown in FIGS. 2 and 5, an atomic force microscope combined with a laser microscope may be applied for observation. An example of such an atomic force microscope is disclosed in Japanese Patent Application Laid-Open No. 2004-69428. The atomic force microscope will be described with reference to FIG.

  The atomic force microscope is integrated with an inverted microscope 270, and the inverted microscope 270 moves the objective lens 272 up and down with a stage 271 on which the sample 211 is placed, an objective lens 272 disposed below the stage 271, and the objective lens 272. A revolver Z moving mechanism 276 that moves the eyepiece 270, an eyepiece lens 273 that cooperates with the objective lens 272 to obtain an optical image of the sample 211, an illumination light source 108 that emits illumination light, and a transmission illumination device 274 that illuminates the sample 211 from above. And an arm 275 for supporting the transmitted illumination device 274.

  The atomic force microscope further includes a cantilever 207 provided with a probe 209 protruding downward on the lower surface of the tip, a displacement detection unit 230 that supports the cantilever 207 and detects the displacement of the cantilever 207, and the cantilever 207 on the sample surface. On the other hand, it has a cylindrical XYZ scanner 213 which is a scanning mechanism for scanning the cantilever 207 in the horizontal direction (X direction and Y direction) and the vertical direction (Z direction).

  The cylindrical XYZ scanner 213 is usually composed of a piezo actuator and is fixed to an arm 275 of the microscope 270. The cantilever 207 is brought close to the sample 211 at the time of measurement, and the probe 209 at the tip is brought into contact with the surface of the sample 211.

  The displacement detection unit 230 includes a laser oscillator 221 that is a light source for displacement measurement that irradiates the back surface of the cantilever with the light beam 231 and a detector 235 that detects the position of the reflected light beam 232 from the back surface of the cantilever. The light beam 231 from the laser oscillator 221 is irradiated with the back surface of the cantilever 207 in focus.

  The detector 235 is composed of, for example, an upper and lower split photodetector (PD), and has an upper sensor and a lower sensor that output a signal corresponding to the received light quantity. The incident position of the reflected light beam 232 can be obtained based on the difference in the amount of light received by the upper sensor and the lower sensor of the two-part PD 235, that is, the difference between the output signals.

  The atomic force microscope further includes an A / D converter 261 that converts an analog signal output from the detector 235 into a digital signal, a data processing unit 262 that processes a digital signal from the A / D converter 261, and a cylinder. Z scan drive control unit 263 and XY scan drive control unit 264 for driving XYZ scanner 213, CPU 257 for controlling the entire system, input unit 109 for giving information to CPU 257 from the outside, and display unit for displaying data 255.

  The data processing unit 262 calculates a difference signal and a sum signal of the outputs of the upper and lower sensors of the two-divided PD 235, and sends this to the CPU 257 as control information. The CPU 257 controls the Z scan drive control unit 263 and the XY scan drive control unit 264 according to the information from the data processing unit 262, and obtains position information of the probe 209 from the Z scan drive control unit 263 and the XY scan drive control unit 264. The data of the sample 211 is acquired and the desired data is displayed on the display unit 255 based on the acquired data.

  Further, the atomic force microscope includes a laser 105 that is a light source that emits an excitation light beam, a light intensity / time designation unit 103 that designates a position, a region, a light amount, and a time of irradiation of the excitation light beam, and an irradiation position / region designation unit 104. And an irradiation position / region control unit 100, an irradiation time control unit 101, an irradiation light amount control unit 102 for controlling the position, region, light amount and time of the excitation light beam irradiation, and imaging for obtaining a whole fluorescent image of the sample 211 And a mirror for directing fluorescence from the sample 211 to the imaging unit 106, for example, a dichroic mirror 107.

  In this atomic force microscope, light from the illumination light source 108 is irradiated to the sample through the cylindrical XYZ scanner 213 by the transmission illumination device 274. This illumination light excites the fluorescent dye of the sample and emits fluorescence. This fluorescent image is picked up by the image pickup unit 106 to obtain an observation image of the entire sample.

  Further, the laser beam from the laser 105 passes through the light intensity / time designating unit 103 and the irradiation position / region designating unit 104 and is then irradiated onto the sample. The irradiation position / area designating unit 104 that optically controls the irradiation position and area is constituted by, for example, a galvanometer mirror or an AOM element, and responds to a signal from the irradiation position / area control section 100 and emits laser light at a corresponding position. Move. If this is used, the laser beam can be raster scanned over the entire observation range. Further, the scanning range is limited by designating the area.

  The control of the time and the amount of light is performed by the light intensity / time specifying unit 103, and the laser light is transmitted only for the specified intensity and time. The light intensity / time designating unit 103 is composed of, for example, an AOTF element. The AOTF element can switch light transmission / blocking at a high speed by a high-speed shutter function, thereby realizing high-precision area designation and time designation.

  This atomic force microscope obtains a fluorescence image of a specific region by scanning an excitation light beam emitted from a laser 105, but the laser 105 emits light that promotes photodenaturation of cells such as red blood cells 22. If not, it can be applied to the observation method of the present embodiment by changing to a laser that emits light that promotes photodenaturation of cells such as red blood cells 22.

  In this case, the light irradiation region for promoting the photodenaturation of the red blood cells 22 is defined by the laser light scanning range of the laser microscope. The scanning range of the laser beam can be arbitrarily changed by the irradiation position / area designating unit 104, and is very small compared to the illumination field of view of a normal optical microscope. Therefore, it is possible to selectively denature only the red blood cells 22 in a minute region of an arbitrary shape and observe with an atomic force microscope.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

  The embodiment has been described by taking the observation with an atomic force microscope as an example. However, the embodiment may be applied to other scanning probe microscopes.

Cytoskeleton, February 2012 69: 101−112; Forced Extension of Delipidated Red Blood Cell Cytoskeleton with Little Indication of Spectrin Unfolding

JP 2004-69428 A

DESCRIPTION OF SYMBOLS 10 ... Optical microscope, 11 ... Mercury lamp, 12 ... Halogen lamp, 13 ... Collimating lens, 14 ... Attenuation filter, 15 ... Dichroic mirror, 16 ... Half mirror, 17 ... Objective lens, 18 ... Observation stage, 19 ... Camera, 21 ... slide glass, 22 ... red blood cell, 23 ... buffer solution, 30 ... atomic force microscope, 31 ... cantilever, 32 ... scanning mechanism, 33 ... displacement detection part, 34 ... control part, 41 ... cytoplasm, 42 ... cell membrane, 43 ... Membrane protein 44 ... cantilever 45 ... probe 100 ... irradiation position / region control unit 101 ... irradiation time control unit 102 ... irradiation light amount control unit 103 ... light intensity / time designation unit 104 ... irradiation position / region Designating part 105 ... Laser 106 ... Imaging part 107 ... Dichroic mirror 108 ... Illumination light source 109 ... Input part 207 ... Can Lever, 209 ... probe, 211 ... sample, 213 ... cylindrical XYZ scanner, 221 ... laser oscillator, 230 ... displacement detection unit, 231 ... light beam, 232 ... reflected light beam, 235 ... detector, 255 ... display unit, 257 ... CPU, 261 ... A / D converter, 262 ... data processing unit, 263 ... Z scan drive control unit, 264 ... XY scan drive control unit, 270 ... microscope, 271 ... stage, 272 ... objective lens, 273 ... eyepiece Lens, 274 ... Transmission illumination device, 275 ... Arm, 276 ... Revolver Z moving mechanism.

Claims (11)

A cell holding step for holding cells that are hardened by light irradiation in a solution;
A light irradiation step of adjusting the hardness of the cells by irradiating the cells held in the solution with light;
An observation method of a scanning probe microscope, comprising an observation step of observing the cells having adjusted hardness in a solution by scanning with a probe.   The observation method according to claim 1, wherein the cell contains hemoglobin.   The observation method according to claim 1, wherein in the light irradiation step, the light irradiation is performed using an optical microscope.   The observation method according to claim 1, wherein the light is light having a wavelength shorter than 505 nm.   The observation method according to claim 3, wherein the light irradiation step defines an irradiation region of the light by an illumination field of view of the optical microscope.   The observation method according to claim 3, wherein the light irradiation step defines an irradiation time of the light according to an illumination time of the optical microscope.   The observation method according to claim 3, wherein the optical microscope is a laser microscope, and the light irradiation step defines an irradiation region of the light according to a scanning range of the laser light of the laser microscope.   The observation method according to claim 3, wherein the optical microscope is a laser microscope, and in the light irradiation step, the irradiation time of the light is defined by the scanning speed and the number of scans of the laser light of the laser microscope.   The observation method according to any one of claims 1 to 8, wherein the cell holding step includes preliminarily adsorbing a substance for enhancing the adsorptivity of cells onto the surface of the transparent member.   The observation method according to claim 9, wherein the substance for enhancing the adsorptivity is such that an amino group appears on a surface to which cells are to be adsorbed.   The observation method according to claim 1, wherein in the observation step, a cell is scanned with a probe from the opposite side of the cell holding portion held in the cell holding step.

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