JP2931268B2 - Laser scan optical device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser scanning optical apparatus which performs laser beam scanning as a fluorescence microscope, an optical drawing apparatus, an integrated circuit repair apparatus, and the like in many fields such as biology, medicine, and semiconductor engineering. .

[0002]

2. Description of the Related Art In an ordinary fluorescence microscope, when observing a three-dimensionally thick sample, an image other than the depth of focus is defocused and superimposed on an image on a focal plane. For this reason, the contrast of the microscopic image is reduced as a whole, and it becomes difficult to quantify the fluorescence intensity. For an approach to addressing this issue,
This will be described below.

FIG. 10 is a configuration diagram showing a configuration of a conventional confocal laser scanning fluorescence microscope. The laser light 2 output from the laser 1 is transmitted through the dichroic mirror 11 after the diameter of the light beam is expanded by the beam expander 4. Note that the laser 1 oscillates a laser beam 2 at a wavelength corresponding to the peak wavelength of the absorption spectrum of the fluorescent dye that labels the sample 15. The beam expander 4 includes two convex lenses 5a and 5b. The dichroic mirror 11 is formed to have a large reflectance with respect to light components in a predetermined wavelength range including the wavelength of the fluorescent light 16 a and a large transmittance with respect to the oscillation wavelength of the laser light 2.

The laser beam 2 emitted by the XY scanner 12 in the direction perpendicular to the optical axis forms an optical spot on the front image plane of the objective lens 14 by the imaging lens 13 and then by the objective lens 14. The light spot narrowed down to the diffraction limit is imaged inside the sample 15. Where XY
The scanner 12 changes the traveling direction of the laser beam 2 within a predetermined angle range and swings the laser beam 2 in two directions orthogonal to each other in a plane. Further, a stage (not shown) on which the objective lens 14 or the sample 15 is set moves parallel to the optical axis. Therefore, inside the sample 15, the light spot of the laser beam 2 is
A three-dimensional scan is executed by executing a scan parallel to the optical axis in addition to a two-dimensional scan (raster scan or the like) in a plane perpendicular to the optical axis. The sample 15 is, for example, a biological sample labeled with a fluorescent dye,
It is excited by the light spot of the laser light 2.

[0005] The fluorescence 16 emitted and emitted from the sample 15
a is the laser beam 2 after being condensed by the objective lens 14.
Travels in the opposite direction along the optical path that has passed. XY scanner 1
The fluorescent light 16a emitted from the light source 2 is reflected by the dichroic mirror 11 in a direction perpendicular to the optical axis, and then collimated by the collimator lens 21.
An image is formed in the 0 pinhole. The fluorescent light 16a emitted from the confocal pinhole plate 30 is such that the fluorescent components emitted from before and after the light spot inside the sample 15 are shielded. This fluorescence 16a is generated by PMT (Phot
o Multiplier Tube) 20 receives light.

The PMT 20 photoelectrically converts the fluorescent light 16a and outputs an electric signal having an intensity corresponding to the light intensity of the fluorescent light 16a. The electric signal output from the PMT 20 is
The data is synchronized with a scanning signal of the XY scanner 12 and stored as image data in a memory of an image reading device (not shown). This image data is processed in accordance with the scanning signal based on a normal method, so that a three-dimensional microscopic image of the sample 15 is obtained.

In such a conventional confocal laser scanning fluorescence microscope, a point light source and a point light detector are ideally arranged at a position conjugate with one point inside the sample, so that the laser beam has a reduced depth of focus. Is formed. In addition, by disposing the pinhole on the photodetector side, the fluorescent component emitted from before and after the light spot inside the sample is removed. Therefore, the defocused image other than the focal plane almost disappears. Therefore, only an image near the focal plane inside the sample is obtained as a microscopic image.

The prior art relating to such a confocal laser scanning fluorescence microscope is disclosed in Japanese Unexamined Patent Publication No. Hei.
No. 05 "and the like.

Next, a conventional two-photon absorption laser scanning fluorescence microscope will be described. In the two-photon absorption excitation laser scanning fluorescence microscope, by using a laser that oscillates a laser beam as a pulse having an extremely short duration, the laser beam forms an image of a light spot having a high energy density. Performs three-dimensional scanning inside the sample in the same manner as in the confocal laser scanning fluorescence microscope described above. Therefore, fluorescence due to excitation based on two-photon absorption is generated only from one point where the light spot is located inside the sample, and no fluorescence is generated from other parts due to excitation based on two-photon absorption. Therefore, a defocused image other than the focal plane does not appear, and the contrast of the microscopic image is improved.

Prior art relating to such a two-photon absorption-excitation laser scanning fluorescence microscope is disclosed in the document "Science, vo.
l.248, pp.73-76,6.April.1990 "," United States Pate
nt, no. 5034613, 1991 ".

Further, in a conventional general laser scanning fluorescence microscope, by replacing the objective lens with an axicon prism, the laser beam interferes on the optical axis and has a focal depth as large as the thickness of the sample. The bundle is converted into a so-called Bessel beam, and the Bessel beam scans the inside of the sample three-dimensionally in the same manner as in the confocal laser scanning fluorescence microscope described above. Therefore, since the image within the depth of focus is not blurred, a microscopic image obtained by projecting a three-dimensional image two-dimensionally is obtained. Alternatively, by injecting laser light into the objective lens as a cylindrical light beam through an aperture having an annular aperture, the laser light forms an image of a Bessel beam having a focal depth as large as the thickness of the sample, The Bessel beam performs three-dimensional scanning inside the sample in the same manner as in the confocal laser scanning fluorescence microscope described above. Therefore, since the image within the depth of focus is not blurred, a microscopic image obtained by projecting a three-dimensional image two-dimensionally is obtained.

FIG. 11A is a configuration diagram showing a conventional light conversion unit for generating a Bessel beam. In this light conversion unit, an aperture 43 having a ring-shaped opening 44 is arranged on the focal plane of the convex lens 46. Here, when the laser light 2 passes through the opening 44 of the aperture 43 as parallel light, a diffracted light 45 is generated as a light beam having a ring-shaped cross-sectional intensity orthogonal to the optical axis. Convex lens 46
The diffracted light 47 that has passed through the optical path travels as a plane wave refracted at a fixed angle with respect to the optical axis, and then forms a conical wavefront that is axially symmetric with respect to the optical axis at the rear focal point of the convex lens 46. For this reason, the diffracted light 45 interferes with each other in the entire region where the wavefront exists near the optical axis, so that the interference generates the Bessel beam 47 having the intensity strengthened by the interference.

FIG. 11B is a graph showing an intensity distribution of the Bessel beam 47 in a cross section orthogonal to the optical axis. The intensity distribution of the Bessel beam 47 is substantially constant within the interference area. Therefore, an elongated linear center beam having a large intensity exists at a position near the optical axis. On the other hand, there is coaxial cylindrical high-order diffracted light having a small intensity at a position away from the optical axis. Therefore, it can be seen that the Bessel beam 47 has a high resolution and a long depth of focus.

FIG. 12A is a configuration diagram showing the shape of the aperture 43. In the vicinity of the rear focal point of the convex lens 46,
The shape of the three-dimensional intensity distribution in the Bessel beam 47 changes depending on the width of the annular zone of the opening 44. Therefore, in the Bessel beam 47, the intensity distribution of the center beam and the higher-order diffracted light changes in accordance with the change in the ratio β (= α ′ / α) of the inner diameter α ′ to the outer diameter α in the opening 44.

FIG. 12B is a graph showing a change in the intensity distribution of the Bessel beam 47 in a plane perpendicular to the optical axis when the ratio of the inner diameter to the outer diameter in the opening 44 changes. FIG. 12C is a graph showing an enlarged main part in the intensity distribution of FIG. here,
The scale on the horizontal axis is 2πNA / λ (where NA: lens 4
6, the numerical aperture of 6, and λ: the wavelength of the laser beam 2). As the ratio β of the inner diameter to the outer diameter of the opening 44 approaches 1, the spot diameter of the center beam becomes smaller, while the intensity of the higher-order diffracted light with respect to the center beam becomes larger. FIGS. 13A and 13B are graphs showing the intensity distribution of the Bessel beam 47 in a plane including the optical axis when the ratio of the inner diameter to the outer diameter in the opening 44 is 0 and 0.7, respectively. is there. Here, the numerical aperture NA of the convex lens 46 is 1.0, and the scales on the vertical axis and the horizontal axis are both normalized based on the wavelength λ of the laser beam 2 and shown. When the ratio β of the inner diameter to the outer diameter at the opening 44 is 0.7 rather than 0, the shapes of the center beam and the higher-order diffracted light become longer in the optical axis direction.

Therefore, it can be seen that the depth of focus and the resolution of the Bessel beam 47 are improved as the shape of the opening 44 becomes a ring shape having a smaller width than a circular shape.

Prior art relating to an annular illumination optical system using an aperture in such a laser scanning fluorescence microscope is disclosed in the document "Optics, Vol. 21, No. 7, pp. 489.
-497, July 1992 ". Further, prior art relating to the generation of a Bessel beam by an axicon prism is described in the document “Transactions of the Laser Microscope Research Society 10th Lecture Meeting, pp. 22-29, 1992.
January "and so on. Further, prior art relating to an annular illumination optical system using an axicon prism is described in detail in “United States Patent, no. 4875992, 1989” and the like.

[0018]

In the above-mentioned conventional confocal laser scanning fluorescence microscope, the fluorescent component emitted from before and after the light spot inside the sample is shielded by the pinhole arranged on the photodetector side. In addition, since the light receiving efficiency of the fluorescence by the photodetector is extremely reduced, there is a problem that the fluorescence intensity, which is originally weak, is further reduced.

The present invention has been made in view of the above-mentioned problems, and has been made in consideration of the above-described problems. A laser scanning optical system capable of detecting fluorescence emitted from a sample while suppressing loss thereof and obtaining a microscopic image with good contrast. It is intended to provide a device.

[0020]

The laser scanning optical apparatus of the present invention irradiates a predetermined sample labeled with a predetermined fluorescent dye with a laser beam and detects fluorescence emitted from the sample, thereby obtaining a sample. This is a laser scanning optical device for obtaining a microscopic image. The laser scanning optical device of the present invention
(A) a light source that oscillates laser light as a pulse having a very short time width, and (b) an optical scanning unit that irradiates the sample while scanning the laser light while changing the traveling direction of the laser light emitted from the light source. (C1) a first photoelectric detector that detects fluorescence emitted from the surface side of the sample based on multiphoton absorption due to laser light irradiation, and (c2) a sample based on multiphoton absorption due to laser light irradiation And a second photoelectric detector for detecting fluorescence emitted from the back surface side of the light emitting element. The laser scanning optical device according to the present invention includes the first and second laser scanning optical devices.
The present invention is characterized in that the sum signal of the respective output signals of the photoelectric detector is accumulated as pixel data in synchronization with the scanning of the laser beam, and a microscopic image of the sample is generated based on the pixel data.

In the laser scanning optical apparatus according to the present invention, the fluorescence when the fluorescent dye is excited at a wavelength corresponding to a fraction of the oscillation wavelength of the laser light is detected. According to the laser scanning optical device of the present invention, not only the fluorescence emitted from the front side of the sample but also the fluorescence emitted from the back side is detected, so the fluorescence emitted from the sample is detected with little loss, As a result, a microscopic image with good contrast can be obtained. In addition, it is possible to use a low-power laser light source, thereby reducing damage to a biological sample or the like.

In the laser scanning optical apparatus according to the present invention, a light conversion unit for forming a light beam of the laser beam incident as parallel light into a cylindrical shape may be provided on the incident side of the light scanning unit. A light converging unit that converges the laser light incident from the optical scanning unit to generate a Bessel beam may be provided on the emission side of the light. Further, the light conversion unit is disposed such that each apex angle is opposite or opposite at a predetermined interval and each optical axis is aligned, and is formed into a shape having the same apex angle angle with a material having the same refractive index. May be composed of two axicon prisms.

With such a configuration including the light conversion unit and the light converging unit, the laser light incident on the light conversion unit as parallel light is refracted at an equal angle with respect to the optical axis by one axicon prism. After forming a conical wavefront, the light beam becomes a divergent light beam having a ring-shaped cross-sectional intensity orthogonal to the optical axis. The laser beam emitted from this axicon prism is refracted by the other axicon prism at an equal angle to the optical axis, and is a cylindrical light beam that is parallel light whose traveling direction is parallel to the optical axis. become. The laser beam output from the light conversion unit in this way is scanned while the traveling direction is changed by the optical scanning unit, and is converged by the light converging unit to become a Bessel beam. In this diffracted light intensity distribution of the Bessel beam, there is an extremely elongated linear central beam having a large intensity in the optical axis direction, and a number of coaxial cylindrical high-order diffracted lights existing around the central beam. Here, since the center beam has an extremely large intensity compared to the higher-order diffracted light, the Bessel beam has a high resolution and a long depth of focus. Therefore, according to the laser scanning optical apparatus having such a configuration, since the cylindrical light beam is formed without impairing the intensity of the laser beam, the sample is scanned by the Bessel beam having a high energy density, a high resolution, and a long depth of focus. Will receive.

Here, since multiphoton absorption occurs only in a portion where the energy level of the laser beam is equal to or higher than a predetermined value, the multi-photon absorption of the laser beam is prevented so that high-order diffraction light accompanying the center beam of the Bessel beam does not cause multiphoton absorption. By performing the output adjustment, fluorescence is generated only from the irradiation position of the center beam of the Bessel beam inside the sample. As a result, a false signal due to high-order diffracted light accompanying the Bessel beam does not occur, and the sample of the sample obtained by accumulating the sum signal of the output signals of the respective photoelectric detectors as pixel data in synchronization with the scanning of the laser beam. The resolution of the microscopic image is improved.

By performing two-dimensional scanning inside the sample with the center beam of the Bessel beam formed linearly, a microscopic image obtained by projecting a three-dimensional image of the sample two-dimensionally has a thickness. Obtained as an integral value in the direction. This eliminates the need to scan the inside of the sample three-dimensionally, so that the scanning time of the light spot is greatly reduced.

The light conversion section may further include a moving means for variably setting the interval between the two axicon prisms. In this case, the outer diameter of the cylindrical light beam emitted from the light conversion unit is determined based on the interval between the two axicon prisms. Here, the annular width, which is the difference between the outer diameter and the inner diameter of the cylindrical light beam, is constant based on the diameter of the light beam of the laser light incident on the incident-side axicon prism. The ratio of the inner diameter to the outer diameter varies continuously according to the distance between the two axicon prisms. Accordingly, the diameter and the intensity of the center beam of the Bessel beam with respect to the higher-order diffracted light change, so that the resolution and the depth of focus can be adjusted.

A beam expander for expanding the diameter of the light beam of the laser beam and a beam reducer for reducing the diameter of the light beam of the laser light are provided on at least one of the incident side and the output side of the light conversion section. May be. If a beam expander or a beam reducer is installed on the incident side of the light conversion unit, the diameter of the light beam of the laser beam incident on the light conversion unit is adjusted, so that the cylindrical light beam emitted from the light conversion unit The orbicular width, which is the difference between the outer diameter and the inner diameter, is determined based on the diameter of the light beam of the laser light incident on the axicon prism on the incident side. Here, since the outer diameter of the cylindrical light beam is constant based on the distance between the two axicon prisms, the ratio of the inner diameter to the outer diameter of the cylindrical light beam is continuously adjusted according to the beam expander or the beam reducer. Fluctuate. Accordingly, the diameter and the intensity of the center beam of the Bessel beam with respect to the higher-order diffracted light change, so that the resolution and the depth of focus can be adjusted.
When a beam expander or a beam reducer is installed on the emission side of the light conversion unit, the outer diameter of the cylindrical light beam emitted from the light conversion unit is adjusted to match the opening diameter of the light converging unit. You. Therefore, there is almost no vignetting of the laser light, that is, the energy utilization efficiency of the laser light increases, and a Bessel beam having a high energy density is generated.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The configuration and operation of an embodiment of the present invention will be described below with reference to the accompanying drawings.
In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description. Also, the dimensional ratios in the drawings do not always match those described.

(First Embodiment) FIG. 1 is a configuration diagram showing a fluorescence microscope which is one embodiment of a laser scanning optical device of the present invention. In this fluorescence microscope, a laser (light source) 1
A plane mirror 3 is arranged along the traveling direction of the laser light 2 output from the optical disk. The laser beam 2 is reflected by the plane mirror 3 in a direction perpendicular to the optical axis. Along the traveling direction of the laser light 2 reflected by the plane mirror 3, a beam expander 4, an axicon compare (optical conversion unit) 6, a beam reducer 9, a dichroic mirror 11, and an XY scanner (optical scanning unit) 1
2 are arranged substantially in a line. This XY scanner 1
The imaging lens 13 and the objective lens 14 extend along the traveling direction of the laser light 2 emitted from the laser beam 2 in a direction perpendicular to the optical axis.
The (light converging portion) and the sample 15 are arranged substantially in a line. The condenser lens 17, the barrier filter 18, and the PMT 19a are arranged substantially in a row along the traveling direction of the fluorescence 16b emitted from the back side of the sample 15. On the other hand, the objective lens 1
4. A PMT 20a is arranged along the traveling direction of the fluorescent light 16a reflected by the dichroic mirror 11 at right angles to the optical axis via the imaging lens 13 and the XY scanner 12.

The laser 1 is a light source that oscillates a laser beam 2 as a pulse having a very short time width. This laser beam 2
Is an optical pulse width of tens to hundreds of fs and tens to hundreds of MHz.
Is a parallel light having a repetition frequency of 2 and a wavelength twice or three times the peak wavelength in the absorption spectrum of the fluorescent dye labeled on the sample 15. The plane mirror 3 is a laser 1
45 with respect to the traveling direction of the laser light 2 emitted from the
It has a mirror surface that is inclined at an angle, and the laser light 2 incident from the laser 1 is reflected by this mirror surface.

The beam expander 4 is composed of two convex lenses 5a and 5b, and expands the light beam of the laser light 2 incident from the plane mirror 3 to a predetermined diameter. These convex lenses 5a and 5b are arranged at predetermined intervals with their optical axes aligned. Further, the beam expander 4 is configured to include a moving means (not shown) for a part of the lens group, and continuously changes the magnification of the laser beam 2 with respect to the diameter of the light beam, that is, changes the zoom ratio in a zoom manner. I can do it.

FIG. 2 is a configuration diagram showing the axicon pair 6 in more detail. FIG. 3 is a perspective view showing the configuration of the axicon prism in the axicon pair 6. The axicon pair 6 is composed of two axicon prisms 7a and 7b. These axicon prisms 7a, 7b
Are formed in a shape in which the bottom surfaces of a cylinder and a cone are combined using materials having the same refractive index. Further, the axicon prisms 7a and 7b are arranged such that the apex angles having the same angle at predetermined intervals are opposed or rearward at predetermined intervals and the optical axes are aligned. The distance between the axicon prisms 7a and 7b is variably set by moving means (not shown).

FIG. 4A is a graph showing an intensity distribution in a plane perpendicular to the optical axis of the laser light 2 incident on the axicon pair 6. FIG. 4B is a configuration diagram showing a case where the distance between the two axicon prisms 7a and 7b is variably set. FIG. 4C is a graph showing an intensity distribution in a plane perpendicular to the optical axis of the laser beam 2 emitted from the axicon pair 6.

In the axicon comparator 6, the axicon prism 7a converts the laser beam 2 incident perpendicularly to the optical axis from the beam expander 4 as parallel light having a sectional intensity based on a Gaussian distribution at a constant angle with respect to the optical axis. And emitted as a conical light beam having a ring-shaped cross-sectional intensity orthogonal to the optical axis. On the other hand, the axicon prism 7b
Refracts the laser beam 2 incident from the axicon prism 7a at a fixed angle with respect to the optical axis, and emits it as a cylindrical light beam 8 whose traveling direction is parallel to the optical axis. In this cylindrical light beam 8, the outer diameter α is determined based on the distance between the axicon prisms 7a and 7b, and the annular zone width, which is the difference between the outer diameter α and the inner diameter α ', is determined.
The value is determined based on the diameter of the light beam of the laser beam 2 incident on the laser beam a. Therefore, the axicon prisms 7a and 7b
The ratio β (= α ′ / α) of the inner diameter α ′ to the outer diameter α in the cylindrical light beam 8 is set continuously, that is, in a zoom manner, in accordance with the interval between them.

The beam reducer 9 has two convex lenses 1
The outer diameter of the laser beam 2 incident as a cylindrical light beam 8 from the axicon pair 6 is reduced so as to match the aperture diameter of the objective lens 14. These convex lenses 10a and 10b are arranged at predetermined intervals with their optical axes aligned. Further, the beam reducer 9 is configured to include a moving means (not shown) for a part of the lens group, and is capable of continuously changing the magnification with respect to the diameter of the light beam of the laser light 2, that is, in a zoom manner. It has become.

The dichroic mirror 11 has a large transmittance with respect to the oscillation wavelength of the laser light 2 incident from the beam reducer 9 and converts the light component in a predetermined wavelength range including the fluorescent light 16 a incident from the XY scanner 12. On the other hand, it has a large reflectance. XY scanner 12
For example, galvanometer type scanner, resonant type scanner, piezo vibration type scanner, rotating polyhedron scanner,
An ultrasonic transducer deflector (Acousto-Optic Deflector) and the like. The XY scanner 12 changes the traveling direction of the laser beam 2 incident from the dichroic mirror 11 within a predetermined angle range and shakes the laser beam 2 in two directions orthogonal to each other in a plane, and also detects the fluorescence 16a incident from the imaging lens 13 Is changed in the same manner as described above so as to be directed to the dichroic mirror 11.

The imaging lens 13 forms an image of the light spot of the laser light 2 incident from the XY scanner 12 on the front image plane of the objective lens 14. Further, the imaging lens 13 emits, as a virtual light source, a light spot formed on the image plane on the rear side thereof as parallel light and the fluorescent light 16a incident thereon. The objective lens 14 converges the incoming laser beam 2 as a virtual light source with the light spot formed on the front image plane as the virtual light source, generates a Bessel beam as a light spot narrowed down to the diffraction limit, and illuminates the inside of the sample 15. Let it pass. The objective lens 14 has a fluorescent light 16a emitted from the surface side of the sample 15.
To form a light spot on the rear image plane of the imaging lens 13.

The sample 15 is, for example, a biological sample labeled with a predetermined fluorescent dye, and is disposed almost at the rear image plane of the objective lens 14 and at the front image plane of the condenser lens 17. The sample 15 is excited based on the multiphoton absorption at the irradiation position of the laser beam 2 incident as a Bessel beam from the objective lens 14, and emits the fluorescence 16a and 16b. The sample 15 is set on a stage (not shown), and is moved in the optical axis direction as the stage moves.

The condenser lens 17 condenses the laser light 2 and the fluorescent light 16b emitted from the back side of the sample 15, and emits them as substantially parallel light. The barrier filter 18 has a large transmittance for light components in a predetermined wavelength range including the fluorescent light 16 b emitted from the sample 15 and has a large absorption for the oscillation wavelength of the laser light 2 transmitted through the sample 15. Have.

PMT (Photo Multiplier Tube) 19
Reference numerals a and 20a denote photoelectric detectors such as a photomultiplier. The PMT 19a receives the fluorescent light 16b emitted from the barrier filter 18, photoelectrically converts the fluorescent light 16b into an electric signal corresponding to the light intensity, and outputs the electric signal. Meanwhile, PM
T20a receives the fluorescent light 16a incident from the dichroic mirror 11, photoelectrically converts the fluorescent light 16a into an electric signal corresponding to the light intensity, and outputs the electric signal.

Next, the operation of the fluorescence microscope of this embodiment will be described. The laser light 2 shown by the solid line is the laser 1
After being emitted from the mirror, the light is reflected by the plane mirror 3 in a direction perpendicular to the optical axis. The laser beam 2 emitted from the plane mirror 3 is expanded by the beam expander 4 while maintaining the traveling direction in the optical axis direction, with the beam diameter being increased to a predetermined value.

In the axicon compare 6, the laser beam 2 emitted as a plane wave collimated from the beam expander 4 is all refracted by the axicon prism 7a at an equal angle with respect to the optical axis to form a conical wavefront. The divergent light beam has a ring-shaped cross-sectional intensity orthogonal to the optical axis. The laser light 2 emitted from the axicon prism 7a is refracted by the axicon prism 7b at an equal angle to the optical axis, and is converted into a cylindrical light beam 8 which is parallel light whose traveling direction is parallel to the optical axis. Become. Here, the beam diameter of the cylindrical light beam bundle 8 shown by a solid line is freely and continuously variable in accordance with the distance between the axicon prisms 7a and 7b, and the image forming lens after passing through the beam reducer 9. It is set so as to match the aperture diameter of the objective lens 14 under the total magnification including the pupil imaging magnification of 13.

The diameter of the laser beam 2 emitted from the axicon compare 6 as a cylindrical beam bundle 8 is reduced to a predetermined value by a beam reducer 9 while maintaining the traveling direction in the optical axis direction. At this time, in the annular cross section orthogonal to the optical axis of the cylindrical light beam 8, the annular width is simultaneously compressed. The laser light 2 emitted from the beam reducer 9 passes through the dichroic mirror 11 and is XY
The light is emitted in a direction perpendicular to the optical axis by the scanner 12, and its traveling direction is changed within a predetermined angle range,
It is swung in two directions orthogonal to each other in a plane.

The laser beam 2 emitted from the XY scanner 12 forms an optical spot on the front image plane of the objective lens 14 by the imaging lens 13. This light spot can be called a virtual light source that is two-dimensionally shaken by the XY scanner 12. The laser beam 2 emitted from this light spot as a conical light beam is transmitted through only the periphery of the objective lens 14 and emitted, and is irradiated on the sample 15 as a Bessel beam which is a light spot narrowed down to the diffraction limit. . This Bessel beam has a diffracted light intensity distribution composed of an extremely elongated linear central beam having a large intensity in the optical axis direction and a number of coaxial cylindrical high-order diffracted lights existing around the central beam. The Bessel beam is X-
It is shaken by the Y scanner 12 to perform a two-dimensional scan inside the sample 15, for example, a raster scan.

In the sample 15, so-called multiphoton absorption occurs only in a portion irradiated with a Bessel beam having a light intensity portion equal to or higher than a predetermined threshold value. As a result, the fluorescent dye labeled on the sample 15 becomes 1/1 of the oscillation wavelength of the laser light 2.
As in the case where the light is excited at the peak wavelength of the absorption spectrum corresponding to 2 or 1/3, the fluorescent light 16a, 16
diverge b.

Laser light 2 transmitted through sample 15 and emitted
Is converted into parallel light whose traveling direction is substantially parallel to the optical axis by the condenser lens 17, and then the barrier filter 1
8 to be absorbed. The fluorescent light 16 b emitted from the back side of the sample 15 is condensed by the condenser lens 17, becomes parallel light whose traveling direction is substantially parallel to the optical axis, passes through the barrier filter 18, and is received by the PMT 19. On the other hand, the fluorescence 16a emitted from the surface side of the sample 15
Travels in the opposite direction on the optical path through which the laser beam 2 is condensed by the objective lens 14 and passes. Then, the fluorescence 16a emitted from the XY scanner 12 is reflected by the dichroic mirror 11 in a direction perpendicular to the optical axis, and
The light is received by T20.

The fluorescence 16a, 16b from the PMTs 19, 20
The electric signals output by the photoelectric conversion corresponding to the light intensity of are summed and synchronized with the scanning signal of the XY scanner 12,
It is stored as pixel data in a memory of an image reading device (not shown). By processing the pixel data in accordance with the scanning signal based on a normal method, a microscopic image obtained by projecting a three-dimensional image of the sample 15 two-dimensionally is obtained.

As described above, in the fluorescence microscope of the present embodiment, the fluorescence diverged from both the front and back sides of the sample is received by the two photoelectric detectors, respectively, and the output signals of these photoelectric detectors are added to form a microscopic image. Have been obtained.
Since the fluorescence emitted from the sample is detected with little loss, a high-contrast microscopic image can be obtained. In addition, since a low-power laser can be used, damage to a biological sample or the like is reduced.

In the fluorescence microscope of this embodiment, the laser light oscillated as an extremely short width pulse is formed into a narrow annular band orthogonal to the optical axis by using two axicon prisms. It is converted into a cylindrical light beam having a sectional intensity. Thus, the intensity of the laser beam is hardly lost when the cylindrical light beam is generated. For this reason, the energy utilization efficiency of the laser light increases, and a high energy density that contributes to multiphoton absorption inside the sample is maintained.

Further, the laser beam oscillated as a very short pulse width passes through only the peripheral portion of the objective lens as a cylindrical light beam, thereby forming an elongated line having a large intensity in the optical axis direction. The inside of the sample is irradiated as a Bessel beam which is a center beam of a shape. As a result, the sample is excited by multiphoton absorption only at the irradiation position of the Bessel beam. Therefore, the fluorescence emitted based on the fluorescent sample labeled on the sample is received by the photoelectric detector with high efficiency without requiring a pinhole. In addition, multiphoton absorption in the sample occurs only in a very thin portion closer to the central region of the Bessel beam by adjusting the energy density of the laser beam. Therefore, the fluorescence microscope has higher resolution and longer depth of focus.

Further, two-dimensional scanning is performed inside the sample with the center beam having a linear image of the Bessel beam. Thus, a microscopic image obtained by projecting a three-dimensional image of the sample in two dimensions is obtained as an integral value in the thickness direction.
Therefore, since it is not necessary to scan the inside of the sample three-dimensionally, the scanning time of the light spot is greatly reduced.

(Second Embodiment) FIG. 5 is a configuration diagram showing a fluorescence microscope which is a second embodiment of the laser scanning optical device of the present invention. This embodiment is configured in substantially the same manner as the fluorescence microscope of the first embodiment. However,
The sample 15 is labeled with a plurality of types of fluorescent dyes,
Each fluorescent dye has a different fluorescence emission wavelength. The sample 1 is placed on the reflection side of the dichroic mirror 11a.
5 along the traveling direction of the fluorescent light 16a emitted from the front side of the PMT 2 and the dichroic mirrors 11b and 11c and the PMT 2
0a are arranged substantially in a line. On the reflection side of these dichroic mirrors 11b and 11c, PMT 20b,
20c are arranged respectively. On the other hand, on the emission side of the barrier filter 18, dichroic mirrors 31b and 31c and the PMT 19a are arranged substantially in a line along the traveling direction of the fluorescence 16b emitted from the back side of the sample 15. PMTs 19b and 19c are arranged on the reflection side of the dichroic mirrors 31b and 31c, respectively.

The dichroic mirror 11a has a large transmittance with respect to the oscillation wavelength of the laser beam 2 incident from the beam reducer 9, and also converts the light component in a predetermined wavelength range including the fluorescent light 16a incident from the XY scanner 12 into the dichroic mirror 11a. On the other hand, it has a large reflectance. The dichroic mirrors 11b and 11c have large reflectances for mutually different wavelength bands so as to respectively include fluorescent components having mutually different emission wavelengths in the fluorescent light 16a emitted from the dichroic mirror 11a. Dichroic mirror 3
1b and 31c have a large reflectance in mutually different wavelength bands so as to include light components having mutually different emission wavelengths in the fluorescent light 16b emitted from the barrier filter 18 respectively.

PMTs 19a to 19c, 20a to 20c
Is a photoelectric detector such as a photomultiplier. PMT
19b receives the fluorescence 16b reflected by the dichroic mirror 31b, photoelectrically converts the fluorescence 16b into an electric signal corresponding to the light intensity, and outputs the electric signal. The PMT 19c receives the fluorescence 16b reflected by the dichroic mirror 31c, photoelectrically converts the fluorescence 16b into an electric signal corresponding to the light intensity, and outputs the electric signal. PM
T19a receives the fluorescence 16b transmitted through the dichroic mirrors 31b and 31c, photoelectrically converts the fluorescence 16b into an electric signal corresponding to the light intensity, and outputs the electric signal. The PMT 20b receives the fluorescence 16a reflected by the dichroic mirror 11b, photoelectrically converts the fluorescence 16a into an electric signal corresponding to the light intensity, and outputs the electric signal. The PMT 20c receives the fluorescence 16a reflected by the dichroic mirror 11c, photoelectrically converts the fluorescence 16a into an electric signal corresponding to the light intensity, and outputs the electric signal. The PMT 20a is a fluorescent light 16a transmitted through the dichroic mirrors 11b and 11c.
Is received, photoelectrically converted into an electric signal corresponding to the light intensity, and output.

Next, the operation of the fluorescence microscope of this embodiment will be described. The fluorescence microscope according to the present embodiment operates in substantially the same manner as the fluorescence microscope according to the first embodiment. However, the fluorescent light 16a emitted from the surface side of the sample 15 is condensed by the objective lens 14, travels in the optical path in which the laser light 2 has passed in the opposite direction, and is reflected by the dichroic mirror 11a in the direction perpendicular to the optical axis. After that, some light components are reflected by the dichroic mirrors 11b and 11c and received by the PMTs 20b and 20c, respectively, and the other light components are received by the PMT 20a. On the other hand, the fluorescent light 16b emitted from the back surface side of the sample 15 is condensed by the condenser lens 17 to become parallel light whose traveling direction is substantially parallel to the optical axis, and after passing through the barrier filter 18, the dichroic mirror 31b , 31c are partially reflected by the PMTs 19b, 19c, respectively.
c and the other light components are converted to PMT
The light is received by 19a.

An electric signal output from the PMTs 20a to 20c by photoelectric conversion corresponding to the light intensity of each light component of the fluorescent light 16a, and an electric signal output from the PMTs 19a to 19c to the fluorescent light 16b
Are combined with an electric signal output by photoelectric conversion corresponding to the light intensity of each light component, and are stored as pixel data in a memory of an image reading device (not shown) in synchronization with a scanning signal of the XY scanner 12. The pixel data is processed in accordance with the scanning signal based on a normal method, so that a microscopic image obtained by projecting the three-dimensional image of the sample 15 two-dimensionally is applied to each of the fluorescent components of the fluorescent light 16a and 16b, that is, the sample 15 Obtained for the emission wavelength of each labeled fluorescent dye.

As described above, according to the fluorescence microscope of the present embodiment, by irradiating a sample labeled with a plurality of types of fluorescent dyes having mutually different emission wavelengths with a Bessel beam, each of the fluorescence emitted from the sample can be obtained. The light component is isolated by the dichroic mirror and received by the photoelectric detector. Thus, when a two-dimensional scan is performed only once inside the sample with the center beam of the Bessel beam formed linearly, a three-dimensional image of the sample is two-dimensionally formed based on the emission wavelength of each fluorescent dye. Obtained simultaneously as a plurality of projected microscopic images. Therefore, it is not necessary to perform three-dimensional scanning inside the sample, and the scanning time of the light spot is greatly reduced.

As described above, the embodiments of the present invention have been described in detail. However, the present invention is not limited to the above embodiments, and various modifications are possible.

For example, in the first and second embodiments,
A dichroic mirror is arranged in an optical path between the beam reducer and the XY scanner. However, as long as the dichroic mirror is disposed at any position in the optical path between the laser and the XY scanner, the same operation and effect as in the above embodiment can be obtained.

In the second embodiment, three dichroic mirrors are used. However, as long as the number corresponds to the type of the fluorescent dye applied to the sample, even if a plurality of dichroic mirrors other than three are arranged, the same operation and effect as in the second embodiment can be obtained.

(First Reference Example) Hereinafter, for reference, some useful laser scanning optical devices as in the present invention will be described. FIG. 6 is a configuration diagram showing a laser scanning optical device according to the first reference example. This laser scanning optical device is a fluorescence microscope configured substantially in the same manner as the present embodiment. However, a condenser lens 17 for measuring the fluorescence emitted from the back side of the sample 15,
The barrier filter 18 and the PMT 19 are not provided. On the reflection side of the dichroic mirror 11, a collimator lens 21 and a stacked sensor array 22 are arranged substantially in a line along the traveling direction of the fluorescence 16a emitted from the surface side of the sample 15. Collimator lens 2
1 is arranged such that the rear focal point is located at the center of the stacked sensor array 22. The stacked sensor array 22 is arranged so that the central axis in the stacking direction coincides with the linear fluorescent image of the fluorescent light 16a.

FIG. 7A is a perspective view showing the configuration of the stacked sensor array 22. FIG. FIG. 7B is a cross-sectional view of the single sensor in the stacked sensor array 22 along the layer thickness direction. The stacked sensor array 22 is configured by stacking a large number of photoelectric detectors coaxially. In each photoelectric detector, a photoelectric detector having a flat cylindrical p-type Si layer 25 on a cylindrical n-type Si layer 24 is formed on a transparent substrate 23. The transparent substrate 23 including the photoelectric detection unit
On top, SiO ₂ layers 26 and 28 are interposed as insulating layers,
A transparent electrode 27 for outputting a signal charge is provided. A metal wiring 29 exposed to the outside is connected to the transparent electrode 27.

Next, the operation of the fluorescence microscope will be described. This fluorescence microscope works in substantially the same manner as the fluorescence microscope of the present embodiment. However, the fluorescent light 16a emitted from the surface side of the sample 15 is condensed by the objective lens 14, travels in the optical path through which the laser light 2 has passed in the opposite direction, and is reflected by the dichroic mirror 11a in a direction perpendicular to the optical axis. After that, a light spot is imaged at the center of the stacked sensor array 22 by the collimator lens 21. This light spot produces a thin linear fluorescent image near the rear focal point of the collimator lens 21, and is received by the stacked sensor array 22. The stacked sensor array 22 converts the fluorescent light 16 a into an electric signal by the stacked photoelectric detectors, and outputs the electric signal from each metal wiring 29.

The electric signal output from each photoelectric detector of the sensor array 22 by photoelectric conversion corresponding to the light intensity is X
The image data is synchronized with the scanning signal of the −Y scanner 12 and stored in parallel as pixel data in a memory of an image reading device (not shown). The pixel data is processed in accordance with the scanning signal in a usual manner, and a microscopic image obtained by dividing the three-dimensional image of the sample 15 into a two-dimensional cross-sectional image equal to the number of photoelectric detectors is obtained at the same time.

According to such a fluorescence microscope, two-dimensional scanning is performed inside the sample with the center beam in which the Bessel beam is linearly imaged, and the fluorescence emitted from the sample is narrowed by the collimator lens. An image is formed on a linear light spot and received by the stacked sensor array. Thus, a three-dimensional image of the sample is obtained as a large number of two-dimensional sectional images divided in the optical axis direction. Therefore, it is not necessary to perform three-dimensional scanning inside the sample, and the scanning time of the light spot is greatly reduced.

(Second Reference Example) Next, an optical drawing apparatus will be described as a laser scanning optical apparatus according to a second reference example. FIG. 8 is a configuration diagram showing such an optical drawing apparatus. In this optical drawing apparatus, an optical modulator 32 and a plane mirror 3 are arranged along a traveling direction of a laser beam 2 emitted from a laser 1 (light source). Axial comparison (light conversion unit) 6, beam reducer 33, and XY scanner (light scanning unit) 12 are arranged along the traveling direction of laser light 2 reflected by plane mirror 3 in a direction perpendicular to the optical axis. They are arranged almost in a line. An imaging lens 13, an aperture stop 35, an objective lens 14 (light converging unit) and an XY stage along the traveling direction of the laser light 2 emitted from the XY scanner 12 in a direction perpendicular to the optical axis. 36 are arranged substantially in a line. The input terminals of the optical modulator 32, the XY scanner 12, and the XY stage 36 include:
The output terminals of the control device 37 are electrically connected to each other. On the XY stage 36, a substrate 38 whose surface is covered with a photosensitive agent 39 is provided.

The optical modulator 32 is an optical modulator that modulates the intensity, frequency, phase, polarization plane, and the like of the laser light 2 incident from the laser 1, and performs continuous irradiation and pulse irradiation of the laser light 2 by intensity modulation. select. The beam reducer 33 includes a convex lens 34a and a concave lens 3
4b, the outer diameter of the laser beam 2 incident as a cylindrical light beam 8 from the axicon pair 6 is reduced so as to match the aperture diameter of the objective lens 14. The convex lens 34a and the concave lens 34b are arranged so that their optical axes coincide. In addition, the beam reducer 33 is configured to include a moving unit (not shown) for a part of the lens group, and can continuously change the zoom ratio with respect to the diameter of the light beam of the laser light 2, that is, in a zoom manner. It has become. The aperture stop 35 has an aperture 35 having a circular aperture.
And restricts the outer diameter of the light beam of the laser light 2 incident as the cylindrical light beam 8 from the imaging lens 13. The XY stage 36 scans the substrate 38 in a direction conjugate to the scanning direction of the laser beam 2 by the XY scanner 12. The control device 37 is a microcomputer or the like, and sets the light modulation of the laser beam 2 by the light modulator 32 and sets the scanning speed and the scanning timing of the XY scanner 12 and the XY stage 36 in synchronization. The substrate 38 is a wafer formed of a semiconductor material or a chip divided from such a wafer, and is arranged on the rear image plane of the objective lens 14. The photosensitive agent 39 is a photoresist or the like, and is exposed with a high contrast at an irradiation position of the laser beam 2 incident as a Bessel beam from the objective lens 14.

Next, the operation of the optical drawing apparatus will be described. The laser light 2 emitted from the laser 1 is optically modulated by the optical modulator 32 and then reflected by the plane mirror 3 in a direction perpendicular to the optical axis. In the axicon pair 6, the laser light 2 emitted from the plane mirror 3 is converted into parallel light whose traveling direction is substantially parallel to the optical axis by the axicon lenses 7a and 7b, and a ring orthogonal to the optical axis. A cylindrical light beam 8 having a band-like cross-sectional intensity is obtained. Here, the diameter of the cylindrical light beam 8 shown by a solid line is freely and continuously variable in accordance with the distance between the axicon prisms 7a and 7b, and the diameter of the imaging lens 13 after passing through the beam reducer 33. The aperture is set to match the aperture diameter of the objective lens 14 under the total magnification including the pupil imaging magnification.

The laser light 2 emitted as a cylindrical light beam 8 from the axicon compare 6 is reduced in diameter by a beam reducer 33 to a predetermined value while maintaining the traveling direction in the optical axis direction. At this time, in the annular cross section orthogonal to the optical axis of the cylindrical light beam 8, the annular width is simultaneously compressed. The laser beam 2 emitted from the beam reducer 33 is emitted by the XY scanner 12 in a direction perpendicular to the optical axis, and its traveling direction is changed within a predetermined angle range, and the laser beam 2 is orthogonal to the plane. Swung in the direction.

The laser beam 2 emitted from the XY scanner 12 forms an optical spot on the front image plane of the objective lens 14 by the imaging lens 13. This light spot is XY
It can also be called a virtual light source that is two-dimensionally shaken by the scanner 12. From this light spot, a cylindrical light beam 8
After the outer diameter of the light beam is set to a predetermined value by the aperture stop 35, the emitted laser light 2 is transmitted through only the peripheral portion of the objective lens 14, and is emitted as a light spot narrowed down to the diffraction limit. The substrate 38 is irradiated as a certain Bessel beam. This Bessel beam has a diffracted light intensity distribution composed of an extremely elongated linear central beam having a large intensity in the optical axis direction and a number of coaxial cylindrical high-order diffracted lights existing around the central beam.

XY scanner 12 and XY stage 3
6 is set to a scanning speed and a scanning timing synchronized with each other based on a control signal output from the control device 37. Therefore, the Bessel beam is changed to the XY scanner 12 and the X-
Two-dimensional scanning on the surface of the substrate 38 by the Y stage 36,
For example, a raster scan is performed. In the photosensitive agent 39, only a portion irradiated with a Bessel beam having a light intensity portion equal to or higher than a predetermined threshold value is exposed. Accordingly, various patterns of the integrated circuit input to the control device 37 are exposed to the photosensitive agent 39 on the surface of the substrate 38.

According to such an optical drawing apparatus, the laser beam passes through only the peripheral portion of the objective lens as a cylindrical light beam, thereby forming an elongated linear image formed with high intensity in the optical axis direction. A Bessel beam is applied to the surface of the substrate as a center beam. As a result, since the Bessel beam has a long focal depth, the photosensitive agent on the substrate is exposed without being affected by the unevenness of the substrate surface. Therefore, it is not necessary to strictly adjust the position of the light spot of the laser beam with respect to the photosensitive agent, so that work efficiency is improved.

The Bessel beam has a coaxial cylindrical peripheral beam around a linear center beam, but has only two levels of ON / OFF as writing light for a photosensitive agent having high contrast. Does not matter. Therefore, since the Bessel beam has a high resolution, exposing a pattern of the focusing circuit to the photosensitive agent on the substrate increases the degree of integration of an integrated circuit formed based on this pattern.

(Third Embodiment) Next, a laser repair apparatus will be described as a laser scanning optical apparatus according to a third embodiment. FIG. 9 is a configuration diagram showing such a laser repair device. This laser repair device is configured in substantially the same manner as the second reference example relating to the optical drawing device. However, unlike the substrate 38, an IC is provided on the XY stage 36.
(Integrated Circuits) A chip 42 is provided. Further, the imaging lens 13 is moved by moving means (not shown).
In the optical path between the lens and the objective lens 14, a deflection prism 40
Is inserted and arranged. In this case, the eyepiece 41 is arranged in a direction in which the reflected light from the IC chip 41 is emitted from the deflection prism 40 via the objective lens 14 and the aperture stop 35.

The deflection prism 40 is at 30 ° with respect to the optical axis.
Or a total reflection surface which makes a declination in a direction such as 45 °
The light incident from the C chip 42 via the objective lens 14 and the aperture stop 35 is reflected. The eyepiece 41 converges light incident from the deflection prism 40 to form an image. The IC chip 42 has an integrated circuit formed in the surface region, and is arranged on the rear image plane of the objective lens 14. The integrated circuit that constitutes the IC chip 42 is one in which a partial failure has been created during manufacturing or has occurred during operation.

Next, the operation of the laser repair device will be described. This laser repair device operates in substantially the same manner as the optical drawing device of the second reference example. However, the laser light 2 emitted through only the peripheral portion of the objective lens 14 is irradiated on the IC chip 42 as a Bessel beam which is a light spot narrowed down to the diffraction limit. In the IC chip 42, only the irradiated portion of the Bessel beam having a light intensity portion equal to or higher than a predetermined threshold value is subjected to etching removal. Therefore, the integrated circuit formed on the IC chip 42 corresponding to the various patterns of the integrated circuit input to the control device 37 is restored.

According to such a laser repair device, the laser beam passes through only the peripheral portion of the objective lens as a cylindrical light beam, thereby forming an elongated linear image formed with high intensity in the optical axis direction. A Bessel beam is applied to the surface of the substrate as a center beam. As a result, since the Bessel beam has a long depth of focus, the integrated circuit in the surface region of the IC chip is etched without being affected by the unevenness of the integrated circuit itself. For this reason, it is not necessary to strictly adjust the light spot of the laser light with respect to the substrate of the laser light, so that work efficiency is improved.

Although the Bessel beam is accompanied by a coaxial cylindrical peripheral beam around a linear central beam, the energy of the peripheral beam with respect to the central beam can be reduced by adjusting the output of the laser. Therefore, since the Bessel beam has a high resolution, a structurally minute obstacle generated in the integrated circuit is repaired.

[0079]

As described above, the laser scanning optical apparatus of the present invention includes the first and second photoelectric detectors for detecting the fluorescence emitted from the front side and the back side of the sample, respectively. Thereby, the fluorescence emitted from the sample is detected with little loss, and the contrast in the microscopic image generated based on the sum of the output signals of the photoelectric detectors is improved. In addition, since it becomes possible to use a low-power laser light source,
Damage to a biological sample or the like can be reduced.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a fluorescence microscope which is a first embodiment of a laser scanning optical device according to the present invention.

FIG. 2 is a configuration diagram showing an axicon compare in the fluorescence microscope of FIG. 1 in more detail;

FIG. 3 is a perspective view showing a configuration of an axicon prism in the axicon pair of FIG. 2;

4A is a graph showing an intensity distribution in a plane perpendicular to an optical axis direction of a laser beam incident on the axicon compare of FIG. 2; FIG. FIG. 3C is a configuration diagram illustrating a case where the distance between the axicon prisms is variably set, and FIG. 3C illustrates the in-plane intensity of the laser light emitted from the axicon pair in FIG. 2, which is perpendicular to the optical axis direction. It is a graph which shows distribution.

FIG. 5 is a configuration diagram showing a fluorescence microscope which is a second embodiment of the laser scanning optical device according to the present invention.

FIG. 6 is a configuration diagram showing a fluorescence microscope according to a first reference example.

7A is a perspective view illustrating a configuration of a stacked sensor array in the fluorescence microscope in FIG. 6, and FIG. 7B is a view along a layer thickness direction of a single sensor in the stacked sensor array in FIG. It is sectional drawing.

FIG. 8 is a configuration diagram illustrating an optical drawing apparatus according to a second reference example.

FIG. 9 is a configuration diagram showing a laser repair device according to a third reference example.

FIG. 10 is a configuration diagram showing a conventional confocal laser scanning fluorescence microscope.

11A is a configuration diagram illustrating a conventional light conversion unit that generates a Bessel beam, and FIG. 11B is a diagram illustrating a Bessel beam emitted from the light conversion unit in FIG. 11A that is perpendicular to the optical axis direction. It is a graph which shows the intensity distribution in a plane.

12A is a configuration diagram showing the shape of the aperture in the light conversion unit in FIG. 11A, and FIG. 12B is a diagram showing the change in the ratio of the inner diameter to the outer diameter at the opening of the aperture in FIG. FIG. 7 is a graph showing a variation of an intensity distribution in a plane perpendicular to the optical axis direction in the Bessel beam in a corresponding case, and FIG. 7C is a graph showing an enlarged main part in the intensity distribution of FIG.

13 (a) and (b) show that the ratio of the inner diameter to the outer diameter at the opening of the aperture of FIG. 12 (a) is 0, respectively.
11 is a graph illustrating an in-plane intensity distribution of a Bessel beam including an optical axis when the ratio is 0.7.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser, 2 ... Laser light, 3 ... Plane mirror, 4 ... Beam expander, 5a, 5b ... Convex lens, 6 ... Axicon compare, 7a, 7b ... Axicon prism, 8 ... Cylindrical light beam bundle, 9 ... Beam reducer , 10a, 10b ... convex lens, 11a to 11c ... dichroic mirror, 12 ...
X-Y scanner, 13: imaging lens, 14: objective lens, 15: sample, 16a, 16b: fluorescence, 17: condenser lens, 18: barrier filter, 19a to 19c ...
PMT, 20a to 20c: PMT, 21: Collimator lens, 22: Stacked sensor array, 23: Transparent substrate, 2
4: n-type Si layer, 25: p-type Si layer, 26: SiO
₂ layers, 27: transparent electrode, 28: SiO ₂ layer, 29: metal wiring, 30: confocal pinhole plate, 31b, 3
1c: dichroic mirror, 32: optical modulator,
33: beam reducer, 34a, 34b: concave lens,
35 eyepiece lens, 36 aperture stop, 37 XY scanner, 38 control device, 39 substrate, 40 photosensitive agent, 4
DESCRIPTION OF SYMBOLS 1 ... Right angle prism, 42 ... IC chip, 43 ... Aperture, 44 ... Aperture, 45 ... Diffraction light, 46 ... Convex lens, 4
7 ... Vessel beam.

Claims (1)

(57) [Claims] 1. A laser scanning optical device for irradiating a predetermined sample labeled with a predetermined fluorescent dye with laser light and detecting fluorescence emitted from the sample to obtain a microscopic image of the sample. A light source that oscillates the laser light as a pulse having a very short time width; and a light scanning unit that irradiates the sample while scanning the laser light while changing a traveling direction of the laser light emitted from the light source. A first photoelectric detector that detects fluorescence emitted from the front side of the sample based on multiphoton absorption due to the irradiation of the laser light, and a back side of the sample based on multiphoton absorption due to the irradiation of the laser light And a second photoelectric detector that detects fluorescence emitted from the light source. The sum signal of the output signals of the first and second photoelectric detectors is converted into pixel data in synchronization with the scanning of the laser light. A laser scanning optical device for accumulating and generating a microscopic image of the sample based on the pixel data.