JP2016202021A - Cell membrane peeling device, peeling method, and observation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To irradiate a cell sample highly accurately and easily with cavitation optimum to cell membrane peeling.SOLUTION: The present invention relates to a cell membrane peeling device in which a recess that is formed so as to be recessed from an end surface of a horn installed to a vibration element of ultrasonic oscillator is provided for the end surface. According to the present invention, a pressure change due to vibration at a tip of the horn attached to the vibration element becomes larger even if an output from an ultrasonic oscillator is low, and desired cavitation is generated even under a low output condition. Because cavitation suitable for cell membrane peeling can be generated, a cell sample can be irradiated with cavitation highly accurately and easily.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a technique for peeling a cell membrane of a cell sample.

  In recent years, with the development of microscopes, various structural analyzes have been carried out in biological research. Regarding the optical microscope, functional changes and intermolecular chemical reactions can be observed by live cell imaging using a confocal laser microscope, a fluorescence microscope, or the like. Since the electron microscope has a molecular / atomic level resolution, it can observe the fine structure of the cell. Furthermore, the tomography method (tilting the sample in an electron microscope, taking images from any angle, and restoring the original three-dimensional image from those images) can visualize the spatial arrangement of the structure. By using the cryo technique, the structure can be observed with high resolution in a state closer to a living cell.

  In parallel with the above methods, sample preparation (pretreatment) methods for various observation purposes have been devised. For example, when proteins are required in cells, they form a complex purposefully and perform various functions. For this reason, proteins are considered to be closely related to many important functions such as functional domains, movement, and signal transmission. Since proteins exist in close contact with the cytoplasmic surface of the cell membrane, the functional structure is elucidated by observing the cytoskeleton and the lining structure of the membrane with various spatial spreads at the ultrastructural level. Can be expected.

  However, the back side of the cell membrane can only be seen in fragments due to the cytoskeleton and soluble proteins that fill the cell. In order to examine the membrane cytoskeleton with a wide field of view, it is necessary to remove the extra cytoplasm filling the cytoplasm. For this reason, a cell membrane detaching method (unroofing) has been devised which leaves most of the cytoplasm, leaving the cell membrane and its membrane cytoskeleton.

  Non-Patent Document 1 describes two cell membrane detachment methods used in recent years. One is a method in which cells are destroyed by a cavitation effect by a commercially available ultrasonic homogenizer (cell crusher) to leave a cell membrane lining structure on the ventral side. The other is a method in which glass or the like with improved adhesion is lightly pressed on the surface of the cell, and the apical (back) side film is peeled off together with the backing structure.

  In the former method, a small probe having a tip diameter of about 2 to 4 mm is placed in a liquid in which a cell sample is immersed, and the sample is irradiated with ultrasonic waves for about 0.5 to 3 seconds. Cavitation is a phenomenon in which a high pressure region and a low pressure region are periodically generated by irradiating a liquid with ultrasonic vibration, and a cavity is instantaneously generated in a low pressure region. The cavity generated by cavitation is compressed and extinguished in a high pressure region, and becomes a very strong shock wave and exerts a large pressure on the surroundings. This physical impact produces actions such as washing, emulsification, dispersion and crushing. When this is used in cell membrane detachment, the cell membrane is broken by directly applying a cavity (bubble) generated by cavitation to the cell. Since ultrasonic waves are used, most of the soluble protein is washed away, so that a structure adhered to a film such as clathrin coating can be clearly observed. In addition, the reproducibility is better than the latter method.

  With the cell membrane detachment method, the cytoplasmic surface of the cell membrane can be observed three-dimensionally, and biochemical events can be morphologically verified. Currently, analysis methods applying this method are being studied.

Jiro Usukura, “Bio-Electron Microscopy Technology Understandable”, Kyoritsu Publishing

  In recent years, with the development of microscope technology, there is an increasing demand for higher precision in sample preparation technology. The cell membrane detachment technique is an indispensable pretreatment method for observing a structure having a spatial extension composed of a membrane cytoskeleton and proteins. Therefore, as a result of intensive studies on the exfoliation of cell membranes with higher accuracy and ease, the inventors of the present application have obtained the following knowledge.

  When the output of the ultrasonic oscillator is high, cavitation increases due to an increase in pressure difference due to ultrasonic vibration. In addition, the total energy due to the ultrasonic vibration increases, and not only the local detachment of the cell membrane but also the cell itself is strongly destroyed and falls off the substrate.

  However, since commercially available ultrasonic homogenizers are intended for cell disruption or emulsification dispersion, where strong ultrasonic vibration is desirable, the output conditions of the apparatus are often too strong to exfoliate the cell membrane.

  Therefore, the present inventor modified a commercially available ultrasonic homogenizer so that the output conditions can be finely adjusted on the low output side, but cavitation did not occur when the output was too low. Moreover, even if it generate | occur | produced, the position and direction where cavitation generate | occur | produced were disjoint. In this case, the cell membrane was not exfoliated, and the backing structure of the cell membrane as the target structure could not be observed.

  Non-Patent Document 1 does not disclose specific technical information such as the output of the ultrasonic oscillator and the tip (probe) shape of the cavitation generating horn.

  The object of the present invention has been made in view of the above-described problems, and relates to irradiating a cell sample with high accuracy and ease of cavitation optimal for cell membrane detachment.

  The present invention relates to a concave portion formed so as to be recessed from an end face on an end face of a horn attached to a vibration element of an ultrasonic oscillator.

  According to the present invention, even if the output of the ultrasonic oscillator is low, the pressure change due to the vibration of the horn tip attached to the vibration element increases, and desired cavitation occurs even under low output conditions. Since cavitation suitable for cell membrane detachment can be generated, it is possible to irradiate the cell sample with high accuracy and ease.

1 is a configuration diagram of a cell membrane peeling apparatus 100 according to Embodiment 1. FIG. 3 is a side view of the horn 103. FIG. It is a perspective view which shows the end surface of the horn front-end | tip part 1031. FIG. It is a side view which shows the example of another shape of the horn 103. It is a perspective view which shows another structural example of the end surface of the horn front-end | tip part. It is a perspective view which shows another structural example of the end surface of the horn front-end | tip part. It is a side view which shows the structure of the cell membrane peeling apparatus 100 which concerns on Embodiment 2. FIG. It is a figure which shows the detailed structure of the horn drive part 603. FIG. It is a flowchart explaining the procedure which observes a cell sample using the cell membrane peeling apparatus 100 demonstrated in Embodiment 1-2. It is a flowchart of the structural analysis procedure which peeled the cell membrane of a cell sample using the cell membrane peeling apparatus 100 demonstrated in Embodiment 1-2, and also combined CLEM method.

<Basic concept of the present invention>
As explained earlier, when the output of the ultrasonic oscillator is high, the pressure difference in the liquid increases and the energy generated by cavitation also increases, so excessive energy is applied to the cell and the cell itself is destroyed. There is a risk. On the other hand, if the output of the ultrasonic oscillator is low, the pressure difference in the liquid is too small and cavitation does not occur. In order to balance these, it is necessary for a skilled worker to repeat trial and error, which hinders efficient observation work.

  The present inventors have provided a recess such as a V-shaped groove, which will be described later, on the end face of a horn (a member that immerses the tip portion in the liquid) attached to the vibration element, so that the output of the ultrasonic oscillator is low and added to the liquid. It has been found that even when the generated energy is relatively small, cavitation suitable for cell membrane detachment can be generated satisfactorily. In the following, the shape of the horn based on the knowledge will be described first, and then a specific configuration for observing a cell sample using this will be described.

<Embodiment 1>
FIG. 1 is a configuration diagram of a cell membrane peeling apparatus 100 according to Embodiment 1 of the present invention. The cell membrane peeling device 100 is a device for peeling a cell membrane of a cell sample, and includes an ultrasonic generator 101, a vibration element 102, and a horn 103. The ultrasonic generator 101 and the vibration element 102 are electrically connected. The vibration element 102 and the horn 103 are brought into close contact with each other at the coupling portion 104 by screw coupling, brazing, soldering, or the like.

  When the cell membrane of the cell sample is peeled off, the sample container 105 is filled with a liquid and the cell sample is immersed, the tip of the horn 103 is immersed in the liquid, and high frequency power is output from the ultrasonic generator 101. The high-frequency power is converted into mechanical vibration by the vibration element 102, and minute ultrasonic vibration is generated from the horn 103 by this mechanical vibration. This ultrasonic vibration causes cavitation in the liquid and peels off the cell membrane.

  FIG. 2 is a side view of the horn 103. The horn 103 is made of, for example, a titanium alloy with good propagation efficiency of ultrasonic vibration. The horn 103 has a cylindrical shape (length: 100 mm) that extends from the horn tip portion 1031 to the coupling portion 104. The size of the coupling portion 104 is 16 mm in diameter (hereinafter abbreviated as φ) and 10 mm in length.

  FIG. 3 is a perspective view showing an end face of the horn tip portion 1031. The horn tip portion 1031 is formed in a cylindrical shape with a diameter of φ4.0 mm or less (for example, φ2.5 mm). For example, a V-shaped groove 1032 having a width of 0.5 mm is formed at the center of the end face of the horn tip 1031. It is considered that a pressure change when the horn 103 vibrates can be increased by providing a concave portion such as a V-shaped groove 1032 on the end face of the horn tip portion 1031. Thereby, even when the power applied to the vibration element 102 (the output of the ultrasonic generator 101) is small, cavitation can be generated. As a result of the experiment, when the horn 103 having the above material and dimensions was used, cavitation could be generated satisfactorily even when the output of the ultrasonic generator 101 was 3 W.

  FIG. 4 is a side view showing another example of the shape of the horn 103. In the shape example shown in FIG. 4, the connecting portion 104 has a diameter of 15 mm and a length of 20 mm. The horn tip portion 1031 has two stages. The upper stage (side closer to the coupling portion 104) has a length of 410 mm and φ40 mm. The lower stage (the side having the end face) has a length of 430 mm and φ2.5 mm.

  FIG. 5 is a perspective view showing another configuration example of the end face of the horn tip portion 1031. In addition to the V-shaped groove 1032 described with reference to FIG. 3, a notch 1033 formed at a location different from the V-shaped groove 1032 on the end surface can be provided on the end surface of the horn tip portion 1031.

  FIG. 6 is a perspective view showing another configuration example of the end face of the horn tip 1031. In addition to the V-shaped groove 1032 described with reference to FIG. 3, a second groove 1034 formed so as to be substantially orthogonal to the V-shaped groove 1032 may be provided on the end surface of the horn tip portion 1031.

<Embodiment 1: Summary>
As described above, the cell membrane peeling apparatus 100 according to the first embodiment includes the horn 103 in which a concave portion such as the V-shaped groove 1032 is formed on the end surface. With this configuration, even when the output applied from the ultrasonic generator 101 to the vibration element 102 is small (for example, 3 W), cavitation suitable for cell membrane separation can be generated satisfactorily. Thereby, even if an operator is not skilled, a quality cell sample can be easily produced.

  In the first embodiment, the overall shape of the horn tip portion 1031 and the configuration example of the end face have been described with reference to FIGS. In any combination of these shapes and end face shapes of the horn tip portion 1031, cavitation could be generated satisfactorily even when the output of the ultrasonic generator 101 was 3 W. The shape of the recess is not necessarily limited to these, and may be any other shape as long as cavitation can be efficiently generated even at a low output (for example, 3 W). However, the V-shaped groove 1032 described in FIG. 3 seems to be preferable from the viewpoint of ease of processing.

<Embodiment 2>
FIG. 7 is a side view showing the configuration of the cell membrane peeling apparatus 100 according to Embodiment 2 of the present invention. In FIG. 7, a sample stage 601 is disposed below the optical microscope 600, and the sample container 105 is placed thereon. A petri dish or the like is used as the sample container 105 in the case of a biological sample. Between the optical microscope 600 and the sample stage 601, a horn 103 and a light 602 for irradiating an observation position are arranged.

  The horn drive unit 603 controls the position, inclination, and rotation of the horn 103. Thereby, cavitation can be generated at a desired position in the sample containers 105 having various shapes. The ultrasonic generator 101 can output an output of 3 W or less stepwise. Thereby, the output according to each sample can be selected.

  The light 602 is connected to the light source 604 through a light transmission line such as an optical fiber, and takes in light from the light source 604. The light driving unit 605 adjusts the irradiation position of the light 602 so that the cavitation generated from the horn 103 can be easily seen from the optical microscope 600 by controlling the position and inclination of the light 602.

  The control device 606 controls the ultrasonic generator 101, the light 602, the horn driving unit 603, the light source 604, and the light driving unit 605. The control device 606 is connected to the computer 607 and controls each unit in accordance with instructions from the computer 607.

  The computer 607 includes a display 607a for displaying an operation screen (Graphical User Interface: GUI) of the apparatus, and an input device 607b such as a keyboard and a mouse. The computer 607, the control device 606, and each component are connected by communication lines.

  In FIG. 7, the control device 606 is connected to the ultrasonic generator 101, the light 602, the horn drive unit 603, the light source 604, and the light drive unit 605, but this connection form is only an example, and is used for communication. Various modifications such as wiring are also included in the present invention. In addition, it is possible to provide a mechanism for the operator to manually move each component.

  The operator observes the sample image in the sample container 105 using the optical microscope 600, picks up a subject (for example, a glass substrate or a grid mesh) on which cells are cultured with tweezers, and applies cavitation to the cell portion. A camera may be attached to the upper part of the optical microscope 600, the camera and the computer 607 may be connected, and the operation may be performed while displaying an observation image on the display 607a. Cavitation generation / stop instructions and output adjustment are performed via the input device 607b. A button or a foot pedal can be connected to the ultrasonic generator 101 to instruct the generation / stop of cavitation by the operator's finger or foot.

  FIG. 8 is a diagram showing a detailed structure of the horn drive unit 603. Here, the figure seen from the end surface side of the horn 103 is shown. The horn 103 is fixed to the hollow portion of the cylindrical rotation mechanism 702 with a fixing screw 701. The rotation mechanism 702 is joined to the rotation mechanism fixing unit 703. The rotation mechanism 702 can rotate ± 180 degrees. Depending on the direction of the concave portion such as the V-shaped groove 1032 provided on the end face of the horn tip portion 1031, the direction in which cavitation is generated from the horn 103 may be different. The rotating mechanism 702 rotates the horn 103, so that the sample can be irradiated with cavitation in an arbitrary direction.

  The rotation mechanism fixing portion 703 is joined to the tilt mechanism 704. The tilt mechanism 704 can tilt the rotation mechanism fixing portion 703 by ± 90 degrees. The tilt mechanism 704 is joined to the Z-axis drive unit 705. The Z-axis drive unit 705 can move along the Z-axis rail 706 to adjust the height of the tilt mechanism 704. The Z-axis rail 706 is joined to the Y-axis drive unit 707. The Y axis drive unit 707 moves along the Y axis rail 708. The Y-axis rail 708 is joined to the X-axis drive unit 709. The X axis drive unit 709 moves along the X axis rail 710. The rails of the XYZ axes are fixed to, for example, an apparatus stage or an optical microscope support unit.

<Embodiment 2: Summary>
Since an apparatus for generating cavitation and a space for carrying out the operation work are relatively large, it is difficult to carry out these works directly under the optical microscope 600. In order to overcome this in the conventional cell membrane detachment method, it is necessary to work by reflecting the sample image once by a mirror and observing the sample image with an optical microscope. Therefore, since the operator has to work while looking at the mirror image, the operation is difficult. According to the second embodiment, since the position, angle, and rotation of the horn 103 can be freely adjusted, the operation can be performed with the horn 103 disposed immediately below the optical microscope 600. Thereby, even if the operator is not skilled in the work, the observation work can be performed efficiently. In addition, cavitation can be applied to samples of various shapes.

  In the second embodiment, the position and angle of the light 602 can be freely adjusted by the light driving unit 605, so that light can be emitted from an angle at which cavitation can be easily seen through the optical microscope 600. Thereby, high-contrast observation can be performed for cavitation generated from various angles.

<Embodiment 3>
FIG. 9 is a flowchart illustrating a procedure for observing a cell sample using the cell membrane peeling apparatus 100 described in the first and second embodiments. The cell membrane detachment method is generally used for cultured cell samples. Hereinafter, each step of FIG. 9 will be described.

(FIG. 9: Step S801)
An operator wash | cleans a culture solution and immerses a cultured cell in the buffer solution put into the petri dish. This operation is performed on the sample stage 601 of the cell membrane peeling apparatus 100. The operator moves the sample container 105 containing the buffer solution under the optical microscope 600 and puts the horn tip 1031 into the buffer solution. The operator grasps the glass substrate or grid mesh with the cultured cells with tweezers, determines the positional relationship between the cells and the horn tip 1031 while confirming with the optical microscope 600, generates cavitation, and irradiates the cells. .

(FIG. 9: Steps S802 to S803)
The operator checks the degree of membrane peeling using the optical microscope 600 after peeling the cell membrane of the cell sample (S802). If film peeling is insufficient, the process returns to step S801, and film peeling is performed again. If film peeling is sufficient, the process proceeds to step S804 (S803).

(FIG. 9: Steps S804 to S805)
Examples of the technique for preparing a sample for observing the structure after peeling the cell membrane using the cell membrane peeling method (S804) and the observation technique (S805) are given below.

(FIG. 9: Steps S804 to S805: Freeze Etching Replica Method)
For example, when performing the spatial molecular construction of proteins on the cytoplasmic surface of the cell membrane, especially the distribution of superficial membrane proteins on the cytoplasmic surface and the three-dimensional analysis of the cytoskeleton, the freeze etching replica method is used. Use. In this method, the sample from which the cell membrane has been peeled is frozen and then cut, and the ice produced by freezing is sublimed by several microns. Next, a metal film such as platinum is vapor-deposited on the cut surface, and this is peeled off to produce a cell surface shape replica, which is observed with an appropriate microscope (for example, an optical microscope, an electron microscope, etc.).

(FIG. 9: Steps S804 to S805: immune freeze etching replica method)
By combining the freeze etching replica method and the immunolabeling method, the localization of the target protein can be clearly confirmed.

(FIG. 9: Steps S804 to S805: Freezing method)
In recent years, in order to observe the intracellular structure closer to the living state, a method (freezing method) in which the movement of cells is instantaneously stopped by rapid freezing and observed in an ice-embedded state (freezing method) has been actively studied. By using this method, it is possible to observe natural structures such as water-containing protein molecules and membrane cytoskeleton. These pretreatments (treatments for freezing cells) are performed on a TEM grid used for TEM observation, for example, when a transmission electron microscope (TEM) is used. The sample frozen on the TEM grid is observed on the TEM grid as it is. In addition, the sample may be observed using a microscope (for example, a scanning electron microscope, an atomic force microscope, or the like) that can generally observe from the μm order to the sub nm order.

<Embodiment 4>
When observing a cell sample using an optical microscope, the localization of the target portion is clarified by an immunolabeling method or the like. On the other hand, there are techniques for continuously observing cells as they are alive, or for observing molecular dynamics with time. For example, in fluorescence microscopy, only target molecules are illuminated with fluorescence, thereby obtaining molecular-specific spatial information such as intracellular localization. In fluorescence microscopy, there are an immunostaining method in which an antibody with fluorescence added to a target molecule is added, and a pretreatment method in which a gene of a fusion protein of a protein and a fluorescent protein is genetically engineered and used as a label.

  In recent years, the Correlative Light and Electron Microscopy (CLEM) method that combines fluorescence microscopy and electron microscopy has been developed. In the CLEM method, the location observed with a fluorescence microscope is observed with an electron microscope, and the observed images obtained from the two methods are compared and correlated to analyze the molecular specific localization with nano-level resolution. Is expected to be. Therefore, Embodiment 4 of the present invention describes a method using cell membrane detachment processing in the CLEM method.

  FIG. 10 is a flowchart of a structural analysis procedure in which the cell membrane of the cell sample is peeled using the cell membrane peeling apparatus 100 described in Embodiments 1 and 2, and the CLEM method is combined. Hereinafter, each step of FIG. 10 will be described.

(FIG. 10: Steps S901 to S902)
The operator applies a fluorescent label to the cultured cells by immunostaining or incorporation of a fluorescent protein (S901). The operator obtains an observation image of the cell sample with a fluorescence microscope (S902). When observing cells while culturing, steps S901 to S902 may be performed in parallel.

(FIG. 10: Steps S801 to S805)
The worker performs steps S801 to S805 described in the third embodiment. When the freezing method is used in the sample preparation process, the sample may be frozen after cell membrane detachment, and observation with a fluorescence microscope and an electron microscope may be performed.

(FIG. 10: Step S903)
An operator compares the observation image acquired with the fluorescence microscope image with the observation image acquired with the electron microscope, and confirms the correlation between both.

<Modification of the present invention>
The present invention is not limited to the embodiments described above, and includes various modifications. The above embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to the one having all the configurations described. A part of the configuration of one embodiment can be replaced with the configuration of another embodiment. The configuration of another embodiment can be added to the configuration of a certain embodiment. Further, with respect to a part of the configuration of each embodiment, another configuration can be added, deleted, or replaced.

  DESCRIPTION OF SYMBOLS 100: Cell membrane peeling apparatus, 101: Ultrasonic generator, 102: Vibration element, 103: Horn, 1031: Horn tip part, 1032: V-shaped groove, 1033: Notch, 1034: Second groove, 104: Coupling part, 105: Sample container, 600: Optical microscope, 601: Sample stage, 602: Light, 603: Horn drive unit, 604: Light source, 605: Light drive unit, 606: Control device, 607: Computer, 701: Fixing screw, 702 : Rotating mechanism, 703: Rotating mechanism fixing part, 704: Inclining mechanism, 705: Z axis driving part, 706: Z axis rail, 707: Y axis driving part, 708: Y axis rail, 709: X axis driving part, 710 : X-axis rail.

Claims (13)

A cell membrane peeling apparatus for peeling a cell membrane of a cell sample by ultrasonic waves,
The cell membrane peeling apparatus comprises an ultrasonic oscillator having a vibration element with a horn attached thereto,
The horn includes a columnar tip having a column shape,
A cell membrane detaching apparatus, wherein a recess formed so as to be recessed from the end surface is provided on an end surface of the columnar tip. The cell membrane peeling apparatus according to claim 1, wherein the recess is formed as a groove provided on the end face. The cell membrane peeling apparatus according to claim 2, wherein the end face has a cutout portion in addition to the groove. The cell membrane peeling apparatus according to claim 2, wherein the end surface has a second groove formed in a direction orthogonal to the groove. The diameter of the columnar tip is 4 mm or less. The cell membrane peeling apparatus according to claim 1. The cell membrane peeling apparatus further includes
An optical microscope for observing the cell sample;
A sample stage for supporting a container containing a liquid in which the cell sample is immersed;
A light for irradiating the position observed by the optical microscope;
The cell membrane peeling apparatus according to claim 1, comprising: 7. The cell membrane according to claim 6, further comprising a horn drive mechanism that moves the horn in three axial directions, tilts the horn, and rotates the horn around a central axis. Peeling device. The cell membrane peeling apparatus according to claim 6, further comprising a light driving mechanism that moves the light in three axial directions and tilts the light. A peeling method for peeling a cell membrane of a cell sample by ultrasound,
A horn provided with a column-shaped tip having a column shape and provided with a recess formed on the end surface of the column-shaped tip is recessed from the end surface. A detachment method comprising generating cavitation by oscillating vibration from a vibration element of a sound wave oscillator, and detaching the cell membrane by the cavitation. An observation method for observing a cell sample,
Place the cell sample on a grid used in sample observation using a transmission electron microscope,
A horn provided with a column-shaped tip having a column shape and provided with a recess formed on the end surface of the column-shaped tip is recessed from the end surface. Cavitation is generated by oscillating vibration from the vibration element of the oscillator, and the cell membrane is peeled off by the cavitation,
Freezing the cell sample from which the cell membrane has been detached on the grid;
An observation method comprising observing the cell sample on the grid with the transmission electron microscope. An observation method for observing a cell sample,
A horn provided with a column-shaped tip having a column shape and provided with a recess formed on the end surface of the column-shaped tip is recessed from the end surface. Cavitation is generated by oscillating vibration from a vibration element of a sound wave oscillator, and the cell membrane is peeled off by the cavitation,
Removing the ice after freezing the cell sample from which the cell membrane has been detached,
Depositing a metal film on the surface of the cell sample;
An observation method comprising peeling the metal film from the cell sample and observing the metal film with a microscope. The observation method further includes:
Observe the cell sample using a fluorescence microscope,
The observation method according to claim 10 or 11, wherein an observation image obtained by the observation is compared with an observation image obtained by observing the cell sample using the fluorescence microscope. A horn attached to a vibration element of an ultrasonic oscillator applied to a cell membrane peeling apparatus,
The horn includes a columnar tip having a column shape,
A horn formed so as to be recessed from the end surface is provided on an end surface of the columnar tip.

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