JP3577514B2 - Total reflection fluorescence microscope - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fluorescence microscope, and more particularly to a total reflection fluorescence microscope capable of observing the direction of one fluorescent dye molecule.
[0002]
[Prior art]
Fluorescent microscopes use a dye that emits light of a longer wavelength than that of a specific color (light of a specific wavelength) when irradiated with excitation light to illuminate the fluorescent dye. It is a combination of an optical system and an optical microscope for observing the fluorescence generated thereby. When a reagent bound to a fluorescent dye is bound to the structure in the cell to be observed and a specific light is applied to the fluorescent dye molecule, the target intracellular structure glows against a dark background. Fluorescence microscopy is a method of applying and observing such a fluorescent dye for staining a microscope specimen.
[0003]
The number of fluorescent dye molecules that can be observed with a general fluorescent microscope is several tens or more, and one fluorescent dye molecule cannot be observed. This is because one fluorescent dye molecule cannot be identified because noise, that is, a signal of light from the surroundings is larger than a signal coming from one fluorescent dye molecule. However, improvements have been made to improve the performance, and a fluorescence microscope capable of visualizing one fluorescent dye molecule has been developed due to the properties of the filter and the quality of the objective lens.
[0004]
In recent years, in order to observe one fluorescent dye molecule, as shown in FIG. 4, a laser light source was used to apply an angle greater than the total reflection angle from the glass 27 side to the sample on the interface between the aqueous solution and the glass 27. A method of irradiating the sample with a laser beam 28 and illuminating the sample with an evanescent field 29 which is non-propagating light generated near the boundary surface (hereinafter, referred to as total reflection illumination) is used. That is, this is a method utilizing the fact that the fluorescent dye molecules emit fluorescence by illumination with an evanescent field.
[0005]
As shown in FIG. 4, the evanescent field 29 attenuates exponentially in a direction perpendicular to the boundary surface, and its attenuation constant depends on the refractive index and the incident angle of the laser beam. Therefore, since the evanescent field illuminates only a local region having a depth of about 150 nm in the aqueous solution from the boundary surface, the total reflection illumination has an extremely small amount of background light as compared with illumination by ordinary light.
[0006]
Furthermore, even under conditions where a large number of fluorescent dye molecules (concentration 〜50 × 10 −9 mol / liter) are present in the aqueous solution, the probability that the fluorescent dye molecules are present on the aqueous solution side near the interface is small. There is little fluorescence emitted from other than one target fluorescent dye molecule fixed on the boundary. Therefore, since noise due to the background light and the fluorescence of the other fluorescent dye molecules is extremely small, it is possible to observe the fluorescence from the single target fluorescent molecule.
[0007]
In the observation of one molecule by the total reflection illumination, for example, a biomolecule such as a protein, DNA, or a substrate, ATP, which is targeted by a fluorescent dye, is bonded to a glass surface, and each molecule is observed as an independent bright spot. I do. When exciting the fluorescent dye, it is necessary that the vibration plane of the dye and the polarization direction of the excitation light match.
[0008]
[Problems to be solved by the invention]
However, in the conventional total reflection illumination, molecules in which the vibration plane of the dye and the polarization direction of the excitation light coincide with each other can be observed brightly, but molecules that do not coincide with each other are dark and cannot be observed, that is, with respect to the polarization direction of the excitation light. In addition, there is a problem that a molecule whose vibration surface targets a dye having an arbitrary direction cannot be observed.
[0009]
In addition, a specific polarization direction does not coincide with the vibration plane of the dye, and observation cannot be performed. However, in this case, the direction of the target dye is unknown. SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional problems, and an object of the present invention is to provide a total reflection fluorescent microscope capable of observing a molecule having a vibrating surface targeting a dye having an arbitrary direction.
[0010]
[Means for Solving the Problems]
Therefore, in order to solve the above-mentioned problem, the present invention relates to an invention according to claim 1, wherein the laser light linearly provided on the optical axis of the laser light so as to be rotatable around the optical axis of the laser light as a rotation axis. A polarization conversion member that converts the light into polarized light, a deflection member that changes the traveling direction of the laser light that is provided rotatably in synchronization with the polarization conversion member with the optical axis as a rotation axis, a condenser lens, and an objective lens. Wherein the polarization conversion member, the deflection member, the condenser lens, the objective lens, and the laser beam total reflection surface are arranged in this order.
The invention according to claim 2 is a laser light source of circularly polarized laser light, and the laser light that is rotatably provided on the optical axis of the laser light around the optical axis of the laser light as a rotation axis is directly polarized. A polarization conversion member for converting, a deflection member for changing the traveling direction of the laser light provided rotatably in synchronization with the polarization conversion member with the optical axis as a rotation axis, a condenser lens, and an objective lens. The laser light source, the polarization conversion member, the polarization member, the condenser lens, the objective lens, and the laser light reflecting surface are arranged in this order, and the rear focal plane of the objective lens is a focal point of the condenser lens. A total reflection surface at a position of a focal plane of the objective lens.
The invention according to claim 3 is the total reflection fluorescence microscope according to claim 1 or 2, wherein the rotation of the polarization conversion member forms a donut-shaped total reflection area in the condenser lens, and Provided is a total reflection fluorescence microscope characterized in that the light can pass through a total reflection area formed on a lens and can be totally reflected on a total reflection surface.
The invention according to claim 4 is the total reflection fluorescence microscope according to claim 3, wherein the polarization direction of the laser light in the evanescent field of the total reflection surface is rotatable. I will provide a.
The invention according to claim 5 provides the total reflection fluorescence microscope according to any one of claims 1 to 4, wherein the polarization conversion member is a quarter-wave plate.
Here, examples of the polarization conversion member include those that convert circularly polarized light into linearly polarized light, those that convert arbitrary linearly polarized light into specific linearly polarized light, and those that convert random polarized light into specific polarized light. In addition, the polarization conversion member is provided with a function of automatically rotating the optical axis of the laser beam as an axis, and not only converts circularly polarized light into linearly polarized light, but also rotates the direction of the linearly polarized light by 360 ° at the same time. Can change. Further, the deflecting member refers to a prism or the like, and can change the irradiation direction of the laser light. By rotating the polarization member and the polarization conversion member in synchronization, it is possible to capture a direction in which the fluorescent dye cannot be observed depending on the specific polarization direction and irradiation direction.
[0011]
Further, the laser light from the laser light source may be any of circularly polarized light, linearly polarized light and random polarized light. The wavelength of the laser light is set to an appropriate wavelength depending on the desired fluorescent dye.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. 1A and 1B show an embodiment of a total reflection fluorescence microscope according to the present invention, wherein FIG. 1A is a configuration diagram, and FIG. 1B is a diagram showing a laser beam total reflection region.
[0013]
As shown in FIG. 1A, a total reflection fluorescence microscope 1 according to an embodiment of the present invention includes a laser light source of a circularly polarized laser beam 2 and a laser beam 2 rotatably provided around an optical axis 3 as a rotation axis.波長 wavelength plate 4 as a polarization conversion member for converting into linearly polarized light, and deflection for changing the traveling direction of the laser beam 2 rotatably provided in synchronization with the 波長 wavelength plate 4 with the optical axis 3 as a rotation axis. It is characterized by comprising a prism 5 as a member, a condenser lens 6 with a focal length f1, an objective lens 7 with a focal length f2, and a total reflection surface 8.
[0014]
The prism 5 is located at the rear focal plane of the condenser lens 6, that is, between the quarter-wave plate 4 and the condenser lens 6. The rear focal plane of the objective lens 7 is located at the position of the focal plane 9 of the condenser lens 6. Therefore, the laser beam 2 incident parallel to the prism 5 is incident parallel to the condenser lens 6, is focused on the focal plane 9 of the condenser lens 6, is incident on the objective lens 7, and is parallel to the total reflection plane 8. Incident on. Here, the distance (f1) between the prism 5 and the condenser lens 6 and the distance (f1) between the condenser lens 6 and the focal plane 9 were made equal. Further, the distance (f2) between the focal plane 9 and the objective lens 7 and the distance (f2) between the objective lens 7 and the total reflection surface 8 were made equal. Note that a configuration may be adopted in which the sample to be observed is provided at the position of the total reflection surface 9 to provide a total reflection surface without providing the total reflection surface 8.
[0015]
When the sample is in water, when the numerical aperture (hereinafter, referred to as NA) of the condenser lens 6 is 1.33 or more, total reflection illumination by an evanescent field is performed. In this case, the prism 5 separates the vertically incident laser beam 2 from the objective lens 7 at a distance of f1 to the objective lens 7. A. It is necessary that the prism has an angle such that the optical path changes by a distance corresponding to 1.33 or more. Note that a mirror may be used instead of the prism 5 for deflection.
[0016]
As shown in FIG. 1B, the laser beam 11 incident on the donut-shaped total reflection area 10 of the condenser lens 6 is transmitted through the donut-shaped total reflection area 12 of the objective lens 7 and is reflected on the total reflection surface 8. Totally reflected. The total reflection surface 8 is an interface between a high refractive index material (left side in FIG. 1A) and a low refractive index material (right side in FIG. 1A), such as an interface between glass and water. .
[0017]
When the refractive index of the low-refractive index material is n1 and the refractive index of the high-refractive index material is n2, that is, n1 <n2, the total reflection surface 8, which is the interface between the low-refractive index material and the high-refractive index material, has a high refractive index. Light incident from the refractive index material side at an incident angle equal to or greater than the total reflection angle θ satisfying sin θ = n1 / n2 is totally reflected by the total reflection surface 8. The total reflection angle θ depends on the refractive index of the material forming the total reflection surface.
[0018]
The quarter-wave plate 4 and the prism 5 are provided so as to be rotatable in synchronization with the optical axis 3 of the laser beam 2 as a rotation axis, whereby the laser beam 11 transmitted through the prism 5 also rotates in the polarization direction. While being converged, it is deflected on the condenser lens 6 so as to draw a circle. Therefore, the laser beam 11 can be moved to the donut-shaped total reflection areas 10 and 12 shown in FIG.
[0019]
That is, as shown in FIG. 2A, the laser beam 2 after the circularly polarized laser beam 2 has passed through the quarter-wave plate 4 and the prism 5 becomes, for example, in the direction represented by the linearly polarized beam 14. It is polarized and passes through the circular transmission area 15 on the condenser lens 6. In such a state, for example, when the quarter-wave plate 4 and the prism 5 are synchronized and rotated counterclockwise in the figure with the optical axis 3 as a rotation axis, as shown in FIG. The region 15 rotates in the rotation direction 17, and the polarization direction 16 also rotates synchronously.
[0020]
When the quarter-wave plate 4 and the prism 5 are rotated in synchronization with each other, a method of synchronization may be selected and synchronized so that each of them can obtain a desired rotation. Instead of rotating the quarter-wave plate 4 to change the polarization direction of the linearly-polarized laser light, the polarization direction is changed by using a polarizer or Electro-Optic Modulators, which is an element for electrically controlling the polarization direction. May be changed. Instead of the prism 5, it is also possible to use Acousto-Optical Modulators, which are elements for electrically controlling the direction of light.
[0021]
Next, total reflection of laser light on the total reflection surface 8 will be described. As shown in FIG. 3A, the laser beam 19 incident on the first incident point 18 of the objective lens 7 from below in the figure is totally reflected on the total reflection surface 8 which is the upper surface of the glass 20, and the objective lens 7 At the second incident point 21 of FIG. Although the polarization direction 22 of the linearly polarized laser light at the first incident point 18 is the direction shown in the figure, the quarter-wave plate 4 and the prism 5 are rotated in synchronization with the optical axis 3 as the rotation axis. The laser beam 19 moves to the third incident point 23, and the polarization direction 22 of the laser beam 19 also changes to the direction shown in FIG. Further, the polarization direction 25 of the evanescent field 24 formed on the sample surface which is the total reflection surface 8 rotates in a plane parallel to the sample surface in synchronization with the polarization direction 22 of the laser light 19.
[0022]
That is, the laser light 19 incident on the objective lens 7 rotates on the objective lens 7 and the polarization direction of the laser light 19 also rotates.
[0023]
As described above, in the total reflection fluorescence microscope 1 according to the embodiment of the present invention, the polarization direction 22 of the laser beam 19 and the incident direction of the laser beam 19 on the sample surface which is the total reflection surface 8 can be changed with time. it can. Since the polarization direction 25 of the evanescent field 24 on the sample surface is determined by the polarization direction of the laser beam 19 and the incident direction of the laser beam 19 on the sample surface, when the laser beam 19 rotates as shown in FIG. The polarization direction 25 of the evanescent field 24 on the sample surface rotates in the sample surface with time.
[0024]
The fluorescent dye molecules placed on the sample surface are brightest when the vibration plane coincides with the polarization direction of the evanescent field, and darkest when shifted by 90 degrees. Therefore, among the plurality of fluorescent dye molecules in the evanescent field 24 on the sample surface, the fluorescent dye molecules whose vibration plane coincides with the polarization direction 25 of the evanescent field are observed, and the rotation of the laser light 19 shown in FIG. That is, as the direction of incidence of the laser beam on the sample surface and its polarization direction 22 are rotated, it becomes possible to observe another fluorophore one by one, and determine in which direction the fluorophore is oriented. can do.
[0025]
For example, a fluorescent dye is bound to F1-ATPase, which is a rotating molecular motor, myosin and dynein, and a proteasome, which is a degrading enzyme having a structure common to them, and observed with a total reflection fluorescence microscope 1. In addition, it is possible to elucidate the mechanism by which biological supramolecules, which are a complex of proteins having various functions, operate, and it becomes possible to use various technologies that apply them.
[0026]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the polarization direction of a laser beam and the incident direction of a laser beam with respect to the sample surface which is a total reflection surface can be changed with time.
Therefore, it is possible to detect the direction of the molecules on the sample surface in the sample surface, and it is possible to use a technique using the direction.
[Brief description of the drawings]
FIGS. 1A and 1B show an embodiment of a total reflection fluorescence microscope according to the present invention, wherein FIG. 1A is a configuration diagram and FIG.
FIG. 2 is a diagram for explaining rotation movement and rotation of a polarization direction of a laser beam shown in FIG. 1;
3A and 3B show the generation of an evanescent field by the total reflection fluorescence microscope of FIG. 1, and FIG. 3A shows the polarization direction of the evanescent field with respect to the polarization direction of the laser beam and the incident direction of the laser beam on the sample surface; () Is a diagram showing a change in the polarization direction of the evanescent field when the laser light is rotationally moved and its polarization direction is rotated.
FIG. 4 is an explanatory diagram of generation of an evanescent field.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Total reflection fluorescence microscope 2 Laser light 3 Optical axis 4 1/4 wavelength plate 5 Prism 6 Condensing lens 7 Objective lens 8 Total reflection surface 9 Focal plane 10 Total reflection area 11 Laser light 12 Total reflection area 13 Circularly polarized light 14 Linear Polarization 15 Transmission area 16 Polarization direction 17 Rotation direction 18 First incidence point 19 Laser light 20 Glass 21 Second incidence point 22 Polarization direction 23 Third incidence point 24 Evanescent field 25 Polarization direction 26 Rotation direction 27 Glass 28 Laser light 29 Evanescent field

Claims (5)

On the optical axis of the laser light, a polarization conversion member that converts the laser light, which is rotatably provided with the optical axis of the laser light as a rotation axis, into linearly polarized light,
A deflecting member that changes a traveling direction of the laser light, which is rotatably provided in synchronization with the polarization conversion member with the optical axis as a rotation axis,
A condenser lens,
An objective lens,
With
A total reflection fluorescent microscope , wherein the polarization conversion member, the deflecting member, the condenser lens, the objective lens, and the laser beam are arranged in this order . A laser light source of circularly polarized laser light,
On the optical axis of the laser light, a polarization conversion member that directly converts the laser light, which is rotatably provided with the optical axis of the laser light as a rotation axis, into polarized light,
A deflecting member that changes a traveling direction of the laser light, which is rotatably provided in synchronization with the polarization conversion member with the optical axis as a rotation axis,
A condenser lens,
An objective lens,
With
The laser light source, the polarization conversion member, the polarization member, the condenser lens, the objective lens and the laser light are arranged in the order of the reflection surface,
The rear focal plane of the objective lens is located at the focal plane of the condenser lens,
The said total reflection surface is in the position of the focal plane of the said objective lens, The total reflection type fluorescence microscope characterized by the above-mentioned. 3. The total reflection fluorescence microscope according to claim 1, wherein a rotation of the polarization conversion member forms a donut-shaped total reflection region in the condenser lens, and a total reflection region formed in the objective lens. 4. A total reflection type fluorescence microscope characterized by being capable of transmitting light through and totally reflecting on a total reflection surface . The total reflection fluorescence microscope according to claim 3, wherein the polarization direction of the laser beam in the evanescent field of the total reflection surface is rotatable . 5. The total reflection fluorescence microscope according to claim 1, wherein the polarization conversion member is a quarter-wave plate.

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