Portable total internal reflection fluorescence microscope using surface plasmon resonance

Abstract

The present invention relates to a portable total reflection fluorescence microscope using surface plasmon resonance, and more specifically, to a portable total reflection fluorescence microscope that can obtain images with a higher contrast ratio than conventional fluorescence microscopes by utilizing the attenuated total reflection phenomenon, can reduce manufacturing costs by fabricating it using a 3D printer, and is easy to carry.

Description

Technology Field

[0001] The present invention relates to a total reflection fluorescence microscope, specifically a total reflection fluorescence microscope utilizing surface plasmon resonance, and more specifically, to a portable total reflection fluorescence microscope that can obtain images with a higher contrast ratio than conventional fluorescence microscopes by utilizing the attenuated total reflection phenomenon, can reduce manufacturing costs by fabricating it using a 3D printer, and is easy to carry. Background Technology

[0002] Total Internal Reflection Fluorescence Microscope (TIRF) is a micro-fluorescence observation instrument that selectively observes only thin regions near the surface by utilizing the annihilation waves generated when light undergoes total internal reflection. Such TIRF microscopes are utilized in various fields such as medicine, biology, and physics, and various studies on fluorescence microscopy are being conducted due to their advantage of being able to image biological tissues and single-cell semiconductor defects in real time.

[0003] However, conventional total reflection fluorescence microscopy technology has the problem that the imaging system is heavy and complex, and requires expensive equipment. The problem to be solved

[0004] The present invention aims to solve the above-mentioned problems by providing a portable total reflection fluorescence microscope that can obtain images with a higher contrast ratio than conventional fluorescence microscopes by utilizing the attenuated total reflection phenomenon, can reduce manufacturing costs by fabricating it using a 3D printer, and is easy to carry. means of solving the problem

[0005] According to one embodiment of the present invention for achieving the above-mentioned purpose, a total reflection fluorescence microscope is provided, comprising: a base; a sample portion including a prism provided on the base and in contact with a sample; a light irradiation portion that irradiates light toward the sample portion; a fluorescence imaging portion provided to acquire a fluorescence image of the sample when light is irradiated from the light irradiation portion; and a spectrum measurement portion provided to acquire fluorescence spectrum data of the sample when light is irradiated from the light irradiation portion. The prism includes an incident surface where light irradiated from the light irradiation portion is incident, a reflective surface provided for the sample to be positioned, and an exit surface where light reflected from the reflective surface is emitted. The sample portion is disposed between the reflective surface of the prism and the sample and includes a thin film that generates surface plasmon resonance when light irradiated from the light irradiation portion is incident, and the thin film generates surface plasmon resonance when light is incident at an attenuated total reflection angle.

[0006] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that a light irradiation unit is positioned to face the incident surface of the prism, a fluorescence imaging unit is positioned to face the reflective surface of the prism, and a spectrum measuring unit is positioned to face the exit surface of the prism.

[0007] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that a light irradiation unit is mounted at a fixed position on a base, and a fluorescence imaging unit and a spectrum measurement unit are each provided to be movable on the base.

[0008] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that the base includes a rail for guiding the positional movement of a fluorescence imaging unit and a spectrum measuring unit, and includes a cart provided so that the fluorescence imaging unit and the spectrum measuring unit can each move along the rail.

[0009] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that the rail has an arc shape and a prism is rotatably mounted on the sample portion.

[0010] In addition, a light irradiation unit according to one aspect of the present invention is characterized by comprising an optical fiber connected to an external light source, a polarizing member that determines the polarization component of light irradiated from the light source, and a first focus adjustment member disposed such that the focus of the light polarized by the polarizing member is formed on a sample.

[0011] In addition, a light source according to one aspect of the present invention is characterized by being connected to the optical fiber so as to be interchangeable.

[0012] In addition, a light source according to one aspect of the present invention is characterized by comprising at least one of a tungsten-halogen lamp and a small laser.

[0013] In addition, a sample portion according to one aspect of the present invention is characterized by being rotatable about a central axis and configured to be vertically movable along the direction of the central axis.

[0014] In addition, a sample section according to one aspect of the present invention is characterized by further including an XYZ stage capable of moving in each of the XYZ directions and a rotation stage capable of rotating the prism to adjust the angle of incidence of light irradiated from the light irradiation section.

[0015] In addition, a fluorescence imaging unit according to one aspect of the present invention comprises a housing, a first objective lens and a second objective lens disposed inside the housing and having different magnifications, a filter member disposed between the first objective lens and the second objective lens and passing light of a specific wavelength range, and a camera that generates an image of a sample using fluorescence emitted from the sample.

[0016] In addition, a filter member according to one aspect of the present invention is arranged to be movable in the direction of a first objective lens and a second objective lens, and is characterized by further including a convex lens.

[0017] In addition, a spectrum measuring unit according to one aspect of the present invention is characterized by comprising an aperture for controlling the amount of light incident on the spectrum measuring unit, a second focusing member and a third focusing member for controlling the focus of light passing through the aperture, an optical fiber connected to an external spectrometer, and an XYZ stage capable of moving in the XYZ direction, respectively.

[0018] In addition, a thin film according to one aspect of the present invention is characterized by being composed of any one of a metal, an organic material, or an organic-inorganic compound.

[0019] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that the cart and rail are formed using a 3D printer.

[0020] In addition, a total reflection fluorescence microscope according to one aspect of the present invention is characterized in that the housing is formed using a 3D printer. Effects of the invention

[0021] According to one embodiment of the present invention, a portable total reflection fluorescence microscope can be provided that is easy to carry and can obtain an image with a higher contrast ratio than a conventional fluorescence microscope by utilizing the attenuated total reflection phenomenon, and can reduce manufacturing costs by manufacturing it using a 3D printer. Brief explanation of the drawing

[0022] FIG. 1 is a perspective view showing a total reflection fluorescence microscope according to one embodiment of the present invention. FIG. 2 is a perspective view showing a light irradiation unit according to one embodiment of the present invention. FIG. 3 is a perspective view showing a first focus adjustment member according to one embodiment of the present invention. FIG. 4 is an exploded view showing a part of a light irradiation unit according to one embodiment of the present invention. FIG. 5 is a perspective view showing a sample portion according to one embodiment of the present invention. FIG. 6 is an enlarged view of a sample attached to a prism according to one embodiment of the present invention. FIG. 7 is a perspective view showing a fluorescence imaging unit according to one embodiment of the present invention. FIG. 8 is a diagram showing the positional relationship inside the housing of a fluorescence imaging unit according to one embodiment of the present invention. FIG. 9 is a perspective view showing a spectrum measuring unit according to one embodiment of the present invention. FIG. 10 is a block diagram showing a total reflection fluorescence microscope according to one embodiment of the present invention. Specific details for implementing the invention

[0023] Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the attached drawings.

[0024] In addition, identical or corresponding components are assigned the same or similar reference numbers regardless of drawing symbols, and redundant descriptions thereof are omitted; furthermore, for the convenience of explanation, the size and shape of each illustrated component may be exaggerated or reduced.

[0025] FIG. 1 is a perspective view showing a total reflection fluorescence microscope according to one embodiment of the present invention.

[0026] Referring to FIG. 1, a total reflection fluorescence microscope (10) according to one embodiment of the present invention may include a base (100), a sample section (200) including a prism (210) provided on the base (100) and in contact with a sample (S), a light irradiation section (300) that irradiates light toward the sample section (200), a fluorescence imaging section (400) provided to acquire a fluorescence image of the sample (S) when light is irradiated from the light irradiation section (300), and a spectrum measurement section (500) provided to acquire fluorescence spectrum data of the sample (S) when light is irradiated from the light irradiation section (300).

[0027] The base (100) may have various shapes, but preferably may be provided in the shape of a disc. As a result, the upper surface of the base (100) can accommodate an arc-shaped rail (110) to be described later, while minimizing weight and size.

[0028] Meanwhile, the prism (210) may include an incident surface (211) into which light irradiated from the light irradiation unit (300) is incident, a reflective surface (212) provided for the sample (S) to be positioned, and an output surface (213) into which light reflected from the reflective surface (212) is emitted.

[0029] At this time, the sample section (200) is positioned between the reflective surface (212) of the prism (210) and the sample (S), and may include a thin film (214) that generates surface plasmon resonance when light irradiated from the light irradiation section (300) is incident.

[0030] At this time, the thin film (214) can generate surface plasmon resonance when light is incident at an attenuated total reflection angle.

[0031] Additionally, the light irradiation unit (300) of the total reflection fluorescence microscope (10) may be positioned to face the incident surface (211) of the prism (210), the fluorescence imaging unit (400) may be positioned to face the reflection surface (212) of the prism (210), and the spectrum measurement unit (500) may be positioned to face the exit surface (213) of the prism (210).

[0032] At this time, the light irradiation unit (300) can be mounted at a fixed position on the base (100).

[0033] In addition, to adjust the angle of incidence of light incident on the sample (S), the sample section (200) may be mounted so that the prism (210) can rotate.

[0034] Additionally, as the prism (210) rotates, the fluorescence imaging unit (400) and the spectrum measuring unit (500) can each be positioned so as to be moved on the base (100).

[0035] Specifically, the sample section (200) can be rotated about a central axis and can be raised and lowered along the direction of the central axis.

[0036] In addition, to receive fluorescence emitted from the sample (S), the fluorescence imaging unit (400) may be configured to adjust the angle with the reflective surface (212) of the prism (210) through positional movement.

[0037] Additionally, in order to receive light reflected from the reflective surface (213) of the prism (210), the spectrum measuring unit (500) may be configured to adjust the angle with the emission surface (214) of the prism (210) through positional movement.

[0038] At this time, the base (100) may include a rail (110) for guiding the positional movement of the fluorescence imaging unit (400) and the spectrum measurement unit (500).

[0039] Additionally, the fluorescence imaging unit (400) and the spectrum measurement unit (500) may each include a cart (460, 560) provided to move along the rail (110).

[0040] At this time, the rail (110) may have an arc shape centered on the sample section (200).

[0041] Additionally, a scale indicating an angle may be drawn along the circumference on the upper surface of the rail (110), and the user can check the rotation angle of the cart (460, 560) through the scale. At this time, the scale may be drawn on the upper surface of the base (100) rather than the upper surface of the rail (110).

[0042] Meanwhile, the light irradiation unit (300) may be positioned on one side of the base (100), but in a direction where the rail (110) is not positioned.

[0043] In other words, the light irradiation unit (300) can be placed in the opening of the arc-shaped rail (110).

[0044] FIG. 2 is a perspective view showing a light irradiation unit according to one embodiment of the present invention, FIG. 3 is a perspective view showing a first focus adjustment member according to one embodiment of the present invention, and FIG. 4 is an exploded view showing a part of the light irradiation unit according to one embodiment of the present invention.

[0045] Referring to FIGS. 2 to 4, a light irradiation unit (300) according to one embodiment of the present invention may include an optical fiber (310) connected to an external light source (30), a polarizing member (320) that determines the polarization component of light irradiated from the light source (30), and a first focus adjustment member (330) that is positioned so that the focus of the light polarized by the polarizing member (320) is formed on a sample (S).

[0046] The optical fiber (310) can transmit light irradiated from an external light source (30) into the light irradiation unit (300).

[0047] Additionally, the light source (30) can be connected to the optical fiber (310) so as to be interchangeable.

[0048] In this way, by using an optical fiber (310) instead of directly mounting the light source (30) to the light irradiation unit (300), the user can easily replace the light source (30) while maintaining the angle of the light irradiation unit (300).

[0049] As a result, when the light source (30) is replaced, the angle of incidence of light can be prevented from being unintentionally changed by the movement of the light irradiation unit (300).

[0050] Meanwhile, the total reflection fluorescence microscope (10) according to an embodiment of the present invention is a device for analyzing a fluorescence image of a sample (S) by fluorescence emitted by surface plasmon resonance and simultaneously analyzing the spectrum of attenuated total reflection light, and the light source (30) required for imaging and spectrum analysis of the sample (S) is different.

[0051] Accordingly, the light source (30) combined with the optical fiber (310) may be provided in multiple numbers.

[0052] At this time, the light source (30) may include at least one of a tungsten-halogen lamp and a small laser.

[0053] Specifically, the tungsten-halogen lamp may have a wavelength range of 210 to 2500 nm. In this case, the tungsten-halogen lamp is used for spectral analysis of the sample (S).

[0054] In addition, the small laser can be replaced depending on the sample (S).

[0055] Specifically, the user may select a small laser of a wavelength such that the light absorption rate of the sample (S) is 10% or more, and the small laser may have a wavelength range of 210 to 2500 nm. At this time, the small laser is used for imaging the sample (S).

[0056] The polarizing member (320) can determine the polarization direction of light irradiated from the light source (30). Specifically, the polarizing member (320) can transmit only the light in a p-polarized state among the light irradiated from the light source (30).

[0057] At this time, the light in the P-polarized state is in a parallel state where the direction of the electric field of the light is aligned parallel to the plane of incidence, and as a result, surface plasmon resonance can be efficiently generated at the interface between the thin film (214) and the medium. Here, the medium is a material in contact with the thin film (214) where the sample (S) is located, and refers to air.

[0058] Meanwhile, as illustrated in FIG. 3, the first focus adjustment member (330) of the light irradiation unit (300) may include a convex lens (331) and a mount (371) having a lens-shaped hole formed in the center so as to be coupled with the lens.

[0059] At this time, the mount (371) may include a fixing groove (371a) for fixing the combined lens and a coupling groove (371b) for fixing the mount (371) to a post (374) to be described later.

[0060] Additionally, the mount may be combined with a polarizing plate or filter instead of a lens in the center, and the polarizing plate and filter combined with the mount in this way may be used as a polarizing member (320) and a filter member (440) to be described later.

[0061] Meanwhile, as shown in FIG. 4, the optical fiber (310), polarizing member (320), and first focus adjustment member (330) of the light irradiation unit (300) can be fixed by a minipost (372) and a collar (373).

[0062] At this time, the collar (373) can adjust and fix the positions of the optical fiber (310), the polarizing member (320), and the first focus adjustment member (330).

[0063] FIG. 5 is a perspective view showing a sample portion according to one embodiment of the present invention, and FIG. 6 is an enlarged view showing a sample attached to a prism according to one embodiment of the present invention.

[0064] Referring to FIG. 5 and 6, a sample section (200) according to one embodiment of the present invention may further include an XYZ stage (220) that can move in each of the XYZ directions and a rotation stage (230) that can adjust the angle of incidence of light irradiated from the light irradiation section (300) by rotating a prism (210).

[0065] The prism (210) may include an incident surface (211) into which light irradiated from the light irradiation unit (300) is incident, a reflective surface (212) provided to be in contact with the sample (S), and an exit surface (213) into which light reflected from the reflective surface (212) is emitted.

[0066] At this time, the prism (210) may include a thin film (214) attached to a reflective surface (212) and disposed between the prism (210) and the sample (S), and the thin film (214) may generate surface plasmon resonance.

[0067] In addition, the thin film (214) can be made of any one of a metal, an organic material, or an organic-inorganic compound, and can satisfy the following mathematical formula 1.

[0068] [Mathematical Formula 1]

[0069]

[0070] At this time, Re(ε m ) represents the real part permittivity of the thin film (214), and Re(ε d ) represents the real part permittivity of the insulator surrounding the sample (214). Here, the insulator surrounding the sample (S) is a medium that is in contact with the thin film (214) and surrounds the sample (S), and refers to air, but is not limited thereto.

[0071] Meanwhile, the rotation stage (230) combined with the post (274) may be provided in a circular shape and may have a scale indicating an angle drawn along the circumference. The user can rotate the prism (210) while checking the angle with the scale, thereby adjusting the angle of incidence of light incident on the incident surface (211).

[0072] Additionally, the XYZ stage (220) positioned between the post (274) and the base (100) can adjust the position of the prism (210), thereby allowing the focus to be shifted by adjusting the distance between the prism (210) and the first objective lens (420) to be described later.

[0073] FIG. 7 is a perspective view showing a fluorescence imaging unit according to one embodiment of the present invention, and FIG. 8 is a diagram showing the positional relationship inside the housing of the fluorescence imaging unit according to one embodiment of the present invention.

[0074] Referring to FIGS. 7 and 8, a fluorescence imaging unit (400) according to one embodiment of the present invention may include a housing (410), a first objective lens (420) and a second objective lens (430) disposed inside the housing (410) and having different magnifications, a filter member (440) disposed between the first objective lens (420) and the second objective lens (430) and passing light of a specific wavelength range, and a camera (450) that generates an image of the sample (S) using fluorescence emitted from the sample (S).

[0075] At this time, the camera (450) may be an EMCCD (Electron Multiplying Charge-Coupled Device) or an sCMOS (Scientific Complementary Metal-Oxide-Semiconductor).

[0076] The filter member (440) may be positioned to be movable in the direction of the first objective lens (420) and the second objective lens (430), and may further include a convex lens.

[0077] At this time, the filter member (440) can pass only light of a specific wavelength, thereby blocking the excitation light source reflected from the sample (S) and transmitting only the fluorescence signal generated from the sample (S) to the camera (450).

[0078] In addition, by moving the filter member (440) including the convex lens, the magnification ratio of the image can be adjusted with the camera (450) and the focus can be adjusted.

[0079] With the above configuration, the fluorescence imaging unit (400) can perform real-time imaging of the sample (S).

[0080] FIG. 9 is a perspective view showing a spectrum measuring unit according to one embodiment of the present invention.

[0081] Referring to FIG. 9, a spectrum measuring unit (500) according to one embodiment of the present invention may include an aperture (510) for controlling the amount of light incident on the spectrum measuring unit (500), a second focusing lens (520) and a third focusing lens (530) for controlling the focus of light passing through the aperture (510), an optical fiber (540) connected to an external spectrometer (50), and an XYZ stage (550) capable of moving in the XYZ direction.

[0082] The aperture (510) blocks edge signals among the diffused light due to light dispersion and transmits only the center signal to the optical fiber (540), thereby improving angular resolution.

[0083] With the above configuration, the spectrum measuring unit (500) can determine the composition and properties of the material within the sample (S) by analyzing the optical properties of the sample (S) by analyzing the spectrum of light totally reflected from the sample (S).

[0084] Meanwhile, the total reflection fluorescence microscope (10) according to one embodiment of the present invention can be manufactured using 3D printing.

[0085] Specifically, the rail (110), cart (460, 560), and housing (410) can be formed using a 3D printer. Additionally, the mount, minipost, collar, and post (274, 374, 474, 574), which each constitute the sample section (200), light irradiation section (300), fluorescence imaging section (400), and spectrum measurement section (500), can also be formed using a 3D printer.

[0086] As a result, the total reflection fluorescence microscope (10) according to one embodiment of the present invention can reduce manufacturing costs compared to conventional total reflection fluorescence microscopes and reduce weight to increase portability.

[0087] FIG. 10 is a block diagram showing a total reflection fluorescence microscope according to one embodiment of the present invention.

[0088] Referring to FIG. 10, a total reflection fluorescence microscope (10) according to one embodiment of the present invention may include a control unit (600). The control unit (600) may be connected to a sample unit (200), a light irradiation unit (300), a fluorescence imaging unit (400), and a spectrum measurement unit (500).

[0089] At this time, the control unit (600) can control the movement and rotation of the sample unit (200), light irradiation unit (300), fluorescence imaging unit (400), and spectrum measurement unit (500).

[0090] Additionally, the control unit (600) can display to the user whether the light source (30) and the spectrometer (50) are connected, and can display to the user the type of the connected light source (30).

[0091] The preferred embodiments of the present invention described above are disclosed for illustrative purposes only, and various modifications and variations of the technical concept of the present invention are possible by those skilled in the art to which the present invention pertains, and such modifications and variations will fall within the scope of protection of the present invention. Explanation of the symbols

[0092] 10: Total Internal Reflection Fluorescence Microscope 30: Light source 50: Spectrometer 100: Bass 110: Rail 200: Sample section 210: Prism 214: Thin film 220: XYZ Stage 230: Rotation Stage 300: Light Investigation Department 310: Optical fiber 320: Polarizing section 330: First focusing member 400: Fluorescence Imaging Section 410: Housing 420: First objective lens 430: Second objective lens 440: Filter element 450: Camera 460: Kart 500: Spectrum measurement unit 510: Aperture 520: Second focus adjustment member 530: Third focusing member 540: Optical fiber 550: XYZ Stage 560: Kart 600: Control unit S: Sample

Claims

Claim 1 A base; a sample section provided on the base and including a prism in contact with a sample; a light irradiation section that irradiates light toward the sample section; a fluorescence imaging section provided to acquire a fluorescence image of the sample when light is irradiated from the light irradiation section; and a spectrum measurement section provided to acquire fluorescence spectrum data of the sample when light is irradiated from the light irradiation section; wherein the prism includes an incident surface where light irradiated from the light irradiation section is incident, a reflective surface provided for the sample to be positioned, and an exit surface where light reflected from the reflective surface is emitted; wherein the sample section is disposed between the reflective surface of the prism and the sample and includes a thin film that generates surface plasmon resonance when light irradiated from the light irradiation section is incident, and wherein the thin film generates surface plasmon resonance when light is incident at an attenuated total reflection angle; and wherein the fluorescence imaging section comprises: a housing; a first objective lens and a second objective lens disposed inside the housing and having different magnifications; and a filter member disposed between the first objective lens and the second objective lens and passing light of a specific wavelength range. A total reflection fluorescence microscope comprising a camera that generates an image of a sample using fluorescence emitted from the sample, wherein the filter member is arranged to be movable in the direction of a first objective lens and a second objective lens, and further comprising a convex lens. Claim 2 A total reflection fluorescence microscope according to claim 1, wherein the light irradiation unit is positioned to face the incident surface of the prism, the fluorescence imaging unit is positioned to face the reflective surface of the prism, and the spectrum measuring unit is positioned to face the exit surface of the prism. Claim 3 In paragraph 2, the light irradiation unit is mounted at a fixed position on a base, and the fluorescence imaging unit and spectrum measurement unit are each provided to be movable on the base, forming a total reflection fluorescence microscope. Claim 4 In paragraph 3, the base includes a rail for guiding the positional movement of the fluorescence imaging unit and the spectrum measuring unit, and the fluorescence imaging unit and the spectrum measuring unit each include a cart provided to move along the rail, in a total reflection fluorescence microscope. Claim 5 In paragraph 4, the rail has an arc shape, and the sample portion is a total reflection fluorescence microscope in which a prism is mounted so as to be rotatable. Claim 6 The total reflection fluorescence microscope according to claim 1, wherein the light irradiation unit comprises: an optical fiber connected to an external light source; a polarizing member that determines the polarization component of light irradiated from the light source; and a first focusing member positioned so that the focus of the light polarized by the polarizing member is formed on a sample. Claim 7 In paragraph 6, the light source is a total reflection fluorescence microscope that is interchangeably connected to the optical fiber. Claim 8 In claim 6, the light source comprises at least one of a tungsten-halogen lamp and a small laser, in a total reflection fluorescence microscope. Claim 9 In claim 1, the sample portion is configured to be rotatable about a central axis and to be vertically movable along the direction of the central axis, forming a total reflection fluorescence microscope. Claim 10 In claim 9, the total reflection fluorescence microscope further comprises: an XYZ stage capable of moving the sample section in each of the XYZ directions; and a rotation stage capable of adjusting the angle of incidence of light irradiated from the light irradiation section by rotating the prism. Claim 11 delete Claim 12 delete Claim 13 The total reflection fluorescence microscope according to claim 1, wherein the spectrum measuring unit comprises: an aperture for controlling the amount of light incident on the spectrum measuring unit; a second focusing member and a third focusing member for controlling the focus of light passing through the aperture; an optical fiber connected to an external spectrometer; and XYZ stages capable of moving in each of the XYZ directions. Claim 14 The total internal reflection fluorescence microscope according to claim 1, wherein the thin film is composed of any one of a metal, an organic material, or an organic-inorganic compound, satisfying the following mathematical formula 1. [Mathematical Formula 1] In the above mathematical formula 1, Re(ε m ) is the real part permittivity of the thin film, and Re(ε d ) is the real part permittivity of the insulator surrounding the sample. Claim 15 In paragraph 4, the cart and the rail are total reflection fluorescence microscopes formed using a 3D printer. Claim 16 In claim 1, the housing is a total reflection fluorescence microscope formed using a 3D printer.

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