JP5278522B2 - Microscope equipment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope device which can acquire an image in high speed, in a super-resolution microscope which illuminates a sample using spatially modulated illumination light, performs arithmetic processing on an obtained image to demodulate the given spatial modulation, and obtains a sample image of high resolution. <P>SOLUTION: A microscope device comprises: an illumination optical system which comprises a light source and phase modulation means 5 provided near a pupil conjugate surface, for performing phase modulation on one part or all of light flux from the light source and which spatially modulates the light flux into a fringe structure near a sample surface and emits the light flux; an imaging optical system for imaging diffraction light of the sample; imaging means 12; and image processing means 13 which performs arithmetic processing on an image imaged by the imaging means 12 to create a sample image. The phase modulation means 5 modulates the phase of the fringe structure, of the illumination light, created near the sample surface. <P>COPYRIGHT: (C)2012,JPO&amp;INPIT

Description

  The present invention relates to a microscope, and more particularly to a high-resolution microscope capable of realizing in-plane super-resolution.

  In the field of observation and measurement of sample microstructure, observation with higher spatial resolution is required. As a method to increase the in-plane resolution of the sample, in order to image the light of high spatial frequency component among the diffracted light from the sample, in the vicinity of the sample surface or at a position conjugate with the sample surface in the illumination optical system, There is a technique in which a certain modulation is applied to diffracted light or illumination light, and demodulation corresponding to the applied modulation is performed at a position substantially conjugate with the sample surface in the imaging optical system. Examples of this include the Lukosz method (W.Lukosz, "Optical systems with resolving powers exceeding the classical limit.II", Journal of the Optical Society of America, Vol.37, PP.932 (1967)). Recently, there are methods disclosed in JP-A-11-242189 and US RE38307.

JP 11-242189 A URRe38307

Journal of the Optical Society of America, Vol.37, PP.932 (1967)

  In the Lukosz method, diffraction gratings each having a conjugate lattice constant are arranged one by one at positions near the sample and substantially conjugate with the sample surface in the imaging optical system, and these are conjugatedly moved. The diffraction grating disposed in the vicinity of the sample surface can cause the diffracted light that cannot originally enter the objective lens of the imaging optical system to reach the image surface. The diffraction grating disposed at a position substantially conjugate with the sample surface in the imaging optical system demodulates the diffracted light by the diffraction grating near the sample surface and forms an image as an original diffraction component. Since light having a high spatial frequency component that did not originally contribute to image formation reaches the image plane by the diffraction grating near the sample surface, a higher spatial resolution than usual can be obtained.

  The sixth embodiment disclosed in Japanese Patent Application Laid-Open No. 11-242189 is an example applied to a fluorescence observation apparatus, and its optical system uses illumination light emitted from a coherent light source by a light beam dividing means such as a diffraction grating. After the division, the illumination light beam is condensed at the pupil position of the objective lens and emitted from the objective lens as a parallel light beam having a different angle, and an overlapping interference fringe is formed in the vicinity of the observation object.

  In this method, spatial modulation is performed by illumination light instead of spatial modulation performed by Lukosz placing a diffraction grating near the sample. As in the Lukosz method, the effect is that diffracted light including the spatial frequency component of the shape information of the observation object that could not be transmitted by the imaging optical system alone can be involved in the imaging. Then, the phase of the divided illumination light beam is relatively modulated, and the interference fringes are moved on the observation object to acquire a plurality of images, thereby enabling image formation by image calculation processing. Specifically, by moving the diffraction grating perpendicular to the optical axis, or in another example, inserting a wedge-shaped prism in one optical path of the illumination light and moving it in a direction perpendicular to the optical axis. Phase modulation is performed.

  In the method disclosed in US RE38307, illumination light from a coherent light source is introduced using an optical fiber, and then split by a light beam splitting means such as a diffraction grating, and then the illumination light beam is condensed at the pupil position of the objective lens, Interference fringes are formed near the observation object. The high-frequency component of the shape information of the observation object that could not be transmitted by the imaging optical system alone can be involved in the imaging by the illumination light modulated in a stripe shape. Similarly, a plurality of images are acquired and image formation is performed by image calculation processing. In this method, in order to form a single image, not only a plurality of images obtained by applying phase modulation to the illumination light beam, but also an image is obtained by changing the direction of the interference fringes of the illumination light.

  The reason is that high-frequency components can participate in imaging only in structures that have the same direction as the interference fringe direction of the illumination light. This is because it is necessary to convert the direction and acquire and combine a plurality of images.

  The present invention relates to imaging using a spatially modulated illumination light up to a high-frequency component of the sample shape, demodulates the spatial modulation given by processing the acquired image, and obtains a high-resolution sample image. An object of the present invention is to provide a microscope apparatus capable of acquiring images at high speed in a resolution microscope.

To solve the above problem,
The present invention
An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane ;
An imaging optical system for imaging light from the specimen;
Imaging means;
A microscope apparatus having image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means,
The phase modulation means comprises an angle modulation means having a reflection surface for deflecting the light beam, and modulates the phase of the fringe structure formed on the specimen by angle modulation of the reflection surface. provide.
The present invention also provides
An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane;
An imaging optical system for imaging light from the specimen;
Imaging means;
Image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means;
A microscope apparatus comprising:
The phase modulation means has a reflecting surface for deflecting the light beam, and modulates the phase of the fringe structure generated near the sample surface by electrically controlling the phase difference or reflectance of the reflecting surface. A microscope apparatus is provided.
The present invention also provides
An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane;
An imaging optical system for imaging light from the specimen;
Imaging means;
Image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means;
A microscope apparatus comprising:
The phase modulation means has a transmission surface that transmits a light beam, and modulates the phase of the fringe structure formed on the specimen by electrically controlling the phase difference or transmittance of the transmission surface. A microscope apparatus is provided.
The present invention also provides
A beam splitting means for splitting the beam from the coherent light source into two beams, causing the two beams to interfere with each other by two beams, illuminating the sample with illumination light spatially modulated into an interference fringe structure, and then spatially modulating the sample; In a structured illumination microscope that involves the diffracted light of the specimen from the illumination light in the image formation and generates the specimen image from the acquired image by image calculation processing.
The light beam splitting unit is disposed in the vicinity of the sample conjugate position in the illumination optical system, the angle modulating unit is disposed in the vicinity of the pupil conjugate position, and a part or all of the light beam split by the light beam splitting unit is The microscope apparatus is characterized in that the phase of the interference fringes generated in the vicinity of the sample surface is modulated by performing angle modulation by the method.

  In the microscope apparatus of the present invention, by arranging the phase modulation means in the vicinity of the pupil conjugate position, the phase of the fringe structure of the illumination light generated in the vicinity of the sample surface can be modulated at high speed.

FIG. 2 is a diagram showing a configuration of the microscope apparatus of the first embodiment. FIG. 3 is a diagram showing a configuration of a folding mirror used in the first embodiment. FIG. 6 is a diagram showing the structure of a diffraction grating used in the second embodiment and how the diffracted light is collected. FIG. 10 is a diagram showing a configuration of a diffraction grating used in the third embodiment. FIG. 10 is a diagram showing a configuration of a folding mirror used in the third embodiment. FIG. 10 is a diagram showing a configuration of a rotary shutter used in the fourth embodiment. These are diagrams showing the structure of the rotary shutter / phase modulation means used in the fifth embodiment. FIG. 10 is a diagram showing a configuration of a microscope apparatus according to a sixth embodiment.

  In the conventional method, since phase modulation is performed by moving an element (such as a diffraction grating) that performs phase modulation in a direction perpendicular to the optical axis, image acquisition cannot be performed at high speed. In particular, when the observation target is a living biological specimen, it is essential to acquire an image at high speed, but the conventional method is insufficient.

  In the Lukosz method, it is necessary to move the two diffraction gratings on the sample side and the imaging plane side in a synchronized manner, and the mechanism becomes very complicated.

  In the method disclosed in Japanese Patent Application Laid-Open No. 11-242189, in order to obtain one final image, it is necessary to calculate from the images obtained by converting the phase of the interference fringes of the illumination light three times. Here, when a biological specimen is targeted, the speed of image acquisition becomes a problem.

  If the objective lens has a magnification of 60 times and NA of 1.45, the magnification relationship between the primary observation image and the illumination system field stop is generally about 0.3 to 0.5 times. In this case, from the sample surface to the field stop surface The magnification is 18 to 30 times. Therefore, NA_fs of the light beam formed on the field stop surface is 0.08 to 0.048 when NA is divided by the magnification. When the pitch of the diffraction grating that generates diffracted light at this angle is calculated, the pitch is 6.6 to 11 μm. That is, to convert the phase by 1/3 to one period, it is necessary to move the diffraction grating by a maximum of 11 μm in the direction perpendicular to the optical axis.

  On the other hand, the maximum drive amount that can be driven stably with a high-accuracy linear actuator is about 10 μm in consideration of the small size because it is built into the illumination device of the microscope, and the repetition drive frequency in that case is the maximum It is about 10kHz. However, if it repeats while stopping every 1/3 period, it becomes substantially 1 Hz, and image acquisition is about 1 second per frame. This is not suitable for shooting moving biological specimens.

  In the method disclosed in US RE38307, it is recommended to further rotate the diffraction grating to change the direction of the interference fringes. If the direction of the interference fringes is converted in three directions every 120 ° and the phase conversion is the same as described above, the movement of the diffraction grating is as follows. That is, image acquisition at the reference position, linearly stepping in the direction perpendicular to the optical axis, acquiring two images, returning to the optical axis position, rotating by 120 ° with a rotary motor, image acquisition, stepping in the direction perpendicular to the optical axis 2 images, return to the optical axis position, rotate to the direction of 240 ° from the reference by the rotation motor, acquire the image, acquire 2 images by stepping in the direction perpendicular to the optical axis, return to the optical axis position, the reference position by the rotation motor Return to. In other words, nine images are obtained by combining rotation and shift, and then one sample image is obtained by arithmetic processing. This method is also difficult to increase the speed of image acquisition.

  The present invention is characterized in that the phase modulation element is placed near the pupil conjugate position. This is because the phase of the interference fringes on the sample surface can be changed by changing the angle of the light beam near the pupil conjugate position. In addition, the position where the two light beams are spatially completely separated is the pupil conjugate position, and since each light beam is condensed, the cross section of the light beam is very small. In addition, noise (0-order diffracted light and unnecessary higher-order diffracted light when a diffraction grating is used as a light beam splitting means) can be removed, and a light beam selection function that allows only the necessary light beam to pass through can be combined. is there. The position where the phase modulation element is disposed is optimal at the pupil conjugate position, but may be within a range (near) slightly shifted from the pupil position.

(First embodiment)
A first embodiment will be described with reference to the drawings. FIG. 1 shows a schematic diagram of a first embodiment of the present invention. Light from a coherent light source (not shown) is guided by the optical fiber 1 and converted into parallel light by the collector lens 2. Diffracted light is generated by the diffraction grating 3 and a pupil conjugate plane is formed by the lens 4. The diffraction grating 3 has a one-dimensional periodic structure in the direction perpendicular to the paper surface of FIG. 1. The diffraction grating 3 may have a structure with concentration (transmittance) or a step (phase difference). This is preferable because the diffraction efficiency of ± first-order light is high.

  Reference numeral 5 denotes a folding mirror disposed in the vicinity of the pupil conjugate plane. 2A is a diagram of the folding mirror 5 viewed from the optical axis direction, and FIG. 2B is a diagram viewed from the direction perpendicular to the optical axis. The reflection surface of the folding mirror 5 is divided into two regions 5a and 5b as shown in FIG. The position where the ± 1st order diffracted light generated by the diffraction grating 3 is collected is in the ring zone of the region 5a, and the region 5b corresponds to the position where the 0th order diffracted light is collected. As for the surface characteristics, 5a is a normal reflecting mirror surface, and 5b is a light shielding portion, which functions to cut 0th-order light. In order to perform phase modulation necessary for image processing, the folding mirror 5 is configured to modulate the angle of the mirror and swing the deflection angle of the light beam. This is done by fixing the end 5c of 5a in FIG. 2 (b) and moving the opposite end 5d stepwise in a direction perpendicular to the reflecting surface by a piezoelectric actuator (not shown). The angle of the mirror 5 is modulated. The method is not limited to this, and a known technique such as a galvanometer mirror can be used. As a result, the angle of passing through the pupil conjugate position of the two light beams can be modulated. As a result, the phase of the interference fringes of the two light beams can be modulated near the sample surface. In FIG. 2, the region 5b is made of a light-shielding film so that the 0th-order light is completely blocked. However, the present invention is not limited to this configuration, and the folding mirror is formed in a donut shape to transmit the 0th-order light toward the sample. It is possible not to proceed.

  Further, second-order or higher-order diffracted light generated by the diffraction grating 3 may be shielded by providing a ring-shaped light-shielding portion outside the reflecting surface 5a, or the diameter of the folding mirror 5 may be reduced by the higher-order diffracted light. It may be smaller than the distance from the optical axis passing through the vicinity so that the high-order diffracted light is not reflected. If neither measure is taken, even if the higher-order diffracted light is folded back by the folding mirror 5 together with the first-order diffracted light, it can be eliminated by the subsequent optical system (for example, the pupil plane P of the objective lens), so that the user is not so concerned. do not have to.

  The illumination light whose direction is changed by 90 degrees by the folding mirror 5 forms a sample conjugate plane at the field stop surface FS by the lens 6 and then passes through the lens 7 and the half mirror 8 which divides and synthesizes the epi-illumination system and the imaging system. Two spots are formed on the pupil P of the objective lens 9. These two spots are formed almost at the outermost periphery of the pupil of the objective lens, and when exiting from the objective lens, illuminate the sample surface as parallel light beams having an angle of maximum NA facing each other. At this time, since the two light beams are coherent, the sample surface is irradiated with a structure of equally spaced interference fringes. This illumination light having a stripe structure is called structured illumination.

  When the specimen is illuminated with this structured illumination light, the periodic structure of the illumination light interferes with the periodic structure of the specimen to generate moire interference fringes, although the moire interference fringes contain high-frequency shape information of the specimen. Since the frequency is lower than the frequency, the light can enter the objective lens. The principle of the structured illumination super-resolution microscope is to acquire an imaged image, and calculate and visualize the unknown sample shape by calculating and restoring the periodic structure of the known illumination light.

  The light from the sample is converted into parallel light through the objective lens, and then transmitted through the half mirror 8 to form a sample image on the imaging surface of the imaging means 12 such as a CCD camera by the second objective lens 11. To do. However, since this acquired image is an image as a result of illumination with the illumination light modulated as described above, the image is processed by the image storage / calculation device 13 and restored by applying reverse modulation. An image can be obtained and a super-resolution image of the specimen can be displayed on the image display device 14.

  When restoring the original image by image processing, it is preferable to shoot the same specimen by modulating the phase of the illumination interference fringe three or more times. This is because the sample structure has three unknown parameters: intensity average value, phase shift amount, and modulation width. In order to obtain the unknowns in the calculation process, more information than the number of unknowns is required. Because it becomes.

In the arrangement of this embodiment, the pitch of the diffraction grating 3 can be expressed by the following equation.
p = (M * βis * f4 * λ) / (NA * f6)
Here, M: magnification of objective lens, βis: primary image → field stop magnification, λ: wavelength used, NA: NA of objective lens, f4, f6: focal length of lenses 4 and 6 in FIG.

  In this embodiment, M = 40, βis = 0.4, λ = 550 [nm], NA = 0.75, f4 = 50 [mm], and f6 = 50 [mm], respectively. In this case, the pitch of the diffraction grating is p = 11.7 μm.

On the other hand, when phase modulation for one wavelength is performed by fixing 5c of the region 5a on the surface of the folding mirror 5 near the pupil conjugate position and pressing 5d perpendicular to the surface, the amount of pressing δ is δ = (√2) * λ
In this embodiment, δ = 0.78 μm. This amount is a necessary stroke to vibrate with the piezoelectric actuator.
Compared with the stroke of 11.7 μm when moving the diffraction grating perpendicular to the optical axis, phase modulation can be performed with a very small amount of movement, so the vibration frequency can be increased and the image acquisition speed is increased. .

  Thus, since the phase modulation of the illumination light can be performed at a high speed, a high-resolution image of a living biological specimen can be obtained.

  Next, the present embodiment is compared with a conventional method in which a phase modulation is performed by moving an element that performs phase modulation in a direction perpendicular to the optical axis.

  In general, a piezoelectric actuator can control the minute expansion of an element corresponding to an applied voltage, but the size of the expansion stroke and the resonance frequency have an inversely proportional relationship. In this example, the resonance frequency is 60 kHz at a stroke of 5 μm, 40 kHz at 15 μm, and the like. That is, if the stroke is increased, the resonance frequency must be decreased.

  In order to move the diffraction grating perpendicularly to the optical axis and give a mutual phase difference of one wavelength, a stroke of about 11 μm is required as previously calculated.

Further, another method disclosed in Japanese Patent Application Laid-Open No. 11-242189, that is, when a phase difference is given by moving one of the two wedge prisms perpendicularly to the optical axis is as follows. Assuming that the phase difference between the optical path containing the wedge prism and the optical path not containing it is m times the wavelength (m: integer) in a certain state, and sliding the wedge prism along the slope from that state, If the movement amount (sliding amount) until the phase difference becomes (m + 1) times is Δx (movement amount perpendicular to the optical axis),
Δx = λ / (tanθ * (1-1 / n))
Here, λ is the wavelength, θ is the apex angle of the wedge prism, and n is the refractive index of the wedge prism. If λ = 550, θ = 10 degrees, and n = 1.5168, respectively, Δx = 9.15 μm. Accordingly, even in this method, the phase modulation stroke requires about 9 μm.

However, if the same phase modulation is applied in the light traveling direction, the stroke can be made shorter. For example, when performing phase modulation with a reflection mirror that converts the traveling direction of light by 180 degrees, the movement amount Δ1 of the reflection mirror corresponding to phase modulation of one wavelength is:
Δ1 ＝ λ / 2
Therefore, if λ = 550 nm, Δ1 = 275 nm. Alternatively, if it is performed at the part of the mirror that bends the optical path at 45 degrees, the amount of movement Δ2 of the bending mirror corresponding to the phase modulation of one wavelength is
Δ2 ＝ λ / sqrt (2)
Therefore, when λ = 550 nm, Δ2 = 389 nm. In any case, it can be seen that a stroke less than one-twentieth of the case of acting vertically on the optical path is sufficient.

  On the other hand, when modulating the phase of the interference fringes of two-beam interference formed in the vicinity of the sample surface by the illumination light, when performing at a position conjugate with the sample surface, the position of the two beams is changed in a plane perpendicular to the optical axis. This is done by shifting the diffraction grating in the prior art described above. In this case, the moving direction is determined by the pitch direction of the grating.

  When using an element that performs phase modulation, such as a wedge prism, which is another method of the above-described prior art, it may be performed where the two light beams are spatially separated, and to some extent from the sample conjugate position. , Anywhere on the optical path.

(Second Embodiment)
A second embodiment of the present invention will be described. The configuration of the optical system is the same as that of the first embodiment, and the configuration of the folding mirror 5 is different. The folding mirror of this embodiment is divided into three regions 5e, 5f, and 5g as shown in FIG. The regions are set so that the + 1st order light diffracted by the diffraction grating 3 passes through 5e, the −1st order light passes through 5f, and the 0th order light passes through 5g. Each region is made of liquid crystal and can electrically control the phase difference and reflectivity during reflection. In the present embodiment, the phase of the interference fringes generated in the vicinity of the sample surface is modulated by giving different phase differences between 5e and 5f at the time of reflection instead of swinging the angle of the light beam. In this embodiment, the example of the liquid crystal has been described, but a liquid crystal having a function of electrically controlling the phase difference and the reflectance may be used instead of the liquid crystal.

(Third embodiment)
A third embodiment of the present invention will be described. The configuration of the optical system is the same as that of the first embodiment, and the difference is the structure of the diffraction grating 3 and the structure of the folding mirror 5.

  In this embodiment, a diffraction grating 13 having the structure shown in FIG. 4A is used instead of the diffraction grating 3 of the first embodiment. It has a three-way periodic structure that is turned 60 degrees from each other. This angle need not be exactly 60 degrees, but is preferably an angle that is roughly divided into three equal parts. FIG. 4B shows a state in which the 0th order light and the ± 1st order light generated by the diffraction grating 13 are condensed at the pupil conjugate position through the lens 4, respectively. In FIG. 2B, 20 represents a zero-order light condensing unit and 21 represents a ± first-order light condensing unit.

  In this embodiment, a folding mirror 15 having the structure shown in FIG. 5A is used instead of the folding mirror 5 of the first embodiment. The areas 15a to 15g of the reflecting surface correspond to the condensing positions of the spots in FIG. Since the folding mirror 15 is arranged at an angle of 45 degrees in the vicinity of the pupil conjugate plane, the spot size is slightly larger than the state of FIG. 4B, but not so much that the light beams intersect with each other. Can be separated by division.

  In the case of the present embodiment, unnecessary zero-order light is removed by a transmission method. That is, it corresponds to the region 15g of the folding mirror 15, and no reflecting member is placed here so that the 0th-order light is transmitted straight. As in the first embodiment, the regions 15a, 15b, and 15c indicated by hatching are fixed at one end in the radial direction, and the other end is pushed and pulled by the piezoelectric actuator, thereby reflecting the light beam at each region. The phase of the interference fringes on the sample can be converted.

  The shape of each region is not limited to this figure. For example, 15a and 15d, 15b and 15e, 15c and 15f are integrated, and the angle of the mirror as shown in the first embodiment in the radial direction is integrated. Modulation may be performed.

  In the configuration of the present embodiment, the method of performing phase modulation by the folding mirror is replaced with a method of electrically modulating the reflectance and the reflection phase difference using liquid crystal as in the second embodiment described above. May be.

  In the first embodiment, the interference fringes generated on the specimen have a one-dimensional periodic structure, but in the present embodiment, the interference fringes are two-dimensional three-direction interference fringes. In this case, a super-resolution image can be acquired regardless of the direction of the period of the fine structure of the specimen.

(Fourth embodiment)
A fourth embodiment of the present invention will be described. This embodiment has the same configuration as that of the third embodiment described above, and is characterized in that a rotary shutter 16 shown in FIG. The rotary shutter 16 has a structure that rotates around its center 16a as a rotation center. The regions 16b and 16c are hollow or transparent with high transmittance glass or the like. The area corresponds to the size of each of the areas 15a to 15f in FIG. 4, and each rotation rotates a pair of light beams 15a and 15d, 15b and 15e, and 15c and 15f, and the other light beams are blocked by the shutter. It is. By rotating this at a high speed, one set can be selected from each of the three sets of ± first-order light generated in the diffraction grating 13. If the timing when the areas 16b and 16c pass and the image capturing timing of the image pickup means are synchronized, the rotary shutter 16 can be kept rotating at a constant speed without stopping. The rotating shutter 16 can be mounted in front of the reflecting surface with the shutter surface parallel to the reflecting surface of the folding mirror, and the shutter surface can be mounted perpendicular to the optical axis before and after folding (the rotation axis matches the optical axis) before and after folding. You may arrange in either. However, it is more convenient to arrange the mirror 15 not far away from the folding mirror 15 because the luminous flux does not spread.

  Further, the shape of the shutter window portion is triangular in FIG. 6, but the shape is not limited to this, and the shape of the shutter may be a fan shape, or the shutter may be a blade shape without leaving a peripheral ring zone.

  According to the method of the present embodiment, since the periodic structure of the illumination light on the sample surface is one-dimensional, the structure that is calculated during image processing is simple, and the direction of the periodic structure of the illumination light changes with time. Super-resolution is possible regardless of the direction of the sample structure.

  Although USRe38307 describes a method for changing the direction of the interference fringes of two-beam interference, it takes time to acquire an image because the diffraction grating is temporarily stopped after being rotated by a predetermined angle. In this embodiment, a periodic structure is created in three directions in advance, and two corresponding spots among six spots in which ± 1st order light in each direction is imaged on the pupil conjugate plane by a group of lenses. The partial light-shielding film is arranged so that the light beam is transmitted and the remaining four spots are shielded. Then, by rotating the light shielding film about the optical axis as a rotation axis, it is not necessary to temporarily stop, and only a spot in the transmission region can be involved in illumination, and an image can be acquired in a short time.

(Fifth embodiment)
The configuration of this embodiment is almost the same as that of the above-described fourth embodiment, and the folding mirror and the rotary shutter are different. The rotary shutter 17 of this embodiment is arranged at the same position as the rotary shutter 16 of the third embodiment, and the structure is as shown in FIG. That is, one of the windows, which is a hollow or uniform transmission member, is divided into three parts, each having a phase difference. 17b is delayed by 1/3 wavelength, and 17c is delayed by 1/3 wavelength phase. For example, it can be realized by using a glass having the same refractive index and changing the thickness considerably to provide a step. Alternatively, it can be realized by depositing a thin film such as MgF2 while controlling the film thickness. In this case, if the light shielding portion 17d is provided with an absorption film, the rotary shutter 17 can be manufactured with a single substrate. Further, only the window portion or the entire rotary shutter can be formed of a transmissive liquid crystal, and the transmittance and phase delay can be electrically generated and used for each corresponding region in FIG. Thereby, only one of the pair of transmitted two light beams can be temporally phase-modulated.

  The folding mirror of this embodiment may be a uniform reflection mirror. This is because the rotary shutter 17 performs phase modulation means and interference fringe direction selection means. Therefore, it is possible to arrange the optical axis from the fiber 1 to the lens 4 in the same direction as the optical axis of the lens 6 without arranging the folding mirror, and the degree of freedom of configuration is widened.

  According to the method of the present embodiment, since the periodic structure of the illumination light on the sample surface is one-dimensional, the structure that is calculated during image processing is simple, and the direction of the periodic structure of the illumination light changes with time. Super-resolution is possible regardless of the direction of the sample structure. Since the movable part is only the rotational movement of the rotary shutter 17, the apparatus is simple.

(Sixth embodiment)
A sixth embodiment of the present invention will be described. FIG. 8 shows a schematic diagram of the sixth embodiment of the present invention.

  The microscope apparatus according to the present embodiment includes a white light source 30 as a light source that emits illumination light with a very short interference length, instead of the coherent light source in the first embodiment, and illumination from the white light source 30. The light is directly incident on the lens 2 without going through the optical fiber 1.

  The microscope apparatus according to the present embodiment includes a concentration type diffraction grating 31 instead of the diffraction grating 3 in the first embodiment. This density type diffraction grating 31 has a striped density pattern (different in transmittance) for converting the illumination light from the white light source 30 into illumination light corresponding to the structured illumination light in the first embodiment. Mask pattern) is provided. This density pattern is preferably a pattern in which the transmittance changes in a sine wave shape because high-order diffracted light does not occur. Thus, the light diffracted by the density type diffraction grating 31 is condensed on the pupil conjugate plane at a position corresponding to the diffraction order via the lens 4. That is, the illumination light from the white light source 30 is split by the density type diffraction grating 31. As described above, also in this embodiment, the same light beam as the illumination light in the first embodiment can be guided to the folding mirror 5.

  In addition, in this embodiment, since it is the same as that of the said 1st Embodiment about parts other than the structure mentioned above, the description is abbreviate | omitted.

  As described above, according to the present embodiment, it is possible to realize a microscope apparatus that exhibits the same effects as those of the first embodiment without using a coherent light source.

DESCRIPTION OF SYMBOLS 1: Optical fiber 2: Collector lens 3: Diffraction grating 4: Lens 5: Folding mirror 9: Objective lens 10: Specimen 12: Imaging means 13: Image storage and calculation device 14: Image display device 30: White light source 31: Density type Diffraction grating

Claims (11)

An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane ;
An imaging optical system for imaging light from the specimen;
Imaging means;
A microscope apparatus having image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means,
The microscope apparatus according to claim 1, wherein the phase modulation means comprises angle modulation means having a reflecting surface for deflecting the light beam, and modulates the phase of the fringe structure formed on the specimen by angle modulation of the reflecting surface . An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane ;
An imaging optical system for imaging light from the specimen;
Imaging means;
A microscope apparatus having image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means,
The phase modulation means has a reflecting surface for deflecting the light beam, and modulates the phase of the fringe structure generated near the sample surface by electrically controlling the phase difference or reflectance of the reflecting surface. A microscope apparatus characterized by that. An illumination optical system in which a light beam from a light source is spatially modulated into a fringe structure to irradiate the sample, and a phase modulation means for modulating the phase of the fringe structure is disposed in the vicinity of the pupil conjugate plane;
An imaging optical system for imaging light from the specimen;
Imaging means;
Image processing means for generating a specimen image by performing arithmetic processing on an image picked up by the image pickup means;
A microscope apparatus comprising:
The phase modulation means has a transmission surface that transmits a light beam, and modulates the phase of the fringe structure formed on the specimen by electrically controlling the phase difference or transmittance of the transmission surface. Microscope device. It said phase modulating means, a microscope apparatus according to any one of claims 1 to 3, characterized in that it comprises a shielding means for shielding unnecessary illumination light to the spatial modulation. The reflective surface is divided into a plurality of regions, and a region where light necessary for the spatially modulated illumination light is reflected is configured to relatively modulate the mutual phase. The microscope apparatus according to claim 2 , wherein unnecessary light is not deflected to the specimen in a region to be reflected. The transmission surface is divided into a plurality of regions, and a region where light necessary for the spatially modulated illumination light is transmitted is configured to relatively modulate the mutual phase, and unnecessary illumination light is transmitted. The microscope apparatus according to claim 3, wherein unnecessary light does not travel to the specimen in a transmitting region. The illumination optical system includes a microscope according to any one of claims 1 to 3, characterized in that arranged near the specimen position conjugate, having three different lifting direction or more periodic structures diffraction grating with each other apparatus. Wherein among the plurality of light beams diffracted by the diffraction grating, at least two, the light beam selecting means for selecting a light beam of an optical axis symmetrically arranged near the pupil conjugate position, the two selected by the beam selecting means, the optical axis microscope apparatus according to claim 7, characterized in that changing the direction of the spatial modulation of the fringe structure by changing the luminous flux of symmetry. 9. The microscope apparatus according to claim 8 , wherein the light beam selection unit is disposed in the vicinity of the angle modulation unit to perform phase conversion of illumination light and direction conversion of spatial modulation. The phase modulation means has shielding means for shielding illumination light unnecessary for spatial modulation,
A region through which one of the two light beams selected by the light beam selecting unit passes is a hollow or uniform transmitting member that changes phase, and a region through which the other beam passes is a phase modulation unit whose phase changes temporally. 9. The microscope apparatus according to claim 8 , wherein the microscope apparatus is arranged in the vicinity of the conjugate position so that the phase modulation of the illumination light beam and the direction change of the spatial modulation can be selected temporally. Has a beam splitter for splitting the light beam from the coherent light source into two light beams, the two light and two light by beam interference in specimens bundle to illuminate the sample with illumination light spatially modulated fringe structure and the spatial modulation In a structured illumination microscope that involves the diffracted light of the specimen from the illumination light in the image formation and generates the specimen image from the acquired image by image calculation processing.
The light beam splitting unit is disposed in the vicinity of the sample conjugate position in the illumination optical system, the angle modulating unit is disposed in the vicinity of the pupil conjugate position, and a part or all of the light beam split by the light beam splitting unit is A microscope apparatus characterized by modulating the phase of interference fringes generated in the vicinity of a specimen surface by angle-modulating with a lens.

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