JP6355127B2 - Fluorescence observation system - Google Patents

Description

  The present invention relates to a fluorescence observation system capable of performing fluorescence observation in real time on the state of a subject that has received physical stimulation by an electric field, a magnetic field, or electromagnetic waves.

  In the biological field and the like, the state of a cell is analyzed by irradiating a cell into which a fluorescent indicator or a gene expressing a fluorescent protein is introduced with excitation light and detecting fluorescence excited by the excitation light. . For example, there is a technique of measuring the pH of a cell by irradiating a cell with pulse excitation light having a specific intensity via a fiber probe and analyzing the fluorescence (see, for example, Patent Document 1).

  On the other hand, fluorescence observation of cells may be performed in a situation where various stimuli such as electric current and heat are received. As a means for giving such a stimulating effect to cells, an electromagnetic field generator generates an electric field, magnetic field, or electromagnetic wave having a desired intensity, and the cell is placed in the electric field, magnetic field, or electromagnetic wave and exposed. Is mentioned.

  When the fluorescence detection device is used in the vicinity of such an electromagnetic field generation device, the fiber probe and the fluorescence detection device main body are also exposed to an electric field, a magnetic field, and electromagnetic waves. Since the fiber probe and the fluorescence detection device include metal parts, these metal parts become sources of current, heat, vibration, and the like. That is, the cells and their fluorescence dynamics are affected by the current, heat, vibration, etc. generated from the metal parts of the fiber probe and fluorescence detection device. Moreover, such a fiber probe and a fluorescence detection apparatus main body also cause the distortion of an electric field, a magnetic field, and electromagnetic wave distribution.

  In this way, current, heat, vibration, etc. generated from metal parts affect cells and fluorescence dynamics, so the state of cells in an electric field, magnetic field, or electromagnetic wave of a desired intensity generated by an electromagnetic field generator is There is a problem that it cannot be observed accurately and in real time.

  In addition, such a problem is not limited to the object for cells, and similarly exists for any specimen containing cells.

JP 2011-185843 A

  In view of the above circumstances, an object of the present invention is to provide a fluorescence observation system that can accurately and in real time observe the state of a subject that has received a physical stimulus by an electric field, a magnetic field, or an electromagnetic wave.

A first aspect for achieving the above object is an electromagnetic field generator that generates an electric field, a magnetic field, or an electromagnetic wave, an excitation light source device that emits excitation light, and the electric field, magnetic field, or electromagnetic wave generated by the electromagnetic field generator. A fiber probe that transmits the fluorescence emitted by irradiating the subject exposed to the excitation light from the excitation light source device, and an imaging unit that images the fluorescence collected by the fiber probe, The fiber probe is formed of a non-conductive and non-magnetic material, and a bundle fiber configured by bundling a plurality of optical fibers, and a lens bonded and fixed to one end surface of the bundle fiber with a non-conductive adhesive. A sleeve that covers the outside of the lens and the bundle fiber and that is formed of a non-conductive and non-magnetic material. In fluorescence observation system characterized in that it is obtained by combining the refractive index of the lens that.

In such a first aspect, the fiber probe disposed close to the subject is formed of a non-conductive material. For this reason, the fiber probe does not become a source of current, heat, vibration, etc. even in an electromagnetic field. Therefore, the fluorescence observation system does not affect the subject or the fluorescence dynamics of the subject and the fluorescence dynamics without causing the influence of the current, heat, vibration, etc. generated from the fiber probe, and the influence of the electromagnetic field of the desired intensity formed by the electromagnetic field generator. Only the fluorescence dynamics detected from the subject can be observed accurately and in real time.
Moreover, since the adhesion part of bundle fiber and a lens is reinforced by providing a sleeve, it can suppress that they remove | deviate or shift | deviate.
Furthermore, the working distance, numerical aperture, and magnification of the lens can be adjusted to desired values.

According to a second aspect of the present invention, in the fluorescence observation system according to the first aspect, the fiber probe is formed of a ceramic or plastic that is a non-conductive, non-magnetic and biocompatible material . It is in the fluorescence observation system characterized by this.

  In the second aspect, the fiber probe can be used in a living body for a long period of time. Thereby, the fluorescence dynamics of the cell in the living body of the subject exposed to the electric field, magnetic field or electromagnetic wave can be detected in real time.

According to a third aspect of the present invention, in the fluorescence observation system according to the first or second aspect , a ceramic or plastic that is a non-conductive, non-magnetic, and biocompatible material that covers the outside of the fiber probe. A fluorescence observation system comprising a tube formed by the above.

In the third aspect, the fiber probe can be used in a living body for a long period of time. Thereby, the fluorescence dynamics of the cell in the living body of the subject exposed to the electric field, magnetic field or electromagnetic wave can be detected in real time.

A fourth aspect of the present invention is the fluorescence observation system according to any one of the first to third aspects, comprising a dichroic mirror that transmits one of excitation light and fluorescence and reflects the other, The excitation light source device is configured such that excitation light is incident on the fiber probe via the dichroic mirror, and the fluorescence incident on the fiber probe is imaged by the imaging means via the dichroic mirror. It is in the fluorescence observation system characterized by this.

In the fourth aspect, since the excitation light and fluorescence can be guided by one fiber probe, the cost of the fiber probe is reduced, and one fiber probe is used rather than a plurality of fiber probes. Is easy to handle.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescence observation system which can observe the state of the subject which received the physical stimulus by an electric field, a magnetic field, and electromagnetic waves more correctly is provided.

1 is a schematic configuration diagram of a fluorescence observation system according to Embodiment 1. FIG. FIG. 3 is a side view showing a main part of the fiber probe according to the first embodiment. FIG. 3 is a cross-sectional view taken along line A-A ′ of FIG. 2. FIG. 4 is a sectional view taken along line B-B ′ in FIG. 3. 6 is a side view showing a main part of a fiber probe according to Embodiment 2. FIG. 6 is a cross-sectional view of a fiber probe according to Embodiment 2. FIG. FIG. 7 is a cross-sectional view taken along line C-C ′ in FIG. 6.

<Embodiment 1>
FIG. 1 is a schematic configuration diagram of a fluorescence observation system according to the present embodiment. The fluorescence observation system 1 includes an electromagnetic field generation device 10, an excitation light source device 20, a fiber probe 30, an imaging device 40, and an analysis device 50. The fluorescence observation system 1 irradiates a subject 60 with excitation light and observes generated fluorescence. is there.

  The subject of the present invention is a cultured cell into which a fluorescent indicator or the like has been introduced, a cell in an animal body, or the like, but is not limited thereto, and is an arbitrary substance containing a fluorescent substance that is excited by excitation light.

  The electromagnetic field generator 10 is a device that generates an electric field, a magnetic field, or an electromagnetic wave (which may be an alternative, or any two or three). Specifically, the electromagnetic field generation device 10 includes an electromagnetic coil 11, a power supply device 12, and a control device 13. The power supply device 12 is a device that supplies a current to the electromagnetic coil 11, and the control device 13 is a device that causes the power supply device 12 to supply a current to the electromagnetic coil 11 so that an electromagnetic field having an input intensity is generated. As described above, the electromagnetic field generation device 10 is configured to generate an electric field, a magnetic field, or an electromagnetic wave corresponding to the intensity from the electromagnetic coil 11 by setting a desired strength in the control device 13.

  The electromagnetic field generator 10 has a set of electromagnetic coils 11, and each electromagnetic coil 11 faces each other and is arranged in the vertical direction. A mounting table 61 on which the subject 60 is mounted is disposed in the middle of the electromagnetic coil 11. By disposing the electromagnetic coil 11 so as to face each other vertically, a uniform electric field, magnetic field, or electromagnetic wave can be generated as indicated by a dotted arrow. Thus, the subject 60 is exposed to the electric field, magnetic field, or electromagnetic wave generated from the electromagnetic coil 11.

  The configuration of the electromagnetic field generator 10 is not limited to that described above, and any configuration that can generate an electric field, a magnetic field, or an electromagnetic wave may be used. For example, one electromagnetic coil 11 may be used without being opposed to each other, or a waveguide may be used instead of the electromagnetic coil 11.

  The excitation light source device 20 is a device that emits excitation light, which is light having a wavelength that can excite a subject. Specifically, the excitation light source device 20 includes a semiconductor light emitting element such as a laser light source or an LED light source that emits excitation light.

  The fiber probe 30 guides excitation light emitted from the excitation light source device 20 to the subject 60 and guides fluorescence emitted from the subject 60 to the imaging device 40.

  Specifically, the fiber probe 30 includes a bundle fiber 31 configured by bundling a plurality of optical fibers, and a lens unit 32 provided with a lens at one end thereof. Details of the lens unit 32 will be described later.

  The fiber probe 30 is arranged so that the excitation light from the excitation light source device 20 is incident from the end opposite to the lens unit 32, and the excitation light is emitted from the lens unit 32 to irradiate the subject. .

  Specifically, a dichroic mirror 70 and an objective lens 71 are disposed between the excitation light source device 20 and the fiber probe 30. The dichroic mirror 70 is disposed so as to reflect the excitation light emitted from the excitation light source device 20 and the reflected excitation light enters the objective lens 71.

  An imaging device 40 is disposed on the side opposite to the excitation light source device 20 with the dichroic mirror 70 interposed therebetween. The dichroic mirror 70 is arranged so that the fluorescence emitted from the fiber probe 30 and reflected through the objective lens 71 is reflected, and the reflected excitation light is incident on the imaging device 40.

  The imaging device 40 is a device having a function of imaging incident fluorescence and displaying it as an image. For example, the imaging device 40 includes a sensor unit that converts and outputs an electrical signal that reflects the intensity of incident fluorescence, an arithmetic unit that converts the electrical signal into pixel information that constitutes an image, and a result of the arithmetic processing. And a monitor for displaying as an image. The objective lens 71 is used to collect fluorescence incident on the imaging device 40.

  An analysis device 50 is connected to the imaging device 40. The analysis device 50 is an information processing device including an arithmetic processing device such as a CPU, a storage device such as a RAM or an HDD, an input device such as a mouse / keyboard, and an output device such as a monitor. Such an analysis device 50 can obtain an image reflecting fluorescence from the imaging device 40 through an interface such as a USB, and can execute software for analyzing the image.

  As described above, the fluorescence observation system 1 according to the present embodiment emits the excitation light from the excitation light source device 20 and causes the excitation light to enter the fiber probe 30 via the dichroic mirror 70 and the objective lens 71. Then, the excitation light is guided by the fiber probe 30 to irradiate the subject 60. The fluorescence emitted from the subject 60 by the excitation light is guided by the fiber probe 30 and is incident on the imaging device 40 via the objective lens 71 and the dichroic mirror 70. In addition, since the excitation light and the fluorescence can be guided by one fiber probe 30, the cost related to the fiber probe 30 is reduced, and the one fiber probe 30 is more preferable than the plurality of fiber probes 30. easy to handle.

  As described above, the subject 60 is exposed to the electric field, magnetic field, or electromagnetic wave generated by the electromagnetic field generator 10. The lens unit 32 is disposed close to the subject 60 in order to irradiate the subject 60 with excitation light and collect fluorescence emitted from the subject 60. That is, the lens unit 32 also collects fluorescence in a state where it is exposed to an electric field, a magnetic field, or an electromagnetic wave.

  Here, the lens portion 32 of the fiber probe 30 will be described in detail with reference to FIGS. 2 is a side view showing the main part of the fiber probe, FIG. 3 is a cross-sectional view taken along the line A-A ′ of FIG. 2, and FIG. 4 is a cross-sectional view taken along the line B-B ′ of FIG.

  The fiber probe 30 is provided with a lens portion 32 at one end on the subject 60 side. The lens unit 32 includes a lens 33 and a sleeve 34.

  The lens 33 is not particularly limited, but is, for example, a graded index (GRIN), and a cylindrical lens can be used. One flat surface 33a of the lens 33 is bonded and fixed to the distal end surface 31a of the bundle fiber 31 with an adhesive or the like, and the other flat surface 33b is disposed so as to face the subject 60 (see FIG. 1).

  The lens 33 collects excitation light from the excitation light source device 20 via the bundle fiber 31. The condensed excitation light is applied to the subject 60. Then, the fluorescence emitted from the subject 60 with respect to the excitation light enters the lens 33. The lens 33 collects the fluorescence and makes it incident on the bundle fiber 31.

  The sleeve 34 is a member formed in a cylindrical shape, and the bundle fiber 31 and the lens 33 are inserted therein. Inside the sleeve 34, an adhesive portion between the bundle fiber 31 and the lens 33 is located, and the bundle fiber 31 and a part of the lens 33 exposed from the sleeve 34 are bonded and fixed to both ends of the sleeve 34 by an adhesive 35. ing.

  Since the sleeve 34 is provided in this manner, the bonded portion between the bundle fiber 31 and the lens 33 is reinforced, so that they can be prevented from being detached or displaced.

  The bundle fiber 31, the lens 33, the sleeve 34, and the adhesive 35 that bonds these components constituting the lens unit 32 are all made of a non-conductive and non-magnetic material. The material of the lens 33 may be glass or plastic which is a non-conductive and non-magnetic material. The material of the bundle fiber 31 may be either glass or plastic which is a non-conductive and non-magnetic material. Further, as the material of the sleeve 34, a ceramic or plastic such as alumina which is a non-conductive and non-magnetic material can be used. Further, as the adhesive 35, a UV curable or epoxy adhesive which is a non-conductive material can be used.

  As described above, in the fluorescence observation system 1 according to the present embodiment, the lens portion 32 of the fiber probe 30 disposed in the vicinity of the subject 60 is formed of a nonconductive material. For this reason, the lens part 32 does not become a generation source of current, heat, vibration, etc. even in an electric field, a magnetic field, or an electromagnetic wave.

  As a result, the fluorescence observation system 1 does not affect the subject 60 with the current, heat, vibration, and the like using the lens unit 32 as a generation source, and the electric field and magnetic field having a desired intensity formed by the electromagnetic field generator 10. Alternatively, the fluorescence dynamics of the subject 60 can be observed after only the influence of the electromagnetic wave is given to the subject 60.

  In addition, the member formed of the non-conductive material may be only the lens unit 32 disposed in the vicinity of the subject 60, and other members and parts may be formed by using a sufficiently long bundle fiber 31 so that the electric field, It can be installed in a remote location that is not affected by magnetic fields or electromagnetic waves. Accordingly, even when an apparatus including metal parts such as the excitation light source apparatus 20, the imaging apparatus 40, or the analysis apparatus 50 is used, it can be avoided that they affect the subject 60, and an electric field, magnetic field, or The fluorescence dynamics of the subject 60 placed in the electromagnetic wave can be observed accurately and in real time.

<Embodiment 2>
In the first embodiment, the lens 33 is a single gradient index lens, but a plurality of lenses may be combined. FIG. 5 is a side view showing a main part of the fiber probe according to the present embodiment. In addition, the same code | symbol is attached | subjected to the same thing as Embodiment 1, and the overlapping description is abbreviate | omitted.

  As shown in the figure, a GRIN lens or plano-convex lens 36 having a refractive index different from that of the lens 33 is bonded to the flat surface 33b of the gradient index lens 33 with an adhesive. In this way, by combining a GRIN lens or plano-convex lens having a different refractive index with the gradient index lens 33, the working distance, the numerical aperture, and the magnification can be adjusted as desired.

  Note that the GRIN lens and the plano-convex lens 36 are also made of glass or plastic which is a non-conductive material, and the adhesive is also a non-conductive material.

<Embodiment 3>
6 is a cross-sectional view of the fiber probe according to the present embodiment (a cross-sectional view corresponding to the cross-sectional view taken along the line AA ′ of FIG. 2), and FIG. 7 is a cross-sectional view taken along the line CC ′ of FIG. is there. In addition, the same code | symbol is attached | subjected to the same thing as Embodiment 1 and Embodiment 2, and the overlapping description is abbreviate | omitted.

  The fiber probe 30 according to the present embodiment includes a tube 37 that covers the outside thereof.

  The tube 37 is made of a non-conductive, non-magnetic and biocompatible material. Biocompatibility means that even when it comes into contact with cells in a living body, it does not show cytotoxicity (cell death or growth inhibition) and has very little interaction and interference with cells. As an example of a biocompatible material, ceramic such as alumina, plastic, or the like can be used. In particular, the tube 37 is preferably formed from a heat-shrinkable material.

  Such a tube 37 covers the outside of the fiber probe 30. Specifically, a part of the plano-convex lens 36 is exposed in the tube 37 and covers the sleeve 34, the adhesive 35, the entire lens 33, and a part of the bundle fiber 31.

  Thus, since the outside of the fiber probe 30 is covered with the tube 37 made of a biocompatible material, the fiber probe 30 can be used in a living body for a long period of time. Thereby, the fluorescence dynamics of the cell in the living body of the subject 60 exposed to the electric field, the magnetic field, or the electromagnetic wave can be detected in real time.

  Note that the tube 37 is not necessarily required if the fiber probe 30 itself is formed from a biocompatible material. That is, each member constituting the fiber probe 30 may be formed of a nonconductive, nonmagnetic, and biocompatible material. Even the fiber probe 30 having such a configuration that does not use the tube 37 can be used in a living body for a long period of time.

<Other embodiments>
The fluorescence observation system 1 according to the first and second embodiments may further include a manipulator for holding the lens unit 32 or adjusting the position of the lens unit 32. Since such a manipulator is disposed near the lens unit 32, the manipulator is exposed to an electric field, a magnetic field, and an electromagnetic wave, and can be a source of current, heat, vibration, and the like. Therefore, for such a manipulator, it is preferable to form a portion exposed to an electric field, a magnetic field or an electromagnetic wave with a non-conductive material.

  Further, in the fluorescence observation system 1 according to the first and second embodiments, the excitation light is guided to the fiber probe 30 and applied to the subject 60, but is not limited to such a configuration. For example, the subject may be configured to irradiate the subject with excitation light directly from the excitation light source device 20. In this case, the excitation light source device 20 is preferably separated to such an extent that it is not affected by the electric field, magnetic field or electromagnetic wave. Further, the excitation light from the excitation light source device 20 may be guided by an optical fiber cable or the like different from the fiber probe 30 to irradiate the subject. Such a portion of the optical fiber on the side of the subject 60 is preferably made of only a non-conductive material because it can be exposed to an electric field, a magnetic field, or an electromagnetic wave and become a source of current, heat, vibration, and the like.

  INDUSTRIAL APPLICABILITY The present invention can be used in the industrial field where fluorescence observation is performed on a state of a cell such as a cultured cell or a cell in a living body when a physical stimulus is applied by an electric field, a magnetic field, or an electromagnetic wave.

DESCRIPTION OF SYMBOLS 1 Fluorescence observation system 10 Electromagnetic field generator 20 Excitation light source device 30 Fiber probe 31 Bundle fiber 33 Lens 34 Sleeve 35 Adhesive 37 Tube 40 Imaging device 50 Analyzing device 60 Subject 70 Dichroic mirror 71 Objective lens

Claims (4)

An electromagnetic field generator for generating an electric field, a magnetic field or an electromagnetic wave;
An excitation light source device that emits excitation light;
A fiber probe that transmits fluorescence emitted by irradiating the subject exposed to the electric field, magnetic field, or electromagnetic wave generated by the electromagnetic field generator with excitation light from the excitation light source device;
An imaging means for imaging fluorescence condensed by the fiber probe;
The fiber probe is
Formed of non-conductive and non-magnetic material ,
A bundle fiber configured by bundling a plurality of optical fibers;
A lens adhered and fixed with a non-conductive adhesive to one end face of the bundle fiber;
Covers the outside of the lens and the bundle fiber and is formed of a non-conductive and non-magnetic material
With a sleeve formed,
The fluorescence observation system , wherein the lens is a combination of lenses having different refractive indexes . In the fluorescence observation system according to claim 1,
The fluorescence probe system is characterized in that the fiber probe is made of ceramic or plastic which is a non-conductive, non-magnetic and biocompatible material . In the fluorescence observation system according to claim 1 or 2 ,
A fluorescence observation system comprising: a tube made of ceramic or plastic that is a non-conductive, non-magnetic, and biocompatible material that covers the outside of the fiber probe. In the fluorescence observation system according to any one of claims 1 to 3 ,
A dichroic mirror that transmits one of excitation light and fluorescence and reflects the other,
The excitation light source device makes excitation light incident on the fiber probe via the dichroic mirror,
The fluorescence observation system, wherein the fluorescence incident on the fiber probe is configured to be imaged by the imaging means via the dichroic mirror.

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