JP3828799B2 - Thin-layer oblique illumination method for optical systems - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a thin-layer oblique illumination method of an optical system equivalent to a microscope that detects light with high sensitivity using a light.
[0002]
[Prior art]
Conventionally, various illumination techniques of an optical microscope for detecting substances and molecules with high sensitivity using light have been proposed, some of which will be described below. FIG. 9 is a schematic view of an epi-illumination method that is currently generally used as illumination for a fluorescence microscope. As shown in the figure, the light from the light source is reflected by a dichroic mirror that reflects the irradiation light and transmits the fluorescence, enters the center of the objective lens, and irradiates the sample observation surface.
[0003]
Further, there is a total reflection illumination method as a method of locally illuminating only the vicinity of the glass surface. Illumination is performed using evanescent light (depth of about 100200 nm) generated upon total reflection. There are a method using a prism and a method using an objective lens. FIG. 10 shows a schematic diagram of an objective lens type total reflection illumination method. When the numerical aperture of the objective lens is NA and the refractive index of the sample solution is n, total reflection illumination can be performed by the objective lens having a numerical aperture (NA) satisfying the formula of NA> n. Conventionally, this method has been used only for a limited amount, but it has been found useful as a method for observing one molecule of a fluorescent dye with a fluorescence microscope, and has recently begun to be used for single-molecule imaging.
[0004]
Further, FIG. 11 shows an outline of a light irradiation switching method proposed by the present inventor and already patented as Japanese Patent No. 3093145. 1 is a sample solution, 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), 5 is irradiation light, 6 is irradiation light, and 7 is a dichroic mirror. The change from epi-illumination to the state of total reflection illumination can be realized only by moving optical components such as mirrors from FIG. 11 (A) → 11 (B) → FIG. 11 (C). Irradiation can be switched by a simple principle without requiring an optical system. Further, in this method, the same state change can be realized by shifting the incident light position or the light source position (for example, the emission position of the optical fiber).
[0005]
As another method for obtaining a sectioning image or a three-dimensional image in a normal optical microscope or a fluorescent microscope using an epi-illumination method, calculation is performed by deconvolution from a continuous image obtained by continuously changing the focus. The method is used. This is to calculate the original three-dimensional image by calculation by knowing in advance what happens when the image of light emitted from one point on the sample deviates from the focus. In addition, this method has a limit when observing a dark part in a bright light.
[0006]
As another method, confocal microscopy is now widely used as a method using a sectioning image or a three-dimensional image. This method is inferior in sensitivity to a high-sensitivity camera. Especially in the observation of a fluorescent sample, the laser light is focused on one point and irradiated, so that the fading of the fluorescent dye is accelerated, or the biological sample There are difficulties such as damaging.
[0007]
[Problems to be solved by the invention]
As described above, the conventional method cannot obtain a high sensitivity and a high S / N ratio in the observation of a thick sample or a dark portion in a bright area. Therefore, the present invention makes it possible to detect substances and molecules with high sensitivity using light using an optical system equivalent to a microscope, and to perform low-level background / high-sensitivity observation in an optical microscope. The purpose is to provide an illumination method.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problem, the thin-layer oblique illumination method of the optical system according to claim 1 of the present invention is such that, in the oblique illumination of the lens optical system using the objective lens , the irradiation radius on the sample observation surface is r, and the sample Where d is the thickness of the illumination light at, and θ is the incident angle of the illumination light at the sample (or the angle between the illumination light and the optical axis of the objective lens), d = 2r · cos θ. As the thickness d of the illumination light becomes 20μm or less in a sample, the light incident on the edge of the objective lens, and the incident angle of the illumination light to the specimen at an angle close to perpendicular to the optical axis of the objective lens, the illumination light objective The configuration is such that the sample is illuminated with a thin layer of light by reducing the half opening angle δφ of the lens incidence and narrowing the irradiation area.
[0009]
This makes it possible to illuminate the sample with thin layered light by narrowing the irradiation area in oblique illumination using an objective lens. In an optical microscope, a low-background / high-sensitivity sectioning image or a three-dimensional image can be displayed. Obtainable.
[0010]
An optical system thin-layer oblique illumination method according to claim 2 of the present invention is oblique illumination of a lens optical system using an objective lens , wherein the irradiation radius on the sample observation surface is r, and the thickness of the illumination light on the sample Where d is the incident angle of the illumination light on the sample (or the angle between the illumination light and the optical axis of the objective lens) is θ, and d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination method that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, an objective lens that has a larger number of apertures than the refractive index of the sample is used. The sample was illuminated with a thinner layer of light by making the incident angle of illumination light on the sample closer to perpendicular to the optical axis of the objective lens and narrowing the irradiation area.
[0011]
This allows thinner as illumination light using an open number of units of a large objective lens, by using an objective lens numerical aperture is larger than the refractive index of the sample, more nearly perpendicular to the optical axis of the illumination light incident angle to the sample Therefore, it can illuminate with thinner light.
[0012]
The thin-layer oblique illumination method of the optical system according to claim 3 of the present invention is oblique illumination of a lens optical system using an objective lens, wherein the irradiation radius on the sample observation surface is r, and the thickness of the illumination light on the sample Where d is the incident angle of the illumination light on the sample (or the angle between the illumination light and the optical axis of the objective lens) is θ, and d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, the sample surface with different heights can be changed by changing the focal position. In the observation, the incident angle of the illumination light on the sample observation surface is kept constant by changing the incident angle of the illumination light to the objective lens.
[0013]
Thus, even if the focal position of the objective lens is changed and the height of the sample observation surface from the cover glass surface is changed, the sample is illuminated at the same incident angle by changing the incident angle of the illumination light to the objective lens. be able to.
[0017]
The thin-layer oblique illumination method for an optical system according to claim 4 of the present invention is an oblique illumination of a lens optical system using an objective lens, which is illuminated on a sample observation surface. The radius of irradiation is r, the thickness of the illumination light on the sample is d, and the incident angle (or illumination) of the illumination light on the sample. If the angle between the bright light and the optical axis of the objective lens is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the objective lens and narrowing the irradiation area , use of multiple incident light, rotation, etc. A thin layer oblique illumination with no bias is performed by moving the incident position.
[0018]
The thin-layer oblique illumination method for an optical system according to claim 5 of the present invention is such that the optical system of claims 1 to 4 is an optical microscope such as a fluorescence microscope or a dark field microscope, or a lens optical system using an objective lens. Is also applicable.
[0019]
Thereby, in the detection using not only an optical microscope but also various microscopes and light, a low background image and a low background signal can be obtained. As a result, an image and a signal with high sensitivity and high S / N ratio can be obtained.
[0020]
A thin layer oblique illumination method of an optical system according to claim 6 of the present invention is a fluorescence microscope, an atomic force microscope, a tunnel microscope, or a photo tunnel microscope using the thin layer oblique illumination method according to any one of claims 1 to 4. In the optical system, a microscope observation using thin-layer light illumination was configured to obtain a continuous image while moving the focal position, and obtain a sectioning image and a three-dimensional image by deconvolution.
[0021]
Thereby, it is possible to obtain a continuous image while moving the focal position from a microscopic observation using thin-layer light illumination, and obtain a sectioning image and a three-dimensional image by deconvolution, and the background light is low and the image quality is high. Further, since the illumination of the sample is local only in a thin layered region, one fluorescent dye molecule can be visualized by observing the obtained fluorescent image with an imaging intensifier CCD which is a high sensitivity camera.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, in the oblique illumination of the lens optical system using the objective lens, by illuminating the sample with a thin layered light, the area to which the illumination light is applied is locally limited, and the background light can be lowered. It relies on being able to obtain images and signals with high sensitivity and high S / N ratio. The optical system can be widely applied not only to an optical microscope such as a fluorescence microscope or a dark field microscope but also to a lens optical system using an objective lens. Next, an embodiment of the present invention will be described in detail below with reference to FIGS.
[0023]
FIG. 1 shows the principle for determining the thickness of illumination light. In the figure, when the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, and the incident angle of the illumination light on the sample (or the angle formed between the illumination light and the objective lens optical axis) is θ.
[Expression 1]
d = 2r · cos θ
From the relational expression, the thickness d of the illumination light in the sample can be obtained.
[0024]
FIG. 2 shows a partially enlarged view related to the objective lens of the optical system. In the figure, 1 is a sample medium (solution or the like), 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), and 5 is irradiation light. Irradiated light from the light source is refracted at right angles by a dichroic mirror (not shown) to become irradiated light 5, and obliquely illuminates the sample medium 1 through the oil 3 and the cover glass 2.
[0025]
2, the distance of the thickness of the illumination light in the sample d, the incident angle of the illumination light in the sample theta, illumination light incident position on the lens, i.e., the lens optical axis of the illumination light in the focal plane after the objective lenses Is X, the incident angle of the illumination light to the objective lens is φ, and the half of the opening angle of the illumination light incident on the objective lens is δφ, the expression d = 2r · cos θ gives the thickness d of the illumination light in the sample. It can be seen that the incident angle θ of the illumination light on the surface is determined by X and φ, and the irradiation radius r on the sample observation surface is determined by δφ. Therefore, reducing the thickness d of the illumination light can be realized by reducing cos θ and r.
[0026]
In order to reduce cos θ, θ should be close to 90 degrees. For this purpose, X is increased. That is, light is incident on the edge of the objective lens, and oblique illumination is performed on the sample at an angle close to perpendicular to the optical axis of the objective lens. Further, the incident angle φ to the objective lens is adjusted so that θ is close to 90 degrees.
[0027]
In order to reduce the irradiation radius r, δφ may be reduced. At this time, if r is reduced, the observation field of view is narrowed. Therefore, it is necessary to reduce r within the range permitted by the observation field of view.
[0028]
Thus, in oblique illumination using the objective lens, the specimen can be illuminated with thin layered light by setting the incident light incident angle on the specimen to an angle close to perpendicular to the optical axis of the objective lens and narrowing the irradiation area. In the optical microscope, a low background / high sensitivity sectioning image or three-dimensional image can be obtained.
[0029]
Although it thin enough illumination light using an open number of units of a large objective lens, by using an objective lens numerical aperture is larger than the refractive index of the sample, more nearly perpendicular to the optical axis of the illumination light incident angle to the sample Therefore, it can illuminate with thinner light. As the numerical aperture (NA) of the objective lens is increased, X in FIG. 2 can be increased, and the incident angle θ of the illumination light is increased. As a result, the thickness d of the illumination light can be reduced. .
[0030]
Next, referring to FIG. 3, if the maximum value of X determined by the numerical aperture NA of the objective lens is X _NA , and the boundary value of X at which total reflection determined by the refractive index n of the sample occurs is X _n , the closed numerical aperture NA In a lens whose refractive index is larger than the refractive index n of the sample, as shown in FIG. 3A, when X is increased, total reflection occurs and the sample cannot be illuminated.
[0031]
However, as shown in FIG. 3B, by adjusting the incident angle φ to the objective lens, the sample can be illuminated obliquely without causing total reflection. When this method is used, the incident angle θ of the illumination light can be made closer to 90 degrees, and the thickness d of the illumination light can be further reduced.
[0032]
Next, the case where the height Z from the cover glass surface of the sample observation surface is changed by changing the focal position of the objective lens will be described with reference to FIG. FIG. 4A shows that the height of the sample observation surface from the cover glass surface is Z at the incident angle φ of the illumination light to the objective lens.
[0033]
FIG. 4B shows that when the incident angle of the illumination light to the objective lens is (φ + Δφ), the height of the sample observation surface from the cover glass surface is (Z + ΔZ). Therefore, even if the focal position of the objective lens is changed, the sample can be illuminated with the same incident angle θ by changing the incident angle φ of the illumination light to the objective lens.
[0034]
Next, a case where the light receiving surface of a light receiving element such as a camera is tilted or a case where the imaging lens is tilted will be described with reference to FIG. In FIG. 5, 1 is a sample medium (solution or the like), 2 is a cover glass, 3 is oil, 4 is an objective lens (lens group), 5 is irradiation light, 8 is a sample observation surface, 9 is an imaging lens, 10 is It is a light receiving element such as a camera. In the figure, the lens system is depicted in a simplified manner, but in actuality, the lens system is configured with a lens group in the middle.
[0035]
FIG. 5A shows that the sample observation surface 8 can be inclined by tilting the light receiving surface of the light receiving element 10 such as a camera, and the oblique illumination light 5 and the sample observation surface 8 can be observed in parallel or substantially parallel. Show.
[0036]
FIG. 5B shows that the sample observation surface 8 can be inclined by tilting the imaging lens 9 so that the oblique illumination light 5 and the sample observation surface 8 can be observed in parallel or substantially in parallel. In the method of tilting the imaging lens 9, the same thing can be done by changing the intermediate optical system.
[0037]
When the sample observation surface 8 in FIG. 5A is tilted, the shape of the illumination light incident on the objective lens 4 can be elongated, and the thin layer light that illuminates the sample can be further reduced. This will be described with reference to FIG. The reference numerals are the same as in the above example.
[0038]
Since FIG. 6A has the same configuration as FIG. 2, description thereof is omitted. FIG. 6B is an enlarged view of the irradiation light of the sample region viewed from above. In the formula d = 2r · cos θ that gives the thickness d of the illumination light, r is the radius of the illumination light in the traveling direction of the cross-sectional shape of the illumination light by the plane parallel to the cover glass. When the sample observation surface is inclined, the size of the observation visual field does not depend on r, but is determined by the radius r ′ in the direction perpendicular to r. Therefore, when elongated incident light having a larger r 'and a smaller r is used, the thickness d of the illumination light can be reduced without narrowing the observation field.
[0039]
In this way, a continuous image can be obtained from a microscope observation using thin layer light illumination while moving the focal position, and a sectioning image and a three-dimensional image can be obtained by deconvolution. According to the present invention, unlike the conventional method, the background light is low and the image quality is high, and the illumination of the sample is local only in a thin layered region.
[0040]
Therefore, there is a great advantage in terms of both calculation and image quality that only the region near the sample observation surface needs to be a deconvolution calculation target. Accordingly, it is possible to perform sectioning images and three-dimensional images of high-sensitivity observation such as observation of a thick sample, observation of a dark portion in a bright state, and observation of one molecule.
[0041]
Further, as shown in FIG. 7, thin layer light illumination with no bias can be performed by using a plurality of incident light or rotationally symmetric incident light, or by moving the incident position by rotation or the like.
[0042]
Next, an example in which the present invention is applied and a thin layer oblique illumination method is used for a fluorescence microscope is shown in FIG. In the figure, the same reference numerals are attached to the same components as in the above example, 11 is an optical filter, 12 is a mirror, 13 is a condensing lens, and R is the inner diameter of the illumination laser light variable aperture. . The laser light is used as illumination light, the laser light is condensed on the rear focal plane of the objective lens 4 by the condensing lens 13, and the illumination light on the sample is converted into parallel light.
[0043]
The incident aperture angle δ φ is changed by changing the variable aperture diameter R to adjust the irradiation radius r. The incident position X is adjusted by moving the mirror 12 and the condenser lens 13 together in the tx direction. Next, when the incident position of the laser beam on the mirror 12 is moved in the tφ direction, the inclination of the optical path after passing through the condensing lens 13 changes, so the incident angle φ to the objective lens 4 is adjusted by tφ. By the above adjustment, the thickness d = 2r · cos θ of the illumination light in the sample can be set to several microns.
[0044]
In an example, when an objective lens having an oil immersion of 100 times NA 1.4 is used and the diameter of the irradiated region on the sample observation surface is 2r = 30 μm, the incident angle θ = 80 ° at the sample, d = 5 μm, and the incident angle θ = At 84 °, d = 3 μm. When an objective lens with an oil immersion of 60 times NA 1.4 is used and the diameter of the irradiated region on the sample observation surface is 2r = 45 μm, the incident angle θ of the sample is 86 ° and d = 3 μm.
[0045]
In this way, one fluorescent dye molecule can be visualized by observing a fluorescent image obtained by thin-layer oblique illumination with an imaging intensifier CCD, which is a high-sensitivity camera.
[0046]
As another application example, in fluorescence microscopy, tilting the sample viewing surface, and elongated illumination light, using a high NA objective lenses, an example of thin thin layer illumination light. In the fluorescence microscope of FIG. 8, the method of tilting the sample observation surface by the method of tilting the imaging lens of FIG. 5 and using the elongated illumination light of FIG.
[0047]
The cross-sectional shape of the illumination light on the cover glass is such that the minor axis 2r = 30 μm and the major axis 2r ′ = 100 μm. In this way, an observation field having a side of 100 μm can be obtained, and the thickness of the illumination light can be reduced without narrowing the observation field.
[0048]
From the equation d = 2r · c o sθ, the thickness d of the layer of the illumination light in a sample, using the objective lens of the oil immersion 60x NA1.4, 2r = 30μm, the incidence angle of the illumination light in the sample theta = 86 At d, d = 2 μm. However, since the thickness d is close to the wavelength of light (0.4 to 0.7 μm for visible light), the spreading of light begins to be observed above and below the light layer due to the diffraction phenomenon.
[0049]
Further, when an objective lens having a large NA of oil immersion 60 times NA 1.45 is used, the incident angle θ of the sample is 87 °, that is, the angle formed with the cover glass is 3 °, and the illumination light and the cover glass are almost parallel. become. By approaching parallel in this way, the influence on the image quality caused by tilting the imaging lens can be ignored. The light thickness d is theoretically less than 2 μm, but the spread of the thickness due to diffracted light is also strong.
[0050]
When the thickness of the illumination light by the thin oblique illumination of the optical system of the present invention and the conventional oblique illumination method is measured and compared, as shown in FIG. 12, for example, when an objective lens having a numerical aperture NA = 1.4 is used, The thickness of the illumination light by the oblique illumination method was about half the thickness.
[0051]
A clear single-molecule imaging was realized even in cells by observation with a fluorescence microscope using the thin-layer oblique illumination method of the present invention. As a result, it became possible to directly observe the movement and change of one molecule. At the same time, by obtaining the fluorescence intensity of one molecule, it was also possible to quantify the number of molecules in the cell from the fluorescence intensity. Furthermore, it was possible to determine the number of binding molecules and the strength of binding in the intercellular interaction from the quantification of the number of molecules.
[0052]
Currently, the application of nanotechnology to biotechnology is attracting strong interest, but the thin-layer oblique illumination method of the present invention that enables highly sensitive detection at the single molecule level will be an important elemental technology in this field. Conceivable. Moreover, it is expected to develop as a new microscope method as a microscope technique.
[0053]
【The invention's effect】
As described above, the thin-layer oblique illumination method of the optical system according to the present invention uses various microscopes and light in addition to an optical microscope by illuminating a sample with thin layered light in oblique illumination using an objective lens. Detection, a low background image and a low background signal can be obtained. As a result, an image and a signal with high sensitivity and high S / N ratio can be obtained.
[0054]
In addition, a continuous image can be obtained from a microscope observation using thin layer light illumination while moving the focal position, and a sectioning image and a three-dimensional image can be obtained by deconvolution, and the background light is low and the image quality is high. In addition, since the illumination of the sample is local only in the thin layered region, by observing the obtained fluorescent image with a high sensitivity camera, one molecule of the fluorescent dye can be visualized and the fluorescence intensity of one molecule can be obtained. It is also possible to quantify the number of molecules in the cell from the fluorescence intensity. Furthermore, it is possible to determine the number of binding molecules and the strength of binding in the intercellular interaction from the quantification of the number of molecules.
[Brief description of the drawings]
FIG. 1 is a principle diagram for determining the thickness of illumination light according to the present invention.
FIG. 2 is a partially enlarged view related to an objective lens of an optical system.
FIG. 3 is a schematic view of a method for adjusting an incident angle to an objective lens.
FIG. 4 is a schematic view of a method for changing the focal position of an objective lens.
FIG. 5 is a schematic view of a method of tilting a light receiving surface or an imaging lens of a light receiving element.
FIG. 6 is a schematic view of a method for elongating the shape of illumination light incident on an objective lens.
FIG. 7 is a schematic diagram of a method of using multiple incident lights or rotationally symmetric incident light.
FIG. 8 is a schematic view of a thin layer oblique illumination method of a fluorescence microscope.
FIG. 9 is a schematic view of an epi-illumination method of a fluorescence microscope.
FIG. 10 is a schematic view of an objective lens type total reflection illumination method.
FIG. 11 is a schematic diagram of a light irradiation switching method.
FIG. 12 is a comparative table of the illumination light thickness of the method of the present invention and the conventional method.
[Explanation of symbols]
1 Sample medium (solution, etc.)
2 Cover glass 3 Oil 4 Objective lens (lens group)
5, 6 Irradiation light 7 Dichroic mirror 8 Sample observation surface 9 Imaging lens 10 Light receiving element 11 Optical filter 12 Mirror 13 Condensing lens

Claims (6)

In oblique illumination of a lens optical system using an objective lens , the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, the incident angle of the illumination light on the sample (or the illumination light and the objective lens optical axis) Is calculated by the following formula : d = 2r · cos θ As the thickness d of the illumination light becomes 20μm or less in a sample, the light incident on the edge of the objective lens, and the incident angle of the illumination light to the specimen at an angle close to perpendicular to the optical axis of the objective lens, the illumination light objective A thin-layer oblique illumination method for an optical system, characterized in that the sample is illuminated with a thin layer of light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area.   2. An objective lens having a numerical aperture larger than the refractive index of the sample is used to illuminate the sample with thinner light by making the incident angle of illumination light on the sample closer to perpendicular to the optical axis. Thin-layer oblique illumination method for optical systems. An oblique illumination of a lens optical system using an objective lens, wherein the irradiation radius on the sample observation surface is r, the thickness of the illumination light on the sample is d, and the incident angle of the illumination light on the sample (or illumination light and objective lens) When the angle between the optical axis and θ is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the lens incidence and narrowing the irradiation area, the sample surface with different heights can be changed by changing the focal position. A thin-layer oblique illumination method for an optical system characterized in that the incident angle of illumination light on the sample observation surface is kept constant by changing the incident angle of illumination light to the objective lens when observing. Oblique illumination of a lens optical system using an objective lens, and illumination on the sample observation surface The radius of irradiation is r, the thickness of the illumination light on the sample is d, and the incident angle of illumination light on the sample (or illumination If the angle between the bright light and the optical axis of the objective lens is θ, it is calculated by the formula d = 2r · cos θ. The light is incident on the edge of the objective lens so that the thickness d of the illumination light in the sample is 20 μm or less, and the incident angle of the illumination light to the sample is set to an angle close to the optical axis of the objective lens. In the thin-layer oblique illumination of the optical system that illuminates the sample with thin layered light by reducing the half-opening angle δφ of the objective lens and narrowing the irradiation area , use of multiple incident light, rotation, etc. A thin-layer oblique illumination method for an optical system, characterized in that thin-layer oblique illumination with no bias is performed by moving an incident position.   5. The thin-layer oblique illumination method for an optical system according to claim 1, wherein the optical system is an optical microscope such as a fluorescence microscope or a dark field microscope, or a lens optical system using an objective lens.   6. The thin layer oblique light of an optical system according to claim 5, wherein, in microscopic observation using thin layer light illumination, a continuous image is obtained while moving a focal position, and a sectioning image and a three-dimensional image are obtained by deconvolution. Lighting method.

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