JP3461448B2 - Specific substance introduction apparatus, observation apparatus using the same, and substance introduction method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an introduction device for bringing a vesicle close to a specific position of a cell sample to introduce a specific substance contained in the vesicle into cells, and further,
The present invention relates to an observation device provided with the introduction device. Furthermore, the present invention relates to a method for introducing a specific substance into cells by bringing the vesicles closer to and contacting the cells.

[0002]

2. Description of the Related Art There are the following three typical methods for introducing a specific substance such as DNA, membrane protein, cytoplasm and intercellular signaling substance into a cell sample. The first method is
First, prepare a substance called a vesicle in which the specific substance is surrounded by a membrane with the same structure as the cell sample, and make it coexist in the culture medium of the cell sample at a specific concentration, and after the cell sample and the vesicles come into contact, the vesicle In this method, the specific substance encapsulated in the vesicles is introduced into the cell sample by waiting until it is taken up by the cell sample.

Here, the vesicle will be described. The vesicle refers to one in which a specific substance to be introduced into a cell sample is incorporated inside or on the lipid membrane, and for example, "liposome" described in JP-A-6-298638 is one of them. .

Japanese Patent Laid-Open No. 6-298638 discloses that
"Liposomes" are described as microvesicles of phospholipid bilayers containing an aqueous phase inside. Further, regarding the specific substance taken into the vesicle, it is described that the water-soluble drug (specific substance) can be taken into the inner aqueous phase and the oil-soluble substance can be taken into the double membrane part. This indicates a state in which the specific substance is taken up inside the lipid membrane or on the lipid membrane. In addition, a state in which a specific substance is taken up by a vesicle is called an internal inclusion.

The second method is to prepare vesicles first, adsorb the vesicles to the tip of the glass tube, and press the vesicles against the cell sample to fuse them. The third method is to include a specific substance in a glass tube with a sharp tip, pierce the tip of the glass tube into a cell sample,
It is a method of injecting a specific substance contained therein into a cell sample.

[0006]

In the first method, it is not known where the specific substance was introduced into the cell sample, which is inconvenient for later observation. In the second method, the size of vesicles that can be adsorbed by the glass tube is at most 10
μm, and vesicles smaller than this cannot be introduced.

In the third method, since the cell membrane of the cell sample is pierced, the cells may be destroyed. Also, the tip of the glass tube is required to be very thin,
There is a limit to the processing.

It is an object of the invention to use vesicles, especially 10 μm.
It is an object of the present invention to provide an apparatus and method for introducing a specific substance that allows even a vesicle smaller than m to be selectively placed in contact with a desired position of a cell sample, thereby introducing the specific substance to the desired position. An object of the present invention is also to provide an observation device and method capable of analyzing the interaction that acts when introducing vesicles into a cell sample.

[0009]

The device of the present invention is a device for introducing a specific substance into a cell by bringing the vesicle close to and in contact with the cell, which can hold the vesicle at its tip and elastically deforms. A cantilever tip with a lever part to obtain, a stage on which a container containing vesicles is placed, a vertical movement mechanism that provides relative vertical movement between the cantilever tip and the stage, and a relative movement between the cantilever tip and the stage. And a horizontal movement mechanism for providing a horizontal movement. Further, the method of the present invention, in order to introduce a specific substance into the cells by bringing the vesicles close to the cells and bringing them into contact with the cells, a step of holding the vesicles at the tip of the cantilever tip having a lever portion capable of elastic deformation, and
Place a container containing vesicles on the stage, give relative vertical movement between the cantilever tip and the stage, and give relative horizontal movement between the cantilever tip and the stage. It includes steps and.

The apparatus preferably further includes a displacement sensor that detects the amount of elastic deformation of the lever portion of the cantilever tip and identifies the force received by the lever portion based on this. Further, the method further includes a step of detecting an elastic deformation amount of the lever portion of the cantilever tip, and a step of specifying a force received by the lever portion based on the step.

Further, the device of the present invention is a device for observing a cell of a specific substance and cells before and after the transfer, and in addition to the above-mentioned device for introducing a specific substance, cells and vesicles are optically And an optical system for observation. In addition, the method of the present invention further includes the step of optically observing the cells and vesicles before and after the introduction of the specific substance into the cells.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 schematically shows the overall structure of an observation device equipped with an introduction device. The observation device of the present embodiment includes an introduction device and an inverted optical microscope,
In the following, the introduction device will be described first, and then the inverted optical microscope will be described.

[Introducing Device] As shown in FIG. 1, a Z stage 20 is provided on the column 4 standing on the base 2. The Z stage 20 has a fixed portion 20a fixed to the support column 4 and a moving portion 20b movably supported with respect to the fixed portion 20a, and the arm 22 fixed to the moving portion 20b includes: An upper end of a cylindrical piezoelectric actuator 24 generally called a tube scanner is fixed. A head 30 is fixed to the lower end of the cylindrical piezoelectric actuator 24. The cylindrical piezoelectric actuator 24 can displace its free end in three dimensions, and thus can move the head 30 in three dimensions.

A cantilever tip 40 is attached to the lower side of the head 30. The cantilever tip 40 has a lever portion 44 extending from the support portion 42, and its tip has
The surface is treated so that the vesicles existing in the liquid can be favorably adsorbed by the intermolecular force. This surface treatment is, for example, hydrophilic treatment or hydrophobic treatment according to the state of the vesicle film. Further, in consideration of the case where a functional group or protein molecule is introduced into the vesicle membrane, a treatment for introducing a functional group or protein molecule corresponding to the tip of the lever portion 44 is performed. Further, in consideration of the case where the vesicle film has an electric charge, a treatment for making the lever portion 44 conductive is performed. Regarding the conductive treatment, instead of treating the surface of the lever portion 44, a conductive material may be used as the material of the lever portion 44.

The head 30 internally has a displacement detection system for detecting the displacement of the lever portion 44 of the attached cantilever tip 40. This displacement detection system is composed of a laser diode 32, a focus lens 34, a total reflection mirror (or dichroic mirror) 36, and a photodiode 38 having a plurality of light receiving regions. The displacement of the lever portion 44 of the cantilever chip 40 is obtained based on the position of the reflected light beam from the lever portion 44 which is incident on the photodiode 38.

The sample table 52 is supported by the support column 4, on which a transparent container such as a petri dish containing vesicles containing a substance such as DNA or a transparent container such as a petri dish containing cell samples is placed. For example, placed one after another. Alternatively,
You may put the container which can separate the inside of one transparent container and can put a vesicle and a cell sample into a different part separately. Furthermore, a slide glass on which the vesicle and the cell sample are placed at different positions may be placed. In the present specification, the description will be made assuming a petri dish as a typical transparent container.

The sample table 52 is preferably movably supported in the horizontal direction, and provides relative horizontal movement between the petri dish and the cantilever tip mounted thereon.
It is used for alignment between the tip of the lever and the vesicle or cell sample.

The alignment between the tip of the lever portion and the vesicle or cell sample does not only move the sample table 52 in the horizontal direction, but also the cylindrical piezoelectric actuator 4
2 may be used. The mechanism for providing relative horizontal movement between the tip of the lever portion and the vesicle or cell sample in this manner is generically referred to as a horizontal movement mechanism.

Further, the sample stage 52 is supported so as to be movable in the vertical direction, constitutes a vertical movement mechanism together with the cylindrical piezoelectric actuator 42, and provides relative movement in the vertical direction between the petri dish and the cantilever tip. Is also possible. This configuration is used to control the distance between the tip of the lever portion whose position has been adjusted by the horizontal movement mechanism and the vesicle or cell sample.

[Inverted Optical Microscope] Next, the elements constituting the inverted optical microscope will be described. An illumination system 10 for transmitted illumination enables observation by a phase difference observation method, and is supported by an arm 6 provided on an upper end of a column 4 standing on the base 2. The illumination system 10 has a light source 12, a collector lens 14, a slider 16, and a condenser lens 18. The slider 16 has a ring opening 16a for observing a phase difference and a light transmitting portion 16b that transmits light, is movable across the optical path, and the ring opening 16a can be arranged in the optical path as necessary.

Below the sample table 52, a plurality of objective lenses can be attached, and a revolver 56 for selectively arranging one of them is disposed below the sample. The revolver 56 is provided with an objective lens 58 having a relatively low magnification for observation by a phase difference observation method and an objective lens 60 having a high magnification and a high NA for fluorescence observation.

A half mirror 62 is arranged below the objective lens arranged under the sample, and another half mirror 64 (or dichroic mirror) is arranged further below the half mirror 62. The half mirror 62 deflects the light from the fluorescent light source 70 toward the objective lens and transmits the light from the objective lens. Half mirror 64
Is a C for partially observing light from the objective lens for observing a microscope image.
It deflects toward the CD camera 66 and transmits the remaining light.

The fluorescence excitation light source 70 includes a light source 72, a collector lens 74, a shutter 76, and a condenser lens 78.
have. Below the half mirror 64, a lens 86 and a photomultiplier 88 are provided to detect fluorescence.
Are arranged. During fluorescence observation, an excitation light filter 82 is placed on the optical path between the excitation light source 70 and the half mirror 62.
And the absorption filter 84 is inserted in the optical path between the half mirrors 62 and 64.

[Introduction of Specific Substance Encapsulated in Vesicle into Cell Sample] The Z stage 20 is operated to raise the head 30 sufficiently, and a solution 114 containing a vesicle 112 encapsulating a specific substance such as DNA is added. A transparent container such as a petri dish 110 is placed on the sample table 52. Z stage 2
2, the head 30 is lowered to lower the head 30, and the cantilever tip 40 is submerged in the solution 114 as shown in FIG. 2, and the tip of the lever portion 44 is placed on the bottom of the petri dish 110 until it enters the depth of focus of the objective lens 58. Get closer.

By observing the vesicles 112 by transmission phase difference observation or differential interference observation, a vesicle 112 suitable for introducing a specific substance into the cell sample is selected, and the tip of the lever portion 44 of the cantilever tip 40 is selected. Vesicle 112
Align on top.

Positioning of the tip end portion of the lever portion 44 and the selected vesicle 112 is carried out by relatively moving both of them using a horizontal movement mechanism. Note that this alignment is performed by the sample table 52 or the cylindrical piezoelectric actuator 42.
It is also possible to move at least one of the above.

The piezoelectric actuator 24 is extended to lower the cantilever tip 40, and the tip portion of the lever portion 44 is pressed against the vesicle 112. As described above, since the tip portion of the lever portion 44 of the cantilever tip 40 is surface-treated so that the vesicles can be favorably adsorbed, the vesicles 112 are adsorbed by the intermolecular force. After adsorption, the head 30 is raised on the Z stage 20.

The petri dish 110 containing the vesicles is removed from the sample table 52, and the petri dish 120 containing the solution 124 of the cell sample 122 into which the specific substance is to be introduced is placed on the sample table 52 instead. The Z stage 20 is operated to lower the head 30, and as shown in FIG.
Are submerged in solution 124.

A cell sample 122 into which a specific substance (hereinafter, simply referred to as a specific substance) contained in the vesicle 112 is introduced by transmission phase difference observation or differential interference observation, and the tip portion of the lever portion 44 of the cantilever tip 40 is selected. , On the selected cell sample 122.

The position of the tip of the lever portion 44 and the selected cell sample 112 is adjusted by moving the both relatively using a horizontal movement mechanism. Note that this alignment is performed by the sample table 52 or the cylindrical piezoelectric actuator 42.
It is also possible to move at least one of the above.

The piezoelectric actuator 24 is extended to lower the cantilever tip 40, and the vesicle 112 attached to the tip of the lever portion 44 is pressed against the cell sample 122. Vesicle 112 pressed against cell sample 122
Is eventually taken up by the cell sample 122, and the specific substance is introduced into the cell sample 122.

At this time, the inside and outside of the cell membrane can be brought into a depolarized state by coexistence of a counter electrode and the like. It is also possible to carry out an operation such as adding Ca ²⁺ to the solvent in the vesicle. By adopting such a configuration, it is possible to artificially create a signal transmission state of nerve cells and the like, so that it becomes possible to introduce a specific substance that induces various states in a cell sample.

In this apparatus, since the lever portion 44 and the cell sample 122 are aligned by using the inverted optical microscope, the specific substance can be introduced into a desired position of the cell sample 122 with high positional accuracy.

When adsorbing the vesicles 112 and introducing the specific substance into the cell sample 122, preferably, the displacement detection system in the head 30 detects the displacement of the tip of the lever portion 44, and the detected displacement information is obtained. Based on this, the force acting on the lever portion 44 is calculated. This makes it possible to know the force with which the lever portion 44 pushes the vesicle 112 when adsorbing the vesicle 112, and the force with which the vesicle 112 pushes the cell sample 122 when introducing a specific substance into the cell sample 122.

The approach for introducing the specific substance into the cell sample 122 is preferably carried out by an automatic proximity program, and the vesicle 112 is applied to the cell sample 12 with an arbitrary force.
It can be pressed against 2. For example, vesicle 112
The force pressing the cell sample 122 against the cell sample 122 can be set to about 0.1 nN, and when the set value is reached, the approach of the two stops. At this time, the lever portion 44 receives the pressing force and stops in a state of being displaced. By continuing to monitor the displacement of the lever portion 44 as it is, it can be determined whether or not the specific substance is introduced into the cell sample 122.

As described above, in this device, the force acting on the lever portion 44 of the cantilever tip 40 can be known, so that it is possible to prevent the vesicle 112 and the cell sample 122 from being destroyed or scratched by an excessive force. it can.

Further, in the conventional devices, there was no way to know the force of pushing the vesicle against the cell sample, but this device can know the force of the vesicle pushing the cell sample at the time of introduction,
Information on the forces acting on the vesicle and the cell sample at the time of introduction will be very useful for elucidating the reaction in which the vesicle 112 is taken up by the cell sample 122.

[Introduction of Specific Substance into Floating Cell Sample]
In the above, the introduction of the specific substance into the cell sample fixed to the bottom of the petri dish was described with reference to FIG. In the introduction of the specific substance into the cell sample floating in the solution in the petri dish, the cell sample 122 is held by using the three-dimensionally positionable capillary 130 supported by the manipulator as shown in FIG. The vesicle 112 adsorbed on the lever portion 44 of the cantilever chip 40 is pressed against the cell sample 122 held by the cell 130. This series of operations is exactly the same except that the positioning of the cell sample 122 is performed by the three-dimensional manipulator.

[Expansion to Atomic Force Microscope] The cantilever tip 40 may include a probe portion 46 at the tip of the lever portion 44. The probe portion 46 has a sharp protrusion 46a protruding from the flat portion 46b, and the height of the protrusion 46a with respect to the flat portion 46b is used as the vesicle 112 for suction.
Smaller than the diameter of.

The vesicle 112 is adsorbed on the boundary between the protrusion 46a and the flat portion 46b. The height of the protrusion 46a is
Since the size smaller than the diameter of the vesicle 112 is selected, when the vesicle 112 is brought close to the cell sample, the sharp protrusion 4 is formed before the vesicle 112 comes into contact with the cell sample.
6a does not touch the cell sample.

The sharp protrusion 46a is used for observation before and after the specific substance is introduced into the cell sample. The present apparatus includes elements necessary for constructing an atomic force microscope, that is, a cylindrical piezoelectric actuator 24, a cantilever tip 40, and a displacement detection system for detecting displacement of the lever portion.
Therefore, by using the cantilever tip 40 having the probe portion 46 shown in FIG. 5, atomic force microscope observation using the protrusion 46a as a probe can be performed, and the cell sample before and after the introduction of the specific substance can be observed with high resolution. can do.

[Illumination system] This apparatus can be used for special observation methods such as transmission bright-field illumination, transmission phase difference illumination, differential interference illumination, and epifluorescence illumination.

[Transparent bright-field illumination] Since the transparent bright-field illumination clearly indicates the position of the cantilever, the position adjustment of the laser beam spot for detecting the displacement of the lever portion 44 irradiated on the back surface of the cantilever is mainly performed on the cantilever. Used for.

[Transmission Phase Difference Illumination] The transmission phase difference illumination is an observation method for providing contrast based on the difference in the refractive index of the sample. This illumination method is applied to an optically transparent sample having a thickness of several μm or less. Effective against It is also effective as a method for observing the morphology of a thin sample (cell) at low resolution, such as finding the boundary of the peripheral portion of a cultured cell.

Since the transmission phase difference illumination uses the ring illumination, the NA of the illumination is not sufficiently used as compared with the NA of the objective lens, which is not an observation method suitable for high resolution observation.

If high resolution observation is to be performed, observation using transmission differential interference illumination is suitable. This illumination is an observation method that contrasts an optically transparent sample based on the positional change of the refractive index distribution. An example of transmission differential interference illumination is described in JP-A-9-15504.
The disclosure content contained in the same document is incorporated herein by reference in its entirety.

In this illuminating method, light from a light source is linearly polarized by a polarizer (polarizer) and made incident on a first Nomarski prism, where it is separated into ordinary light and extraordinary light, passed through a condenser lens, and the sample The transmitted ordinary and extraordinary rays are combined by the second Nomarski prism through the objective lens, and the combined ordinary and extraordinary rays are made to interfere through the analyzer to form a differential interference image on the image plane. is doing.

Transmission differential interference illumination can be realized by adding this configuration to the apparatus of FIG. This differential interference illumination is not limited in principle to the illumination NA as compared with the phase-difference illumination, so that all NAs of the objective lens can be used and high-magnification observation is possible. Therefore, it can be used when it is desired to confirm the morphology of even a fine structure of a sample.

The above-mentioned transmission phase difference illumination and transmission differential interference illumination are collectively referred to as transmission contrast illumination. [Epi-illumination] Epi-illumination is used to confirm that the vesicle contains a sufficient amount of the target substance. Further, it is suitable for observing the reaction / state change of the sample after the vesicle is brought into contact with the sample (cell). Furthermore,
Fluorescently stain a specific part of the sample you want to contact with the vesicle,
Alternatively, for example, GFP (Green
Introducing DNA that expresses Flourescent Protein),
It can be used to identify a fluorescent portion or a fluorescent cell that produces GFP.

As the fluorescence microscope, it is effective to use a scanning laser microscope to observe the vesicles in addition to the structure shown in FIG. In addition, transmission phase difference illumination, transmission differential interference illumination, and epi-fluorescence illumination are not only for observing the sample, but also for position adjustment to attach the vesicle to the tip of the probe and the probe with the vesicle attached to the tip of the probe. It is used to adjust the position of the sample and the sample.

As described above, the fluorescent illumination is suitable for observing and measuring the reaction / state change between the sample and the vesicle, and the transmission contrast illumination is suitable for observing and measuring the morphology / structure of the sample. Therefore, when it is desired to measure the morphology of the sample and the reaction of the sample by the vesicles in association with each other, an illumination method combining fluorescence illumination and transmission contrast illumination is preferable. The present invention is not limited to the above-described embodiments, but includes all implementations performed within the scope of the invention.

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided an introducing device and an introducing method capable of introducing a specific substance by pressing even a vesicle containing the specific substance, particularly a vesicle smaller than 10 μm, to a desired position of a cell sample. .

[Brief description of drawings]

FIG. 1 schematically shows the overall configuration of an observation device equipped with an introduction device according to the present invention.

FIG. 2 shows a state in which a vesicle is adsorbed on a lever portion of a cantilever tip.

FIG. 3 shows how vesicles adsorbed on the lever portion of a cantilever chip are introduced into a cell sample.

FIG. 4 shows how vesicles are introduced into a floating cell sample.

FIG. 5 shows a structure of a lever portion of a cantilever tip that enables extension to an atomic force microscope.

[Explanation of symbols]

20 Z stage 24 Piezoelectric actuator 30 heads 40 cantilever tip 42 Support 44 Lever part 52 sample table

Continuation of the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12M 1/00-3/10 C12N 15/00-15/90 G01B 21/30 G02B 21/00 BIOSIS / MEDLINE / WPID S (STN) PubMed

Claims (6)

(57) [Claims] 1. A device for introducing a specific substance into a cell by bringing the vesicle close to and in contact with the cell, the cantilever tip having a lever portion capable of holding the vesicle at its tip and elastically deformable, A stage on which a container containing vesicles is placed, a vertical movement mechanism that provides relative vertical movement between the cantilever tip and the stage, and a horizontal movement mechanism that provides relative horizontal movement between the cantilever tip and the stage. A specific substance introducing device having a moving mechanism. 2. The specific substance introduction according to claim 1, further comprising a displacement sensor that detects the amount of elastic deformation of the lever portion of the cantilever tip and specifies the force received by the lever portion based on this. apparatus. 3. An apparatus for observing a specific substance into cells and cells before and after the introduction, and the specific substance introducing apparatus according to claim 1 and an optical for optically observing cells and vesicles. An observation device for cells and vesicles having a system. 4. A step of holding a vesicle at the tip of a cantilever tip having a lever portion that can be elastically deformed in order to introduce a specific substance into the cell by bringing the vesicle close to and in contact with the cell, and the vesicle on the stage. The step of placing the container in which it is placed, the step of giving a relative vertical movement between the cantilever tip and the stage, and the step of giving a relative horizontal movement between the cantilever tip and the stage. Method of introducing specific substances including. 5. The method for introducing a specific substance according to claim 4, further comprising the step of detecting the amount of elastic deformation of the lever portion of the cantilever tip, and based on this, the force received by the lever portion is specified. A method for introducing a specific substance including steps. 6. The method for introducing a specific substance according to claim 4, further comprising the step of optically introducing the specific substance into cells and observing the cells and vesicles before and after the introduction.