JP2015203708A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device capable of capturing a high contrast image of a sample even when an optical axis of illumination light intersects with an optical axis of observation light.SOLUTION: A microscope device 1 includes: an objective lens 4 for collecting observation light emitted from a sample S under observation; an illumination unit 8 which emits sheet-like illumination light for illuminating the sample along an axis substantially perpendicular to an optical axis of the objective lens; an optical member 5 which bends the sheet-like illumination light emitted by the illumination unit to direct the same toward the objective lens along an axis that is substantially parallel to but different from the optical axis of the objective lens and which transmits the observation light collected by the objective lens; and an image capturing unit 7 which receives the observation light passing through the optical member and captures an image. The objective lens reflects the illumination light along an axis at an angle with the optical axis of the objective lens before outputting the illumination light.

Description

  The present invention relates to a microscope apparatus that irradiates a specimen with illumination light from a direction substantially intersecting an observation optical axis, receives light generated from the specimen, and observes the specimen image.

  Conventionally, in the fields of medicine and biology, a microscope for illuminating and observing a specimen is used for observing cells and the like. In the industrial field, microscopes are used for various purposes such as quality control of metal structures, research and development of new materials, inspection of electronic devices and magnetic heads, and the like. In addition to visual observation using an eyepiece lens, observation of a specimen with a microscope is known by taking an image of a specimen using an image sensor such as a CCD or CMOS image sensor and observing the captured image on a monitor display. Yes.

  In recent years, in a microscope, a technique for removing disturbance light from other than the observation focal plane and obtaining an observation image with high contrast has been proposed (see, for example, Patent Document 1). In Patent Document 1, an observation image is obtained by irradiating a specimen with sheet-like illumination light and detecting light in a direction substantially orthogonal (crossing) with the optical axis of the illumination light as observation light.

JP 2012-108491 A

  However, in the technique disclosed in Patent Document 1, when observing a specimen stored in a cup-shaped container such as a dish used for culturing cells, the optical axis of illumination light or the optical axis of observation light is used. Since any one of the light will pass through the side surface of the container, the light is scattered by the side surface, etc., it is not possible to illuminate the sample, or to obtain the observation light emitted from the sample, In some cases, a specimen image with high contrast could not be obtained.

  The present invention has been made in view of the above, and provides a microscope apparatus capable of acquiring a high-contrast sample image even when the optical axis of illumination light and the optical axis of observation light intersect each other. The purpose is to do.

  In order to solve the above-described problems and achieve the object, a microscope apparatus according to the present invention includes an objective lens that captures observation light emitted from a specimen to be observed, and sheet-like illumination light that illuminates the specimen. An illumination unit that emits along an axis substantially orthogonal to the optical axis of the objective lens, and a sheet-like illumination light emitted by the illumination unit is bent to be substantially parallel to the optical axis of the objective lens. An imaging device that advances the illumination light toward the objective lens along different axes, and that passes through the observation light captured by the objective lens, and receives and images the observation light that has passed through the optical member. And the objective lens refracts and emits the illumination light along an axis inclined with respect to the optical axis of the objective lens.

  In the microscope device according to the present invention, the illumination unit includes a cylindrical lens.

  In the microscope device according to the present invention, the illumination unit includes an optical fiber scanner.

  The microscope device according to the present invention is characterized in that, in the above invention, the imaging unit is configured such that a normal line of a light receiving surface that receives the observation light is inclined with respect to an optical axis of the observation light. .

  The microscope apparatus according to the present invention is characterized in that, in the above-described invention, the microscope device includes a tilting unit that tilts the light receiving surface of the imaging unit at an arbitrary angle with respect to the optical axis of the objective lens.

  The microscope apparatus according to the present invention further includes an imaging lens that forms an image of the observation light that has passed through the optical member, and a light reflecting member that reflects the light that has passed through the imaging lens. The imaging unit receives the observation light reflected by the light reflecting member.

  Moreover, the microscope apparatus according to the present invention is characterized in that, in the above-described invention, the microscope apparatus includes a tilting unit that tilts the reflecting surface of the light reflecting member at an arbitrary angle with respect to the optical axis of the objective lens.

  According to the present invention, even when the optical axis of the illumination light and the optical axis of the observation light intersect, there is an effect that a sample image with high contrast can be acquired.

FIG. 1 is a schematic diagram showing an overall configuration of a microscope apparatus according to an embodiment of the present invention. FIG. 2 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the embodiment of the present invention, and is a diagram illustrating an illumination optical system. FIG. 3 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the embodiment of the present invention, and is a diagram illustrating an observation optical system. FIG. 4 is a schematic diagram illustrating the configuration of the main part of the microscope system according to the first modification of the present embodiment, and is a diagram illustrating another example of the configuration of the emission mechanism. FIG. 5 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the second modification of the present embodiment, and is a diagram illustrating an observation optical system. FIG. 6 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the third modification of the present embodiment, and is a diagram illustrating the observation optical system. FIG. 7 is a schematic diagram illustrating a configuration of a main part of a microscope apparatus according to Modification 4 of the present embodiment, and is a diagram illustrating an observation optical system.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment. The drawings referred to in the following description only schematically show the shape, size, and positional relationship so that the contents of the present invention can be understood. That is, the present invention is not limited only to the shape, size, and positional relationship illustrated in each drawing.

  First, an inverted microscope apparatus according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram showing the overall configuration of the microscope apparatus according to the present embodiment. As shown in FIG. 1, a microscope apparatus 1 is an inverted microscope for observing a specimen S by imaging observation light from a specimen S specifically stained with a fluorescent dye, for example. A main body 2 that forms the foundation of the apparatus 1, a stage 3 on which a specimen S is placed, and an objective lens 4 are provided. In the present embodiment, the specimen S is held in a cup-shaped container 100 such as a dish.

  The main body 2 has a concave shape, and holds an objective lens support 4 a that supports the stage 3 and the objective lens 4. The objective lens support 4a holds the objective lens 4 at a position where the optical axis of the objective lens 4 coincides with the observation optical axis N10. The main body 2 may be provided with a control board that controls the entire microscope apparatus 1. In some cases, the control board relays the power from the outside to each part, or the power supply is built in itself and relays to each part.

  The main body 2 holds a dichroic mirror 5 (optical member) that reflects the illumination light that illuminates the specimen S and transmits the light (observation light) taken in by the objective lens 4, and the observation that has passed through the dichroic mirror 5. An imaging lens 6 that forms an image by forming an image of light and an imaging unit 7 that includes an imaging device that receives and photoelectrically converts the light imaged by the imaging lens 6 are provided. The imaging unit 7 is realized using a CCD image sensor, a CMOS image sensor, or the like.

  The main body 2 has a half mirror (not shown) for branching the light imaged by the imaging lens 6 and a lens barrel (FIG. 1) for entering one of the lights branched by the half mirror. And a binocular unit (not shown) for visually observing the specimen image of the specimen S formed by the light that is attached to the barrel and formed into an image. May be. Of the light branched from the light imaged by the imaging lens 6, the other light enters the imaging unit 7.

  The main body 2 is provided with an illumination unit 8 that emits illumination light that illuminates the specimen S. The illumination unit 8 includes a light source unit 8 a that emits illumination light, and a light projecting tube 8 b that causes the light source unit 8 a to enter the dichroic mirror 5. In the light source unit 8a, a halogen lamp or the like is used as a light source. The light source can be applied in place of a halogen lamp or a laser diode. In addition, the light emitted from the light source unit 8a is transmitted to the light projecting tube 8b, converted into a sheet shape, and the sheet-shaped illumination light (sheet illumination light) is illuminated substantially orthogonal to the observation optical axis N10. An emission mechanism 81 that emits along the axis N20 is provided.

  Illumination light emitted from the light source unit 8a includes excitation light for exciting the fluorescent dye that stains the specimen S, and excitation light in a wavelength band corresponding to the wavelength band of the fluorescent dye is selected. The excitation light may be selected by using a filter that transmits light of a predetermined wavelength, or by switching the wavelength of illumination light by exchanging the light source.

  The dichroic mirror 5 bends the light in the wavelength band of the illumination light emitted from the illumination unit 8 along the observation optical axis N10 and transmits the light in the fluorescence wavelength band emitted from the sample S.

  In the present embodiment, in the imaging unit 7, the light receiving surface that receives light is orthogonal to the observation optical axis N10.

  In the microscope apparatus 1 having the above-described configuration, the sheet illumination light traveling along the illumination optical axis N20 from the illumination unit 8 is bent toward the objective lens 4 by the dichroic mirror 5, and the sample S ( Irradiate container 100). At this time, the sheet illumination light tilts with respect to the observation optical axis N10 and enters the sample S to illuminate the sample S. The observation light emitted from the specimen S by being irradiated with the sheet illumination light is taken into the objective lens 4, imaged by the imaging lens 6, and photoelectrically converted by the imaging unit 7 to generate an electrical signal. By displaying an image generated based on the electrical signal from the imaging unit 7 on the display device 200, the sample image can be observed.

  FIG. 2 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the present embodiment, and is a diagram illustrating an illumination optical system. FIG. 3 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the present embodiment, and is a diagram illustrating an observation optical system.

  The emission mechanism 81 transmits the illumination light emitted from the light source and emits the radial illumination light, and converts the radial illumination light emitted from the optical fiber 811 into unidirectional illumination light (parallel light). And a cylindrical lens 812 that emits light. The emission mechanism 81 emits substantially sheet-shaped light (sheet illumination light) as illumination light by the conversion of illumination light by the cylindrical lens 812. The sheet illumination light preferably has a sheet surface formed by light orthogonal to the observation optical axis N10.

  The sheet illumination light emitted from the emission mechanism 81 enters the dichroic mirror 5. The sheet illumination light is bent by the dichroic mirror 5 along the observation optical axis N <b> 10 (in a substantially parallel direction) and enters the objective lens 4. Here, the sheet illumination light bent by the dichroic mirror 5 travels on an axis that is substantially parallel to the observation optical axis N10 and different from the observation optical axis N10. For this reason, the sheet illumination light enters the end side of the objective lens 4 and travels on an axis that is refracted and inclined with respect to the observation optical axis N10 when it is emitted to the outside. The axis inclined by the refraction is an axis passing through the sample S and intersects the observation optical axis N10. The axis inclined by refraction is an angle at which the inclination angle with respect to the bottom surface of the container 100 is transmitted through the container 100 (not totally reflected at the interface). In other words, the sheet illumination light is incident at an angle that is not totally reflected by the bottom surface of the container 100. The sheet illumination light emitted from the objective lens 4 irradiates the specimen S (see FIG. 2).

  The specimen S emits observation light (fluorescence) when the fluorescent dyes F1 and F2 are excited by irradiation with sheet illumination light, for example (see FIG. 4). Observation light emitted from the specimen S (fluorescent dyes F1 and F2) is taken into the objective lens 4 and travels along the observation optical axis N10 (solid line in FIG. 4). Thereafter, the observation light passes through the dichroic mirror 5, is imaged by the imaging lens 6, and enters the imaging unit 7. The observation light incident on the imaging unit 7 is photoelectrically converted by the image sensor, and an electric signal including information constituting the observation image of the sample S is generated.

  Thus, the illumination light traveling on the optical axis shifted from the optical axis of the objective lens 4 (observation optical axis N10) by being reflected by the dichroic mirror 5 is incident on the objective lens 4 and is tilted by refraction. Is incident on the specimen S, so that the specimen S is irradiated with the illumination light without being interfered with by the side surface of the container 100, and the light (observation light) emitted by the illumination light is emitted. ) Can be taken into the objective lens 4.

  In addition, since the illumination light that illuminates the specimen S has a sheet shape, the specimen S can be illuminated over a wider range than when the specimen S is illuminated using a point light source.

  According to the present embodiment described above, illumination light traveling on the optical axis shifted from the optical axis of the objective lens 4 (observation optical axis N10) is incident on the objective lens 4 and travels on the axis inclined by refraction. Since the sheet illumination light to be incident on the specimen S, a specimen image with high contrast can be obtained even when the optical axis of the illumination light and the optical axis of the observation light intersect.

(Modification 1 of embodiment)
FIG. 4 is a schematic diagram illustrating the configuration of the main part of the microscope system according to the first modification of the present embodiment, and is a diagram illustrating another example of the configuration of the emission mechanism. In the above-described embodiment, the emission unit 12 includes the optical fiber 811 and the cylindrical lens 812, and the optical fiber 811 emits radial illumination light. However, in the first modification, from the optical fiber scanner, The emission mechanism 82 is used. In the first modification, the light source unit 8a will be described as having a laser diode and emitting linear illumination light.

  The emission mechanism 82 transmits an illumination light emitted from the light source and emits a radial illumination light, and a condensing lens 814 that collects and emits the linear illumination light emitted from the optical fiber 813. A permanent magnet 815 provided in the optical fiber 813 and electromagnetic coils 816a and 816b provided at positions facing each other via the permanent magnet 815 constitute an optical fiber scanner.

  In the emission mechanism 82, the electromagnetic coil 816a, 816b is energized to vibrate the optical fiber 813 in one direction (the axial direction passing through each central axis of the electromagnetic coils 816a, 816b), and the sheet traveling on the vibration surface Shaped illumination light is emitted (for example, see JP2013-54185A). The emission mechanism 82 has been described as condensing the illumination light emitted from the optical fiber 813 with the condensing lens 814, but may be emitted through the cylindrical lens 812, or may be a cylindrical lens. The light may be emitted to the dichroic mirror 5 without using a lens such as 812.

(Modification 2 of embodiment)
FIG. 5 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the second modification of the present embodiment, and is a diagram illustrating an observation optical system. In the above-described embodiment, the light receiving surface of the imaging unit 7 has been described as being orthogonal to the observation optical axis N10. However, in the second modification, according to the optical axis of the sheet illumination light incident on the sample S, The normal line N30 of the light receiving surface is inclined with respect to the optical axis of the objective lens 4 (observation optical axis N10).

  When the optical axis of the sheet illumination light incident on the specimen S is inclined with respect to the observation optical axis N10, for example, each fluorescence emitted by the fluorescent dyes F1 and F2 at different positions relative to the observation optical axis N10. Are different from each other in the imaging position formed by the imaging lens 6. For this reason, it is preferable that the angle of the normal line N30 of the light receiving surface with respect to the observation optical axis N10 is adjusted so that each imaging position is positioned on the light receiving surface of the imaging unit 7. Thereby, a clearer image can be obtained.

  The imaging unit 7 is rotatable by a driving unit (tilting unit) (not shown) that is an axis on the reflecting surface, passes through the observation optical axis N10, and is orthogonal to the observation optical axis N10. The light receiving surface may be inclined at an arbitrary angle according to the angle of illumination light and the light emitted from the specimen S. The drive means includes, for example, a motor that is a drive source and a rotating member that rotates the imaging unit 7 with the power generated by the motor. The rotating member is realized using, for example, a gonio stage, and is preferably arranged so that the rotation center of the gonio stage is located on the observation optical axis N10. The motor is driven and controlled under the control of the control unit. For example, the fluorescence from the fluorescent dyes F1 and F2 is focused, and the angle of the light receiving surface where each fluorescent light is focused is calculated. The imaging unit 7 is rotated so that the angle is set.

(Modification 3 of embodiment)
FIG. 6 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the third modification of the present embodiment, and is a diagram illustrating the observation optical system. In the above-described embodiment, the light receiving surface of the image pickup unit 7 is described as being on the observation optical axis N10. However, in the third modification, the image pickup unit 7 is provided at a position off the observation optical axis N10.

  In the third modification, a reflection mirror 9 that is provided on the optical path ahead of the imaging lens 6 and reflects the light that has passed through the imaging lens 6 is provided as means for causing the observation light to enter the imaging unit 7. Observation light (imaged light) can be incident on the imaging unit 7 provided at a position off the observation optical axis N10 by the reflection mirror 9. Thereby, the arrangement position of the imaging unit 7 is limited in the main body unit 2 and the shape of the main body unit 2 is changed even when the imaging unit 7 cannot be arranged on the observation optical axis N10 or when the imaging unit 7 cannot be rotated. The observation light can be made incident on the image pickup unit 7.

  Further, in the third modification, the imaging positions formed by the imaging lens 6 are different as in the second modification described above (a plurality of imaging positions are not on one straight line orthogonal to the observation optical axis N10). In this case, the reflection mirror 9 is rotated about an axis on the reflection surface that passes through the observation optical axis N10 and is orthogonal to the observation optical axis N10, thereby reflecting the light reception surface of the imaging unit 7 and the reflection surface. The relative position of the mirror 9 with respect to the reflecting surface may be changed. This is a case where the image formation position is different or the image formation position is changed in different specimens S by tilting the reflection surface of the reflection mirror 9 at an arbitrary angle with respect to the optical axis of the objective lens 4. Even a clear image can be obtained. The drive means includes, for example, the motor and the rotating member as described above, and is driven under the control of the control unit. When a gonio stage is used as the rotating member, it is preferable that the gonio stage is disposed so that the rotation center of the gonio stage is located on the light receiving surface of the imaging unit 7.

(Modification 4 of embodiment)
FIG. 7 is a schematic diagram illustrating a configuration of a main part of a microscope apparatus according to Modification 4 of the present embodiment, and is a diagram illustrating an observation optical system. In the third modification described above, the reflection mirror 9 is described. However, in the fourth modification, the variable shape mirror 10 is provided instead of the reflection mirror 9.

  The variable shape mirror 10 has a plurality of variable portions 10a whose surface can be independently deformed (inclined), and is formed by arranging a plurality of variable portions 10a in a matrix, for example. In the deformable mirror 10, the reflection angle of light can be changed by driving each variable portion 10a and adjusting each angle under the control of a control mechanism (not shown). Thereby, the image formation positions of a plurality of fluorescence can be adjusted by individually driving the variable portions 10a without rotating the entire reflection mirror 9 as described above.

  In the above-described embodiment, the container 100 has been described as containing only the specimen S. However, the container 100 may further contain a culture solution, water (physiological saline), or the like. In this case, it is preferable to adjust the inclination angle of the optical axis of the sheet illumination light incident on the sample S by the refractive index of the liquid.

  In the above-described embodiment, the dichroic mirror 5 is used. However, instead of the dichroic mirror 5, a mirror that bends the illumination light emitted from the illumination unit 8 toward the objective lens 4 is used as an optical member. It may be.

  The above-described embodiments are merely examples for carrying out the present invention, and the present invention is not limited to these. In addition, the present invention can form various inventions by appropriately combining a plurality of constituent elements disclosed in the embodiments and modifications. It is obvious from the above description that the present invention can be variously modified according to specifications and the like, and that various other embodiments are possible within the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Main body part 3 Stage 4 Objective lens 5 Dichroic mirror 6 Imaging lens 7 Imaging part 8 Illumination part 9 Reflection mirror 10 Shape variable mirror 10a Variable part 811 813 Optical fiber 812 Cylindrical lens 814 Condensing lens 815 Permanent magnet 816a 816b Electromagnetic coil

Claims (7)

An objective lens that captures observation light emitted from a specimen to be observed;
An illumination unit that emits sheet-like illumination light for illuminating the specimen along an axis substantially orthogonal to the optical axis of the objective lens;
The sheet-like illumination light emitted from the illumination unit is bent, and the illumination light travels toward the objective lens along an axis substantially parallel to the optical axis of the objective lens and different from the optical axis. An optical member for passing the observation light taken in by the objective lens;
An imaging unit that receives and images observation light that has passed through the optical member;
With
The microscope apparatus, wherein the objective lens refracts and emits the illumination light along an axis inclined with respect to the optical axis of the objective lens.   The microscope apparatus according to claim 1, wherein the illumination unit includes a cylindrical lens.   The microscope apparatus according to claim 1, wherein the illumination unit includes an optical fiber scanner.   The microscope apparatus according to claim 1, wherein a normal line of a light receiving surface that receives the observation light is inclined with respect to an optical axis of the observation light. .   The microscope apparatus according to claim 1, further comprising a tilting unit that tilts the light receiving surface of the imaging unit at an arbitrary angle with respect to the optical axis of the objective lens. An imaging lens that forms an image of the observation light that has passed through the optical member;
A light reflecting member that reflects light that has passed through the imaging lens;
Further comprising
The microscope apparatus according to claim 1, wherein the imaging unit receives the observation light reflected by the light reflecting member.   The microscope apparatus according to claim 6, further comprising a tilting unit that tilts the reflecting surface of the light reflecting member at an arbitrary angle with respect to the optical axis of the objective lens.

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