JP2015094887A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a confocal microscope which uses optical fibers for illumination and photoreception systems.SOLUTION: A confocal microscope 10 comprises a polarization-maintaining fiber bundle combiner 12, a beam collimator 14, a polarized beam splitter 16, a scanner 18, a wideband 1/4 wavelength plate 20, an objective lens 22, an imaging lens 24, and a fiber bundle combiner 26. Only reflected light from a focal position of the objective lens 22 located above a sample 2 efficiently travels against a direction of incident light. The reflected light goes through the wideband 1/4 wavelength plate 20 to have a polarization direction thereof turned by 90 degrees, and then is reflected by the polarized beam splitter 16. The reflected light reflected by the polarized beam splitter 16 enters an end face of the fiber bundle combiner 26 via the imaging lens 24. The end face of the fiber bundle combiner 26 is located at a position conjugate with the focal position of the objective lens 22 located above the sample 2.

Description

  The present invention relates to a confocal microscope using an optical fiber as an illumination optical system.

  Conventionally, by irradiating a sample to be observed (specimen) with irradiation light, the light beam that has passed through a pinhole at a position conjugate with the sample is detected by a photodetector among the observation light from the sample. A confocal microscope that obtains a two-dimensional image of a sample is known. By providing a pinhole in a confocal microscope, it is possible to detect only observation light from only a thin layer in the sample in the same direction as the traveling direction of irradiated light, and obtain an image of that portion.

  In particular, the ability to adjust the observation range in the thickness direction of the sample to be observed, more specifically, the ability to adjust the observation range in the thickness direction of the sample is called sectioning resolution. It is said that the sectioning resolution is higher. For example, in a confocal microscope, the smaller the pinhole opening, the thinner the observation range in the thickness direction of the sample to be detected by the observation light, so the sectioning resolution increases.

Special table 2009-522605 gazette JP 2004-212434 A JP 2001-185756 A

Patent Documents 1 to 3 disclose techniques using an optical fiber for a confocal microscope. However, although those described in these documents use an optical fiber as a light source, a structure as a photodetector is not specifically suggested. Therefore, when irradiating a sample with a plurality of light beams at the same time, it is difficult to arrange a pinhole having a small opening at a conjugate position.
Accordingly, an object of the present invention is to provide a confocal microscope equipped with an optimal light receiving system when an optical fiber bundle is used as an illumination optical system.

  The confocal microscope of the present invention is an illumination that condenses the light having different wavelengths emitted from the end faces of a plurality of optical fibers that respectively transmit light having different wavelengths from a light source onto the observation surface of a sample to be observed as illumination optical light. An optical system, a scanner that scans the illumination light on the observation surface, an imaging optical system that collects the observation light from the sample, and a light condensing position of each wavelength of the illumination light in the sample. And a detection optical system including a plurality of optical fibers on which end faces are arranged at conjugate positions and the observation light is incident from the end faces.

  The present invention can provide a confocal microscope using an optical fiber bundle for the illumination optical system and the light receiving system. Thereby, the configuration on the illumination optical system side and the light receiving system side can be optimized according to each wavelength, so that high definition and high resolution can be realized.

It is a figure explaining the fiber bundle combiner confocal microscope which is embodiment of this invention. It is a figure for demonstrating a polarization preservation type fiber bundle combiner. It is a figure for demonstrating another polarization preservation type | mold fiber bundle combiner. It is a figure explaining the optical path for every wavelength.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram illustrating a fiber bundle combiner confocal microscope according to an embodiment of the present invention. The confocal microscope 10 includes a polarization preserving fiber bundle combiner 12, which is a light source, a beam collimator 14, a polarization beam splitter 16, a scanner 18, a broadband quarter wavelength plate 20, an objective lens 22, an imaging lens 24, and a light receiving optical system. A polarization preserving fiber bundle combiner 26 is provided. The output position of each color (R, G, B, IR (infrared light)) of the polarization preserving fiber bundle combiner 12 functions as a pinhole. Although not shown, in order to section in the thickness direction (z direction), the sample 2 is placed on a movable stage having a resolution of submicron or less in the z direction. The configurations of the polarization preserving fiber bundle combiner 12 and the polarization preserving fiber bundle combiner 26 will be described in detail later.

  The light emitted from the end face of each optical fiber of the polarization preserving fiber bundle combiner 12 is converted into parallel light by the beam collimator 14 from the light source. Although the light source light is converted into parallel light by the beam collimator 14, it is inclined with respect to the optical axis depending on the emission position. The light converted into parallel light by the beam collimator 14 passes through the polarization beam splitter 16, and the light deflected by the scanner 18 passes through the broadband quarter-wave plate 20 and the objective lens 22, and the observation surface on the observation object. Is focused on the focal point of the objective lens 22 on the sample 2. By driving the scanner 18, the sample 2 can be two-dimensionally scanned by the light beam from the light source. The exit position (exit end face) of the polarization preserving fiber bundle combiner 12 and the position of the focal point 4 of the objective lens 22 on the sample 2 are in a conjugate positional relationship. Here, in order to section the thickness direction data of the sample 2 or the surface shape, two-dimensional scanning is performed while shifting the movable stage in the z direction (not shown).

  Only the reflected light from the focal position of the objective lens 22 on the sample 2 is directed to the direction opposite to the incident light with high efficiency as observation light. Then, the polarization direction of the reflected light is changed by 90 degrees by the broadband quarter wavelength plate 20 and reflected by the polarizing beam splitter 16. The reflected light reflected by the polarization beam splitter 16 is incident on the end face of the polarization preserving fiber bundle combiner 26 of the light receiving optical system via the imaging lens 24. The position of the end face of each optical fiber of the polarization preserving fiber bundle combiner 26 is in a conjugate positional relationship with the position of the focal point 4 of the objective lens 22 on the sample 2. The end face of each optical fiber of the polarization preserving fiber bundle combiner 26 functions as a pinhole.

  Here, the configuration of the polarization preserving fiber bundle combiner 12 and the polarization preserving fiber bundle combiner 26 used in the present invention will be described with reference to FIGS. FIG. 2 is a diagram for explaining the polarization preserving fiber bundle combiners 12 and 26. FIG. 2A is a broken perspective view of the ferrule 33. FIG. 2B is a cross-sectional view of the fiber bundle fixed by the ferrule 33. FIG. 2C is a diagram for explaining which fiber each fiber 32 shown in FIG. 2B is.

The ferrule 33 is configured in a cylindrical shape by, for example, zirconia. The ferrule 33 fixes a total of seven fibers, one R fiber 31r, one G fiber 31g, and one B fiber 31b, and four D fibers 31d, in a cylindrical through hole 33a. In FIG. 1, a part of the D fiber is replaced with an IR fiber. In FIG. 1, the R fiber, G fiber, B fiber, and IR fiber are illustrated as protruding from the ferrule 33, but are aligned at the end of the ferrule 33 as shown in FIG. May be arranged.

Each fiber 31 has a core 311 and a clad 312 that covers the periphery of the core. The core 311 is formed at the center of the core of the fiber 31 and transmits laser light. The clad 312 is formed on the outer periphery of the core 312 and has a refractive index lower than that of the core 311.
In the plurality of fibers 31, the other six fibers are concentrically arranged so as to surround the G fiber 31 g as a center. Further, the R fiber 31r, the G fiber 31g, and the B fiber 31b are arranged so as to be aligned in the A direction in FIG. The diameter of each fiber 31 is substantially equal, and the distance between two adjacent cores 311 is also substantially equal. The ferrule 33 fixes the fiber bundle bundled in such an arrangement.

  In the polarization preserving fiber bundle combiner 12 for illumination, the R fiber 31r, the G fiber 31g, and the B fiber 31b transmit laser beams emitted from R, G, and B laser diodes (not shown) that are light sources. The D fiber 31d is a dummy fiber.

  The ferrule 33 is an example of a fixture, and the R fiber 31r, the G fiber 31g, the B fiber 31b, and the D fiber 31d are bundled and fixed at the end opposite to the laser light source (not shown). The RGB laser beams are emitted from the emission end face of each optical fiber 31 at the end of the ferrule 33.

  FIG. 3 is a diagram for more specifically explaining the polarization preserving fiber bundle combiners 12 and 26 used in FIG. In the example shown in FIG. 3, one of the dummy D fibers shown in FIG. 2 is a fiber that outputs infrared rays. This fiber is called IR fiber. The IR fiber is an example of an infrared irradiation / light receiving fiber. The position of the IR fiber may be any of the four locations (other than the RGB fiber) where the D fiber was present. 3A shows a cross-sectional view of the fiber bundle combiner 12 of the illumination optical system, and FIG. 3B shows the arrangement of the polarization preserving fiber bundle combiner 26 for the detector (light receiving optical system). In FIG. 3B, the R fiber, G fiber, B fiber, and IR fiber are connected to the R light receiving element, the G light receiving element, the B light receiving element, and the IR light receiving element, respectively.

  FIG. 4 is a diagram for explaining an optical path for each wavelength. In FIG. 4, for simplicity, only the optical path of blue light emitted from the B fiber and the optical path of infrared light emitted from the IR fiber are shown. As an illumination optical system, each of the R, G, B, and IR laser light emitted from the R, G, B, and IR laser light sources is supplied with a beam collimator 14, a polarization beam splitter 16, a scanner 18, and a telecentric relay optical system 28. Then, the light enters the objective lens 22 and is condensed as illumination light at the focal point on the sample 2. The positions of the different fibers serving as the R, G, B, and IR laser light sources are angle-converted by the beam collimator 16. The scanner 18 scans the focal point of the objective lens 22 around the home position of the MEMS by the MEMS.

In order to prevent vignetting between the position of the scanner 18 and the position of the entrance pupil of the objective lens 22, a bilateral telecentric relay optical system 28 having a 4f arrangement is used.
Then, as the light receiving optical system, the reflected light reflected from the sample 2 passes through the optical path opposite to the incident light and reaches the incident end face of each optical fiber of the polarization preserving fiber bundle combiner 26 as observation light. The incident end face of the polarization preserving fiber bundle combiner 26 has a function as a pinhole. Then, the position of the focal point on the sample 2 and the incident end face of the polarization preserving fiber bundle combiner 26 are in a conjugate relationship. That is, the output light forms an image at the position of the polarization preserving fiber bundle combiner 26 of the light receiving optical system conjugate with the position of the polarization preserving fiber bundle combiner 12 of the illumination optical system.

  The fiber bundle combiner confocal microscope R laser light, G laser light, B laser light, and IR laser light according to the embodiment of the present invention are transmitted by individual optical fibers constituting the fiber bundle combiner on the illumination optical system side and the light receiving system side. Is transmitted. By using individual optical fibers optimized for individual wavelengths, it is possible to design optimally for each wavelength. In this embodiment, each optical fiber is bundled with a ferrule. However, a cylindrical ferrule is not used, and each optical fiber may be arranged and fixed in a straight line.

  For example, since the wavelength can be spatially separated from the light receiving side, it is not necessary to use a color filter or the like, and simultaneous scanning of R laser light, G laser light, B laser light, and IR laser light is possible.

  If the sample 2 is an eyeball such as a human, a scanning laser ophthalmoscope can be configured by an optical system equivalent to the present invention.

2 Sample 4 Focus 10 Confocal microscope 12 Polarization preserving fiber bundle combiner 14 Beam collimator 16 Polarizing beam splitter 18 Scanner 20 Broadband quarter wave plate 22 Objective lens 24 Imaging lens 26 Polarization preserving fiber bundle combiner 28 Telecentric optical system

f Focal length B Blue G Green R Red IR Infrared color

Claims (3)

A light source comprising a plurality of optical fibers for transmitting light of different wavelengths,
An illumination optical system that condenses the light having different wavelengths emitted from the end face of the light source as illumination light on the observation surface of the observation object;
A light receiving optical system including a plurality of optical fibers that collect and receive the observation light from the observation object;
The end face of the light source and the condensing position where the illumination light is condensed on the observation surface of the observation object are in a conjugate position,
A confocal microscope characterized in that a condensing position where the illumination light is condensed on an observation surface of the observation object and an end surface where the light receiving optical system receives light are in a conjugate position.   The confocal microscope according to claim 1, wherein the light having different wavelengths is red, blue, and green laser light.   The confocal microscope according to claim 2, wherein the light having different wavelengths further includes infrared light.

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