JPH02267513A - Con-focal laser scan fluorescence microscope - Google Patents

Abstract

PURPOSE:To obtain this microscope being small in size and light in weight, to simplify its adjustment and to reduce its cost by using a semiconductor laser as a light source, and also, integrating all of a waveguide, an electrode and a mode splitter, a branch waveguide and a photodetector into the same substrate. CONSTITUTION:A semiconductor laser 2 is used as a light source of a con-focal microscope, and a waveguide 3 for generating a second higher harmonic, an electrode 4 and mode splitters 5, 8, branch waveguides 6, 12 and a photodetector 15 are all integrated into the same substrate 1. By generating a second higher harmonic by this semiconductor laser 2 and the waveguide 3 and the electrode 4, a laser light beam of a small size and short wavelength can be realized instead of a conventional Ar laser or He-Cd laser. Also, since the laser light source 2 and the photodetector 15 are integrated into the same substrate 1, it is unnecessary to provide a mirror and a prism, the device is simplified, its adjustment is simplified and its cost is reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a microscope, and more particularly to a confocal laser scanning fluorescence microscope.

[Summary of the invention]

In a laser scanning microscope, a leading waveguide creates a second harmonic light source of a semiconductor laser and at the same time constitutes a confocal type microscope. The fundamental wavelength is removed by a mode splitter, and only the necessary fluorescence from the object is captured by a second mode splitter into a photodetector. It is small, lightweight, and easy to manufacture.

[Conventional technology]

Conventionally, a confocal laser scanning fluorescence microscope has the following configuration.

Conventionally, argon lasers (Ar lasers), helium cadmium lasers (He--Cd lasers), and the like have been used as short wavelength laser light sources for laser scanning fluorescence microscopes. Furthermore, since the laser light source and the photodetector were located apart from the microscope main body, the laser light source was guided by reflection and refraction using mirrors, prisms, etc. from the microscope to the microscope and from the microscope to the photodetector. Furthermore, in order to spectroscopically measure the fluorescence output from the test object, a separate optical system dedicated to spectroscopic measurement has been added.

[Problem to be solved by the invention]

Conventional confocal laser scanning fluorescence microscopes use short wavelength laser light sources that are extremely large and expensive.

Furthermore, since the laser light source and photodetector are separated from the microscope body, it is necessary to construct an optical system with mirrors, prisms, etc. to guide the light, making the device large and difficult to adjust. Furthermore, in order to spectroscopically measure the light output from the microscope, it was necessary to add an optical system dedicated to spectroscopically measuring, which resulted in a large device and difficulty in adjustment.

The present invention was made in view of such conventional problems,
The aim is to create a confocal laser scanning fluorescence microscope that is small, lightweight, and easy to adjust.

[Means to solve problems]

The confocal laser scanning fluorescence microscope of the present invention includes a light source consisting of a semiconductor laser (2), a waveguide (3) and an electrode (4) for producing a second harmonic (S), and a mode splitter (5).
), a branching waveguide (6), a second mode splitter (8), a second branching waveguide (12), and a photodetector (1
4), and the objective lens (l).
O).

[For production]

In the present invention, instead of the extremely large and expensive Ar laser or He-Cd laser, the second harmonic is generated from the semiconductor laser (2), the waveguide (3), and the electrode (4). A short wavelength laser light source can be realized, and
Furthermore, by assembling all the optical systems on the same substrate (1) and also connecting the semiconductor laser (2) and photodetector (15) to the substrate (1), the device can be overwhelmingly miniaturized and simplified. made possible. Furthermore, spectroscopic measurement is also possible using the second mode splitter (8) and mode converter (13) that are incorporated in the same substrate (1) in the same way as the optical system. Furthermore, not only is it easier to assemble and adjust the optical system, but also the connection of the light source and photodetector simplifies assembly and adjustment, resulting in a significant reduction in manufacturing costs.

In addition, in the confocal laser scanning fluorescence microscope of the present invention, the end face (3a) of the waveguide, which is the exit position of the light from the optical system, and the light reflected from the test object (11) are connected to the waveguide of the optical system. Since the incident point is the same as that of the laser beam, it automatically becomes a confocal laser scanning microscope, eliminating the need to adjust the position of the pinhole detector that was previously required.

〔Example〕

FIG. 1 shows an embodiment of the invention. In Figure 1, (1) is a substrate made of lithium niobate (LiN), which has electro-optic effects, nonlinear optical effects, and birefringence.
A crystal such as bOs) or a polymer material is used.

(2) is a semiconductor laser, and the emitted light is transmitted to the substrate (1).
The light enters the waveguide (3) formed above and propagates.

The waveguide (3) is a channel waveguide obtained by subjecting the material forming the substrate (1) to metal diffusion or the like. The light emitting portion of the semiconductor laser (2) is directly connected to or close to the end face of the waveguide (3). Note that a lens system or a light guiding optical fiber may be interposed between the light emitting portion of the semiconductor laser (2) and the entrance of the waveguide (3).

A nonlinear polarized wave excited by the incident laser beam is generated in the waveguide (3), and a second harmonic wave is generated. for example,
In a channel waveguide made of LiNbO5 with 2-cut X propagation, the nonlinear optical constant (Is
The second harmonic of 7M mode is generated via +. At this time, in order to efficiently extract the second harmonic, the fundamental wave and the second
It is necessary to achieve phase matching between harmonics. For this purpose, phase matching is achieved by controlling the voltage applied to the electrode (4) along the waveguide (3). The above is well known.

In the present invention, a new substrate (1) is added to the above configuration.
).

(5) is the fundamental wave traveling in the waveguide (3) and the second wave.
Separates the harmonics and guides the fundamental wave to the branch waveguide (6),
It is a mode splitter. As the mode splitter (5), other than the directional coupler shown in the figure in which two channel waveguides run in parallel, a mode splitter using a double mode region or the like may be used.

(6a) is the end face of the branch waveguide, and the branch waveguide (6)
The waveguide is coated with a light-absorbing material, such as a black paint with a refractive index almost the same as that of the waveguide, so as to absorb the fundamental wave propagating through the waveguide. The end face (6a) of the branch waveguide is not limited to the above-mentioned one, but may be polished diagonally or cut into an uneven shape, as long as it has a structure that allows the fundamental wave to escape to the outside and disappear. It is sufficient that the fundamental wave propagated through the branch waveguide (6) is not reflected at the end face (6a) of the branch waveguide and returned to the semiconductor laser (2).

(3a) is the end face of the waveguide (3), which emits the second harmonic from which the fundamental wave has been removed by the mode splitter (5) from the substrate (1) to the external objective lens (lO).

(lO) is an objective lens, and (11) is a test object.

The end face (3a) of the waveguide and the test object (11) are in a conjugate relationship with respect to the objective lens (10).

(8) is the second mode splitter, which transmits the second harmonic wavelength range and returns to the light source, but the visible light wavelength range (
500-700 nm) is designed to be power coupled to the second branch waveguide (12).

(12) is a second branching waveguide, which sends visible light from the object (11) combined by the second mode splitter (8) to the photodetector (15).

(15) is a photodetector. The light receiving portion of the photodetector (15) is directly coupled to the output end face of the second branch waveguide (12). The output end face and the light receiving section or the case of the light receiving section may be fixed with adhesive or the like. In addition, the photodetector (
15) may be somewhat distant from the output end face of the branch waveguide (12), and a lens system, a light guiding optical fiber, etc. may be interposed between the output end face and the photodetector.

The operation of this embodiment will be explained using FIG.

One of the TE mode light and 7M mode light (for example, TB mode light) that entered the waveguide (3) from the semiconductor laser (2) is transmitted to the part of the waveguide (3) where the electrode (4) is loaded. When acted as a phase compensation device, a phase matching condition is obtained and the second harmonic is Tl! It is generated as the other of mode light and 7M mode light (for example, 7M mode light). The residual part of the fundamental wave and the second harmonic are both transferred to the waveguide (3).
to reach the mode splitter (5). In the mode splitter (5), the remaining part of the fundamental wave of the TE mode light is combined and proceeds through the branching waveguide (6), where it is absorbed or scattered at the end face (6a) of the branching waveguide. The second harmonic of the 7M mode light that was not coupled in the mode splitter (5) further travels through the waveguide (3) and reaches the end face (3a) of the waveguide.
Emits from. The emitted second harmonic is passed through the objective lens (lO
) to create a spot on the test object (11).

The end face (3a) of the waveguide, the objective lens (10), and the test object (11) constitute a confocal type microscope.

In other words, the out-of-focus image of the spot on the test object (11) is redirected from the end face (3a) of the waveguide to the waveguide (
3), a confocal type is constructed that cuts out the out-of-focus areas.

When the test object (11) is a fluorescent object, the test object (11)
The reflected light contains fluorescent light with a much longer wavelength than the incident light. The reflected light reaches the end face (3a) of the waveguide again by the objective lens (10) and enters the waveguide (3). The fluorescent portion of the reflected light is combined by a second mode splitter (8) and guided to a second branch waveguide (12). However, the light coupled by the second mode splitter (8) is limited to a specific wavelength.

The second mode splitter (8)
Directly reflected light from the harmonic test object (11) passes through the waveguide (
3) and returns to the semiconductor laser (2). However, the second
Even if the harmonics return to the laser, the wavelengths of the output light and the returned light are considerably different, so they do not become output noise of the semiconductor laser (2).

The desired fluorescent light output is output from the second branch waveguide (12) and incident on the photodetector (15), where it becomes an electrical signal.

Note that a scanning system necessary for a laser scanning microscope, such as a galvano mirror, a polygon mirror, or a moving stage, is not shown.

Furthermore, the configuration of the relationship between the end face (3a) of the waveguide, the objective lens (10), and the test object (11) is a so-called confocal system, because the cross section of the waveguide is sufficiently small. (several μm).

Furthermore, if a large number of confocal laser scanning fluorescence microscopes of the present invention are arranged side by side, one-dimensional scanning can be achieved by laser switching or second harmonic generation voltage switching, leading to an improvement in scanning speed. .

[Second Embodiment] FIG. 2 shows a second embodiment of the present invention. In FIG. 2, the same symbols as in FIG. 1 represent the same effects. In FIG. 2, electrodes are attached to the surface of the substrate (1) on both sides of the second mode splitter (8).

By gradually changing the voltage applied to the electrodes of the second mode splitter (8), the wavelength that can be coupled also changes gradually, making it possible to perform spectroscopic measurements of the reflected light from the test object (11).

[Third Embodiment] FIG. 3 shows a third embodiment of the present invention. In FIG. 3, the same symbols as in FIG. 1 represent the same effects. Furthermore, (13) is a mode converter, and (I4) is a polarizing filter. Here, the mode converter (13) refers to the TE mode light.
This is a polarization state conversion device that converts M mode light and 7M mode light to TE mode light, but since the conversion efficiency has extremely high wavelength dependence, it is also used as a wavelength selection filter.

In FIG. 3, when it is desired to perform spectroscopic measurement of the fluorescent light reflected from the test object (11), instead of the spectroscopic measurement using the second mode splitter (8) of the second embodiment, the mode converter (13 ), the wavelength selectivity will be much sharper, and the wavelength selectivity will be much sharper.
There is no need to worry about fluorescent light returning to the screen. Polarizing filter (14)
is for cutting out light that has not been mode converted.

〔Effect of the invention〕

In the present invention, instead of using an extremely large and expensive A laser or He-Cd laser, the second harmonic is generated from a semiconductor laser (2), a waveguide (3), and an electrode (4). A compact short wavelength laser light source can be realized, and
Furthermore, by assembling all the optical systems on the same substrate (1) and also connecting the semiconductor laser (2) and photodetector (15) to the substrate (1), the device can be overwhelmingly miniaturized and simplified. made possible. Furthermore, spectroscopic measurement is also possible using the second mode splitter (8) and mode converter (13) that are incorporated in the same substrate (1) in the same way as the optical system. Furthermore, not only is the assembly and adjustment of the optical system easier (the connection of the light source and photodetector simplifies the assembly and adjustment of the entire microscope, but the manufacturing cost of the entire microscope is significantly reduced).

In addition, in the confocal laser scanning fluorescence microscope of the present invention, the end face (3a) of the waveguide, which is the exit position of the light from the optical system, and the light reflected from the test object (11) are connected to the waveguide of the optical system. Since the incident point is the same as the one where the light enters again, a confocal laser scanning fluorescence microscope with good performance is automatically obtained, and the position adjustment of the pinhole detector, which was necessary in the past, is no longer necessary.

[Brief explanation of drawings]

FIG. 1 is a block diagram of a first embodiment of the present invention, FIG. 2 is a block diagram of a second embodiment of the present invention, and FIG. 3 is a block diagram of a third embodiment of the present invention. [Explanation of symbols of main parts] (1)...Substrate, (2)...Semiconductor laser, (3)...Waveguide, (3a)...End face of waveguide, (4)...・Electrode, (5)...Mode splitter, (6)...Branch waveguide, (8)...Second mode splitter, (10)...
Objective lens, (11)...Test, (12)...Second branch waveguide, (15)...Photodetector.

Claims (3)

[Claims] (1) A light source consisting of a semiconductor laser, a waveguide and electrode that generates a second harmonic, a mode splitter, a branching waveguide, a second mode splitter, a second branching waveguide, and a photodetector. and an objective lens, characterized in that the end face of the waveguide and the test object are arranged in a confocal relationship with the objective lens.
Confocal laser scanning fluorescence microscope. (2) The microscope according to claim 1, characterized in that the second mode splitter has spectroscopy ability.
Confocal laser scanning fluorescence microscope. (3) A confocal laser scanning fluorescence microscope according to claim 1, wherein a mode converter is provided in the second branch waveguide.