JP3671227B2 - Total reflection type fluorescence microscope and illumination optical system - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a total reflection fluorescent microscope that excites a fluorescent dye in an evanescent field.
[Prior art]
The fluorescence microscope is a microscope that observes a sample by exciting a fluorescent dye that emits light having a wavelength longer than the wavelength of the irradiated light and observing the fluorescence.
[0002]
More specifically, a fluorescent dye is first bound to the sample. The fluorescent dye is excited by light having a polarization component in the same direction as the vibration moment and emits fluorescence. A microscope for observing the fluorescence is a fluorescence microscope.
[0003]
Among such fluorescent microscopes, a fluorescent microscope that totally reflects light irradiated on a sample surface is called a total reflection fluorescent microscope. A total reflection type fluorescence microscope, for example, makes light incident on a sample surface at the boundary between an aqueous solution and glass at an angle larger than the critical angle, totally reflects the incident light, and generates an aqueous solution by an evanescent field generated in the vicinity of the sample surface. It is the microscope which illuminates the inside sample.
[0004]
In a conventional total reflection type fluorescence reflection microscope, an evanescent field is created by irradiating a sample surface with laser light from one direction (for example, Non-Patent Document 1). Here, the polarization direction of the evanescent field is determined by the polarization direction of the laser beam and the incident direction of the laser beam on the sample surface, but a conventional total reflection fluorescent reflection microscope in which the sample surface is irradiated with the laser beam from one direction. In this case, it is impossible to excite a dye having a vibration moment in a certain direction to emit fluorescence. For example, when the interface where the laser beam is totally reflected is defined as the xy plane and the light beam of the totally reflected laser beam is on the xz plane, for example, the direction of polarization of the laser beam is polarized in the y-axis direction. The polarization component of the generated evanescent field is only in the y-axis direction. In addition, when the laser light traveling in the xz plane is polarized to an axis that is perpendicular to the traveling direction and is in the xz plane, the polarization component of the generated evanescent field is mainly negligible in the z-axis direction. Only the polarization component in the x-axis direction. That is, regardless of the polarization of the incident laser light, the generated evanescent field mainly includes the polarization components in the y-axis direction and the z-axis direction, and the polarization component in the x-axis direction is Since it is hardly contained, only a fluorescent dye whose vibration moment is directed to the y-axis or z-axis can be sufficiently excited.
[0005]
As a result, in the conventional total reflection type fluorescence reflection microscope in which the sample surface is irradiated with laser light from one direction, the sample cannot be observed depending on the direction of the sample.
[0006]
[Non-Patent Document 1]
Tokunaga, M., Kitamura, K., Saito, K., Iwane, AH, and Yanagida, T. (1997) .Single molecule imaging of fluorophores and biological reactions achieved by objective-type total internal reflection fluorescence microscopy. Research Communications 235, 47-53
[0007]
[Problems to be solved by the invention]
The present invention achieves one or more of the objects described below.
[0008]
An object of the present invention is to observe whether or not a sample exists regardless of the direction of the sample bound to the fluorescent dye.
[0009]
Another object of the present invention is to make it possible to count the number of fluorescent dyes gathered at one place because one molecule of the fluorescent dye can be visualized regardless of the direction of the sample bound to the fluorescent dye. For example, if a biomolecule labeled with a fluorescent dye is observed, it is an object to quantitatively determine information about what unit the biomolecule is associated with. Another object of the present invention is to provide a total reflection fluorescence microscope or illumination optical system capable of quantifying association / separation dynamics by changing the conditions of an aqueous solution while observing the same biomolecule.
[0010]
An object of the present invention is to provide a total reflection fluorescence microscope or illumination optical system that can be observed even when a sample is in water.
Further, the present invention differs from NMR and X-ray analysis after crystallization in that a total reflection fluorescence microscope or illumination optical system capable of analyzing changes in the three-dimensional structure of biomolecules such as proteins, DNA and RNA having activity in aqueous solution The purpose is to provide.
[0011]
[Means for Solving the Problems]
The present invention relates to a total reflection fluorescent microscope for exciting a fluorescent dye in an evanescent field, a light source for emitting light for creating the evanescent field, a diffracting means for diffracting light, a condensing means, and an objective lens And a total reflection fluorescence microscope.
[0012]
The present invention is a total reflection type fluorescence microscope that excites a fluorescent dye in an evanescent field, a light source that emits light for creating the evanescent field, a diffraction unit that diffracts the light emitted from the light source, A first condensing unit that condenses the diffracted light, a second condensing unit that condenses the light that has passed through the first condensing unit, and a sample that is irradiated with light that has passed through the second condensing unit. Provided is a total reflection fluorescent microscope including an objective lens.
[0013]
The present invention also provides an illumination optical system that excites a fluorescent dye in an evanescent field, a light source that emits light for creating the evanescent field, a diffraction unit that diffracts the light, a condensing unit, and an objective lens An illumination optical system is provided.
[0014]
The present invention is an illumination optical system that excites a fluorescent dye in an evanescent field, a light source that emits light for creating the evanescent field, a diffraction unit that diffracts the light emitted from the light source, and the diffracted light A first condensing means for condensing light, a second condensing means for condensing the light transmitted through the first condensing means, and an objective lens for irradiating the sample with the light transmitted through the second condensing means; An illumination optical system is provided.
[0015]
The light emitted from the light source is preferably laser light that is circularly polarized light or random polarized light.
[0016]
Further, it is preferable that the light emitted from the light source is diffracted in an annular shape by the diffraction means.
[0017]
It is preferable to have a mask that blocks a part of the transmitted light so that the light irradiated on the sample has a ring shape with little light leakage.
[0018]
The mask is preferably disposed between the first condenser lens and the second condenser lens.
[0019]
It is preferable that the diffraction means rotates. More preferably, the rotation axis of the rotating diffraction means does not coincide with the axis of light emitted from the light source.
[0020]
Preferably, the diffractive means is a diffractive diffuser, and the diffractive diffuser vibrates while maintaining the direction of the surface.
[0021]
Further, it is preferable that the annular light is incident on the back focal plane of the objective lens, and the principal ray of the annular light travels on the cylindrical surface.
[0022]
It is preferable that the focal point of the light transmitted through the second condenser lens is located on the back focal plane of the objective lens.
[0023]
Here, the annular light travels while having the thickness of the ring, and light having the center of the thickness of the ring as the optical path is called a principal ray. The back focal plane refers to a position where two lights traveling in parallel are collected by the objective lens when they enter the objective lens from the sample surface.
Embodiment
Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.
[0024]
FIG. 1 is a diagram showing an illumination optical system of a total reflection fluorescent microscope according to the present invention.
[0025]
The illumination optical system 100 of the total reflection fluorescent microscope includes, from the left, a laser light source 1, a diffractive diffuser 2 as a diffractive means, a first condensing lens 3 as a first condensing means, a mask 6, The second condensing lens 4 as the second condensing means and the objective lens 5 are included. A sample surface 7 is on the right side of the objective lens 5.
[0026]
FIG. 2 is a diagram showing the configuration of the mask 6. The mask 6 includes an annular slit portion 10 and a light shielding portion 11 shielded by aluminum vapor deposition.
[0027]
In the mask 6, the annular slit portion 10 surrounded by two concentric circles having different diameters transmits light, and the light shielding portion 11 which is the other portion blocks light.
[0028]
Next, the optical path of the laser light emitted from the laser light source 1 and reaching the sample surface 7 in the total reflection type fluorescence microscope will be described.
The light emitted from the laser light source 1 is preferably laser light that is circularly polarized light or random polarized light.
[0029]
Laser light emitted from the laser light source 1 is incident on a diffractive diffuser 2. The laser light incident on the diffraction diffusion plate 2 is diffracted in an annular shape, and the diffracted light spreads in a conical shape. Here, as the diffraction diffusion plate 2, ThorLab D0740A (a 635 nanometer wavelength laser with an opening angle of 10.3 degrees) is used.
[0030]
The diffraction diffusion plate 2 rotates about an axis parallel to the optical axis of the laser light emitted from the laser light source 1. The rotation speed is preferably faster than the time resolution of the apparatus for recording the fluorescence signal. Usually, it is preferably 10 revolutions or more per second. For example, in the case of normal video recording at 33 milliseconds / frame, the rotational speed of the diffraction diffusion plate 2 is preferably 30 revolutions or more per second. When the diffraction diffusion plate 2 is not rotated, the laser beams that have passed through different parts of the condenser lens are overlapped again, so that interference fringes are generated on the sample surface 7 and, as a result, uneven excitation of the fluorescent dye may occur.
[0031]
Therefore, by rotating the diffraction diffusion plate 2, the pattern of the interference fringes can be temporally changed, and a uniform light intensity on the sample surface 7 can be realized. As a result, it is possible to prevent uneven excitation of the fluorescent dye due to the interference fringes.
[0032]
In addition, the rotation axis does not coincide with the axis of light emitted from the laser light source 1. This is because the laser light strikes different portions of the diffraction diffusion plate during one rotation, and thus the interference fringe pattern can be effectively erased. The rotation axis and the light axis are preferably separated from each other within the size range of the diffraction diffusion plate 2. In general, it is preferable that the distance is 2 mm or more. For example, when the size of the diffraction diffusion plate 2 is a disk shape having a diameter of about 5 mm, the distance between the rotation axis of the diffraction diffusion plate and the light axis is preferably 2 to 4 mm.
[0033]
In addition to rotating the diffraction diffusion plate 2 as described above, the excitation unevenness can be prevented by vibrating the diffraction diffusion plate 2 while maintaining the direction of the surface of the diffraction diffusion plate 2. The vibration amplitude is preferably larger within the size range of the diffraction diffusion plate 2. The frequency of the vibration is preferably faster than the time resolution of the apparatus for recording a fluorescence signal.
[0034]
It is preferable that the light spread by the diffraction diffusion plate 2 travels as a hollow conical shape, that is, an annular light. This annular light is collected by the first condenser lens 3. For example, a green laser beam having a wavelength of 532 nm can be used as the laser beam, and a diffraction diffuser plate of Thor Lab D0740A can be used as the diffraction diffuser plate 2. Moreover, as the 1st condensing lens 3, the achromatic lens of Melles Griot company whose focal distance is 100 mm can be used, for example.
[0035]
The light condensed by the first condenser lens 3 enters the mask 6. With the configuration of the mask 6 having the annular slit portion 10, the light that has passed through the mask 6 travels almost completely as annular light and enters the second condenser lens 4. For example, the light that has passed through the diffraction diffusion plate 2 includes leaking light that travels straight through the center, but the leaked light is shielded by the mask 6. As shown in FIG. 2, the mask 6 has an annular slit portion 10 that can transmit light between two concentric circles having an inner diameter of 14.6 mm and an outer diameter of 14.9 mm, for example. And the board comprised from the light-shielding part 11 by which aluminum was vapor-deposited can be used.
[0036]
In order to condense the light with the back focal plane, it is preferable to design the light traveling along the principal ray to be collected with the e-BFP. Further, the mask 6 is desirably placed at the position of e-BFP (conjugate surface of the back focal plane). This is because the light leaking can be cut most efficiently when placed at a position where the light is condensed. [0037]
The annular light travels while having the thickness of the ring, and the light that passes through the center of the thickness of the ring is referred to as a principal ray 20. Before entering the second condenser lens 4, the principal ray 20 of the annular light is focused at e-SP (conjugate surface of the sample surface).
[0038]
The optical system is designed so that the principal ray 20 of the annular light travels on the cylindrical surface by the second condenser lens 4. This is because, on the sample surface 7, the incident light from all directions overlaps in the same region. Further, as shown in FIG. 1, the second condenser lens 4 is designed to focus on the back focal plane 8 of the objective lens 5. This is because each portion of light incident from all directions on the sample surface 7 becomes parallel light. This is because the components in the xyz-axis direction of the evanescent field are not uniform in the illuminated region unless the light is parallel light.
[0039]
As the second condenser lens 4, for example, an achromatic lens manufactured by Sigma Koki Co., Ltd. having a focal length of 220 mm can be used.
[0040]
The annular light that has passed through the second condenser lens 4 and passed through the back focal plane 8 is condensed and irradiated by the objective lens 5 so as to be focused on the sample surface 7. The sample surface 7 is irradiated with annular light, that is, the sample surface 7 is irradiated with light simultaneously from various directions. The generated evanescent field has all three-dimensional (x, y, z) polarization components. As a result, any fluorescent dye having vibrational moments in different directions can be excited to emit fluorescence, so that the sample can be observed regardless of the direction of the sample.
[0041]
The sample surface 7 is constituted by, for example, a boundary surface between an aqueous solution (sample) containing a protein bound to a fluorescent dye and glass in contact with the aqueous solution. The sample surface 7 is irradiated from the glass side so that the light projected from the objective lens 5 is totally reflected, and an evanescent field is generated in the vicinity of the sample surface 7 on the aqueous solution side. Since the penetration depth of the evanescent wave in the evanescent field is shallow, only a fluorescent dye very close to the surface of the sample is excited to emit light, and an ultrathin optical cross section is created. On the other hand, the fluorescence is minimized outside the evanescent field. As a result, an extremely high contrast image is formed on the observation target.
[0042]
The light emitted from the fluorescent dye is observed in the same manner as in a normal fluorescent microscope. For example, a dichroic mirror (not shown) that reflects light having a red wavelength and transmits light having a green wavelength is disposed between the second condenser lens 4 and the objective lens 5. Thereby, the red light obtained by emitting the fluorescent dye is transmitted through the objective lens 5 and reflected by the dichroic mirror, and the light can be analyzed.
[0043]
【The invention's effect】
The total reflection fluorescence microscope of the present invention makes it possible to observe whether the sample exists regardless of the direction of the sample bound to the fluorescent dye.
[0044]
With the total reflection fluorescent microscope of the present invention, one molecule of the fluorescent dye can be visualized regardless of the direction of the sample bound to the fluorescent dye, and thus the number of fluorescent dyes collected in one place can be counted. As a result, by observing a biomolecule labeled with a fluorescent dye, it is possible to quantitatively determine information about what unit the biomolecule is associated with. For example, while observing the same biomolecule, the association / separation dynamics can be quantified by changing the conditions of the aqueous solution.
[0045]
The total reflection fluorescence microscope of the present invention can be observed even when the sample is in water. Therefore, the protein in a state where the activity is maintained can be observed as it is.
[0046]
According to the present invention, unlike NMR and post-crystallization X-ray analysis, it is possible to analyze changes in the three-dimensional structure of biomolecules such as proteins, DNA, and RNA that are active in an aqueous solution. In particular, the microscope of the present invention is suitable for targeting one molecule.
[Brief description of the drawings]
FIG. 1 is a diagram showing an illumination optical system of a total reflection type fluorescence microscope. FIG. 2 is a diagram showing a configuration of a mask.
DESCRIPTION OF SYMBOLS 1 Laser light source 2 Diffraction diffusing plate 3 1st condensing lens 4 2nd condensing lens 5 Objective lens 6 Mask 7 Sample surface 8 Back focal plane 10 Annular slit part 11 Light-shielding part 20 Main light ray 100 Illumination optics of a total reflection type fluorescence microscope system

Claims (14)

A total reflection fluorescence microscope for exciting a fluorescent dye in an evanescent field,
A light source that emits light for creating the evanescent field;
Diffracting means for diffracting light emitted from the light source in an annular shape;
First condensing means for condensing the diffracted light;
Second light collecting means for collecting light transmitted through the first light collecting means;
An objective lens for irradiating the sample with light transmitted through the second light collecting means;
Total reflection type fluorescence microscope.   The total reflection fluorescence microscope according to claim 1, further comprising a mask that blocks a part of the transmitted light so that the light irradiated on the sample has an annular shape. The total reflection fluorescence microscope according to claim 1, wherein the diffraction unit rotates. The total reflection fluorescent microscope according to claim 3, wherein a rotation axis of the rotating diffraction unit does not coincide with an axis of light emitted from a light source. The total reflection fluorescence microscope according to any one of claims 1 to 4, wherein the diffraction means is a diffraction diffusion plate, and the diffraction diffusion plate vibrates while maintaining the direction of the surface. The total reflection fluorescent microscope according to claim 1, wherein annular light is incident on a back focal plane of the objective lens, and a principal ray of the annular light travels on a cylindrical surface. The focus of the light which permeate | transmitted the said 2nd condensing lens is located on the back focal plane of the said objective lens. 6. The total reflection fluorescent microscope according to any one of 6 above. An illumination optical system that excites a fluorescent dye in an evanescent field,
A light source that emits light for creating the evanescent field;
Diffracting means for diffracting light emitted from the light source in an annular shape;
A first light collecting the diffracted light Condensing means;
The first A second light that condenses the light transmitted through the light collecting means; Condensing means;
The second An illumination optical system including an objective lens that irradiates a sample with light transmitted through a condensing unit. The illumination optical system according to claim 8, further comprising a mask that blocks part of the transmitted light so that the light irradiated on the sample has a ring shape. The illumination optical system according to claim 8 or 9, wherein the diffraction means rotates. The illumination optical system according to claim 10, wherein a rotation axis of the rotating diffraction unit does not coincide with an axis of light emitted from a light source. The illumination optical system according to any one of claims 8 to 11, wherein the diffractive means is a diffractive diffusion plate, and the diffractive diffusion plate vibrates while maintaining the direction of the surface. The illumination optical system according to any one of claims 8 to 12, wherein annular light is incident on a back focal plane of the objective lens, and a principal ray of the annular light travels on a cylindrical surface. On the back focal plane of the objective lens, the second lens The illumination optical system according to claim 8, wherein a focal point of light transmitted through the condenser lens is located.

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