JP2005316289A - Illumination apparatus of microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an illumination apparatus of a microscope with which an image of a local illumination diaphragm does not become a twin image. <P>SOLUTION: The illumination apparatus is provided with a local illumination optical system 2 which generates local illumination light, an illumination optical system 3 for fluorescence observation which generates illumination light for observation, a dichroic mirror 13 for illumination light synthesis which synthesizes an optical path of the local illumination light and an optical path of the illumination light for observation and a dichroic mirror 5 for fluorescence observation arranged on an optical axis 1a of an observation optical system 1 which forms an sample image M by imaging light from a sample S. The local illumination optical system 2 includes the local illumination diaphragm 12 and the illumination optical system 3 for fluorescence observation includes a visual field diaphragm 18. The dichroic mirror 13 for illumination light synthesis has a surface 13a and a backside 13b and the backside 13b has a tilt angle toward the surface 13a. The surface 13a reflects the local illumination light emitted from the local illumination optical system 2 and transmits excited light emitted from the illumination optical system 3 for fluorescence observation. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an illumination device for a microscope.

  In studies that analyze the dynamics and functions of proteins and organelles in living cells, experiments have been widely conducted in which light is irradiated to specific sites in the cells and the reaction caused by the irradiation is observed. In this experiment, a fluorescent specimen obtained by labeling a specific substance in a cell to be observed with a fluorescent reagent mainly used for light irradiation reaction by antibody staining or gene introduction is used. And according to the mode of the light irradiation reaction of various fluorescent reagents, an experimental technique utilizing the feature has been devised.

  A representative example of the experimental technique is an experiment (hereinafter referred to as a caged experiment) using a reagent called “Caged compound” (hereinafter referred to as a caged reagent). The Caged reagent has a function of binding to a protein and inactivating the activity of the protein, but the binding to the protein is released and suppressed by irradiation with UV light (light having a central wavelength of about 360 nm). Protein activity is activated. If this feature is utilized, only the portion irradiated with UV light can be activated, and therefore, it is widely used as a technique for controlling the location and time at which intracellular proteins are desired to be activated.

  In the Caged experiment, UV light is irradiated to a desired location at a desired timing in the course of fluorescence observation, and how the fluorescence dynamics change before and after the observation and how it propagates into the cell is observed. . Therefore, in order to perform a caged experiment, a local illumination optical system for releasing the caged reagent that locally irradiates UV light at a desired position in the observation range of the specimen, and for fluorescence observation that irradiates the entire observation range with excitation light. Two types of illumination optical systems are required: an illumination optical system. In particular, when the activation phenomenon after the release of the caged reagent occurs within an early time of 1 second or less, it is necessary to be able to simultaneously observe fluorescence while releasing the caged reagent.

Japanese Patent Application Laid-Open No. 7-56092 discloses an illuminating device that enables fluorescence observation while simultaneously releasing the caged reagent. This illumination apparatus includes a local illumination optical system for releasing a caged reagent having a local illumination stop, a fluorescence observation illumination optical system for excitation light irradiation having a field stop, and a local illumination emitted from the local illumination optical system. A dichroic mirror as an illumination light combining means for combining the optical path of the light and the optical path of the fluorescence observation illumination light emitted from the fluorescence observation illumination optical system, and the local illumination light and the fluorescence observation illumination light on the specimen Can be irradiated simultaneously.
Japanese Unexamined Patent Publication No. 7-56092

  Since the illumination device of Japanese Patent Laid-Open No. 7-56092 is configured for caged experiments, it is premised on performing local illumination with UV light. Therefore, a dichroic mirror is used as a synthesis means with fluorescence observation illumination by visible light excitation. Used. On the other hand, when looking at experimental methods other than the caged experiment, the light required for local illumination is not limited to UV light. For example, an experimental technique called FRAP (Fluorescence Recovery After Photobleaching) is to observe the appearance of a fluorescently labeled substance flowing in from the periphery after irradiating the desired position of the specimen with a strong excitation light and fading it. This is a technique for visualizing the movement and diffusion of substances such as specific proteins. In FRAP, excitation light having the same wavelength is used for local illumination for fading and illumination for fluorescence observation. Therefore, in this case, it is appropriate to use a half mirror instead of a dichroic mirror as the combining means.

  However, when the dichroic mirror of the illumination device disclosed in Japanese Patent Laid-Open No. 7-56092 is changed to a half mirror, the following problems occur. FIG. 7 schematically shows a part of an optical system in which the dichroic mirror of the illumination device disclosed in Japanese Patent Laid-Open No. 7-56092 is changed to a half mirror. In FIG. 7, most of the light that has passed through the local illumination stop 45 of the local illumination optical system is reflected by the front surface 35 a of the half mirror 35 that is the combining means, and part of the light is reflected by the half mirror 35. Then, the light passes through the surface 35a and reaches the back surface 35b of the half mirror 35, and a part thereof is reflected by the back surface 35b of the half mirror 35. The reflected light from the back surface 35b of the half mirror 35 becomes light shifted in parallel to the reflected light from the front surface 35a of the half mirror 35. Both the reflected light from the back surface 35 b of the half mirror 35 and the reflected light from the front surface 35 a of the half mirror 35 are projected onto the sample surface S through the projection lens 36, the dichroic mirror 25, and the objective lens 24. For this reason, the projection position of the reflected light from the back surface 35 b of the half mirror 35 is shifted from the projection position of the reflected light from the front surface 35 a of the half mirror 35. As a result, the image of the local illumination stop 45 projected onto the sample surface S becomes a double image.

  Even when a caged experiment is performed using the illumination device disclosed in Japanese Patent Laid-Open No. 7-56092, there are the following problems. Before conducting a caged experiment, confirm that the position where the caged reagent is released in the specimen, that is, the projected position of the image of the local illumination diaphragm on the specimen surface, matches the position desired by the experimenter. I want to. However, since the caged reagent is released when the UV light is irradiated, it is necessary to perform illumination with visible light that does not release the caged reagent in order to confirm the projection position of the image of the local illumination stop in advance. However, the dichroic mirror as a combining means has a characteristic of reflecting UV light and transmitting visible light. More specifically, when the light passing through the local illumination stop is visible light, the light reflected from the front surface of the dichroic mirror that is the combining means is less than 5%, and most of the light of the dichroic mirror. The light passes through the front surface and reaches the back surface of the dichroic mirror. Similarly, about 5% of light is reflected by the back surface of the dichroic mirror. This reflectance is a typical physical property value on an uncoated glass surface. As a result, the image of the local illumination stop projected onto the sample surface becomes a double image, as in the case of using the half mirror described above.

  The present invention has been made in consideration of such a situation, and an object of the present invention is to provide an illumination device for a microscope in which an image of a local illumination stop does not become a double image.

  The present invention is directed to a microscope illumination apparatus.

  The illumination device of the present invention includes an observation illumination optical system that generates observation illumination light, a local illumination optical system that generates local illumination light, and an illumination that combines the optical path of the observation illumination light and the optical path of the local illumination light. This is a mirror for light synthesis, and the mirror for illumination light synthesis has a front surface and a back surface, and the front surface reflects at least the first illumination light which is one of observation illumination light and local illumination light and for observation. At least the second illumination light that is the other of the illumination light and the local illumination light is transmitted, the back surface transmits at least the second illumination light that transmits the front surface, and the back surface has an inclination angle with respect to the front surface. Illuminating light synthesizing mirror and a dichroic mirror for fluorescence observation placed on the optical axis of the microscope's observation optical system that forms the sample image by imaging the light from the specimen. Observation light and local illumination light And a dichroic mirror for fluorescence observation for guiding the specimen to the optical axis coaxial with the system.

  Another illumination device of the present invention combines an observation illumination optical system that generates observation illumination light, a local illumination optical system that generates local illumination light, and an optical path of observation illumination light and an optical path of local illumination light. The illumination light combining cemented prism has a cemented surface, and the cemented surface reflects at least one of the observation illumination light and the local illumination light and at least the other of the observation illumination light and the local illumination light. Fluorescent observation dichroic mirror placed on the optical axis of the microscope's observation optical system that forms a specimen image by forming light from the specimen by forming a cemented prism for illuminating light synthesis that is transmitted through and incident from the mirror for illumination light synthesis And a fluorescence observation dichroic mirror for guiding the observation illumination light and the local illumination light to the specimen coaxially with the optical axis of the observation optical system.

  ADVANTAGE OF THE INVENTION According to this invention, the illuminating device of the microscope in which the image of a local illumination stop does not become a double image is provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[First embodiment]
FIG. 1 shows a configuration of a microscope provided with the illumination device according to the first embodiment of the present invention. The microscope of the present embodiment includes a microscope main body and an illumination device incorporated in the microscope main body. The microscope main body includes an observation optical system 1 that forms an image of a sample image M by imaging light from the sample S. The specimen S includes a substance that is inactivated by the caged reagent and at the same time is labeled with a fluorescent reagent.

  The observation optical system 1 includes an objective lens 4, an absorption filter 6, and an imaging lens 7. The objective lens 4, the absorption filter 6, and the imaging lens 7 are arranged in order from the sample S side on the optical axis 1 a of the observation optical system 1. Observation light from the specimen S enters the objective lens 4, passes through the objective lens 4, becomes a parallel light beam, passes through the absorption filter 6, and enters the imaging lens 7. The specimen image M is formed on a predetermined imaging plane. The sample image M is observed or measured by an eyepiece, a camera, or a photometric device (not shown) arranged at a position conjugate with the sample image M.

  The illumination device includes a local illumination optical system 2 that generates local illumination light, a fluorescence observation illumination optical system 3 that is an observation illumination optical system that generates observation illumination light, an optical path of the local illumination light, and an observation illumination light. An illumination light combining dichroic mirror 13 that is an illumination light combining mirror that combines the optical paths of the two, and a correction wedge glass plate 13c that is a correction optical element that corrects a deviation between the optical path of the observation illumination light and the optical path of the local illumination light. A field stop projection lens 19 and a fluorescence observation dichroic mirror 5 disposed on the optical axis 1 a of the observation optical system 1.

  The local illumination optical system 2 includes a local illumination light source 8, a collect lens 9, a shutter 10, a band pass filter 11, and a local illumination stop 12. The light emitted from the local illumination light source 8 is shaped into a substantially parallel light beam by the collect lens 9 and enters the bandpass filter 11 when the shutter 10 is opened. The bandpass filter 11 selectively transmits only light in the UV wavelength band for releasing the caged reagent, and selectively transmits light in the wavelength band of fluorescence emitted by the fluorescent reagent in the sample S. And a band-pass filter changer 11x that selectively places one of the UV band-pass filter 11u and the fluorescence band-pass filter 11a on the optical axis 2a of the local illumination optical system 2. The band pass filter 11 transmits only light in a wavelength band necessary for desired local illumination. In other words, the bandpass filter 11 constitutes an illumination wavelength changing unit that changes the wavelength of the local illumination light in accordance with the required illumination wavelength. As a result, local illumination light is generated. The local illumination light transmitted through the band pass filter 11 enters the local illumination stop 12. The local illumination stop 12 is disposed at a position optically conjugate with the sample S, and regulates the illumination range of the local illumination light on the sample S. The local illumination stop 12 can arbitrarily select the shape of the opening from a circular pinhole or an elongated slit, and can select the size of the opening. The local illumination stop 12 is also arranged to be movable in the direction of the optical axis 2a of the local illumination optical system 2 and the direction perpendicular to the optical axis 2a by the local illumination stop operating unit 12x. That is, in the local illumination stop 12, the aperture shape, the aperture size, the position in the optical axis direction, and the aperture position in the plane perpendicular to the optical axis are variable. The local illumination light that has passed through the local illumination stop 12 enters the front surface 13a of the dichroic mirror 13 for synthesizing illumination light.

  The fluorescence observation illumination optical system 3 includes an excitation light source 14, a collect lens 15, a shutter 16, an excitation filter 17, and a field stop 18. The light emitted from the excitation light source 14 is shaped into a substantially parallel light beam by the collect lens 15 and enters the excitation filter 17 when the shutter 16 is opened. The excitation filter 17 selectively transmits only light having a wavelength band necessary for exciting the fluorescent reagent on the specimen S. As a result, excitation light that is observation illumination light is generated. The excitation light that has passed through the excitation filter 17 enters the field stop 18. The field stop 18 is disposed at a position optically conjugate with the sample S, and regulates the illumination range of the excitation light with respect to the sample S. The field stop 18 can be moved in a direction perpendicular to the optical axis 3a of the fluorescence observation illumination optical system 3 by the field stop operating part 18x, and the diameter of the opening can be changed by a field stop opening / closing part (not shown). That is, the field stop 18 has a variable aperture size and an aperture position in a plane perpendicular to the optical axis. The excitation light that has passed through the field stop 18 passes through the correction wedge glass plate 13c and enters the illumination light combining dichroic mirror 13 from the back surface 13b side.

  The local illumination optical system 2 and the fluorescence observation illumination optical system 3 are arranged so that their optical axes 2a and 3a are substantially orthogonal to each other.

  The illumination light combining dichroic mirror 13 is configured as a wedge-shaped glass plate. Accordingly, the illumination light combining dichroic mirror 13 has a front surface 13a and a back surface 13b, and the back surface 13b is not parallel to the front surface 13a but has a predetermined inclination angle. The front surface 13a of the illumination light combining dichroic mirror 13 reflects at least the local illumination light emitted from the local illumination optical system 2 and transmits at least the excitation light emitted from the fluorescence observation illumination optical system 3. As a result, the optical path of local illumination light and the optical path of excitation light that is observation illumination light are combined.

  More specifically, the illumination light combining dichroic mirror 13 has a front surface 13a that is approximately 45 degrees with respect to the optical axis 2a of the local illumination optical system 2 and the optical axis 3a of the illumination optical system 3 for fluorescence observation. It is arranged to have a slope of. The front surface 13a of the illumination light combining dichroic mirror 13 reflects almost 100% of UV light incident at an incident angle of approximately 45 degrees and transmits 95% or more of visible light incident at an incident angle of approximately 45 degrees. An interference film coat having wavelength characteristics is applied.

  The illumination light combining dichroic mirror 13 and the correcting wedge glass plate 13c are integrally inserted and removed by the mirror operating unit 13x. That is, the illuminating device inserts the illumination light combining dichroic mirror 13 and the correcting wedge glass plate 13c integrally onto the optical axis 3a of the fluorescence observation illumination optical system 3, or on the optical axis 3a of the fluorescence observation illumination optical system 3. Further, a mirror operating unit 13x that is retracted from the projector is further provided.

  The correcting wedge glass plate 13 c is disposed between the fluorescence observation illumination optical system 3 that generates excitation light, which is fluorescence observation illumination light that passes through the illumination light synthesis dichroic mirror 13, and the illumination light synthesis dichroic mirror 13. Yes. The correcting wedge glass plate 13c has a front surface and a back surface, and the back surface has an inclination angle with respect to the front surface. The inclination angle of the back surface with respect to the front surface of the correcting wedge glass plate 13c is equal to the inclination angle of the back surface 13b with respect to the front surface 13a of the illumination light combining dichroic mirror 13. Further, the correcting wedge glass plate 13c and the illumination light combining dichroic mirror 13 are arranged so that the directions in which the thickness increases are opposite to each other.

  For this reason, the excitation light emitted from the fluorescence observation illumination optical system 3 is refracted by passing through the correction wedge glass plate 13c, but then in the reverse direction by passing through the illumination light combining dichroic mirror 13. Refracted. As a result, the traveling direction of the excitation light after passing through the illumination light combining dichroic mirror 13 is corrected substantially parallel to the traveling direction of the excitation light before entering the correction wedge glass plate 13c. Therefore, the optical path of excitation light when the dichroic mirror 13 for illumination light synthesis and the wedge glass plate 13c for correction are integrally retracted from the optical axis 3a of the illumination optical system 3 for fluorescence observation by the mirror operating unit 13x, and fluorescence observation When inserted on the optical axis 3a of the illumination optical system 3, the optical path of the excitation light is substantially parallel. As a result, the illumination light combining mirror and the correcting wedge glass plate are integrally inserted on the optical axis 3a of the fluorescence observation illumination optical system 3 and retracted from the optical axis 3a of the fluorescence observation illumination optical system 3. The positional deviation of the projected image of the field stop on the specimen is extremely small.

  The excitation light from the fluorescence observation illumination optical system 3 passes through the correction wedge glass plate 13c and the illumination light combining dichroic mirror 13 and travels in a direction substantially parallel to the optical axis 3a of the fluorescence observation illumination optical system 3, The light passes through the aperture projection lens 19 and enters the fluorescence observation dichroic mirror 5. As will be described later, the local illumination light from the local illumination optical system 2 is reflected by the illumination light combining dichroic mirror 13 to be fluorescent, although the details of the behavior vary depending on the type of filter of the bandpass filter 11 as will be described later. The light advances in a direction substantially parallel to the optical axis 3 a of the observation illumination optical system 3, passes through the field stop projection lens 19, and enters the fluorescence observation dichroic mirror 5.

  The fluorescence observation dichroic mirror 5 is formed of an optically transparent parallel plate. Therefore, the dichroic mirror 5 for fluorescence observation has the front surface 5a and the back surface 5b, and the front surface 5a and the back surface 5b are parallel to each other. The fluorescence observation dichroic mirror 5 is arranged such that its front surface 5a has an inclination of approximately 45 degrees with respect to the optical axis 1a of the observation optical system 1. The optical axis 1a of the observation optical system 1 is substantially orthogonal to the optical axis 3a of the fluorescence observation illumination optical system 3. Accordingly, the fluorescence observation dichroic mirror 5 guides the observation illumination light and the local illumination light incident from the illumination light combining dichroic mirror 13 to the sample S coaxially with the optical axis 1 a of the observation optical system 1.

  The front surface 5a of the fluorescence observation dichroic mirror 5 reflects almost 100% of light having a relatively short wavelength including the wavelength band of the excitation light selected by the excitation filter 17 with respect to incident light of approximately 45 degrees. In addition, an interference film coat having a wavelength characteristic that transmits 95% or more of light having a relatively long wavelength including the fluorescence wavelength band emitted by the fluorescent reagent on the specimen S excited by the excitation light is applied. The excitation light reflected by the front surface 5 a of the fluorescence observation dichroic mirror 5 passes through the objective lens 4 and then irradiates the range on the sample S regulated by the field stop 18. The fluorescence emitted from the fluorescent reagent in the sample S excited by the excitation light passes through the objective lens 4, the fluorescence observation dichroic mirror 5, the absorption filter 6, and the imaging lens 7, and is transmitted by the objective lens 4 and the imaging lens 7. An image is formed to form a sample image M.

  2 shows the wavelength transmittance characteristics of the fluorescence observation dichroic mirror 5, the absorption filter 6, the UV bandpass filter 11u, the fluorescence bandpass filter 11a, the illumination light synthesis dichroic mirror 13 and the excitation filter 17, and the fluorescent reagent in the sample S. Shows an example of the relationship between the excitation spectrum Ex and the fluorescence spectrum Em.

  The behavior of the local illumination light from the local illumination optical system 2 is classified into two types according to the wavelength.

  When the UV bandpass filter 11u is arranged on the optical axis 2a of the local illumination optical system 2, the local illumination light from the local illumination optical system 2 is almost 100 on the front surface 13a of the illumination light combining dichroic mirror 13. % Light is reflected and travels in a direction parallel to the optical axis 3a of the illumination optical system 3 for fluorescence observation, passes through the field stop projection lens 19, and is almost 100% on the front surface 5a of the dichroic mirror 5 for fluorescence observation. Is reflected, passes through the objective lens 4, is irradiated onto the illumination range restricted by the local illumination stop 12 on the specimen S, and the caged reagent in the illumination range is released.

  Further, when the fluorescent bandpass filter 11 a is arranged on the optical axis 2 a of the local illumination optical system 2, the local illumination light from the local illumination optical system 2 is transmitted through the front surface 13 a of the illumination light combining dichroic mirror 13. Only about 5% of light is reflected, and the remaining light is transmitted through the front surface 13a. The light reflected by the front surface 13a travels in a direction parallel to the optical axis 3a of the fluorescence observation illumination optical system 3, passes through the field stop projection lens 19, and enters the front surface 5a of the fluorescence observation dichroic mirror 5. Only about 5% light is reflected, passes through the objective lens 4 and is irradiated to the illumination range restricted by the local illumination stop 12 on the sample S. A very small component of the light irradiated to the specimen S is totally reflected at the boundary surface between the cover glass and the aqueous solution of the specimen S, and the objective lens 4, the dichroic mirror 5 for fluorescence observation, the absorption filter 6, and the imaging lens 7 are reflected. The sample image M is formed by being transmitted and imaged by the objective lens 4 and the imaging lens 7.

  On the other hand, less than 5% of the 95% light transmitted through the front surface 13a of the illumination light combining dichroic mirror 13 is reflected by the back surface 13b of the illumination light combining dichroic mirror 13. As described above, this reflectance is a typical physical property value on a glass surface that is not coated. However, since the back surface 13b of the illumination light combining dichroic mirror 13 has an inclination angle with respect to the front surface 13a, the light reflected by the back surface 13b is the optical axis 3a of the illumination optical system 3 for fluorescence observation. And is completely excluded from the observation optical system 1 before reaching the objective lens 4 through the field stop projection lens 19 and the fluorescence observation dichroic mirror 5. In other words, the inclination angle of the back surface 13b with respect to the front surface 13a of the illumination light combining dichroic mirror 13 is the back surface 13b of the local illumination light that is transmitted through the front surface 13a of the illumination light combining dichroic mirror 13. The angle at which the reflected light is completely excluded from the observation optical system 1 before reaching the objective lens 4. This angle is determined depending on the focal length of the field stop projection lens 19 and the distance from the illumination light combining dichroic mirror 13 to the field stop projection lens 19.

  On the other hand, less than 5% of the 95% light transmitted through the front surface 5a of the fluorescence observation dichroic mirror 5 is reflected by the back surface 5b of the fluorescence observation dichroic mirror 5, and the front surface 5a. Is incident on the objective lens 4 as light deviated in parallel with the light reflected by. However, since the light incident on the objective lens in parallel forms an image at the same position, the projection image of the local illumination stop 12 on the sample S matches. From the above, the projection image of the local illumination stop 12 on the specimen S does not become a double image.

  This observation mode can be described as a state in which the projected image on the sample S of the local illumination stop 12 is observed in the incident bright field in the same wavelength band as the fluorescence emitted by the fluorescent reagent on the sample S. The total light amount of the incident bright-field observation image of the local illumination stop 12 at the position of the sample image M is a faint one-10,000 or less of the total light amount of the local illumination light transmitted through the local illumination stop 12. This is sufficiently larger than the total light amount of the fluorescence observation image, which is the main purpose of the experiment. In addition, since the wavelength band of the light transmitted through the fluorescent bandpass filter 11a is out of the excitation wavelength range of the fluorescent reagent on the specimen S, the fading of the fluorescent reagent can be extremely reduced. In addition, since the wavelength band of the light transmitted through the fluorescent bandpass filter 11a is far from the UV wavelength range, there is no concern that the caged reagent is released.

  Next, an operation procedure of the microscope according to this embodiment described above will be described. The shutter 10 and the shutter 16 are closed except when the specimen S is observed. First, the shutter 16 of the illumination optical system 3 for fluorescence observation is opened, and the objective lens 4 is focused while observing the fluorescence image of the sample S. The observation range of the fluorescent image is set by operating the field stop operating unit 18x to align the projected image of the field stop 18 with the center of the field of view, and operating the field stop opening / closing unit to reduce the diameter of the projected image of the field stop 18. This is done by adjusting to a desired size. Next, the bandpass filter changer 11x is operated to place the fluorescent bandpass filter 11a on the optical axis 2a of the local illumination optical system 2, and then the shutter 10 is opened. The image observed at this time is in a state in which the reflected bright field projection image of the local illumination stop 12 shines in the fluorescent image of the sample S. The wavelength range of the light of the incident bright field projection image of the local illumination stop 12, that is, the wavelength range of the light transmitted through the fluorescence bandpass filter 11a, is as described above from the excitation wavelength range of the fluorescent reagent on the sample S. Since it is off, there is very little discoloration of the fluorescent reagent, and since it is away from the UV wavelength range, there is no fear that the caged reagent is released. While viewing this image, the local illumination stop operating unit 12x is operated to focus the projected image of the local illumination stop 12 on the sample S and move to a desired position on the sample S, that is, a position where the caged reagent is to be released. Let At this time, the specimen S itself may be moved. When the position is determined, the shutter 10 of the local illumination optical system 2 is closed, and the bandpass filter changer 11x is operated to place the UV bandpass filter 11u on the optical axis 2a of the local illumination optical system 2, and then the shutter at a desired timing. Release 10 and release caged.

  When performing normal fluorescence observation that does not require local illumination as in the caged experiment, the mirror operating unit 13x is operated to integrally observe the illumination light combining dichroic mirror 13 and the correcting wedge glass plate 13c. By retracting from the optical axis 3a of the illumination optical system 3, the efficiency of the observation illumination can be increased. Here, when the mirror operating unit 13x is inserted and retracted, the optical path of the excitation light slightly shifts, so that the projected image of the field stop 18 slightly shifts laterally. This shift is caused by operating the field stop operating unit 18x. Can be easily corrected.

  According to the present embodiment described above, the projection image on the specimen S of the local illumination stop 12 does not become a double image regardless of the wavelength of the local illumination light. In addition, the position of the projected image of the local illumination stop 12 on the specimen S can be confirmed and accurately adjusted to a desired position before performing the local illumination with UV light to release the caged reagent.

[Modification of First Embodiment]
Although the first embodiment of the present invention has been described above, the present embodiment can be modified as described below.

  The local illumination light source 8 and the excitation light source 14 are usually composed of a high-intensity arc light source such as mercury or xenon, but the local illumination light source 8 and the excitation light are light sources that can blink at high speed, such as an LED light source. By configuring the light source 14, the shutter 10 and the shutter 16 can be omitted.

  It is also possible to reverse the arrangement of the local illumination optical system 2 and the fluorescence observation illumination optical system 3 by inverting the wavelength characteristics of the interference film coating on the front surface 13a of the illumination light combining dichroic mirror 13. .

  Further, the fluorescence observation dichroic mirror 5, the absorption filter 6, the fluorescence bandpass filter 11a, and the excitation filter 17 may be combined with any wavelength characteristic depending on the wavelength characteristics of the fluorescent reagent in the sample S. A pattern may be prepared and one of them may be combined and a pattern may be selectively inserted into the optical path.

  In addition to the above, various modifications can be made without departing from the spirit of the present embodiment.

[Second Embodiment]
FIG. 3 shows a configuration of a microscope including the illumination device according to the second embodiment of the present invention. 3, members indicated by the same reference numerals as those shown in FIG. 1 are the same members, and detailed description thereof is omitted.

  Since the aperture of the local illumination stop 12 is very small, the focal position shift of the projected image on the sample S due to the difference in wavelength of the local illumination light is conspicuous due to the influence of chromatic aberration due to the combination of the objective lens 4 and the field stop projection lens 19. There is a case. In particular, aberrations in the UV wavelength band increase depending on the type of the objective lens 4. Accordingly, when the projected image of the local illumination stop 12 is blurred when the fluorescent bandpass filter 11a is switched to the UV bandpass filter 11u, the local illumination stop operating unit 12x is connected to the optical axis 2a of the local illumination optical system 2. It is necessary to correct the focus by moving in the direction of.

  As can be seen from FIG. 3, the illumination apparatus of the present embodiment includes the local illumination optical system 2 according to the wavelength selection state of the bandpass filter 11 that is an illumination wavelength changing unit in addition to the configuration of the illumination apparatus of the first embodiment. Is provided with a local illumination stop correction unit 20 that moves the local illumination stop 12 along the optical axis 2a to match a position optically conjugate with the sample S. The local illumination stop correction unit 20 receives an electrical signal indicating the wavelength selection state of the bandpass filter changer 11x, and correspondingly moves the local illumination stop operating unit 12x to a designated position in the direction of the optical axis 2a. The position of the illumination stop 12 is corrected.

  An operation procedure of the microscope of this embodiment will be described. Since the specimen S is necessary for the purpose of confirming the focal point of the objective lens 4, a specimen that does not interfere with UV light or excitation light is used. First, the shutter 16 of the illumination optical system 3 for fluorescence observation is opened, and the objective lens 4 is focused while observing the specimen S. Next, the shutter 16 is closed, and the bandpass filter changer 11x is operated to place the fluorescent bandpass filter 11a on the optical axis 2a of the local illumination optical system 2, and then the shutter 10 is opened. The image observed at this time is only the reflected bright field projection image of the local illumination stop 12. While viewing this image, the local illumination stop operating unit 12x is operated to focus the projected image of the local illumination stop 12 on the sample S, and the in-focus position is stored in the local illumination stop correcting unit 20. Next, the bandpass filter changer 11x is operated to place the UV bandpass filter 11u on the optical axis 2a of the local illumination optical system 2, and the local illumination stop operating unit 12x is similarly operated to project the local illumination stop 12. The image is focused on the sample S, and the in-focus position is stored in the local illumination stop correction unit 20. Thereafter, the local illumination stop correction unit 20 performs the local illumination stop simply by operating the bandpass filter changer 11x to place the fluorescent bandpass filter 11a or the UV bandpass filter 11u on the optical axis 2a of the local illumination optical system 2. The operating unit 12x is automatically moved to a position corresponding to the filter disposed on the optical axis 2a.

  According to the present embodiment described above, even when the focal position of the projection image of the local illumination stop 12 on the sample S is shifted due to switching of the wavelength of the local illumination light, the focal position is corrected by the local illumination stop correction unit 20. Is automatically corrected. After confirming the position of the projection image of the local illumination stop 12 on the sample S at the transmission wavelength of the fluorescent bandpass filter 11a, the projection image of the local illumination stop 12 is not blurred when switching to the UV bandpass filter 11u. The caged reagent at the projection image position confirmed in advance can be canceled accurately.

[Third embodiment]
FIG. 4 shows a configuration of a microscope including the illumination device according to the third embodiment of the present invention. In FIG. 4, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted. The specimen S includes a substance labeled with a fluorescent reagent and is used for the FRAP experiment.

  In the configuration of this embodiment, the UV bandpass filter 11u in the local illumination optical system 2 is changed to the excitation filter 11e, and the illumination light synthesis dichroic mirror 13 is changed to the illumination light synthesis half mirror 130 from the configuration of the second embodiment. It is a thing. That is, the illumination device of this embodiment includes an excitation filter 11e and an illumination light combining half mirror 130 instead of the UV bandpass filter 11u and the illumination light combining dichroic mirror 13 of the second embodiment. Furthermore, in the illumination device of this embodiment, instead of the mirror operating unit 13x of the second embodiment, the illumination light combining half mirror 130 and the correcting wedge glass plate 13c are integrated with the optical axis 3a of the fluorescence observation illumination optical system 3. A mirror operating unit 130x that is inserted above or retracted from the optical axis 3a of the fluorescence observation illumination optical system 3 is provided.

  The excitation filter 11e has the same wavelength transmittance characteristics as the excitation filter 17 in the illumination optical system 3 for fluorescence observation, and selectively selects only light in a wavelength band necessary for exciting the fluorescent reagent on the excitation sample S. Transparent to.

  The illumination light combining half mirror 130 is configured as a wedge-shaped glass plate. Therefore, the illumination light combining half mirror 130 has a front surface 130a and a back surface 130b, and the back surface 130b is not parallel to the front surface 130a but has a predetermined inclination angle. The front surface 130a of the illumination light combining half mirror 130 reflects at least the local illumination light emitted from the local illumination optical system 2 and transmits at least the excitation light emitted from the fluorescence observation illumination optical system 3. As a result, the optical path of local illumination light and the optical path of excitation light that is observation illumination light are combined.

  More specifically, the illumination light combining half mirror 130 has a front surface 130a of approximately 45 degrees with respect to the optical axis 2a of the local illumination optical system 2 and the optical axis 3a of the fluorescence observation illumination optical system 3. It is arranged to have a slope of. The front surface 130a of the illumination light combining half mirror 130 is provided with a dielectric multilayer coating having a reflectance characteristic that reflects approximately 95% of incident light of approximately 45 degrees regardless of wavelength. .

  FIG. 5 shows the wavelength transmittance characteristics of the dichroic mirror 5 for fluorescence observation, the absorption filter 6, the excitation filter 11 e, the fluorescence bandpass filter 11 a, the illumination light synthesis half mirror 130, and the excitation filter 17 described above. An example of the relationship between the excitation spectrum Ex and the fluorescence spectrum Em of the fluorescent reagent is shown.

  The behavior of the excitation light from the illumination optical system 3 for fluorescence observation is the same as that of the first embodiment except that the amount of light passing through the illumination light combining half mirror 130 and entering the field stop projection lens 19 is reduced to approximately 5%. This is the same as described in.

  The behavior of the local illumination light from the local illumination optical system 2 is as follows. When the excitation filter 11e is arranged on the optical axis 2a of the local illumination optical system 2, the local illumination light from the local illumination optical system 2 is approximately 95% on the front surface 130a of the illumination light combining half mirror 130. The light is reflected and the remaining light passes through the front surface 130a. Nearly 95% of the light reflected by the front surface 130a travels in a direction parallel to the optical axis 3a of the fluorescence observation illumination optical system 3, passes through the field stop projection lens 19, and passes through the field observation projection dichroic mirror 5. Nearly 100% of the light is reflected by the front surface 5 a, passes through the objective lens 4, and is irradiated onto the illumination range restricted by the local illumination stop 12 on the sample S. As a result, if the local illumination light source 8 and the excitation light source 14 have substantially the same light amount, the light amount per unit area of the excitation light applied to the sample S is the same as the light amount from the local illumination optical system 2 and fluorescence observation. The ratio of the amount of light from the illumination optical system 3 is approximately 95: 5. Therefore, the excitation light in the illumination range restricted by the local illumination stop 12 has a relatively excessive intensity, contributing to the fading of the fluorescent reagent.

  On the other hand, less than 5% of the almost 5% light transmitted through the front surface 130 a of the illumination light combining half mirror 130 is reflected by the back surface 130 b of the illumination light combining half mirror 130. As described above, this reflectance is a typical physical property value on a glass surface that is not coated. However, since the back surface 130b of the illumination light combining half mirror 130 has an inclination angle with respect to the front surface 130a, the light reflected by the back surface 130b of the illumination light combining half mirror 130 is fluorescence observation. The illumination optical system 3 deviates from the direction parallel to the optical axis 3a and is completely excluded from the observation optical system 1 before reaching the objective lens 4 through the field stop projection lens 19 and the fluorescence observation dichroic mirror 5. The In other words, the inclination angle of the back surface 130b with respect to the front surface 130a of the illumination light combining half mirror 130 is the back surface 130b of the local illumination light transmitted through the front surface 130a of the illumination light combining half mirror 130. The angle at which the reflected light is completely excluded from the observation optical system 1 before reaching the objective lens 4. This angle is determined depending on the focal length of the field stop projection lens 19 and the distance from the illumination light combining half mirror 130 to the field stop projection lens 19.

  The behavior when the fluorescent bandpass filter 11a is arranged on the optical axis 2a of the local illumination optical system 2 is such that the reflectance at the front surface 130a of the illumination light combining half mirror 130 is approximately 95%. Except for this, the contents are the same as those described in the first embodiment.

  Next, an operation procedure of the microscope according to this embodiment described above will be described. The shutter 10 and the shutter 16 are closed except when the specimen S is observed. First, the shutter 16 of the illumination optical system 3 for fluorescence observation is opened, and the objective lens 4 is focused while observing the fluorescence image of the sample S. The observation range of the fluorescent image is set by operating the field stop operating unit 18x to align the projected image of the field stop 18 with the center of the field of view, and operating the field stop opening / closing unit to reduce the diameter of the projected image of the field stop 18. This is done by adjusting to a desired size. Next, the bandpass filter changer 11x is operated to place the fluorescent bandpass filter 11a on the optical axis 2a of the local illumination optical system 2, and then the shutter 10 is opened. The image observed at this time is in a state in which the reflected bright field projection image of the local illumination stop 12 shines in the fluorescent image of the sample S. The wavelength range of light of the incident bright field projection image of the local illumination stop 12, that is, the wavelength range of the light transmitted through the fluorescent bandpass filter 11a is out of the excitation wavelength range of the fluorescent reagent on the specimen S. The scarlet color is extremely small. While viewing this image, the local illumination stop operating unit 12x is operated to focus the projected image of the local illumination stop 12 on the sample S and move to a desired position on the sample S, that is, a position where the caged reagent is to be released. Let At this time, the specimen S itself may be moved. When the position is determined, the shutter 10 of the local illumination optical system 2 is closed, and the bandpass filter changer 11x is operated to place the excitation filter 11e on the optical axis 2a of the local illumination optical system 2, and then the shutter 10 is moved at a desired timing. Open and fade the fluorescent reagent.

  When performing normal fluorescence observation that does not require local illumination as in the FRAP experiment, the mirror operating unit 13x is operated to integrally observe the illumination light combining half mirror 130 and the correcting wedge glass plate 13c. By retracting from the optical axis 3a of the illumination optical system 3, the efficiency of the observation illumination can be increased. Here, when the mirror operating unit 13x is inserted and retracted, the optical path of the excitation light slightly shifts, so that the projected image of the field stop 18 slightly shifts laterally. This shift is caused by operating the field stop operating unit 18x. Can be easily corrected.

  According to the present embodiment described above, the projection image on the specimen S of the local illumination stop 12 does not become a double image regardless of the wavelength of the local illumination light. In addition, it is possible to confirm the position of the projected image of the local illumination stop 12 on the specimen S and accurately match it to a desired position before performing the local illumination with strong excitation light to fade the fluorescent reagent. .

[Modification of Third Embodiment]
Although the third embodiment of the present invention has been described above, the present embodiment can be modified as described below.

  The local illumination light source 8 and the excitation light source 14 are usually composed of a high-intensity arc light source such as mercury or xenon, a gas laser light source, or a solid laser light source, but can be rapidly blinked like an LED light source or an LD light source. By configuring the local illumination light source 8 and the excitation light source 14 with a simple light source, the shutter 10 and the shutter 16 can be omitted.

  It is also possible to reverse the arrangement of the local illumination optical system 2 and the fluorescence observation illumination optical system 3 by inverting the wavelength characteristics of the dielectric multilayer coating on the front surface 130a of the illumination light combining half mirror 130. It is. In this case, as described above, a typical physical property value on an uncoated glass surface is almost 95% of transmittance with respect to incident light at 45 degrees. Can be composed of glass plate.

  Further, the fluorescence observation dichroic mirror 5, the absorption filter 6, the fluorescence bandpass filter 11a, the excitation filter 11e, and the excitation filter 17 may be combined with any wavelength characteristic depending on the wavelength characteristics of the fluorescent reagent on the specimen S. A plurality of combination patterns may be prepared in advance, and one of these combination patterns may be selectively inserted into the optical path.

  In addition to the above, various modifications can be made without departing from the spirit of the present embodiment.

[Fourth embodiment]
FIG. 6 shows a configuration of a microscope including the illumination device according to the fourth embodiment of the present invention. In FIG. 6, the members indicated by the same reference numerals as those shown in FIG. 1 are the same members, and detailed description thereof will be omitted.

  In the configuration of the present embodiment, the illumination light combining half mirror 130 is changed to the illumination light combining half prism 131 and the correcting wedge glass plate 13c is changed to the correcting convex lens 131c from the configuration of the third embodiment. That is, the illuminating device of this embodiment replaces the illumination light combining half mirror 130, the correction wedge glass plate 13c, and the mirror operating unit 13x of the third embodiment with the optical path of the local illumination light and the optical path of the observation illumination light. Illuminating light synthesizing half prism 131, synthesizing illumination light synthesizing half prism 131, illuminating light synthesizing half prism 131, correction convex lens 131 c that is a correction lens for correcting a change in optical path length due to insertion / retraction of illumination light synthesizing prism, A prism operating unit 131x that integrally inserts the half prism 131 and the correcting convex lens 131c onto the optical axis 3a of the illumination optical system 3 for fluorescence observation or retracts from the optical axis 3a of the illumination optical system 3 for fluorescence observation; It has.

  The illumination light combining half prism 131 is composed of a cubic cemented prism formed by bonding two right-angled isosceles triangular prisms, and has a cemented surface 131a. The joint surface 131 a of the illumination light combining half prism 131 reflects at least the local illumination light emitted from the local illumination optical system 2 and transmits at least the excitation light emitted from the fluorescence observation illumination optical system 3. As a result, the optical path of local illumination light and the optical path of excitation light that is observation illumination light are combined.

  More specifically, the illumination light combining half prism 131 has a joint surface 131a inclined at approximately 45 degrees with respect to the optical axis 2a of the local illumination optical system 2 and the optical axis 3a of the fluorescence observation illumination optical system 3. Are arranged to have. Further, in the illumination light combining half prism 131, the surface on which the local illumination light from the local illumination optical system 2 is incident is orthogonal to the optical axis 2a of the local illumination optical system 2, and the excitation light from the fluorescence observation illumination optical system 3 is received. The incident surface is disposed so as to be orthogonal to the optical axis 3 a of the fluorescence observation illumination optical system 3. As a result, the surface of the illumination light combining half prism 131 from which the local illumination light and the excitation light are emitted is orthogonal to the optical axis 2 a of the local illumination optical system 2. The joint surface 131a of the illumination light combining half prism 131 is provided with a dielectric multilayer coating having a reflectance characteristic that reflects approximately 95% of incident light of approximately 45 degrees regardless of wavelength.

  The correcting convex lens 131 c is disposed between the fluorescence observation illumination optical system 3 that generates excitation light that passes through the illumination light combining half prism 131 and the illumination light combining half prism 131. The correcting convex lens 131c corrects the change in the air-converted value of the optical path length due to the insertion / retraction of the illumination light combining half prism 131, so that the projection position of the field stop 18 of the fluorescence observation illumination optical system 3 coincides with the sample S. Let In other words, the correcting convex lens 131c is optically conjugate with the sample S when the illumination light combining half prism 131 is inserted, and with the sample S when the illumination light combining half prism 131 is retracted. Make the position the same. As a result, the defocus of the projected image of the field stop on the specimen is extremely reduced when the illumination light combining half prism 131 and the correction convex lens 131c are inserted and retracted.

  The behavior of the illumination light and the observation light in this embodiment is almost the same as that described in the third embodiment except that there is no reflected light from the back surface 130b of the illumination light combining half mirror 130. In other words, in the present embodiment, in the illumination light combining half prism 131, the reflecting surface for guiding the local illumination light from the local illumination optical system 2 to the fluorescence observation dichroic mirror 5 is only the joint surface 131a. Therefore, there is no factor that the local illumination light is guided to the fluorescence observation dichroic mirror 5 by the other reflection surface of the joint surface 131a. Therefore, the projection image of the local illumination stop 12 on the sample S does not become a double image.

  The operation procedure of this embodiment is substantially the same as that described in the third embodiment, except for the following procedures for insertion / retraction of the prism operating unit 131x. More specifically, when the prism operating unit 131x is inserted and retracted, the projection magnification of the field stop 18 changes by the amount of the correction convex lens 131c, so the projection range of the field stop 18 is slightly enlarged or reduced. The change in size can be easily corrected by operating the field stop opening / closing part.

  According to the present embodiment described above, the projection image on the specimen S of the local illumination stop 12 does not become a double image regardless of the wavelength of the local illumination light. In addition, the position of the projection image of the local illumination stop 12 on the specimen S can be confirmed and accurately adjusted to a desired position before the fluorescent reagent is faded by performing local illumination with strong excitation light. .

[Modification of Fourth Embodiment]
Although the fourth embodiment of the present invention has been described above, the present embodiment can be modified as described below.

  The local illumination light source 8 and the excitation light source 14 are usually composed of a high-intensity arc light source such as mercury or xenon, a gas laser light source, or a solid laser light source, but can be rapidly blinked like an LED light source or an LD light source. By configuring the local illumination light source 8 and the excitation light source 14 with a simple light source, the shutter 10 and the shutter 16 can be omitted.

  It is also possible to reverse the wavelength characteristics of the dielectric multilayer coating on the joint surface 131a of the illumination light combining half prism 131 and to reverse the arrangement of the local illumination optical system 2 and the fluorescence observation illumination optical system 3.

  The fluorescence observation dichroic mirror 5, the absorption filter 6, the fluorescence bandpass filter 11a, the excitation filter 11e, and the excitation filter 17 can be combined with any wavelength characteristic depending on the wavelength characteristics of the fluorescent reagent on the sample S. It is possible to prepare a plurality of combination patterns and selectively insert them into the optical path.

  Further, the coating of the joint surface 131a of the illumination light combining half prism 131 may be an interference film coating to form a dichroic prism. For example, as described in the first embodiment, an interference coating having a wavelength characteristic that reflects almost 100% of UV light with respect to incident light of approximately 45 degrees and transmits approximately 95% or more of visible light is applied. It can also be configured for Caged experiments.

  In addition to the above, various modifications can be made without departing from the spirit of the present embodiment.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

[Conclusion]
The present invention is directed to an illumination device for a microscope, and includes the illumination devices listed in the following items.

  1. The illumination device of the present invention includes an observation illumination optical system that generates observation illumination light, a local illumination optical system that generates local illumination light, and an illumination that combines the optical path of the observation illumination light and the optical path of the local illumination light. This is a mirror for light synthesis, and the mirror for illumination light synthesis has a front surface and a back surface, and the front surface reflects at least the first illumination light which is one of observation illumination light and local illumination light and for observation. At least the second illumination light that is the other of the illumination light and the local illumination light is transmitted, and the back surface has an inclination angle with respect to the front surface, and the light from the sample is imaged. Fluorescent observation dichroic mirror placed on the optical axis of the microscope observation optical system that forms the specimen image. The observation illumination light and local illumination light incident from the illumination light synthesis mirror are coaxial with the optical axis of the observation optical system. Dichroic for fluorescence observation that guides light to the specimen And an error.

  2. In another illumination device according to the first aspect of the present invention, in the first aspect, the inclination angle of the back surface with respect to the front surface of the illumination light combining mirror is transmitted through the front surface of the illumination light combining mirror in the first illumination light. The angle at which the light reflected by the surface is completely excluded from the observation optical system before reaching the objective lens of the observation optical system.

3. Another illuminating device of the present invention is the illuminating optical system for observation according to the first aspect, wherein the observation illumination optical system includes a field stop that regulates an illumination range of the observation illumination light, and the field stop is disposed at a position optically conjugate with the sample. The aperture size and the aperture position in the plane perpendicular to the optical axis are variable,
The local illumination optical system includes a local illumination stop that regulates the illumination range of the local illumination light, and the local illumination stop is disposed at a position optically conjugate with the sample, and the aperture shape, aperture size, optical axis direction position, and light. The opening position in a plane perpendicular to the axis is variable.

  4). Another illuminating device of the present invention includes a correction optical element that corrects a deviation between the optical path of the observation illumination light and the optical path of the local illumination light according to the first item, and the correction optical element is an illumination light combining mirror. The correction optical element has a front surface and a back surface, and the back surface is relative to the front surface. The tilt angle of the back surface relative to the front surface of the correction optical element is equal to the tilt angle of the back surface relative to the front surface of the illumination light combining mirror, and the correction optical element and the illumination light combining mirror are Thickness increasing directions are arranged in opposite directions.

  5). Another illumination device of the present invention is the illumination device according to item 3, wherein the front surface of the illumination light combining mirror reflects at least the local illumination light and transmits at least the observation illumination light. The illumination device further includes the observation illumination light. The correction optical element for correcting the deviation of the optical path of the local illumination light and the optical path of the local illumination light, and the illumination light combining mirror and the correction optical element are integrally inserted on the optical axis of the observation optical system or the optical axis of the observation optical system A mirror operating unit that retracts from above is provided, and the correction optical element is disposed between the observation illumination optical system that generates observation illumination light that passes through the illumination light synthesis mirror and the illumination light synthesis mirror. The correction optical element has a front surface and a back surface, the back surface has an inclination angle with respect to the front surface, and the inclination angle of the back surface with respect to the front surface of the correction optical element is an illumination light composition. Of the back surface with respect to the front surface of the mirror Equal to angle, mirror lighting photosynthesis correcting optical element has a direction of extension of the thickness are arranged opposite to each other.

  6). Another illuminating device of the present invention is the illuminating device according to the third aspect, further including an illumination wavelength changing unit that changes the wavelength of the local illumination light according to a necessary illumination wavelength, and the illumination device further includes: A local illumination stop correction unit is provided that moves the local illumination stop along the optical axis of the local illumination optical system in accordance with the wavelength selection state of the illumination wavelength changing means to match the position optically conjugate with the sample.

  7). Another illumination device of the present invention combines an observation illumination optical system that generates observation illumination light, a local illumination optical system that generates local illumination light, and an optical path of observation illumination light and an optical path of local illumination light. The illumination light combining cemented prism has a cemented surface, and the cemented surface reflects at least one of the observation illumination light and the local illumination light and at least the other of the observation illumination light and the local illumination light. Fluorescent observation dichroic mirror placed on the optical axis of the microscope's observation optical system that forms a specimen image by forming light from the specimen by forming a cemented prism for illuminating light synthesis that is transmitted through and incident from the mirror for illumination light synthesis And a fluorescence observation dichroic mirror for guiding the observation illumination light and the local illumination light to the specimen coaxially with the optical axis of the observation optical system.

8). Another illumination device according to the present invention is the illumination device according to item 7, wherein the observation illumination optical system includes a field stop that regulates an illumination range of the illumination light for observation, and the field stop is disposed at a position optically conjugate with the sample. The aperture size and the aperture position in the plane perpendicular to the optical axis are variable,
The local illumination optical system includes a local illumination stop that regulates the illumination range of the local illumination light, and the local illumination stop is disposed at a position optically conjugate with the sample, and the aperture shape, aperture size, optical axis direction position, and light. The opening position in a plane perpendicular to the axis is variable.

  9. Another illuminating device of the present invention is the illuminating light combining cemented prism according to item 7, wherein the cemented surface of the illuminating light combining cemented prism reflects at least local illumination light and transmits at least observation illumination light. A prism operating unit that inserts the lens on the optical axis of the observation optical system or retracts it from the optical axis of the observation optical system, and a correction lens that corrects a change in the optical path length due to the insertion / retraction of the cemented prism for illumination light synthesis, The correction lens is disposed between the observation illumination optical system for generating observation illumination light that passes through the illumination light combining cemented prism and the illumination light combining cemented prism. It is inserted on the optical axis of the observation optical system integrally with the optical mirror, or is retracted from the optical axis of the observation optical system.

  10. Another illuminating device of the present invention is the illuminating device according to the eighth aspect, wherein the local illuminating optical system further includes illumination wavelength changing means for changing the wavelength of the local illuminating light according to a necessary illumination wavelength, A local illumination stop correction unit is provided that moves the local illumination stop along the optical axis of the local illumination optical system in accordance with the wavelength selection state of the illumination wavelength changing means to match the position optically conjugate with the sample.

The structure of the microscope provided with the illuminating device of 1st embodiment of this invention is shown. Wavelength transmittance characteristics of the fluorescence observation dichroic mirror, absorption filter, UV bandpass filter, fluorescence bandpass filter, illumination light synthesis dichroic mirror, and excitation filter in Fig. 1, and the excitation and fluorescence spectra of the fluorescent reagent in the sample An example of the relationship is shown. The structure of the microscope provided with the illuminating device of 2nd embodiment of this invention is shown. The structure of the microscope provided with the illuminating device of 3rd embodiment of this invention is shown. Figure 4 shows the relationship between the wavelength transmittance characteristics of the fluorescence observation dichroic mirror, absorption filter, excitation filter, fluorescence bandpass filter, illumination light synthesis half mirror, and excitation filter, and the excitation spectrum and fluorescence spectrum of the sample fluorescence reagent. An example is shown. The structure of the microscope provided with the illuminating device of 4th embodiment of this invention is shown. 1 schematically shows a part of an optical system in which a dichroic mirror of a lighting device disclosed in Japanese Patent Laid-Open No. 7-56092 is changed to a half mirror.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Observation optical system, 1a ... Optical axis, 2 ... Local illumination optical system, 2a ... Optical axis, 3 ... Fluorescence observation illumination optical system, 3 ... Observation illumination optical system, 3a ... Optical axis, 4 ... Objective lens, 5 ... Dichroic mirror for fluorescence observation, 5a ... Front surface, 5b ... Back surface, 6 ... Absorption filter, 7 ... Imaging lens, 8 ... Local illumination light source, 9 ... Collect lens, 10 ... Shutter, 11 ... Band Pass filter, 11a ... Fluorescent band pass filter, 11e ... Excitation filter, 11u ... UV band pass filter, 11x ... Band pass filter changer, 12 ... Local illumination stop, 12x ... Local illumination stop operating unit, 13 ... Dichroic mirror for illumination light synthesis , 13a ... front surface, 13b ... back surface, 13c ... wedge glass plate for correction, 13x ... mirror operating part, 14 ... excitation light source, 15 ... collect lens DESCRIPTION OF SYMBOLS 16 ... Shutter, 17 ... Excitation filter, 18 ... Field stop, 18x ... Field stop operation part, 19 ... Field stop projection lens, 20 ... Local illumination stop correction part, 130 ... Half mirror for illumination light composition, 130a ... Front surface , 130b ... back surface, 130x ... mirror operating part, 131 ... half prism for illumination light synthesis, 131a ... cemented surface, 131c ... convex lens for correction, 131x ... prism operating part.

Claims (6)

A microscope illumination device,
An observation illumination optical system for generating observation illumination light;
A local illumination optical system for generating local illumination light;
An illumination light combining mirror that combines the optical path of the observation illumination light and the optical path of the local illumination light. The illumination light combining mirror has a front surface and a back surface, and the front surface includes the observation illumination light and the local illumination light. Reflects at least the first illumination light that is one of the illumination light and transmits at least the second illumination light that is the other of the observation illumination light and the local illumination light, and the back surface has an inclination angle with respect to the front surface. A mirror for illumination light synthesis,
Fluorescent observation dichroic mirror placed on the optical axis of the microscope's observation optical system that forms the specimen image by imaging the light from the specimen. Observation illumination light and local illumination light incident from the illumination light combining mirror An illumination device for a microscope, comprising: a dichroic mirror for fluorescence observation that guides light to a specimen coaxially with the optical axis of the observation optical system.   2. The optical system according to claim 1, wherein an inclination angle of the back surface with respect to the front surface of the illumination light combining mirror is a light for observing light reflected by the back surface that is transmitted through the front surface of the illumination light combining mirror among the first illumination light. An illuminating device that is an angle that is completely excluded from the observation optical system before reaching the objective lens of the system. 2. The observation illumination optical system according to claim 1, further comprising a field stop that regulates an illumination range of the observation illumination light. The field stop is disposed at a position optically conjugate with the sample, and is perpendicular to the aperture size and the optical axis. The position of the opening in a simple plane is variable,
The local illumination optical system includes a local illumination stop that regulates the illumination range of the local illumination light, and the local illumination stop is disposed at a position optically conjugate with the sample, and the aperture shape, aperture size, optical axis direction position, and light. An illumination device in which an opening position in a plane perpendicular to an axis is variable.   2. The correction optical element according to claim 1, wherein the correction optical element corrects a deviation between the optical path of the observation illumination light and the optical path of the local illumination light, and the correction optical element generates the second illumination light that passes through the illumination light combining mirror. The correction optical element has a front surface and a back surface, and the back surface has an inclination angle with respect to the front surface, and the correction optical element is disposed between the illumination optical system and the illumination light combining mirror. The inclination angle of the back surface with respect to the front surface of the element is equal to the inclination angle of the back surface with respect to the front surface of the illumination light combining mirror, and the direction in which the thickness of the correction optical element and the illumination light combining mirror spread is opposite to each other. Arranged in the lighting device. A microscope illumination device,
An observation illumination optical system for generating observation illumination light;
A local illumination optical system for generating local illumination light;
The illumination light combining cemented prism combines the optical path of the observation illumination light and the optical path of the local illumination light. The illumination light combining cemented prism has a cemented surface, and the cemented surface has at least one of the observation illumination light and the local illumination light. An illumination light combining cemented prism that reflects and transmits at least the other of the observation illumination light and the local illumination light;
Fluorescent observation dichroic mirror placed on the optical axis of the microscope's observation optical system that forms the specimen image by imaging the light from the specimen. Observation illumination light and local illumination light incident from the illumination light combining mirror An illumination device for a microscope, comprising: a dichroic mirror for fluorescence observation that guides light to a specimen coaxially with the optical axis of the observation optical system. 6. The observation illumination optical system according to claim 5, comprising a field stop that regulates an illumination range of the observation illumination light, the field stop is disposed at a position optically conjugate with the sample, and is perpendicular to the aperture size and the optical axis. The position of the opening in a simple plane is variable,
The local illumination optical system includes a local illumination stop that regulates the illumination range of the local illumination light, and the local illumination stop is disposed at a position optically conjugate with the sample, and the aperture shape, aperture size, optical axis direction position, and light. An illumination device in which an opening position in a plane perpendicular to an axis is variable.

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