JP2013054102A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a high resolution image by a microscope device having an optical scan optical system.SOLUTION: A microscope device 1 includes a light source 2 oscillating laser light L with which a sample S is irradiated, a scan optical system having a plurality of pinholes 6P through which a part of the laser light L passes and scanning the sample S with the laser light L, a microscope optical system having an objective lens 12 focusing the light after passing through the pinholes 6P of the laser light L on the sample S and guiding return light R emitted from a focal point to the pinholes 6P, a camera 16 detecting the return light R emitted from the sample S, and a relative movement mechanism 11 scanning the scan optical system, to relatively move the image of the return light R formed by the scan optical system with respect to the camera 16.

Description

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

  A fluorescence microscope is used as a microscope apparatus. A fluorescence microscope introduces a fluorescent dye or a fluorescent protein into a sample and irradiates a laser beam as excitation light to cause the sample to fluoresce. Then, an image of the sample is acquired by observing this fluorescence. Although a microscope device is required to obtain a high resolution, Patent Document 1 discloses a technique for artificially improving the resolution of an observation image by shifting pixels.

  Further, Japanese Patent Application Laid-Open No. 2003-228561 discloses a Nipo disk-type confocal microscope that scans a sample with excitation light at high speed using a pinhole disk and a microlens disk. Many pinholes are arranged on the pinhole disk, many microlenses are arranged on the microlens disk, and the pinhole disk and the microlens disk are rotated to scan the sample with excitation light at high speed. Can do.

JP 2009-25362 A JP 2011-22327 A

  The microscope (microscope system) of Patent Document 1 is configured by a microscope optical system (microscope) and a microscope image pickup device (image pickup device). Then, a parallel flat glass for pixel shifting is provided inside the microscope optical system, and the inclination of the parallel flat glass is changed back and forth and left and right, so that the position of the image formed on the imaging element is changed back and forth and left and right. I try to shift it to

  On the other hand, the microscope of Patent Document 2 has a scanning optical system as well as a microscope optical system and a camera (imaging device). The scanning optical system includes a pinhole disk and a microlens disk, and is provided between the microscope optical system and the camera. That is, unlike the microscope of Patent Document 1, the microscope is composed of a microscope optical system, a camera, and a scanning optical system.

  The microscope of Patent Document 1 realizes pixel shift by adjusting the two optical systems of the microscope optical system and the imaging device. Thereby, the resolution of the observation image can be improved in a pseudo manner. However, when realized by a three-part optical system of a microscope optical system, a camera, and a scanning optical system, such as a Niipou disc type confocal microscope as in Patent Document 2, a pixel shifting technique is simply applied. I can't.

  Therefore, an object of the present invention is to obtain a high resolution image with a microscope apparatus having an optical scanning optical system.

  In order to solve the above problems, a microscope apparatus of the present invention includes a light source that oscillates excitation light that irradiates a sample, and a plurality of openings that allow a part of the excitation light to pass therethrough. A scanning optical system for scanning, and a microscope optical system that has an objective lens that focuses the light that has passed through the opening of the excitation light with the sample, and that guides fluorescence generated from the focus to the opening. A detection unit that detects fluorescence generated from the sample, and a relative movement mechanism that relatively moves the fluorescence image formed on the scanning optical system by scanning the scanning optical system with respect to the detection unit. , Provided.

  According to this microscope apparatus, the relative movement mechanism moves the return light (fluorescence) image formed in the scanning optical system relative to the detection unit. By relatively moving the image of the return light, pixel shift can be performed in the detection unit. Thereby, a high-resolution image can be obtained with a microscope having a scanning optical system.

  The scanning optical system includes a pinhole disk having a plurality of pinholes as the opening, and a rotating unit that rotates the pinhole, and the rotating unit rotates the pinhole disk to rotate the pinhole disk. Scanning the focal point with the sample, forming a fluorescence image of the sample on the pinhole disk surface, and detecting the fluorescence image relayed by a relay optical system that transmits the fluorescence image on the pinhole An image sensor as a unit is provided.

  By rotating the pinhole disk, the focal point can be scanned in a direction orthogonal to the optical axis direction of the sample. The image of the return light when scanned passes through the pinhole, is relayed by the relay optical system, and is scanned by the image sensor. Thereby, cross-sectional information of the sample can be obtained.

  Further, the relative movement mechanism moves the photographing lens disposed between the objective lens and the pinhole disk in a direction orthogonal to the optical axis direction, thereby causing the fluorescence formed on the pinhole disk. An image is moved relative to the image sensor.

  By moving the photographic lens in a direction perpendicular to the optical axis direction, the image of the return light formed on the pinhole disk can be moved relative to the image sensor.

  Further, when the wavelength of the fluorescence is λ, the numerical aperture of the objective lens is NA, and the moving pitch of the photographing lens is s, s ≦ (1/2) × 0.61 × (λ / NA). As described above, the relative movement mechanism moves the photographing lens.

  By setting the moving pitch s of the photographic lens as in the above formula, it is possible to obtain an image with a high resolution so as to obtain a limit value of the optical resolution with a microscope having a scanning optical system.

  In addition, a control unit that switches between a high-resolution mode in which the relative movement mechanism operates and a high-speed mode in which the relative movement mechanism does not operate is provided.

  The high resolution mode in which pixel shifting is performed can obtain a high resolution, but the processing takes time. When high resolution is not required, the processing speed can be increased by selecting a high-speed mode in which pixel shifting is not performed.

  Further, the relative movement mechanism moves the relay optical system in a direction orthogonal to the optical axis direction to move the image formed on the pinhole disk relative to the image sensor. .

  By moving the relay optical system in the direction orthogonal to the optical axis direction, the image formed on the pinhole disk can be moved relative to the image sensor relative to the sh.

  In the present invention, the relative movement mechanism relatively moves the return light image formed in the scanning optical system with respect to the detection unit. For this reason, it is possible to perform pixel shifting in the detection unit by relatively moving the image of the return light. Thereby, a high-resolution image can be obtained with a microscope having a scanning optical system.

It is a block diagram of the microscope apparatus of embodiment. It is a block diagram of a pinhole disk and a micro lens disk. It is a figure explaining a pinhole and a scanning pitch. It is a figure explaining the focus before and behind the movement of a photographic lens. It is a figure explaining the image of an image sensor when a photographic lens is moved. It is a figure when rearrangement of a pixel is performed. It is a block diagram of the microscope apparatus of the modification 1. It is a figure explaining the change of the image of the image sensor of the modification 2.

  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 uses a sample S as an observation target, and includes a light source 2, a fiber 3, a collimating lens 4, a microlens disk 5, a pinhole disk 6, a connecting drum 7, a motor 8 dichroic mirror 9, a photographing lens 10, and a relative movement mechanism 11. And an objective lens 12, a front relay lens 13, a fluorescent filter 14, a rear relay lens 15, a camera 16, a control unit 17, and a monitor 18.

  The light source 2 oscillates laser light L as excitation light. The fiber 3 is an optical fiber and guides the laser light L. A collimating lens 4 is disposed on the emission side of the fiber 3, and the laser light L emitted from the fiber 3 becomes parallel light by the collimating lens 4. A microlens disc 5 is disposed in front of the collimating lens 4.

  As shown in FIG. 2, the microlens disk 5 is a rotating disk in which a large number of microlenses 5M are arranged in a spiral shape. The pinhole disk 6 is a rotating disk in which a large number of pinholes 6P are arranged in the same pattern as the microlens 5M. The pinhole 6P is an opening through which only light within the focal range of the sample S to be observed passes. The micro lens 5M has a function of condensing the laser light L in the corresponding pinhole 6P.

  The microlens disk 5 and the pinhole disk 6 are connected by a connecting drum 7, and the connecting drum 7 is connected to a motor 8. When the rotational force is applied by the motor 8, the microlens disk 5 and the pinhole disk 6 rotate integrally. The connecting drum 7 and the motor 8 constitute a rotating part.

  Each of the microlens disk 5 and the pinhole disk 6 constitutes a scanning optical system (scanning plate). The laser light L is scanned on the horizontal plane (XY plane) of the sample S by the scanning optical system. Thereby, a tomographic image of a predetermined region of the sample S can be acquired.

  As shown in FIGS. 1 and 2, a dichroic mirror 9 is provided between the microlens disk 5 and the pinhole disk 6. The dichroic mirror 9 is an optical element having a characteristic of transmitting the wavelength of the laser light L oscillated by the light source 2 and reflecting the wavelength of the fluorescence of the sample S.

  A photographing lens (tube lens) 10 is arranged at a position where the laser light L is transmitted through the pinhole 6P. The photographing lens 10 makes the laser light L parallel light. The photographing lens 10 is provided with a relative movement mechanism 11 that minutely moves the photographing lens 10 in a direction orthogonal to the optical axis direction (Z direction) of the laser light L.

  The relative movement mechanism 11 may be any mechanism that moves the photographic lens 10 in a direction orthogonal to the optical axis direction. For example, an actuator that drives a holder that clamps the photographic lens 10 may be applied. it can.

  An objective lens 12 is disposed in the optical path of the laser light L that has been collimated by the photographing lens 10. The objective lens 12 causes the laser beam L to focus on the sample S to be observed. The photographing lens 10 and the objective lens 12 constitute a microscope optical system.

  A front relay lens 13 is disposed in the optical path of the fluorescence (return light R) reflected by the dichroic mirror 9, and the return light R becomes parallel light by the front relay lens 13. A fluorescent filter 14 is disposed in the optical path of the return light R. The fluorescent filter 14 transmits only the fluorescent wavelength component of the sample S. A rear relay lens 15 is disposed in the optical path of the return light R. The parallel light return light R is focused on the image sensor 16C of the camera 16 by the rear relay lens 15.

  The control unit 17 is a computer connected to the camera 16 and performs predetermined arithmetic processing based on an electrical signal output from the camera 16. Thereby, an image of the sample S is generated. The generated image is displayed on the monitor 18. The control unit 17 controls the light source 2, the motor 8, the relative movement mechanism 11, and the camera 16.

  Next, the operation will be described. The control unit 17 oscillates a laser beam L having a wavelength for exciting the sample S from the light source 2. This laser light L becomes excitation light. The laser light L is focused on the fiber 3 and emitted from the other end of the fiber 3 with a spread. The laser light L is converted into parallel light by the collimating lens 4. The laser light L that has become parallel light is applied to each microlens 5M of the microlens disk 5.

  As shown in FIG. 2, the laser light L collected by the individual microlenses 5M of the microlens disk 5 passes through the dichroic mirror 9. This is because the dichroic mirror 9 has an optical characteristic of transmitting the laser light L. The laser beam L is focused at the pinhole 6P corresponding to the microlens 5M.

  The laser light L that has passed through the pinhole 6P is converted by the photographing lens 10 into parallel light having an inclination corresponding to the position of the pinhole 6P. The parallel laser beam L is incident on the objective lens 12 and forms a focal point F at a corresponding position on the focal plane in accordance with the inclination thereof. Therefore, the laser beam L incident on the sample S forms a focal point F corresponding to the inclination. In FIG. 1, the light beam at the center of the optical axis is indicated by a solid line, and the light beam shifted from the optical axis is indicated by a broken line.

  In the sample S, a fluorescent dye or a fluorescent protein excited by the laser light L is introduced. Therefore, when the laser beam L is focused on the sample S, fluorescence is generated. This fluorescence becomes return light R. The return light R enters the dichroic mirror 9 through the same optical path as the laser light L like the objective lens 12, the photographing lens 10, and the pinhole 6P.

  The dichroic mirror 9 has a characteristic of reflecting the light having the fluorescence wavelength of the sample S. Therefore, the return light R is reflected. The return light R is incident on the front relay lens 13. The image of the return light R that has passed through the pinhole 6P is formed at the same magnification on the image pickup device 16C of the camera 16 by the action of the front relay lens 13 and the rear relay lens 15. At this time, only the fluorescent component is selectively transmitted through the return light R by the action of the fluorescent filter 14, and the components other than the fluorescent component are removed.

  At this time, the control unit 17 controls the motor 8 to rotate. Thereby, the microlens disk 5 and the pinhole disk 6 which are scanning optical systems rotate integrally. As shown in FIG. 2, the light passing through the pinhole 6P is scanned on the corresponding focal plane of the sample S, and each return light R passes through the pinhole 6P again. Then, the light is reflected by the dichroic mirror 9 and scanned by the image sensor 16 </ b> C of the camera 16.

  Thereby, the information on the focal plane of the sample S is projected on the camera 16 and the sample S can be observed. At this time, light other than the focal plane hardly passes through the pinhole 6P and therefore does not reach the camera 16. Therefore, the image acquired by the camera 16 is an image of only light within the focal range of the sample S, that is, a confocal image. In other words, the microscope apparatus 1 is a confocal microscope.

  Then, the motor 8 rotates the microlens disk 5 and the pinhole disk 6 so that the laser beam L can be scanned over the sample S at high speed. Thereby, the tomogram of the sample S can be scanned at high speed, and a predetermined tomographic image can be generated at high speed.

  Here, the control unit 17 controls the relative movement mechanism 11 to relatively move the photographing lens 10 in a direction orthogonal to the optical axis direction of the laser light L. Thereby, the image of the return light R (fluorescence) formed on the pinhole disk 6 (scanning optical system) is moved relative to the camera (detection unit) 16.

  The moving pitch s for moving the photographing lens 10 by the relative movement mechanism 11 is “s ≦ (1/2) × 0.61 ×, where λ is the wavelength of the return light R and NA is the numerical aperture of the objective lens 12. (Λ / NA) ”. This is “σ = 0.61 × (λ / NA)” when the limit value of the optical resolution is σ, and the sampling pitch s for obtaining information of the limit value of the optical resolution is “s ≦ (1 / 2) × σ ”.

  Here, it is assumed that the magnification of the objective lens 12 is 20, the numerical aperture NA is 0.75, and the wavelength λ of the return light R is 550 nm. In this case, the limit value σ of the optical resolution is σ = 0.45 μm. However, since the magnification of the objective lens 12 is 20 times, the limit value σ is enlarged and projected onto the image sensor 16C of the camera 16 at σ = 9 (= 0.45 × 20) μm. That is, the limit value σ of the optical resolution is σ = 9 μm.

  On the other hand, FIG. 3 shows the scanning pitch P1 at which the focal point F of the sample S is scanned. As shown in this figure, since the focal point F is continuous in the broken line direction (X direction), scanning can be performed with extremely high resolution. However, there is a limit to the pitch in the direction orthogonal to this direction (scanning pitch P1). Here, it is assumed that the scanning pitch P1 is 7 μm. Further, it is assumed that the pixel size of the image sensor 16C of the camera 16 is also 7 μm.

  The limit value σ of the optical resolution is σ = 9 μm, and the sampling pitch s at the limit value σ is “s ≦ (1/2) × σ = 4.5 μm”. Therefore, since the scanning pitch P1 and the pixel size are both 7 μm and the sampling pitch s (= 4.5 μm) is not reached, information on the limit value of the optical resolution is not obtained. As a result, an image with high resolution cannot be obtained.

  Therefore, the photographing lens 10 is slightly moved using the relative movement mechanism 11. The moving amount of the taking lens 10 is the moving amount of the sampling pitch s, that is, “s ≦ (1/2) × 0.61 × (λ / NA)”. In the above example, “s ≦ 4.5 μm” may be satisfied, but here, it is assumed that the pixel size of the image sensor 16C is half of this range. That is, “s = 3.5 μm”.

  The relative movement mechanism 11 moves the photographing lens 10 in a direction orthogonal to the optical axis of the return light R. The direction orthogonal to the optical axis of the return light R is a plane, and this plane has two directions (XY directions) orthogonal to each other. When both the scanning pitch P1 and the pixel size do not reach the sampling pitch s, the relative movement mechanism 11 moves the photographing lens 10 in two directions of the XY directions. On the other hand, when either one has not reached the sampling pitch s, the relative movement mechanism 11 moves the photographing lens 10 in any one of the XY directions.

  Here, since neither the scanning pitch P1 nor the pixel size has reached the sampling pitch s, the relative movement mechanism 11 moves the photographing lens 10 in two directions in the XY directions. FIG. 4 shows the relationship between the pinhole disk 6 and the photographic lens 10. In the figure, the solid line shows the state before moving the photographic lens 10 in the X direction, and the broken line shows the state after moving in the X direction.

  Before the movement, the focal point of the taking lens 10 is at the point 21 of the pinhole disk 6. When the pinhole 6P is located at this point 21, the laser light L emitted from the focal point is parallel to the optical axis and is focused on the objective lens 12. The return light R also passes through the same optical path. At this time, the relative movement mechanism 11 moves the photographing lens 10 in the X direction by a sampling pitch s (that is, 3.5 μm: X1 in FIG. 4).

  By moving the photographic lens 10 in the X direction, the focal point of the photographic lens 10 changes to a point 22. When the pinhole 6P is positioned at the point 22, the laser light L emitted from this focal point is parallel to the optical axis and is focused on the objective lens 12. The return light R also passes through the same optical path. Since both of the laser beams L that have passed through these photographing lenses 10 are light parallel to the optical axis, both of them are condensed at the same point after passing through the objective lens 12.

  That is, the information observed at the point 21 moves to the point 22 by moving the photographing lens 10 in the X direction by the sampling pitch s. Similarly, the other points of the pinhole disk 6 also move in the X direction by the sampling pitch s, so that the image projected on the pinhole disk 6 also moves in the X direction by moving the photographing lens 10. .

  This image is formed at the same magnification on the image sensor 16C of the camera 16 by the action of the front relay lens 13 and the rear relay lens 15. Therefore, the image formed on the pinhole disk 6 is moved in the X direction by moving the photographing lens 10, and the movement of this image is also moved in the image sensor 16C. Here, the fact that the image moves in the X direction with respect to the image sensor 16C means that the image sensor 16C has moved in the −X direction relative to the image. That is, the field of view observed by the image sensor 16C is taken as a field of view moved by the sampling pitch s in the -X direction from the field of view before the movement.

  In FIG. 4, the movement is only in one direction (X direction). However, as described above, since the photographing lens 10 moves in two directions XY, the image of the return light R is two directions in XY in the image sensor 16C. Moved to.

  Here, the image sensor 16C will be described. FIG. 5A shows four pixels A1, B1, C1, and D1 of the image sensor 16C. These pixel sizes are 7 μm and do not reach the sampling pitch s (= 4.5 μm) for obtaining the limit value of the optical resolution. First, one image is formed in this state.

  Next, the control unit 17 controls the relative movement mechanism 11 to move the photographing lens 10 in any direction (XY direction) orthogonal to the optical axis (Z direction) of the return light R by the sampling pitch s or less. Let As described above, the pixel is moved by half the pixel size (= 3.5 μm). Here, the photographic lens is first moved 3.5 μm in the −Y direction. As a result, the field of view to be photographed is moved by 3.5 μm in the Y direction.

  The broken line in FIG. 5B indicates an image before movement, and the solid line indicates an image after movement. That is, an image shifted by 3.5 μm in the Y direction is acquired. Next, from the state of FIG. 5B, the relative movement mechanism 11 moves the photographing lens 10 by 3.5 μm in the −X direction (perpendicular to the Y direction). As a result, as shown in FIG. 5C, an image deviated by 3.5 μm in the X direction and 3.5 μm in the Y direction from the initial state (the state of the broken line in the figure) is acquired. Then, the relative movement mechanism 11 moves the photographing lens 10 by 3.5 μm in the Y direction from the state of FIG. Thereby, as shown in FIG.5 (d), the image which shifted | deviated 3.5 micrometers in the X direction from the initial state (state of the broken line in a figure) is acquired.

  The control unit 17 controls the relative movement mechanism 11 to acquire images when shifted by 3.5 μm in the XY directions, and acquires a total of four images. The control unit 17 rearranges the pixel information based on the acquired four images. Thereby, as shown in FIG. 6, pixel information composed of 16 pixels in a pseudo manner is obtained.

  However, each pixel size is originally coarse. For example, the pixel information of C1 includes information on peripheral pixel information (A3, B4, B3, C2, C3, C4, D3, D2). The control unit 17 is a computer that performs a predetermined calculation, and artificially improves the resolution of the observation image by performing image processing such as difference processing and differentiation processing. By performing these processes for all pixels, a high-resolution image is generated.

  As described above, the relative movement mechanism 11 moves the photographic lens 10. The relative movement mechanism 11 moves the image of the return light R formed on the pinhole disk 6 relative to the image pickup device 16C, whereby the microscope optical system having the objective lens 12, the camera 16, and the pinhole disk 6 are moved. In a microscope apparatus configured with three members including a scanning optical system, it is possible to acquire an image with a high resolution that reaches the limit value of the optical resolution.

  In the above, as shown in FIGS. 5A to 5D, the photographing lens 10 is moved so that one pixel is divided into four. That is, although the taking lens 10 is moved so as to be divided into two in the X direction and the Y direction, the number of pixel divisions may be set arbitrarily. For example, nine images of 3 × 3 may be set and nine image acquisitions may be performed.

  The relative movement mechanism 11 moves the photographing lens 10 to move the image of the return light R formed on the pinhole disk 6 relative to the image sensor 16C. However, the camera 16 itself is moved. It may be. However, since a high-sensitivity imaging camera generally used for biological fluorescence observation is heavy due to the mounting of a cooling mechanism or the like, it is preferable to move the imaging lens 10 as in this embodiment rather than moving the camera 16.

  Moreover, you may make it shift the position of an imaging image back and forth, right and left by changing inclination to front and rear and right and left using a parallel plate glass. However, when parallel flat glass is used, since the inclination is changed to a distance, it is difficult to accurately maintain the correspondence. In addition, it is difficult to perform an operation for converting the inclination into a distance. For this reason, in the present embodiment in which the photographing lens 10 is simply moved in the XY directions, the moving distance of the photographing lens 10 becomes the moving distance of the image in the camera 16 as it is, which is accurate and simple.

  The control unit 17 may have a high resolution mode and a high speed mode. In the high resolution mode, as described above, the relative movement mechanism 11 moves the photographing lens 10 and acquires an image at that time. The control unit 17 rearranges the image information to obtain a high resolution image.

  In the high resolution mode, a predetermined time is required for the relative movement mechanism 11 to move the photographing lens 10 and for the control unit 17 to perform arithmetic processing for rearrangement of image information. That is, the processing is delayed in order to obtain a high resolution image.

  For example, when a high resolution image is obtained for recording, a high resolution mode is set in the control unit 17. Thereby, the relative movement mechanism 11 is controlled to obtain a high resolution image. On the other hand, when it is not necessary to obtain an image with such a high resolution, for example, when performing normal observation, the high-speed mode is set in the control unit 17.

  As a result, the control unit 17 does not operate the relative movement mechanism 11 and does not rearrange the image information. Therefore, although an image with high resolution cannot be obtained, the processing speed can be increased. Therefore, by enabling the control unit 17 to set either the high resolution mode or the high speed mode, an appropriate mode can be used according to the purpose.

  Next, Modification 1 will be described. FIG. 7 shows a microscope apparatus 1 according to the first modification. In the microscope apparatus 1, the relative movement mechanism 11 moves the rear relay lens 15, not the photographing lens 10, in a direction orthogonal to the optical path of the return light R. Other configurations are the same as those of the above-described embodiment.

  However, the magnification of the objective lens 12 is 40 times, the NA is 0.95, and the wavelength (that is, the fluorescence wavelength) λ of the return light R is 550 nm. As a result, the limit value σ of the optical resolution is “σ = 0.35 μm”. Since the magnification of the objective lens 12 is 40 times, it is enlarged and projected onto the image sensor 16C, and the limit value σ becomes “σ = 14 μm = 0.35 × 40”. At this time, the sampling pitch s for obtaining the limit value of the optical resolution is “s ≦ (1/2) × σ = 7 μm” using the above-described equation.

  Here, it is assumed that the scanning pitch P1 of the pinhole disk 6 is P1 = 7 μm, and the pixel size of the image sensor 16C is 14 μm. Therefore, since the scanning pitch P1 satisfies the expression of the sampling pitch s, information on the limit value of the optical resolution can be obtained. On the other hand, the pixel size cannot acquire information on the limit value of the optical resolution.

  Therefore, the relative movement mechanism 11 moves the image of the return light R formed on the pinhole disk 6 in one direction with respect to the imaging element 16C. Although the photographing lens 10 may be moved as described in the embodiment, in the first modification, the rear relay lens 16 is moved. Also by this, the image of the return light R formed on the pinhole disk 6 can be moved relative to the image sensor 16C.

  In the first modification, an image sensor 16C having a relatively large pixel size of 14 μm is used. In general, the camera 16 having the image pickup element 16C having a large pixel size has high sensitivity and is suitable for capturing a high-speed phenomenon. Moreover, since the light quantity of excitation light can be reduced, it has many merits, such as being able to reduce the influence which it has on a living cell.

  Therefore, when the pixel size is large, the rear relay lens 16 (of course, the photographic lens 10 may be used) is moved in the direction orthogonal to the optical axis of the return light R as in the first modification, thereby Do a shift. As a result, an image having high resolution can be generated by a microscope apparatus having three units of a microscope optical system, a camera, and a scanning optical system.

  In addition, when the magnification of the relay lens is changed from the same magnification and the image in the pinhole 6P is reduced and projected, the pixel size may be relatively large. can do.

  In the first modification, the relative movement mechanism 11 is attached to the rear relay lens 15 and moved. However, the relative movement mechanism 11 is attached to the front relay lens 13 and is orthogonal to the optical path of the return light R. You may make it move.

  Next, Modification 2 will be described. In the second modification, as in the embodiment, it is assumed that the magnification of the objective lens 12 is 20, the numerical aperture NA is 0.75, and the wavelength λ of the return light R is 550 nm. Therefore, the limit value σ of the optical resolution is σ = 0.45 μm, and the magnification of the objective lens 12 is 20 times, so the limit value σ is σ = 9 μm. Therefore, the sampling pitch s for obtaining the limit value of the optical resolution is “s ≦ 4.5 μm”.

  On the other hand, in the second modification, the scanning pitch P1 of the pinhole disk 6 is P1 = 7 μm, and the pixel size of the image sensor 16C is 3.5 μm. Accordingly, the pixel size has reached the sampling pitch s, but the scanning pitch P1 has not reached the sampling pitch s. Since the pinhole disk 6 is continuously rotated, scanning is performed with high resolution in the X direction in FIG.

  FIGS. 8A to 8C show a 16-pixel image sensor 16C. Since the scanning pitch of the pinhole disk 6 is coarse, almost the same image information is transmitted to the A row and the B row, for example. Similarly, almost the same image information is transmitted to the C and D rows.

  First, an image in the state of FIG. Next, the relative movement mechanism 11 moves the photographic lens 10 by the sampling pitch s (3.5 μm) in the −Y direction orthogonal to the optical axis direction, so that the image in the state of FIG. get.

  The image in the state of FIG. 8B is equivalent to an image obtained by moving the image sensor 16C by 3.5 μm in the Y direction. This is indicated by E line, F line, G line, and H line. Since the scanning pitch of the pinhole disk 6 is coarse, almost the same image information is transmitted to the E row and the F row, for example. Similarly, almost the same image information is transmitted to the G row and the H row.

  Then, the control unit 17 performs calculations such as difference and differentiation based on the images in FIGS. 8A and 8B and rearranges the images. Thereby, a high resolution image as shown in FIG. 8C is obtained. By performing this process for all the pixels, a high-resolution image of the sample S can be obtained.

  Therefore, as in the second modification, when the pixel size reaches the sampling pitch s but the scanning pitch P1 does not reach the sampling pitch s, the pixel is moved by moving the photographing lens 10 in one direction. Shifting can be performed to obtain a high-resolution image. The movement of the photographic lens 10 moves the image of the return light R formed on the pinhole disk 6 relative to the camera 16, so that the microscope apparatus has three members: a microscope optical system, a camera, and a scanning optical system. With this, an image with high resolution can be obtained.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Light source 5 Micro lens disk 5M Micro lens 6 Pinhole disk 6P Pinhole 7 Connection drum 8 Motor 10 Shooting lens 11 Relative movement mechanism 12 Objective lens 13 Front side relay lens 15 Rear side relay lens 16 Camera 16C Image pick-up element 17 Control Part F Focus L Laser light R Return light S Sample

Claims (6)

A light source that oscillates excitation light that irradiates the sample;
A scanning optical system having a plurality of openings for allowing a part of the excitation light to pass through, and scanning the sample with the excitation light;
A microscope optical system having an objective lens that focuses the light that has passed through the opening of the excitation light with the sample, and that guides fluorescence generated from the focus to the opening;
A detection unit for detecting fluorescence generated from the sample;
A relative movement mechanism for moving the fluorescence image formed in the scanning optical system relative to the detection unit by scanning the scanning optical system;
A microscope apparatus comprising: The scanning optical system includes a pinhole disk having a plurality of pinholes as the opening, and a rotating unit that rotates the pinhole, and the rotating unit rotates the pinhole disk to focus the focus. Scan with the sample to form a fluorescent image of the sample on the pinhole disk surface,
The microscope apparatus according to claim 1, further comprising: an imaging element as the detection unit that detects the fluorescence image relayed by a relay optical system that transmits a fluorescence image on the pinhole. The relative movement mechanism moves the photographic lens disposed between the objective lens and the pinhole disk in a direction orthogonal to the optical axis direction, thereby causing the fluorescence image formed on the pinhole disk to move. The microscope apparatus according to claim 2, wherein the microscope apparatus is moved relative to the image sensor. When the wavelength of the fluorescence is λ, the numerical aperture of the objective lens is NA, and the moving pitch of the photographing lens is s,
s ≦ (1/2) × 0.61 × (λ / NA)
The microscope apparatus according to claim 3, wherein the relative movement mechanism moves the photographic lens so that The microscope apparatus according to any one of claims 1 to 4, further comprising a control unit that switches between a high-resolution mode in which the relative movement mechanism operates and a high-speed mode in which the relative movement mechanism does not operate. The relative movement mechanism moves the relay optical system in a direction orthogonal to an optical axis direction, thereby moving an image formed on the pinhole disk relative to the imaging element. 2. The microscope apparatus according to 2.

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