JP2013041142A - Microscopic device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an image having a high contrast based on a refractive index distribution of a cell and obtain a fluorescent image and a differential interference image by one microscopic device.SOLUTION: A microscopic device 1 according to the present invention includes: a light source that oscillates laser beam L which irradiates on a specimen S; a Wollaston prism 21 that separates the laser beam L into two deflections the directions of which are different from each other; a first object lens 22 that condenses the two deflections into two alienated points of the specimen S; a second object lens 24 that converts the two deflections having transmitted the specimen S into a parallel light; a corner cube 25 that retro-reflects the two deflections having passed the second object lens 24; and a polarizer 20 in which the laser beam L incident on the Wollaston prism 21 is converted into linear polarization and the two deflections retro-reflected by the corner cube 25 and having transmitted the specimen S are interfered with each other.

Description

  The present invention relates to a microscope apparatus that irradiates a sample with illumination light and observes an image of the sample.

  The microscope apparatus is used for irradiating a sample with illumination light and observing an image thereof. Examples of the microscope apparatus include a phase contrast microscope, a differential interference microscope, and a fluorescence microscope. When the observation target is a biological sample, generally, the bright field microscope, the phase contrast microscope, and the differential interference microscope perform observation by transmitting light to the sample that is the observation target. A bright-field microscope observes the distribution of light absorption. Generally, a sample such as a cell observes cell information of a cell having a low degree of light absorption and a low contrast. A phase contrast microscope and a differential interference microscope image a refractive index distribution, and a cell shape can be observed with relatively high contrast using a refractive index difference of cells or the like.

  The fluorescence microscope irradiates illumination light to a sample into which a fluorescent dye or fluorescent protein is introduced to generate fluorescence, and observes the sample based on the generated fluorescence. This type of microscope is mainly used to observe functional information of cells. The fluorescence microscope performs observation by performing epifluorescence. In particular, a confocal microscope having a high resolution in the depth direction is frequently used as a fluorescence microscope.

  Patent Document 1 discloses a confocal laser scanning transmission microscope as a microscope that transmits light through a sample for observation. The microscope of this document transmits the laser beam twice through the sample by providing an infinity corrected objective lens and a corner cube reflector. Thereby, an image with enhanced contrast is obtained.

Japanese Patent Laid-Open No. 6-18785

  Like the microscope of Patent Document 1, the contrast due to the light absorption can be coordinated by transmitting the laser light twice through the sample to double the light absorption of the sample. However, when the sample is a cell, in general, since the amount of light absorbed by the cell is extremely small, sufficient contrast cannot be obtained even if laser light is transmitted through the sample twice.

  The microscope of Patent Document 1 is a bright field microscope that transmits light to a sample and can observe the distribution of absorption of light, but observe the distribution of refractive index like a phase contrast microscope or differential interference microscope. Can not do it. Moreover, the microscope of patent document 1 cannot observe the functional information of a sample like a fluorescence microscope. When it is desired to observe both the cell shape and the function information for the same sample to be observed, the microscope disclosed in Patent Document 1 can observe only a shape having a low contrast based on absorption of light from the cell.

  Therefore, an object of the present invention is to obtain a high-contrast image based on the refractive index distribution of cells, and to obtain a fluorescence image and a differential interference image with a single microscope apparatus.

  In order to solve the above problems, a microscope apparatus of the present invention includes a light source that oscillates illumination light that irradiates a sample, a polarization separation element that separates the illumination light into two polarized light beams having different polarization directions, and the two polarized light beams. A first objective lens that collects light at two spaced points of the sample, a second objective lens that converts the two polarized lights that have passed through the sample into parallel light, and the two objective lenses that have passed through the second objective lens A retroreflective optical system that retroreflects polarized light, and polarized light that converts the illumination light incident on the polarization separation element into linearly polarized light, and that is retroreflected by the retroreflective optical system and interferes with two polarized lights that have passed through the sample. And a child.

  According to this microscope apparatus, two polarized lights separated by the polarization separating element are retroreflected by the retroreflective optical system. Thereby, two polarized light passes through two different points of the sample twice. For this reason, the phase difference between the two polarizations is doubled, and a high-contrast image can be obtained.

  The polarization separation element, the second objective lens, the retroreflective optical system, and the polarizer are movably moved from the optical path of the illumination light, and the polarization when the first mode is set. A separation element, the second objective lens, the retroreflective optical system, and the polarizer are inserted into the optical path of the illumination light, and when the second mode is set, the polarization separation element, the second objective lens, and the And a controller that controls the moving mechanism so as to retract the retroreflective optical system and the polarizer from the optical path of the illumination light.

  Switching between the first mode and the second mode is performed by the moving mechanism and the control unit. In the first mode, an optical system capable of obtaining a differential interference image can be constructed, and in the second mode, an optical system capable of obtaining a fluorescence image can be constructed. As a result, the differential tentative image and the fluorescence image can be obtained with one microscope apparatus.

  The moving mechanism is a half that transmits or reflects the illumination light from the light source to the sample and reflects or transmits return light from the sample when the control unit is set to the first mode. When the second mode is set in the control unit, a mirror and a dichroic mirror that transmits or reflects the wavelength of the illumination light from the light source and reflects or transmits the wavelength of the fluorescence of the sample are switched. It is characterized by that.

  By switching between the half mirror and the dichroic mirror, it is possible to easily switch between using as a differential interference microscope and using as a fluorescence microscope.

  A pinhole disk in which a plurality of pinholes that transmit light within the focal range of the sample are arranged; and a plurality of lenses that are arranged in the same pattern as the pinhole and collect the illumination light in the pinhole. It is characterized by comprising an arrayed lens disk, and a rotating part for integrally rotating the pinhole disk and the lens disk.

  When the rotating part integrally rotates the pinhole disk and the lens disk, the illumination light can be scanned over the sample, and an image can be generated at high speed.

  Further, a λ / 4 wavelength plate is provided between the polarization separation element and the polarizer.

  By providing the λ / 4 wavelength plate, a high-contrast image can be obtained even for crossed Nicols that become dark when there is no phase difference between the two polarizations.

  Further, a pupil relay lens system for relaying a pupil position of the second objective lens is provided between the second objective lens and the retroreflective optical system.

  Even when the second objective lens and the retroreflective optical system are too close together, the retroreflective optical system can be provided at the pupil position of the second objective lens by providing the pupil relay lens system, and the first A predetermined interval can be provided between the two objective lenses and the retroreflective optical system.

  In the present invention, the illumination light is separated into two polarizations by the polarization separation element, and the two polarizations are transmitted through the sample twice by retroreflection by the retroreflection optical system. Thereby, it is possible to transmit twice between two points separated from each other of the sample, and the phase difference can be doubled, so that a high contrast image can be obtained. Further, the control unit controls the moving mechanism, so that a differential interference image and a fluorescence image can be obtained with one microscope device.

It is a block diagram of the microscope apparatus of the differential interference mode of embodiment. It is a block diagram of a pinhole disk and a micro lens disk. It is a block diagram of the microscope apparatus of the fluorescence mode of embodiment. It is a block diagram of the microscope apparatus of the modification 1. FIG. 10 is a configuration diagram of a differential interference mode microscope apparatus according to a second modification. FIG. 10 is a configuration diagram of a fluorescence mode microscope apparatus according to a second modification. FIG. 10 is a configuration diagram of a differential interference mode microscope apparatus according to Modification 3. FIG. 10 is a configuration diagram of a fluorescence mode microscope apparatus according to a third modification. It is a figure of the 1st objective lens and the 2nd objective lens of modification 4 and 5.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows a microscope apparatus 1 according to an embodiment. The microscope apparatus 1 includes a scanning optical system 2 and a microscope optical system 3. As shown in the figure, the scanning optical system 2 includes a light source 4, a fiber 5, a collimating lens 6, a microlens disk 7, a pinhole disk 8, a connecting drum 9, a motor 10, a half mirror 11a, a first relay lens 12, and a mirror. 13, a second relay lens 14, a camera 15, a control unit 16, and a monitor 17.

  The light source 4 is a light source that oscillates the laser light L. The laser light L becomes illumination light for observing the sample S that is an observation target. The fiber 5 is an optical fiber and guides the laser light L. A collimating lens 6 is disposed on the emission side of the fiber 5, and the laser light L emitted from the fiber 5 becomes parallel light by the collimating lens 6.

  A microlens disk 7 is disposed in front of the collimating lens 6. The micro lens disk 7 and the pinhole disk 8 are connected by a connecting drum 9, and the connecting drum 9 is connected to a motor 10. The microlens disk 7 and the pinhole disk 8 are rotating disks, and when the rotational force is applied to the connecting drum 9 by the motor 10, the microlens disk 7 and the pinhole disk 8 rotate integrally.

  As shown in FIG. 2, the pinhole disk 8 is formed with a large number of spiral pinholes 8 </ b> P arranged in multiple rows (four in the figure). The pinhole 8P is a minute opening that allows only light within the focal range of the sample S to pass through. The microlens disk 7 is formed by arranging a large number of spiral microlenses 7M in the same pattern as the pinhole 8P. The micro lens 7M has a function of condensing the laser light L in the corresponding pinhole 8P.

  As shown in FIGS. 1 and 2, a half mirror 11 a is provided between the microlens disk 7 and the pinhole disk 8. The half mirror 11a is an optical element that divides incident light into transmission and reflection. The first relay lens 12 is provided at a position where light is reflected by the half mirror 11a.

  The light relayed by the first relay lens 12 is reflected by the mirror 13 and guided to the second relay lens 14. This light is condensed on the camera 15 by the second relay lens 14. The camera 15 converts the received light into an electrical signal and outputs it to the control unit 16. The control unit 16 is a computer that controls the entire microscope apparatus 1, and generates an image of the sample S based on an electrical signal input from the camera 15. The generated image is displayed on the monitor 17.

  Next, the microscope optical system 3 will be described. As shown in FIG. 1, the microscope optical system 3 includes a photographing lens 18, a mirror 19, a polarizer 20, a Wollaston prism 21, a first objective lens 22, a dish 23, a second objective lens 24, and a corner cube 25. Is configured.

  The taking lens 18 converts the laser light L that has passed through the pinhole 8P into parallel light. The parallel light is reflected by the mirror 19 and guided to the polarizer 20. The polarizer 20 generates linearly polarized light having a predetermined polarization direction. The Wollaston prism 21 is a polarization separation element having birefringence, and separates the illumination light that has passed through the polarizer 20 into two polarized light beams having a predetermined opening angle. The polarization directions of the two polarized lights are orthogonal to each other.

  At this time, the plane of polarization generated by the polarizer 20 is inclined 45 degrees with respect to the optical axis of the crystal of the Wollaston prism 21. As a result, the two polarized light beams orthogonal to each other are separated by a predetermined opening angle by the Wollaston prism 21.

  The dish 23 is a mounting portion on which the sample S is mounted. The two polarized lights (laser light L) separated by the Wollaston prism 21 are focused at two points on the sample S that are slightly separated from each other. Then, the two polarized light beams transmitted through the sample S are guided to the second objective lens 24 and collected on the corner cube 25. The corner cube 25 is a retroreflective optical system in which a cube is formed by combining three mirrors at right angles to each other. As a result, the two polarized lights are reflected (retroreflected) in the original direction.

  Here, the first moving mechanism is attached to the half mirror 11a. A second moving mechanism is attached to the polarizer 20, the Wollaston prism 21, the second objective lens 24 and the corner cube 25. A moving mechanism is constituted by the first moving mechanism and the second moving mechanism. The first moving mechanism moves the half mirror 11a in a direction perpendicular to the paper surface of FIG.

  When the first moving mechanism moves the half mirror 11a from the optical path of the laser light L, the dichroic mirror 11b is positioned in the optical path of the laser light L. That is, the first moving mechanism selectively positions either the half mirror 11a or the dichroic mirror 11b in the optical path of the laser light L (between the microlens disk 7 and the pinhole disk 8). The dichroic mirror 11b is an optical element having a characteristic of transmitting the light of the wavelength of the laser light L and reflecting the wavelength of the fluorescence of the sample S (wavelength longer than the wavelength of the laser light L).

  The second moving mechanism moves the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 in a direction orthogonal to the paper surface of FIG. 1 or a direction orthogonal to the optical path of the laser light L. As a result, the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 are retracted from the optical path of the laser light L.

  As the first moving mechanism and the second moving mechanism, any moving means can be used as long as each optical component can be moved. For example, a guide rail is provided in a clamp mechanism that clamps the half mirror 11a, the dichroic mirror 11b, the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25, and is moved by a driving means such as an actuator. May be.

  3, the first moving mechanism and the second moving mechanism are operated from the state of FIG. 1 to position the dichroic mirror 11 b in the optical path of the laser light L, and the polarizer 20, the Wollaston prism 21, and the second objective lens. The state where 24 and the corner cube 25 were retracted is shown.

  Next, the operation will be described. Here, there are two modes, a differential interference mode (first mode) and a fluorescence mode (second mode). The differential interference mode is a mode in which the microscope apparatus 1 is used as a differential interference microscope. That is, in order to observe the shape of the cell or the like, the sample S is observed by transmitting the laser light L. The fluorescence mode is a mode in which the microscope apparatus 1 is used as a fluorescence microscope. That is, in order to observe the function information of the cells and the like, the sample S is irradiated with the laser light L and the fluorescence is observed.

  The control unit 16 performs switching between the differential interference mode and the fluorescence mode. That is, either the differential interference mode or the fluorescence mode is set in the control unit 16 that is a computer. Based on this setting, the first moving mechanism and the second moving mechanism operate. When the differential interference mode is set, the first moving mechanism positions the half mirror 11a on the optical path of the laser light L. The second moving mechanism positions the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 on the optical path of the laser light L. That is, the configuration shown in FIG.

  On the other hand, when the fluorescence mode is set, the first moving mechanism positions the dichroic mirror 11b on the optical path of the laser light L. The second moving mechanism retracts the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 from the optical path of the laser light L. That is, the configuration is as shown in FIG.

  First, a case where the differential interference mode is set in the control unit 16 will be described. In this case, the first moving mechanism positions the half mirror 11a on the optical path of the laser beam L, and the second moving mechanism moves the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 to the laser beam. It is located on the L optical path.

  Then, the laser light L is oscillated from the light source 4. The laser light L is condensed on the fiber 5 and emitted from the end of the fiber 5 with a spread. The laser light L is converted into parallel light by the collimating lens 6 and enters the microlens 7M of the microlens disk 7.

  The microlens 7M functions as a condensing lens, and the laser light L is focused on the pinhole 8P corresponding to the microlens 7M after passing through the half mirror 11a. The laser beam L that has passed through the pinhole 8P is incident on the taking lens 18. The laser light L is converted by the photographing lens 18 into parallel light having an inclination corresponding to the position of the pinhole 8P. The parallel light is reflected by the mirror 19, the optical path is converted by 90 degrees, and enters the polarizer 20.

  The polarizer 20 converts the laser light L into linearly polarized light having a polarization plane having an inclination of 45 degrees with respect to the optical axis of the crystal of the Wollaston prism 21. This linearly polarized laser beam L enters the Wollaston prism 21. The Wollaston prism 21 is separated into two polarized light beams whose vibration directions are orthogonal to each other from the birefringence of the crystal, and refracted in opposite directions on the bonded surfaces. As a result, the light is separated into two polarized lights orthogonal to each other and travels with a predetermined opening angle. In FIG. 1, the central optical axes of the two polarized lights are shown, but in actuality, the light shift (shear amount) is extremely small.

  The laser light L that has been polarized by the Wollaston prism 21 is condensed by the first objective lens 22 in a state where the optical axes are parallel to each other, and is focused on the focal plane of the sample S. The two polarized light beams that have passed through the sample S are converted by the second objective lens 24 into parallel light having inclinations corresponding to the respective focal positions. Note that the focal planes of the first objective lens 22 and the second objective lens 24 are at the same position.

  That is, when the focal point is at the center of the optical axis, the two polarized lights become parallel light parallel to the optical axis, and become parallel light having an inclination with respect to the optical axis as the focus is shifted from the optical axis center. The two polarized lights that have become parallel light are retroreflected by the corner cube 25 in the same direction as the incident direction. Therefore, the light travels back along the same optical path as the incident light, and enters the second objective lens 24 again.

  The two polarized lights are again focused on the same part of the sample S by the second objective lens 24. Thereafter, the two polarized lights return on the same optical path and are guided to the Wollaston prism 21 by the first objective lens 22. The Wollaston prism 21 refracts light according to the polarization direction and acts so that the traveling directions of the two polarizations overlap.

  In addition, the light that has passed through the Wollaston prism 21 enters the polarizer 20, and the polarizer 20 causes interference between the two polarized lights. When there is no phase difference in the optical path of the two polarized lights, the two polarized lights are strengthened by interference. On the other hand, when there is a phase difference between the optical paths of the two polarized light beams, the two polarized light beams are weakened due to less interference components.

  The two polarized lights are focused at two points on the sample S that are slightly separated from each other. Depending on whether or not a phase difference is generated between the two points, it is determined whether the intensity of the light is increased or decreased, thereby causing a contrast. Here, the case of open Nicol in which the light intensity is shown brightly when there is no phase difference between the two polarized lights is shown.

  The light that has passed through the polarizer 20 (referred to as return light R) is reflected by the mirror 19 and collected by the photographing lens 18 in the pinhole 8P. Since the pinhole 8P passes only light within the focal range of the sample S, the image generated by the camera 15 is a confocal image. The return light R that has passed through the pinhole 8P is reflected by the half mirror 11a.

  Then, the return light R reflected by the half mirror 11 a is relayed by the first relay lens 12 and reflected by the mirror 13. Then, the image is formed on the camera 15 by the second relay lens 14. The camera 15 converts the formed return light R into an electric signal and outputs it to the control unit 16. The control unit 16 performs predetermined image processing based on the input electrical signal.

  Here, the control unit 16 controls the motor 10 to apply a rotational force. Thereby, the connecting drum 9 rotates and the microlens disk 7 and the pinhole disk 8 rotate integrally. A number of microlenses 7M are arranged on the microlens disk 7, and a number of pinholes 8P having the same pattern are arranged on the pinhole disk 8.

  Therefore, by rotating the microlens disk 7 and the pinhole disk 8 integrally, as shown in FIG. 2, the pinhole 8P through which the laser light L passes changes sequentially with rotation. Thereby, the laser beam L scans the focal plane of the sample S on a horizontal plane. The return light R passes through the pinhole 8P again, is reflected by the half mirror 11a, and is scanned on the imaging surface of the camera 15.

  Therefore, the laser light L can be scanned at high speed in the horizontal plane direction of the sample S by integrally rotating the microlens disk 7 and the pinhole disk 8. Thereby, since the camera 15 outputs the electrical signal of the return light R sequentially scanned, the control unit 16 can generate an image of the sample S at high speed. Then, the generated image of the sample S is displayed on the monitor 17.

  At this time, the laser light L applied to the sample S and the return light R imaged on the camera 15 pass through the pinhole 8P. Since the pinhole 8P passes only light within the focal point of the sample S and does not allow light other than that to pass through, the return light R imaged on the camera 15 is only light within the focal range of the sample S. Thereby, a high-resolution confocal image is generated in the depth direction.

  Accordingly, in the differential interference mode, the laser light L is separated into two polarized lights by the Wollaston prism 21 to focus on the sample S, and the two polarized lights that have passed through the sample S are converted into the second objective lens 24 and the corner cube 25. Is retroreflected. Thereby, the two polarized lights are again focused on the sample S. That is, the two polarized light beams pass through the sample S twice.

  As a result, the phase difference generated between two spaced points of the sample S is doubled. The two polarized lights whose phase difference is doubled interfere with each other by the polarizer 20 and are converted into return light R and imaged by the camera 15. Accordingly, the phase difference of the return light R detected by the camera 15 is also doubled, and the obtained contrast is emphasized.

  That is, instead of simply transmitting the laser beam L twice through the sample S and observing the distribution of the absorption of the light, the two polarizations for detecting the phase difference are transmitted through the sample S twice. Can be doubled. Therefore, a portion where light is strengthened and a portion where light is weakened due to the phase difference between the two optical paths are clearly identified, and an image with good contrast can be acquired. Thereby, even when the sample S has a small refractive index difference like a cell, an image with high contrast can be obtained.

  The above is the differential interference mode. When it is desired to observe the cell function information, the control unit 16 is used to set the fluorescence mode. Thereby, the first moving mechanism arranges the dichroic mirror 11b in the optical path of the laser beam L. The second moving mechanism retracts the polarizer 20, the Wollaston prism 21, the second objective lens 24, and the corner cube 25 from the optical path. That is, the configuration of FIG. 3 is obtained.

  In this state, the control unit 16 oscillates the laser light L from the light source 4. This laser light L is condensed by the microlens 7M of the microlens disk 7 through the fiber 5 and the collimating lens 6. Since the dichroic mirror 11b has a characteristic of transmitting the laser beam L, the laser beam L transmits the dichroic mirror 11b and focuses on the pinhole 8P.

  Then, the light is collimated by the photographing lens 18, reflected by the mirror 19, and incident on the first objective lens 22. The laser light L incident on the first objective lens 22 is focused on the sample S mounted on the dish 23. A fluorescent dye, fluorescent protein, or the like is introduced into the sample S, and the laser light L has a wavelength at which the fluorescent dye, fluorescent protein, or the like reacts.

  Therefore, when the laser beam L is focused on the sample S, the sample S generates fluorescence. This fluorescence becomes return light R. The return light R is focused on the pinhole 8P of the pinhole disk 8 via the first objective lens 22, the mirror 19, and the photographing lens 18. Then, only light within the focal range of the sample S passes through the pinhole 8P and enters the dichroic mirror 11b. Thereby, the generated image becomes a confocal image.

  The dichroic mirror 11b reflects the wavelength of fluorescence (return light R). Therefore, the reflected return light R forms an image on the camera 15 via the first relay lens 12, the mirror 13, and the second relay lens 14. Then, the return light R imaged on the camera 15 is converted into an electric signal and output to the control unit 16.

  Similarly to the case described above, the microlens disk 7 and the pinhole disk 8 are integrally rotated by rotating the motor 10. As a result, the sample S can be scanned with the laser light L, and an image can be generated at high speed.

  Therefore, a fluorescence image can be obtained by setting the fluorescence mode in the control unit 16. Since only the differential interference mode or the fluorescence mode is set in the control unit 16, the first moving mechanism and the second moving mechanism move the optical components so as to be inserted and removed from the optical path. It becomes possible to easily switch between the fluorescence microscope. That is, it is possible to obtain both differential interference images and fluorescent images with a simple configuration and simple operation using one (one) microscope apparatus 1.

  In the above, the first moving mechanism is controlled to switch between the half mirror 11a (differential interference mode) and the dichroic mirror 11b (fluorescence mode), but the first moving mechanism may not be switched. That is, the dichroic mirror 11b is fixedly arranged, and the dichroic mirror 11b is used even in the differential interference mode.

  However, in this case, a dichroic mirror 11b having a function equivalent to that of a half mirror is used. That is, in the dichroic mirror 11b, the light source 4 is selected so as to oscillate the laser light L in a wavelength band having a predetermined reflectance and a predetermined transmittance.

  The corner cube 25 is an optical system in which a cube is formed by combining three mirrors at right angles to each other. As the mirror, a mirror that is made by polishing glass to be totally reflected may be used, or a mirror that is provided with a reflective film may be used. In short, any optical element can be used as long as it performs retroreflection to return incident light in the incident direction.

  Further, the Wollaston prism 21 may be a Nomarski prism, or another element having birefringence may be used.

  In addition, by using a polarization beam splitter instead of the half mirror 11a and inserting a Faraday rotator or the like in front of the polarizer 20, the polarization planes of the laser light L and the return light R are changed by 90 degrees, and light is efficiently emitted. You may make it collect | recover.

  In addition, it is desirable to insert a fluorescence filter that selectively transmits only the fluorescence wavelength component of the sample S in the front stage of the camera 15.

  In FIG. 1, the light incident on the Wollaston prism 21 indicates light parallel to the optical axis. However, when light having an inclination with respect to the optical axis is incident by the scanning optical system 2, Similarly, the light is divided into two optical paths, and is condensed at two slightly separated points on the focal plane, and returns to the same optical path by the action of the second objective lens 24 and the corner cube 25.

  Next, Modification 1 will be described. FIG. 4 shows a microscope apparatus 1 according to the first modification, in which a λ / 4 wavelength plate 26 is added to the microscope apparatus 1 of the above-described embodiment. The λ / 4 wavelength plate 26 is inserted between the polarizer 20 and the Wollaston prism 21. Other configurations are the same as those of the embodiment.

  The embodiment is open Nicol that becomes bright when there is no phase difference when the two polarized light beams pass through the sample S, but in the first modification, there is no phase difference when the two polarized light beams pass through the sample S. It becomes crossed Nicol which becomes dark.

  In the first modification, the laser light L transmitted through the polarizer 20 enters the λ / 4 wavelength plate 26. When transmitted through the polarizer 20, the laser light L is linearly polarized light and is converted into circularly polarized light by the action of the λ / 4 wavelength plate 26. At this time, the optical axis of the crystal of the Wollaston prism 21 and the polarization plane of the laser light L are arranged to be inclined by 45 degrees.

  The laser light L that has become circularly polarized light is separated by the Wollaston prism 21 into two linearly polarized light beams whose vibration directions are orthogonal to each other from the birefringence of the crystal, and refracted in opposite directions on the bonding surface.

  As a result, when the light is emitted from the Wollaston prism 21, it is separated into two linearly polarized light beams whose vibration directions are orthogonal to each other, and proceed with being separated with a predetermined opening angle. Then, the light advances to the first objective lens 22, the sample S, the second objective lens 24, and the corner cube 25, retroreflects, and enters the Wollaston prism 21 again. At this time, the two polarized light beams pass through the sample S twice.

  When the two polarized light beams are superposed by the Wollaston prism 21, if there is no phase difference between the two optical paths, the circularly polarized light rotates in the opposite direction to that when entering the sample S. This circularly polarized light is converted into linearly polarized light by the λ / 4 wavelength plate 26 and enters the polarizer 20.

  When the two polarized light beams interfere with each other in the polarizer 20, there is no light that cancels each other and passes through. Therefore, when there is no phase difference between the two light paths of the sample S that are slightly separated from each other, the light intensity becomes dark. On the other hand, when there is a phase difference between the two optical paths, the polarization state changes according to the phase difference, and a part of the light passes through the polarizer 20. Thereby, when the phase difference is produced in the optical path of two polarized light, the intensity | strength of light becomes bright.

  In the case where there is no phase difference between the two polarizations, the reason why the light cancels out in the polarizer 20 and there is no light passing through is that the polarization plane of the return light R is shifted by 90 degrees from the polarization plane of the polarizer 20. This can also be explained from the fact that it cannot pass through the polarizer 20.

  Therefore, in the first modification, even in the case of crossed Nicols, the phase difference can be doubled by transmitting the two polarized light beams through the sample S twice. Thereby, an image with high contrast can be obtained.

  In the first modification, the polarization plane may be rotated 45 degrees using a Faraday rotator or the like instead of the λ / 4 wavelength plate 26. In this case, the polarizer 20 and the optical axis of the Wollaston prism 21 are arranged in the same direction, and the polarization plane is rotated 45 degrees by the Faraday rotator.

  Next, a second modification will be described. In the second modification, the configurations of the scanning optical system 2 and the microscope optical system 3 are different from those in the embodiment. First, the scanning optical system 2 will be described. The scanning optical system 2 includes a light source 31, a half mirror 32a, a scanning optical unit 33, a scanning system pupil relay lens 34, a focusing lens 35, a pinhole 36, a detector 37, a control unit 16, and a monitor 17. . The control unit 16 and the monitor 17 are the same as those in the embodiment.

  Moreover, the microscope optical system 3 adds a first pupil relay lens 41 and a second pupil relay lens 42 in addition to the configuration of the embodiment. The first pupil relay lens 41 and the second pupil relay lens 42 constitute a pupil relay lens system, and the pupil relay lens system is provided between the second objective lens 24 and the corner cube 25.

  As in the embodiment, the first mirror is attached to the half mirror 32a, and the half mirror 32a and the dichroic mirror 32b can be switched with respect to the optical path of the laser light L. FIG. 6 shows the case where the dichroic mirror 32b is arranged. Further, as shown in the figure, the second moving mechanism causes the polarizer 20, the Wollaston prism 21, the second objective lens 24, the first pupil relay lens 41, the second pupil relay lens 42, and the corner cube 25 to be laser light. It is possible to retreat from the L optical path.

  In the above configuration, first, the differential interference mode will be described. The control unit 16 sets the differential interference mode. Accordingly, the first moving mechanism arranges the half mirror 32a in the optical path of the laser light L. Further, the second moving mechanism arranges the polarizer 20, the Wollaston prism 21, the second objective lens 24, the first pupil relay lens 41, the second pupil relay lens 42, and the corner cube 25 in the optical path of the laser light L. That is, the configuration shown in FIG.

  A parallel laser beam L is oscillated from the light source 31. The laser beam L is reflected by the half mirror 32 a and guided to the scanning optical unit 33. The scanning optical unit 33 includes a first variable mirror 33a that can rotate around one axis, and a second variable mirror 33b that can rotate around an axis substantially orthogonal to the axis of the first variable mirror 33a.

  The laser light L is scanned in the horizontal plane direction of the sample S by the first variable mirror 33a and the second variable mirror 33b. The light that has passed through the scanning optical unit 33 exits the scanning optical system 2 from the scanning system pupil relay lens 34 and enters the photographing lens 18 of the microscope optical system 3. Then, it passes through the polarizer 20 and the Wollaston prism 21 and is separated into two polarized lights.

  The two separated polarized lights are focused on the sample S of the dish 23 by the first objective lens 22. The two polarized light beams pass through the sample S and are guided from the second objective lens 24 to the corner cube 25 by the pupil relay lens system of the first pupil relay lens 41 and the second pupil relay lens 42.

  Then, the sample is retroreflected by the corner cube 25 and passes through the sample S again through the first pupil relay lens 41, the second pupil relay lens 42, and the second objective lens 24. As a result, the two polarized light beams pass through the sample S twice. Then, the light enters the Wollaston prism 21 and the polarizer 20 from the first objective lens 22, and the two polarized lights interfere with each other.

  The interfering return light R is reflected by the mirror 19 and enters the half mirror 32 a from the photographing lens 18 through the scanning pupil relay lens 34 and the scanning optical unit 33. Then, the light passes through the half mirror 32 a and is converged on the detector 37 by the focusing lens 35. At this time, the pinhole 36 is provided on the optical path, and the pinhole 36 is disposed in a positional relationship conjugate with the focal point of the first objective lens 22. Therefore, only the light within the focal range of the sample S of the return light R passes. Thereby, the generated image becomes a confocal image.

  The detector 37 converts the return light R into an electric signal and outputs it to the control unit 16. The control unit 16 performs predetermined image processing based on the input electrical signal and generates an image of the sample S. The image at this time is a differential interference image based on the phase difference between two points of the sample S that are slightly separated from each other. Since the phase difference is doubled, the image is a high contrast image.

  Here, the pupil position of the second objective lens 24 is very close to the second objective lens 24. Since the optical system of the lens including the second objective lens 24 is generally mounted inside the lens barrel, the pupil position of the second objective lens 24 may be located inside the lens barrel. The corner cube 25 can be arranged at the pupil position (back focal position) of the second objective lens 24 to obtain the highest light utilization efficiency. However, the pupil position of the second objective lens 24 is located inside the lens barrel. Therefore, the corner cube cannot be arranged at the pupil position of the second objective lens 24.

  Therefore, a first pupil relay lens 41 and a second pupil relay lens 42 that relay the pupil position of the second objective lens 24 are provided. Thereby, the pupil position of the second objective lens 24 is relayed, and the pupil position can be relayed to the vicinity of the corner cube 25 at a position away from the second objective lens 24. Thereby, almost all of the light emitted from the pupil of the second objective lens 24 is incident on the second objective lens 24 again by retroreflection, so that high light utilization efficiency can be obtained.

  The above is the differential interference mode. Next, the fluorescence mode will be described. The control unit 16 sets the fluorescence mode. As a result, the first moving mechanism switches from the half mirror 32a to the dichroic mirror 32b. The second moving mechanism retracts the polarizer 20, the Wollaston prism 21, the second objective lens 24, the first pupil relay lens 41, the second pupil relay lens 42, and the corner cube 25 from the optical path of the laser light L. That is, the configuration shown in FIG.

  The dichroic mirror 32b has an optical characteristic of reflecting the wavelength of the laser light L oscillated by the light source 31 and transmitting the fluorescence of the sample S (wavelength longer than the wavelength of the laser light L). Therefore, the laser beam L oscillated by the light source 31 is reflected by the dichroic mirror 32b. The laser light L is scanned by the scanning optical unit 33 and focused on the sample S through the scanning pupil relay lens 34, the photographing lens 18, the mirror 19, and the first objective lens 22.

  The sample S is fluorescent by the laser light L to become return light R and returns to the dichroic mirror 32b. Since the dichroic mirror 32 b transmits fluorescence, the return light R is transmitted and detected by the detector 37 via the focusing lens 35 and the pinhole 36. By detecting the return light R, the control unit 16 generates a fluorescence image of the sample S. In addition, it is desirable to arrange a fluorescence filter for selecting a fluorescence wavelength in the previous stage of the detector 37.

  Therefore, by switching the differential interference mode and the fluorescence mode by the control unit 16, the differential interference image and the fluorescence image can be easily and simply obtained with one microscope apparatus. In addition, an image with high contrast can be obtained, and by arranging the first pupil relay lens 41 and the second pupil relay lens 42, the pupil position can be relayed, and high light utilization efficiency can be obtained. It becomes like this.

  Next, a third modification will be described. The microscope apparatus of Modification 3 is configured by a microscope optical system 50 and does not have a scanning optical system. FIG. 7 shows a microscope optical system 50 of the third modification.

  The microscope optical system 50 includes a light source 51, an illumination lens 52, a wavelength selection filter 53, a half mirror 54a, a polarizer 20, a Wollaston prism 21, a first objective lens 22, a dish 23, a second objective lens 24, and a telecentric imaging lens. 55, a reflection mirror 56, a photographic lens 18, and a camera 57. Among these, the polarizer 20, the Wollaston prism 21, the first objective lens 22, the dish 23, the second objective lens 24, and the photographing lens 18 are the same as those in the implementation system.

  A first moving mechanism is attached to the half mirror 54a, and either one of the half mirror 54a and the dichroic mirror 54b can be selected. The polarizer 20, the Wollaston prism 21, the second objective lens 24, the telecentric imaging lens 55, and the reflection mirror 56 can be retracted from the optical path by the second moving mechanism.

  A control unit (not shown) (corresponding to the control unit 16 in the embodiment) can set the differential interference mode and the fluorescence mode. When the differential interference mode is set, as shown in FIG. 7, the half mirror 54a is selected, and the polarizer 20, the Wollaston prism 21, the second objective lens 24, the telecentric imaging lens 55, and the reflection mirror 56 are placed on the optical path. To place.

  On the other hand, when the control unit 16 sets the fluorescence mode, as shown in FIG. 8, the dichroic mirror 54b is selected, the polarizer 20, the Wollaston prism 21, the second objective lens 24, the telecentric imaging lens 55, the reflection The mirror 56 is retracted from the optical path.

  First, the differential interference mode will be described. When the differential interference mode is set, the first moving mechanism and the second moving mechanism operate so as to have the configuration shown in FIG. The light source 51 emits illumination light L. The illumination light L is adjusted by the illumination lens 52 so that the sample S is illuminated uniformly. The illumination light L that has passed through the illumination lens 52 passes through the wavelength selection filter 53.

  The wavelength selection filter 53 passes only the wavelength component irradiated to the sample S. The illumination light L that has passed through the wavelength selection filter 53 passes through the half mirror 54a. Then, it is separated into two polarized light by the polarizer 20 and the Wollaston prism 21. The two separated polarized lights are focused on two points that are slightly separated from each other on the sample S mounted on the dish 23 by the first objective lens 22.

  The two polarized light beams transmitted through the sample S are condensed by the second objective lens 24 and then converted into parallel light by the telecentric imaging lens 55. The illumination light L that has become the parallel light is reflected by the reflection mirror 56 and passes through the sample S from the telecentric imaging lens 55 and the second objective lens 24 again.

  As a result, the two polarized light beams are transmitted twice through two points that are slightly separated from the sample S. Then, the light enters the Wollaston prism 21 and the polarizer 20 from the first objective lens 22 and interferes therewith. Then, the two interfered polarized lights become return light R, reflected by the half mirror 54a, and imaged on the camera 57 by the photographing lens 18. The camera 57 converts the formed return light R into an electrical signal. The camera 57 is connected to a control unit (not shown), and performs image processing based on an electrical signal input from the camera 57. Thereby, a differential interference image can be generated.

  On the other hand, when the fluorescence mode is selected, the first moving mechanism and the second moving mechanism operate so as to have the configuration shown in FIG. The illumination light L emitted from the light source 51 is adjusted so that the sample S is uniformly illuminated by the illumination lens 52. Further, only the wavelength that excites the fluorescence of the sample S is selected by the wavelength selection filter 53. The illumination light L enters the dichroic mirror 54b.

  The dichroic mirror 54b has an optical characteristic of transmitting the light having the wavelength of the illumination light L and reflecting the fluorescence of the sample S. Therefore, the illumination light L passes through the sample S. Then, the first objective lens 22 focuses on the sample S mounted on the dish 23. Thereby, the sample S fluoresces and the return light R is generated.

  The return light R enters the dichroic mirror 54b from the first objective lens 22. The return light R incident on the dichroic mirror 54 b is reflected and imaged on the camera 57 by the photographing lens 18. Then, by detecting the return light R imaged on the camera 57, a fluorescent image is generated.

  In the third modification, a telecentric imaging lens 55 and a reflection mirror 56 are used as a retroreflective optical system without using a corner cube. Thereby, a retroreflective optical system can be realized.

  Also, a confocal image cannot be obtained as compared with the embodiment. However, the configuration is very simple compared to the embodiment. That is, even in the configuration as in the fourth modification, an image with high contrast can be obtained by transmitting two polarized lights through the sample S twice.

  Further, the first moving mechanism and the second moving mechanism are controlled by the control unit so that two types of images, that is, a differential interference image and a fluorescence image can be easily and simply acquired using one microscope apparatus. become. As in the above-described embodiment, it is desirable to arrange a fluorescence filter for selecting the fluorescence wavelength in the front stage of the camera 57.

  Next, Modification 4 will be described. FIG. 9 a) shows a fourth modification, in which the first objective lens 22, the dish 23, and the second objective lens 24 are shown. The first objective lens 22 is a dry objective lens. Here, a 20 × dry objective lens, a numerical aperture of 0.75, and a working distance of about 0.6 mm are used. Of course, numerical values other than these may be used.

  Since the working distance of the first objective lens 22 is very short, when a similar dry objective lens is used as the second objective lens 24, a dish in which the sample S is mounted between the first objective lens 22 and the second objective lens 22 is used. It becomes difficult to arrange 23.

  Therefore, a water immersion objective lens is used as the second objective lens 24. As the water immersion objective lens, it is desirable to use, for example, an electrophysiological objective lens that does not require a cover glass. For example, it is desirable to use a 40 × water immersion lens, a numerical aperture of 0.8, and a working distance of about 3.3 mm.

  By using such a water immersion objective lens as the second objective lens 24, a sufficient working distance can be secured, and the dish 23 on which the sample S is mounted can be easily disposed. Further, when the sample S is a biological sample of a cell, since it is often in the culture medium W, it is preferable to use a water immersion objective lens.

  Next, Modification 5 will be described. FIG. 9B) shows a fifth modification, in which the first objective lens 22, the dish 23, and the second objective lens 24 are shown. Similar to the modified example 4, as the first objective lens 22, a dry objective lens having a magnification of 20 times, a numerical aperture of 0.75, and a working distance of about 0.6 mm is used. Of course, numerical values other than these may be used.

  As the second objective lens 24, an objective lens that works optimally on the wavelength of the laser light L used in the differential interference mode is used. That is, a lens that functions as an objective lens is used only for light having the wavelength of the laser light L oscillated by the light source 4 used in the differential interference mode.

  In general, when an objective lens corresponding to a wide wavelength band of visible light is used, the structure is complicated because the lens is composed of a large number of lenses, and the working distance is shortened. Therefore, when an objective lens having a short working distance is used, it becomes difficult to arrange the dish 23 on which the sample S is mounted.

  Therefore, the second objective lens 24 uses an objective lens specialized for the wavelength of the laser light L used in the differential interference mode. As a result, it is only necessary to fulfill the function as an objective lens at a specific wavelength. Therefore, the second objective lens 24 can be realized with a small number of lenses, for example, one aspherical lens or two to three spherical lenses. .

  Thereby, a sufficient working distance can be secured. For example, the second objective lens 24 having a numerical aperture of 0.7 and a working distance of about 20 mm can be used. Therefore, since a sufficient working distance can be secured, the dish 23 on which the sample S is mounted can be easily arranged.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Scan optical system 3 Microscope optical system 4 Light source 6 Collimating lens 7 Micro lens disk 8 Pinhole disk 9 Connection drum 10 Motor 11a Half mirror 11b Dichroic mirror 15 Camera 16 Control part 20 Polarizer 21 Wollaston prism 22 1st Objective lens 23 Dish 24 Second objective lens 25 Corner cube 26 λ / 4 wavelength plate 31 Light source 32a Half mirror 32b Dichroic mirror 33 Scanning optical unit 41 First pupil relay lens 42 Second pupil relay lens L Laser beam R Return beam S Sample

Claims (6)

A light source that oscillates the illumination light applied to the sample;
A polarization separation element that separates the illumination light into two polarizations having different polarization directions;
A first objective lens for condensing the two polarized lights at two spaced points of the sample;
A second objective lens that converts the two polarized lights transmitted through the sample into parallel light;
A retroreflective optical system that retroreflects the two polarized light beams that have passed through the second objective lens;
A polarizer that converts the illumination light incident on the polarization separation element into linearly polarized light, and retroreflected by the retroreflective optical system and interferes with two polarized light beams transmitted through the sample;
A microscope apparatus comprising: A moving mechanism for removably moving the polarization separation element, the second objective lens, the retroreflective optical system, and the polarizer from the optical path of the illumination light;
When the first mode is set, the polarization separation element, the second objective lens, the retroreflective optical system, and the polarizer are inserted into the optical path of the illumination light, and when the second mode is set, the A control unit for controlling the moving mechanism so as to retract the polarization separation element, the second objective lens, the retroreflective optical system, and the polarizer from the optical path of the illumination light;
The microscope apparatus according to claim 1, further comprising: The moving mechanism includes a half mirror that transmits or reflects the illumination light from the light source to the sample and reflects or transmits return light from the sample when the control unit is set to the first mode. When the second mode is set in the control unit, the dichroic mirror that transmits or reflects the wavelength of the illumination light from the light source and reflects or transmits the wavelength of the fluorescence of the sample is switched. The microscope apparatus according to claim 2, characterized in that: A pinhole disk in which a plurality of pinholes that transmit light within the focal range of the sample are arranged;
A lens disk arranged in the same pattern as the pinhole, and a plurality of lenses arranged to collect the illumination light in the pinhole; and
A rotating unit that integrally rotates the pinhole disk and the lens disk;
The microscope apparatus according to claim 3, further comprising: The microscope apparatus according to claim 4, wherein a λ / 4 wavelength plate is provided between the polarization separation element and the polarizer. The microscope apparatus according to claim 4, wherein a pupil relay lens system that relays a pupil position of the second objective lens is provided between the second objective lens and the retroreflective optical system.

Images