JP5743209B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus that performs observation by irradiating an observation target with laser light and detecting return light from the observation target.

  2. Description of the Related Art Conventionally, a microscope apparatus has been used in which a sample into which a fluorescent dye or fluorescent protein is introduced is irradiated with laser light to generate fluorescence, and the sample is observed based on the generated fluorescence. One type of microscope apparatus of this type is a structured illumination microscope (Structured Illumination Microscopy). The structured illumination microscope generates a super-resolution image exceeding the Rayleigh limit spatial frequency by causing interference fringes formed using a diffraction grating to interfere with each other.

  There is a technique disclosed in Non-Patent Document 1 as a structured illumination microscope. This technique will be described with reference to FIG. FIG. 6 shows a structured microscope (hereinafter, microscope apparatus 100). The microscope apparatus 100 includes a fiber 101, a collimating lens 102, a polarizer 103, a diffraction grating 104, a first imaging lens 105, a dichroic mirror 106, an objective lens 107, a second imaging lens 108, and a CCD 109. .

  The fiber 101 is connected to a laser light source (not shown) and emits the guided laser beam L. Laser light L emitted from the fiber 101 enters the collimating lens 102. The collimating lens 102 is provided at the focal position of the output end of the fiber 101, and converts the laser light L as divergent light into parallel light.

  This laser light L is incident on the polarizer 103. The polarizer 103 converts the laser light L into light having a certain polarization direction (linearly polarized light). The polarized laser beam L is incident on the diffraction grating 104. The diffraction grating 104 diffracts the laser light L into zero-order light (non-diffracted light) and ± first-order light (diffracted light). The diffraction angle is determined by the coefficient of the diffraction grating 104.

  The diffracted laser light L enters the imaging lens 105, and the laser light L emitted from the imaging lens 105 enters the dichroic mirror 106. The dichroic mirror 106 is an optical element that transmits the wavelength of the laser light L and reflects the wavelength at which the sample S to be observed is fluorescent. Accordingly, the laser light L passes through the dichroic mirror 106.

  The laser beam L incident on the objective lens 107 is focused on the sample S. The laser light L is diffracted light, and becomes structural illumination having periodic intensity modulation by combining and interfering on the surface of the sample S. By irradiating the laser beam L, the fluorescent dye or fluorescent protein introduced into the sample S fluoresces and becomes return light R.

  The return light R enters the dichroic mirror 106 through the objective lens 107. The return light R is reflected by the dichroic mirror 106 and enters the second imaging lens 108. The second imaging lens 108 returns the light R to the CCD 109 and condenses it.

  The sample S is irradiated with diffracted structural illumination. Then, due to the interaction between the structure of the sample S and the illumination structure, the image of the return light R formed on the CCD 109 becomes a moire image. The diffraction grating 104 is provided with a mechanism for moving and rotating (moving and rotating mechanism) (not shown), and performs parallel translation five times in the direction in which the laser light L is diffracted by 1/5 period. Then, the diffraction grating 104 is rotated by 120 degrees for a total of three times for each parallel movement. That is, a total of 15 movements and rotations are performed.

  A moire image is acquired every time this movement and rotation is performed. Therefore, a total of nine moire images are acquired. Spatial frequency components exceeding the “Rayleigh limit spatial frequency” are reproduced by performing image processing on the obtained nine moire images using Fourier transform or the like. Then, this spatial frequency component is added to reconstruct the image. The reconstructed image is generated as a super-resolution image having a spatial frequency exceeding the “Rayleigh critical spatial frequency”.

"Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination" Mats G.I.Gustafsson, et.al, Biophysical Journal vol 94 pp.4957-4970 (2008)

  A general structured illumination microscope is a method called a “Wide-Field illumination method”, which is one of methods for illuminating the entire surface of the sample S simultaneously. That is, this is a technique for super-resolution of a predetermined tomography of the sample S2, and a technique for super-resolution of an image in a predetermined plane. Therefore, there is no resolution in the optical axis direction.

  As disclosed in Non-Patent Document 1, in order to obtain resolution in the optical axis direction, image processing using a complex algorithm is performed on the cross section of the sample S using 15 moire images. There is a need.

  Accordingly, an object of the present invention is to obtain an image having a three-dimensionally excellent resolution without performing image processing using a complicated algorithm using a microscope apparatus.

In order to solve the above problems, a microscope apparatus of the present invention includes a light source that oscillates a laser beam irradiated on an observation target, and a pinhole that allows a laser beam within the focal range of the observation target to pass through the laser beam. A spatial modulation unit that performs spatial modulation to form interference fringes in the planar direction of the observation target with respect to the laser light that has passed through the pinhole, and the laser light spatially modulated by the spatial modulation unit A microscope optical system for focusing on, an observation unit for observing the return light from the observation target, a moving rotation mechanism for moving and rotating the spatial modulation unit, and between the movement rotation mechanism and the microscope optical system And a relay optical system that relays the laser beam .

  According to this microscope apparatus, the resolution in the optical axis direction can be obtained by passing the laser light through the pinhole. In addition, since the laser light is spatially modulated by the spatial modulation unit, the resolution in the plane direction can be increased. As a result, since means for increasing the resolution in the optical axis direction and the plane direction are arranged in the optical path of the laser light, an image with a three-dimensionally high resolution can be obtained in a small number of image acquisition times, that is, in a short time.

  The scanning means includes a pinhole disk in which a plurality of the pinholes are arranged, a lens disk arranged in the same pattern as the pinholes, and a plurality of lenses in which the laser beams are condensed on the pinholes; And a rotating unit that integrally rotates the pinhole disk and the lens disk.

  By rotating the pinhole disk and the lens disk integrally, the laser beam can be scanned at a high speed, and an image can be acquired in a short time. By arranging pinholes on the pinhole disk, a confocal image with high resolution in the optical axis direction can be generated at high speed.

  A polarization adjusting element that converts the laser light emitted from the light source into one polarization direction; and a polarization beam splitter that transmits the polarization direction of the laser light and reflects light in the polarization direction orthogonal to the polarization direction. And a λ / 4 wavelength plate provided between the observation object that reflects the laser light and the polarizing beam splitter.

  When the object to be observed is not a material that emits fluorescence like a sample but a reflector such as an IC chip or an optical disk, a three-dimensional structure is provided by providing a polarization adjusting element, a polarizing beam splitter, and a λ / 4 wavelength plate. It becomes possible to perform observation with high resolution.

  A housing that houses the microscope optical system, a moving and rotating mechanism that moves and rotates the spatial modulation unit, and a relay optical that relays the laser light provided between the housing and the moving and rotating mechanism. And a system.

  When the distance between the moving and rotating mechanism and the housing that houses the microscope optical system is close, the moving and rotating mechanism of the moving and rotating mechanism may interfere with the housing. For this reason, by providing the relay optical system, the distance between the moving and rotating mechanism and the housing can be separated and interference can be eliminated.

  The zoom lens further includes a zoom lens that changes the magnification of the image to be observed so as to form an interference fringe having a period synchronized with the numerical aperture of the objective lens provided in the microscope optical system.

  In order to synchronize the numerical aperture of the objective lens of the microscope with the period of the interference fringes formed by the spatial modulation unit, the zoom lens changes the magnification. Thereby, the numerical aperture and the interference fringes can be synchronized, and an accurate super-resolution image can be obtained.

  In the present invention, a pinhole and a spatial modulation unit are provided in the optical path from the laser beam oscillated from the light source to the observation target, and an image with high resolution in the optical axis direction and the planar direction can be obtained. Since spatial modulation is performed on the light passing through the pinhole, a high-resolution image can be obtained three-dimensionally in a short time with a small number of times.

It is a block diagram of the microscope apparatus of embodiment. It is a block diagram of the microscope apparatus of a 1st modification. It is a conceptual diagram of the microscope apparatus of the 2nd modification. It is a block diagram of the microscope apparatus of a 3rd modification. It is a block diagram of the microscope apparatus of a 4th modification. It is a block diagram of the conventional microscope apparatus.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a microscope apparatus 1 of the present embodiment. The observation target of the microscope apparatus 1 is a sample S into which a fluorescent protein, a fluorescent dye, or the like is introduced. When the sample S is illuminated, fluorescence is generated. As will be described later, the observation target is not limited to the sample S, and may be, for example, a reflector.

  The microscope apparatus 1 is a structured illumination microscope (Structured Illumination Microscopy). The microscope apparatus 1 includes a light source 2, a condenser lens 3, a fiber incident end 4, a fiber 5, a fiber exit end 6, a collimator lens 7, a scanning unit 8, a telecentric optical system 9, a spatial modulation unit 10, and a microscope optical system 11. An imaging lens 12 and a camera 13 are provided.

  The light source 2 is a laser light source that oscillates the laser light L. The laser light L is light irradiated to the sample S and has a wavelength at which a fluorescent protein or a fluorescent dye introduced into the sample S reacts. Note that lamp light may be used instead of the laser light L. In this case, the light source 2 uses a lamp. The laser light L oscillated from the light source 2 is condensed on the fiber incident end 4 by the condenser lens 3.

  The fiber incident end 4 is an incident end of the fiber 5. The fiber 5 guides the laser light L. A fiber exit end 6 is provided at the exit end of the fiber 5, and the guided laser beam L is emitted as divergent light. The diverged laser beam L enters the collimating lens 7. The collimating lens 7 converts the divergent laser beam L into parallel light. For this purpose, the fiber exit end 6 is disposed at the focal position of the collimating lens 7. The laser beam L converted into parallel light by the collimating lens 7 enters the scanning unit 8.

  The scanning unit 8 is a scanning unit that scans the laser light L in a plane direction, and includes a lens disk 21, a pinhole disk 22, a connecting drum 23, and a dichroic mirror 24. The lens disk 21 is a disk-shaped disk in which a plurality of lenses (microlenses) 21R are arranged. A plurality of lenses 21 </ b> R are spirally arranged in the lens disk 21. Each lens 21R has a function of condensing the laser light L.

  The pinhole disk 22 is a disk-shaped disk in which a plurality of pinholes 22P are arranged. Pinholes 22P are arranged on the pinhole disk 22 in the same pattern as the lenses R formed on the lens disk 21. That is, they are arranged in a multi-spiral pattern. And the pinhole 22P is formed in the condensing position of the lens 21R. The pinhole 22P is a minute opening through which laser light L and return light R described later pass, and only light within the focal range of the sample S passes therethrough. Thereby, the microscope apparatus 1 can be used as a confocal microscope with high resolution in the optical axis direction.

  The lens disk 21 and the pinhole disk 22 are integrally attached to the connecting drum 23. The connecting drum 23 is connected to a motor (not shown), and the rotational force of the motor is applied to the connecting drum 23. Thereby, the lens disk 21 and the pinhole disk 22 rotate integrally.

  A dichroic mirror 24 is disposed between the lens disk 21 and the pinhole disk 22, that is, between the lens 21R and the pinhole 22P. The dichroic mirror 24 transmits light in a predetermined wavelength range and reflects light in a predetermined wavelength range. Here, it is assumed that the light having the wavelength of the laser light L is transmitted and the light having the wavelength of the return light R described later is reflected. Therefore, the laser light L that has passed through the lens R passes through the dichroic mirror 24 and enters the pinhole 22P.

  The laser light L that has passed through the scanning unit 8 becomes light only within the focal range of the sample S and enters the telecentric optical system 9. The telecentric optical system 9 has a first relay lens 25 and a second relay lens 26. The first relay lens 25 is provided at the focal position of the lens R, and the laser light L that has been diverged by the lens R is incident on the first relay lens 25.

  The first relay lens 25 converts the diverging laser beam L into parallel light and emits it to the second relay lens 26. The second relay lens 26 condenses the laser light L that is parallel light. The distance between the first relay lens 25 and the second relay lens 26 is the sum of the focal lengths of both lenses.

  The spatial modulation unit 10 is disposed at the position of the laser light L condensed by the second relay lens 26. The spatial modulation unit 10 performs spatial modulation (intensity modulation or phase modulation) on the laser light L. By performing this spatial modulation, bright and dark interference fringes are generated in the laser light L. As the spatial modulation unit 10, for example, a diffraction grating can be applied. A rotational movement mechanism 27 is attached to the spatial modulation unit 10, and the spatial modulation unit 10 can be rotated and moved by the rotational movement mechanism 27.

  As the spatial modulation unit 10, for example, a pattern in which the transmittance is periodically changed to high or low may be formed, or a pattern in which the thickness of the glass material is periodically changed may be formed. In any case, the spatial modulation unit 10 has a predetermined planar shape, and forms a pattern that generates bright and dark interference fringes. Laser light L scanned by the scanning unit 8 is incident on the spatial modulation unit 10. At this time, the laser beam L is scanned over a predetermined range of the spatial modulation unit 10, and the spatial modulation unit 10 is provided with a plane that covers the entire scanning range of the laser beam L.

  The laser beam L that has been spatially modulated by the spatial modulation unit 10 enters the microscope optical system 11. The microscope optical system 11 has an imaging lens 28 and an objective lens 29. The imaging lens 28 converts the laser light L, which is divergent light, into parallel light having a predetermined inclination. The laser beam L converted into parallel light by the imaging lens 28 is focused on the sample S by the objective lens 29. Therefore, the sample S is arranged at the focal position of the objective lens 29.

  The sample S is mounted on a dish or the like (not shown), and the laser beam L fluoresces when focused on the sample S. This fluorescence becomes return light R and returns along the same optical path as laser light L. The laser light L applied to the sample S is spatially modulated by the spatial modulation unit 10 and is applied to the sample S as structural illumination having periodic intensity modulation.

  The sample S absorbs the laser light L and emits fluorescence as return light R. The return light R is captured by the objective lens 29, is incident on the spatial modulation unit 10 via the imaging lens 28, and is subjected to spatial modulation. Then, the light passes through the telecentric optical system 9 and enters the dichroic mirror 24 from the pinhole 22P of the pinhole disk 22. That is, the return light R follows the same optical path as the laser light L and is guided to the dichroic mirror 24.

  Since the dichroic mirror 24 has an optical characteristic of reflecting the return light R, the return light R is reflected to the side. And it condenses on the imaging surface of the camera 13 as an observation part with the imaging lens 12. FIG. Note that the surface of the pinhole disk 22 and the imaging surface of the camera 13 are in a conjugate plane.

  The above is the configuration. Next, the operation will be described. Laser light L is oscillated from the light source 2. The laser light L is guided through the fiber 5 from the fiber incident end 4 and is emitted from the fiber exit end 6. Then, the light is converted into parallel light by the collimator lens 7 and enters the scanning unit 8.

  The scanning unit 8 applies a rotational force to the connecting drum 23 so that the lens disk 21 and the pinhole disk 22 rotate integrally. The lens 21R and the pinhole 22P are arranged in a multi-spiral pattern, and the two are in a corresponding positional relationship. Therefore, when the lens disk 21 and the pinhole disk 22 rotate integrally, the position of the laser beam L irradiated to the sample S changes at high speed. Thereby, the laser beam L can be scanned in the plane direction of the sample S (direction orthogonal to the optical axis).

  Only the light within the focal range of the sample S passes through the laser light L that has passed through the pinhole 22P. That is, an image based on the image of the return light R condensed on the camera 13 becomes a confocal image. Accordingly, an image with high resolution in the optical axis direction of the laser beam L can be obtained. The pinhole disk 22 rotates and the laser beam L is scanned, but in any case, since the laser beam L passes through the pinhole 22P, the image based on the image received by the camera 13 is always high in the optical axis direction. It becomes a confocal image of resolution.

  The laser light L that has passed through the pinhole 22P is incident on the spatial modulation unit 10 via the telecentric optical system 9. That is, the laser beam L that generates a confocal image having high resolution in the optical axis direction is incident on the spatial modulation unit 10. The spatial modulation unit 10 generates bright and dark interference fringes and focuses the sample S by the objective lens 29 of the microscope optical system 11. As a result, the laser light L having bright and dark interference fringes is combined and interfered at the focal point of the sample S, resulting in structural illumination having periodic intensity modulation.

  The sample S fluoresces by the laser light L to generate return light R. The frequency of the image of the return light R and the frequency of the structural illumination image are superimposed so that the image acquired by the camera 13 is a moire image. It becomes. At this time, the moire image is an image using the laser light L for generating a confocal image that has passed through the pinhole 22P, that is, has a high resolution in the optical axis direction.

  Therefore, a moire image is acquired by the camera 13. This moire image is an image obtained when the spatial modulation unit 10 is in one state. A rotational movement mechanism 27 is attached to the spatial modulation unit 10. Accordingly, the spatial modulation unit 10 can be rotated and moved. By rotating or moving the spatial modulation unit 10, the position and direction of the interference fringes generated in the laser light L change.

  After one moiré image is acquired, the rotational movement mechanism 27 rotates the spatial modulation unit 10 by 120 degrees. Thereby, the direction of the interference fringes can be rotated by 120 degrees. In this state, a moire image is acquired using the laser light L. Then, the direction of the interference fringes is further rotated 120 degrees to obtain a moire image. Thereby, three moire images (images obtained by rotating the interference fringes by 120 degrees each) are obtained.

  Thereafter, the rotational movement mechanism 27 rotates the spatial modulation unit 10 by 120 degrees to return to the initial angle. Then, the spatial modulation unit 10 is moved in the fringe direction of the interference fringes by 1/5 period. Thereby, the position (phase) of the interference fringes can be shifted by 1/5 period. In this state, a moire image is acquired.

  Thereafter, the rotational movement mechanism 27 rotates the spatial modulation unit 10 by 120 degrees to acquire respective moire images. Further, the rotational movement mechanism 27 shifts the spatial modulation unit 10 by 1/5 period and rotates it by 120 degrees to acquire each moiré image. By repeating the above operation five times, a total of 15 moire images are acquired. Instead of performing three rotations for each movement, five movements may be performed for each rotation.

  The camera 13 is connected to image processing means (not shown), and the 15 acquired moire images are output. Then, the image processing means performs image processing by performing Fourier transform on the 15 moire images. Thereby, the resolution of the sample S in the planar direction can be increased. That is, by performing image processing on 15 moiré images, it is possible to generate a super-resolution image having extremely high resolution in the plane direction exceeding the “Rayleigh limit spatial frequency”.

  The fifteen moire images use the laser light L that has passed through the pinhole 22P, that is, the fifteen moire images are confocal images. Therefore, the image has a high resolution in the optical axis direction. Then, by performing image processing to superimpose 15 moiré images, the image has a high resolution even in the planar direction. That is, an image with a high resolution in three dimensions can be obtained.

  The laser light L that has passed through the pinhole 22P is incident on the spatial modulation unit 10. That is, the pinhole 22P for obtaining a high resolution in the optical axis direction with respect to the optical path of the laser light L and the spatial modulation unit 10 for obtaining a high resolution in the plane direction are arranged. Accordingly, each moiré image is a confocal image with high resolution in the optical axis direction, and an image with high resolution can be obtained not only in the plane direction but also in the optical axis direction only by processing nine moiré images. .

  In the present embodiment, the laser light L that has passed through the pinhole 22P is incident on the spatial modulation unit 10, so that an image with high resolution in the optical axis direction can be obtained without obtaining images of planes above and below the plane of interest. Can be obtained. As a result, it is possible to acquire an image with a high three-dimensional resolution without performing image processing to which a complex algorithm is applied.

  In the present embodiment, the scanning unit 8 is used as a means for scanning the laser light L in the plane direction of the sample S. However, any means may be used as long as the scanning can be performed in the plane direction of the sample S. For example, the laser beam L may be scanned in the plane direction using a galvanometer mirror. However, the spatial modulation unit 10 is disposed at a position where the laser light L after being scanned by the galvanometer mirror is incident.

  Next, a first modification will be described. FIG. 2 shows a microscope apparatus 1 according to a first modification. Unlike the microscope apparatus 1 shown in FIG. 1, a polarization adjusting element 31 is provided between the collimating lens 7 and the scanning unit 8. Further, a polarizing beam splitter 32 is provided in place of the dichroic mirror 24, and a λ / 4 wavelength plate 33 is added between the first relay lens 25 and the second relay lens 26 of the telecentric optical system 9. Other configurations are the same as those of the microscope apparatus 1 of FIG.

  In this first modification, the observation target is not the sample S into which a fluorescent dye or fluorescent protein is introduced, but the reflector S1. As the reflector S1, for example, the surface of an IC chip, an optical disk, or the like can be applied. Any material that reflects the laser light L with high reflectance can be used as an observation target.

  The polarization adjusting element 31 adjusts the polarization direction of the laser light L so as to be one polarization direction (linearly polarized light). For example, one polarization plane is converted into light that vibrates, such as a λ / 2 wavelength plate or a polarizing plate. At this time, the polarization direction of the laser light L is matched with the polarization direction in which the transmittance of the polarization beam splitter 32 is maximized.

  The polarization beam splitter 32 is an optical element that transmits light having a predetermined polarization direction and reflects light having a polarization direction orthogonal to the polarization direction. The polarization beam splitter 32 transmits the laser beam L having the polarization direction adjusted by the polarization adjusting element 31, and reflects the polarization direction (return light R) in the orthogonal direction.

  The λ / 4 wavelength plate 33 is an optical element that converts linearly polarized light into circularly polarized light. The λ / 4 wavelength plate 33 is disposed at the focal position of the first relay lens 25. The λ / 4 wavelength plate 33 has a function of rotating the polarization plane of the laser light L by 45 degrees. As a result, the laser light L transmitted through the λ / 4 wavelength plate 33 changes from linearly polarized light to circularly polarized light.

  This circularly polarized laser beam L is incident on the reflector S1 by the microscope optical system 11. The reflector S1 reflects the laser light L. As a result, return light R is generated. The return light R passes through the microscope optical system 11 and enters the λ / 4 wavelength plate 33 again. Thereby, the return light R is converted from circularly polarized light to linearly polarized light. At this time, the polarization plane of the return light R rotates 45 degrees again. As a result, the return light R transmitted through the λ / 4 wavelength plate 33 changes from circularly polarized light to linearly polarized light. In addition, when viewed from the original laser beam L, the polarization plane is rotated 90 degrees.

  The return light R enters the polarization beam splitter 32. The return light R has a polarization plane rotated by 90 degrees from the time of the laser light L, that is, the polarization direction is orthogonal. For this reason, the return light R is reflected by the polarization beam splitter 32. Then, the return light R is condensed to the camera 13 to obtain an image. At this time, since the laser beam L passes through the pinhole 22P and is subjected to the action of the spatial modulation unit 10, the generated image is an image having a three-dimensionally high resolution.

  Therefore, in the first modification, the material to be fluoresced like the sample S is not an observation object, but the reflector S1 is the observation object. By simply providing the polarization adjusting element 31, the polarization beam splitter 32, and the λ / 4 wavelength plate 33, the reflector S1 such as an IC chip or an optical disk can be set as an observation target.

  Next, a second modification will be described. In the second modified example, as shown in FIG. 3, a multistage lens system 41 and a housing 42 are provided. The multistage lens system 41 includes a telecentric optical system 9, a spatial modulation unit 10, and a relay optical system 43. The housing 42 is a case that houses the microscope optical system 11 therein.

  The relay optical system 43 of the multistage lens system 41 has the same configuration as the telecentric optical system 9. That is, the first relay lens 44 and the second relay lens 45 are provided. The laser light L subjected to the spatial modulation action by the spatial modulation unit 10 is converted into parallel light by the first relay lens 44 and converted into light condensed by the second relay lens 45.

  Therefore, although there is no particular effect on the laser light L, a predetermined interval is provided between the spatial modulation unit 10 and the housing 42. That is, by providing the relay optical system 43 between the spatial modulation unit 10 and the housing 42, the spatial modulation unit 10 and the housing 42 are separated from each other.

  A rotational movement mechanism 27 is attached to the spatial modulation unit 10 to move and rotate the spatial modulation unit 10. At this time, if the spatial modulation unit 10 and the casing 42 are close to each other, the rotational movement mechanism 27 physically interferes with the casing 42 when the spatial modulation unit 10 is moved or rotated. Therefore, by simply interposing the relay optical system 43 in which two lenses are arranged, no interference occurs between the rotational movement mechanism 27 and the housing 42.

  From this point, although only one relay optical system 43 may be provided, a plurality of relay optical systems 43 may be provided. However, when the number of the relay optical systems 43 is increased, the optical path lengths of the laser light L and the return light R are increased by that amount, causing problems such as light quantity loss. Therefore, it is desirable to provide only one relay optical system 43.

  Next, a third modification will be described. In the third modification, a zoom lens 46 is added to the relay optical system 43 as shown in FIG. The zoom lens 46 is movable in the optical axis direction, and adjusts the magnification of an image captured by the camera 13. Other points are the same as those of the second modification described above.

  The objective lens 29 of the microscope optical system 11 has a predetermined numerical aperture (NA). On the other hand, the spatial modulation unit 10 generates bright and dark interference fringes in the laser light L. The interference fringes have a periodic light and dark pattern. Although interference occurs between the samples S due to the period of the bright and dark interference fringes, the effect of the interference changes depending on the numerical aperture of the objective lens 29.

  If the period of the interference fringes is synchronized with the numerical aperture of the objective lens 29, the interference fringes can be caused to interfere with each other highly, and the period of the moire image can be shortened. On the other hand, if the period of the interference fringes is not synchronized with the numerical aperture of the objective lens 29, the interference fringes cannot interfere with each other highly, and a desired moiré image cannot be acquired.

  Therefore, a zoom lens 46 is provided in order to obtain a period of interference fringes synchronized with the numerical aperture of the objective lens 29. The zoom lens 46 is provided on the rear stage side of the spatial modulation unit 10 and acts on the spatially modulated laser light L to change the magnification ratio. Thereby, the frequency of the structural illumination with which the sample S is irradiated can be arbitrarily adjusted.

  The zoom lens 46 is movable in the optical axis direction, and the magnification is changed so that the period of the interference fringes is synchronized with the numerical aperture of the objective lens 29 (the position of the zoom lens 46 is changed). Thereby, since the numerical aperture of the objective lens 29 and the period of the interference fringe are synchronized, the sample S can be largely interfered, and a moire image with a short period can be obtained. That is, a super-resolution image can be obtained.

  Next, a fourth modification will be described. FIG. 5 shows a fourth modification. The optical path separation optical system 50 in FIG. 5 is provided between the pinhole disk 22 and the microscope optical system 11. That is, it is provided at the same position as the multistage lens system 41 described in the second modification of FIG.

  The optical path separation optical system 50 includes a first relay lens 51, a first dichroic mirror 52, a second relay lens 53, a third relay lens 54, a first reflection mirror 55, a second dichroic mirror 56, a fourth relay lens 57, and a fifth relay lens. A relay lens 58, an optical path length adjusting element 59, a sixth relay lens 60, and a second reflecting mirror 61 are provided.

  The first relay lens 51 converts the laser light L that has passed through the pinhole 22P of the pinhole disk 22 into parallel light. The laser light L converted into parallel light by the first relay lens 51 enters the first dichroic mirror 52. The first dichroic mirror 52 has a characteristic of transmitting light having the wavelength of the laser light L and reflecting light having the wavelength of the return light R. Therefore, the laser light L passes through the first dichroic mirror 52.

  The transmitted laser light L is condensed on the spatial modulation unit 10 by the second relay lens 53, receives the action of spatial modulation of the spatial modulation unit 10, and enters the third relay lens 54. The laser light L is converted into parallel light by the third relay lens 54 and reflected by the first reflecting mirror 55. The reflected laser light L enters the second dichroic mirror 56.

  The second dichroic mirror 56 has a characteristic of reflecting the laser beam L and transmitting the return beam R. Therefore, the laser light L is reflected and enters the fourth relay lens 57. The laser light L that has passed through the fourth relay lens 57 becomes divergent light and enters the microscope optical system 11. Return light R is generated from the sample S and enters the fourth relay lens 57 from the microscope optical system 11.

  The return light R becomes parallel light by the fourth relay lens 57 and enters the second dichroic mirror 56. The return light R is transmitted through the second dichroic mirror 56. Then, the return light R is condensed on the optical path length adjusting element 59 by the fifth relay lens 58. The optical path length adjusting element 59 is an optical window for adjusting the optical path length, and gives the same optical path length as that of the spatial modulation unit 10 to the return light R.

  The sixth relay lens 60 converts the return light R whose optical path length is adjusted by the optical path length adjusting element 59 into parallel light, and the return light R that has become parallel light is reflected by the second reflecting mirror 61. The reflected return light R is incident on the first dichroic mirror 52. Since the return light R is reflected by the first dichroic mirror 52, the reflected return light R travels toward the pinhole 22 </ b> P of the pinhole disk 22.

  In the example described above, both the laser beam L and the return beam R are subjected to the spatial modulation action of the spatial modulation unit 10. Therefore, it is necessary to perform image processing so that a super-resolution image of the sample S can be generated when the return light R undergoes spatial modulation. An image processing unit connected to the camera 13 is provided with a system for performing this image processing.

  On the other hand, in a system based on the premise that the spatial modulation unit 10 acts only on the laser light L, accurate image processing cannot be performed when the return light R is subjected to spatial modulation. Therefore, the optical path between the laser light L and the return light R can be partially separated by separating and combining the laser light L and the return light R with the first dichroic mirror 52 and the second dichroic mirror 56. By arranging the spatial modulation unit 10 in the optical path of only the laser beam L, spatial modulation can be applied only to the laser beam L.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Light source 8 Scan part 9 Telecentric optical system 10 Spatial modulation part 11 Microscope optical system 12 Imaging lens 13 Camera 21 Lens disk 21R Lens 22 Pinhole disk 22P Pinhole 24 Dichroic mirror 27 Rotation movement mechanism 31 Polarization adjustment element 32 Polarization Beam splitter 33 λ / 4 wavelength plate 41 Multi-stage lens system 42 Housing 43 Relay optical system 46 Zoom lens 50 Optical path separation optical system 52 First dichroic mirror 56 Second dichroic mirror

Claims (5)

A light source that oscillates a laser beam irradiated on an observation target;
A pinhole that allows laser light within the focal range of the observation target to pass through the laser light; and
A spatial modulation unit that performs spatial modulation to form interference fringes in the planar direction of the observation target with respect to the laser light that has passed through the pinhole;
A microscope optical system that focuses the laser beam spatially modulated by the spatial modulation unit on the observation target;
An observation unit for observing the return light from the observation target;
A moving and rotating mechanism for moving and rotating the spatial modulation unit;
A relay optical system that is provided between the moving rotation mechanism and the microscope optical system and relays the laser beam;
A microscope apparatus comprising: A pinhole disk in which a plurality of the pinholes are arranged;
A lens disk arranged in the same pattern as the pinhole, and arranged with a plurality of lenses for condensing the laser light in the pinhole;
A rotating unit that integrally rotates the pinhole disk and the lens disk;
The microscope apparatus according to claim 1, further comprising: A polarization adjusting element that converts the polarization direction of the laser light emitted from the light source into one polarization direction;
A polarization beam splitter that transmits the polarization direction of the laser light and reflects light in the polarization direction orthogonal to the polarization direction;
A λ / 4 wavelength plate provided between the observation object that reflects the laser light and the polarizing beam splitter;
The microscope apparatus according to claim 1 or 2, further comprising: A housing for housing the microscope optical system ;
It said relay optical system, a microscope device according to any one of claims 1 to 3, characterized in that provided between the housing and the moving rotation mechanism. Any of claims 1 to 4, further comprising a zoom lens for changing the magnification of the microscope optical system provided with an objective lens the observation target image such that the interference fringes of period synchronized to the numerical aperture of the The microscope apparatus according to claim 1 .

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