JP2010072014A - Microscope device - Google Patents

Abstract

Provided is a microscope apparatus that can easily scan illumination light and is suitable for observation of a deep part of a living body.
A light projecting optical system for projecting a line-shaped condensed light onto an object, a detection optical system for receiving light generated by the line-shaped condensed light and forming an image on a line CCD, and a line-shaped And a biaxial stage 12 that scans the light collection 7. The main axis of the cylindrical lens 7 of the projection optical system and the main axis of the objective lens 8 of the detection optical system intersect at a fixed angle, and the line CCD 11 has an intensity distribution on the line of the light generated by the line-shaped light collection 7. Is detected. The controller 15 acquires image information on a plane obtained by scanning the line-shaped light collection 7 with the biaxial stage 12 based on a signal from the line CCD 11.
[Selection] Figure 1

Description

  The present invention relates to a projection optical system that projects line-shaped illumination light onto an object, a detection optical system that receives light generated by the line-shaped illumination light and forms an image on a detector, and line-shaped illumination. And a scanning unit that scans light, and a microscope apparatus that acquires image information on a plane obtained by scanning a line-shaped illumination light by the scanning unit based on a signal from a detector.

  In microscopic observation, when performing deep observation of a living body, the SN of an image deteriorates due to the influence of return light from other than the focal plane. This phenomenon cannot be removed sufficiently even if a confocal microscope is used.

  Techniques (USP 5,973,828, USP 5,969,854, USP 6,423,956, DE4326473) for solving this problem have been proposed.

  In these technologies, the horizontal resolution is reduced by using two objective lenses and combining the focal regions with each other and combining the confocal microscope technology to obtain information on only those intersecting regions. Although it is a little worse, it mainly improves the resolution in the depth direction (see FIG. 2 of USP 5,973,828).

Further, Non-Patent Document 2 describes that the following effects are obtained when these configurations are configured with lenses having a relatively low NA.
(1) The resolution in the optical axis direction is improved.
(2) Since NA is small, it is difficult for aberrations to occur even in deep body observation.
(3) Small diameter is easy to manufacture.
(4) WD can be lengthened and is suitable for deep observation.
(5) Since the two optical paths are separate, even if deep observation is performed, it is difficult to be affected by noise light such as return light from other than the observation surface.

FIG. 7 of USP 5,973,828 discloses a scanning mechanism that scans the focal position while maintaining the positional relationship between the two objective lenses. Non-Patent Document 1 and Non-Patent Document 2 disclose a method of scanning an object by moving it on a stage or the like.
U.S. Pat.No. 5,973,828 U.S. Pat.No. 5,969,854 U.S. Patent No. 6,423,956 German patent No. 4364773 `` Confocal microscope with large field and working distance '', Applied Optics, Vol.38, No.22, pp.4870 `` Dual-axis confocal microscope for high-resolution in vivo imaging '', Optics Letters, Vol.28, No.6, pp414

  However, it is conceivable that the mechanism for scanning the focal position while maintaining the positional relationship of the focal point of the light passing through the two angled objective lenses is complicated and it takes time to scan. In addition, when applying a method of scanning by moving a stage or the like, it is considered that a very long scanning time is actually required.

  An object of the present invention is to provide a microscope apparatus that can easily scan illumination light and is suitable for observation of a deep part of a living body.

The microscope apparatus of the present invention includes a light projecting optical system that projects line-shaped illumination light onto an object, a detection optical system that receives light generated by the line-shaped illumination light and forms an image on a detector, A scanning unit that scans the line-shaped illumination light, and obtains image information on a plane obtained by scanning the line-shaped illumination light by the scanning unit based on a signal from the detector In the apparatus, the main axis of the objective lens of the light projecting optical system and the main axis of the objective lens of the detection optical system intersect at a constant angle, and the detector is configured to detect the light generated by the line-shaped illumination light. It is characterized by detecting an intensity distribution on the line.
According to this microscope apparatus, the main axis of the objective lens of the light projecting optical system and the main axis of the objective lens of the detection optical system intersect at a fixed angle, and the detector can detect the line of light generated by the line-shaped illumination light. Since the intensity distribution above is detected, the illumination light can be easily scanned, and a microscope apparatus suitable for observation of the deep part of the living body can be obtained.

  The light generated by the line-shaped illumination light may be fluorescence from the object.

  The light generated by the line-shaped illumination light may be reflected light or scattered light from the object.

  The angle may be 90 degrees.

  The angle may be in a range of 30 degrees to 180 degrees.

  The projection optical system may irradiate near infrared laser light.

  The light projecting optical system may project the linear illumination light by causing parallel light to enter a cylindrical lens.

  The line-shaped illumination light may be on a focal plane of the detection optical system.

  The light projecting optical system may project the line-shaped illumination light by forming an image of a slit on the symmetrical object.

  You may irradiate the said line-shaped illumination light on the line which the focal plane of the said projection optical system and the focal plane of the said detection optical system cross | intersect.

  The scanning unit may scan the line-shaped illumination light in a direction perpendicular to the line.

  The scanning unit may scan the illumination light by moving all or part of the light projecting optical system and the detection optical system.

  A slit may be disposed on the imaging surface of the detection optical system.

  The detector may be a line sensor.

  The detector may be a two-dimensional sensor, and the two-dimensional sensor may be moved in accordance with detection light in synchronization with scanning by the scanning unit.

  According to the microscope apparatus of the present invention, the main axis of the objective lens of the light projecting optical system and the main axis of the objective lens of the detection optical system intersect at a constant angle, and the detector can detect the light generated by the line-shaped illumination light. Since the intensity distribution on the line is detected, the illumination light can be easily scanned, and a microscope apparatus suitable for observation of the deep part of the living body can be obtained.

  Hereinafter, embodiments of a microscope apparatus according to the present invention will be described.

  Hereinafter, the microscope apparatus according to the first embodiment will be described with reference to FIGS.

  FIG. 1 is a block diagram illustrating the configuration of the microscope apparatus according to the first embodiment.

  As shown in FIG. 1, an optical fiber 2 that guides light from a light source (not shown) is disposed in the optical head 1 of the microscope apparatus of this embodiment. Here, the light from the light source is preferably near-infrared light for observation of the deep part of the living body, and the optical fiber 2 preferably has a small core diameter, such as a single mode fiber. In front of the optical fiber 2 is a collimating lens 3.

  Further, in the optical head 1, a reflection mirror 4, a cylindrical lens 5, an aperture window 6, an objective lens 8, a mirror 9, and an imaging lens 10 are disposed along the optical path.

  The cylindrical lens 5 is composed of a cylindrical surface having an axis in the Y direction (axis perpendicular to the paper surface in FIG. 1). Here, the object-side focal point of the objective lens 8 is approximately at the midpoint (the midpoint of the line width) of the line-shaped condensed light 7 formed when parallel light is introduced along the principal axis of the cylindrical lens 5. Each optical component is arranged so as to come.

  A relatively small NA of the objective lens 8 is used. Further, the line CCD 11 is disposed at the condensing position of the imaging lens 10 as shown in FIG. Here, the pixels of the line CCD 11 are arranged in the Y direction (direction perpendicular to the paper surface in FIG. 1).

  As shown in FIG. 1, the optical head 1 can be moved in the X direction and the Z direction by a biaxial stage 12.

  Further, the line CCD 11 and the biaxial stage 12 are connected to the controller 15 via signal lines indicated by broken lines, and a monitor 16 is connected to the controller 15.

  Next, the operation of the microscope apparatus of this embodiment will be described.

  Near-infrared light from the light source is emitted from the tip of the optical fiber 2, converted into parallel light by the collimator lens 3, reflected by the mirror 4, and then transmitted through the aperture window 6 by the action of the cylindrical lens 5. It becomes the light collection 7 of the shape. Here, the light collection 7 has a line shape extending in the Y direction (direction perpendicular to the paper surface in FIG. 1).

  FIG. 2 is a diagram schematically showing a condensing point.

  As shown in FIG. 2, the condenser 7 is located on the focal plane 8 </ b> A of the objective lens 8. When there is an object such as a living body in the condensed light 7 and scattered light or fluorescence is emitted, these lights are converted into parallel light by the action of the objective lens 8. The parallel-converted light is reflected by the mirror 9, and the line-shaped condensed light 7 is imaged on the line CCD 11 by the action of the imaging lens 10, and the intensity distribution on the line of the condensed light 7 is reflected by the line CCD 11. Detected. Since the pixel of the line CCD 11 can be regarded as sufficiently small, it becomes a confocal optical system, and the influence of light other than the focal plane 8A is reduced. Moreover, you may arrange | position a slit on the line CCD11 as needed.

  FIG. 3 is an enlarged view of the line-shaped light collection 7.

  FIG. 3 shows the beam spread by the point spread function (PSF) of the cylindrical lens 5 and the beam by the point spread function (PSF) when parallel light is introduced into the objective lens 8 from the opposite direction along its principal axis. The spread of is shown. Since only the light in the region where the beam shapes overlap by the two lenses shown by hatching in FIG. 3 is projected onto the line CCD 11, the line CCD 11 has a good resolution in the optical axis direction and is on the line of the light collection 7. Information is conveyed.

  When obtaining scattered light from the condenser 7, the configuration shown in FIG. 1 may be used. However, when obtaining fluorescence information from the object, a fluorescent filter (not shown) is appropriately inserted near the front surface of the line CCD 11. It is desirable.

  Here, by moving the biaxial stage 12 in the X direction (FIG. 1) by the controller 15, the line-shaped light collection 7 is scanned in the X direction. Information on the line-shaped light collection 7 at each position at this time is captured by the line CCD 11. The controller 15 can display the XY cross-sectional image on the monitor 16 using the position signal of the biaxial stage 12 and the signal from the line CCD 11, or can record the XY cross-sectional image on a recording device inside the controller 15. is there.

  Further, by moving the biaxial stage 12 in the Z direction (FIG. 1) by the controller 15, the linear light collection 7 is scanned in the Z direction. Information on the line-shaped light collection 7 at each position at this time is captured by the line CCD 11. The controller 15 can display the YZ cross-sectional image on the monitor 16 using the position signal of the biaxial stage 12 and the signal from the line CCD 11, or can record the YZ cross-sectional image on a recording device inside the controller 15. is there.

  In the above embodiment, the cylindrical lens and the objective lens are used as the objective lens, but the present invention is not limited to this, and other lens systems having the same function may be used. A reflective optical system such as a concave mirror may be used.

  As described above, in the microscope apparatus of the present embodiment, the object-side focal point of the objective lens 8 of the detection optical system is positioned on the line where the light projecting optical system condenses, and the optical axes of both optical systems are angled. Since it is arranged with a mark, information on the line can be obtained with excellent resolution in the optical axis direction. The angle between the optical axes is preferably selected in the range of 30 degrees to 180 degrees, and more preferably 90 degrees.

  Further, since the light projecting light path and the light collecting light path are separated, the light other than the light collecting position is difficult to return, and an image with a good SN ratio can be obtained.

  Further, since the cylindrical lens 5 having a small NA is used as the objective lens of the light projecting optical system, WD can be taken and the aberration is small, so that it is suitable for deep observation.

  Furthermore, since the image is obtained by scanning the lines as described above, the scanning method is easy and high-speed scanning is possible.

  Furthermore, since near infrared light is used as the light source, a microscope apparatus suitable for deep observation can be obtained.

  Hereinafter, the microscope apparatus according to the second embodiment will be described with reference to FIGS.

  FIG. 4 is a block diagram illustrating the configuration of the microscope apparatus according to the second embodiment.

  As shown in FIG. 4, the optical head 1A of the microscope apparatus of the present embodiment has an optical fiber 21, a collimator lens 22, a cylindrical lens 23, and a condensing point of the cylindrical lens 23 for guiding near-infrared light from a light source (not shown). A slit 24, an imaging lens 25, a reflection mirror 26, a reflection mirror 27, and an objective lens 28 are disposed along the optical path of the light projecting optical system.

  Further, the object side focal point 29 of the objective lens 28 and the objective lens 30 are arranged so as to coincide with each other. The angle formed by the optical axes of the two objective lenses is 90 degrees. These objective lens 28 and objective lens 30 have a relatively small NA. The angle between the optical axes may not be 90 degrees, but is preferably selected in the range of 30 degrees to 180 degrees.

  Further, the reflection mirror 31 and the reflection mirror 32 are arranged as shown in FIG.

  Here, the reflection mirror 27, the reflection mirror 31, the objective lens 28, and the objective lens 30 are integrated as an X drive unit 33. The X drive unit 33 is movable in the X direction by the X drive stage 34. The X drive unit 33, the X drive stage 34, the reflection mirror 26, and the reflection mirror 32 are integrated as a Z drive unit 36, and are configured to be movable in the Z direction by the Z drive stage 37.

  Further, as shown in FIG. 1, an imaging lens 39, a slit 40 provided at an imaging position of the imaging lens 39, a relay lens 41, and a relay lens 42 are arranged in the optical path. A high-sensitivity camera 43 as a two-dimensional sensor is disposed at the focal position of the relay lens 42.

  The high sensitivity camera 43 is movable in the X direction by the X drive stage 44.

  The X drive stage 34, the Z drive stage 37, the second X drive stage 44, and the high-sensitivity camera 43 are connected to the controller 46 via signal lines indicated by broken lines, and a monitor 47 is connected to the controller 46. Has been.

  Next, the operation of the microscope apparatus of this embodiment will be described.

  Near-infrared light from the light source is emitted from the tip of the optical fiber 21, converted to parallel light by the collimator lens 22, and condensed on the slit 24 by the cylindrical lens 23. The light that can pass through the slit 24 is converted into parallel light corresponding to the position of the slit 24 by the imaging lens 25. The parallel light passes through the objective lens 28 after being reflected by the reflection mirror 26 and the reflection mirror 27. Here, the slit 24 is projected onto the imaging 29 by the action of the imaging lens 25 and the objective lens 28.

  Here, the slit 24 extends in the Y direction (direction perpendicular to the paper surface in FIG. 1), and the imaging 29 also has a line shape extending in the Y direction.

  FIG. 5 is a diagram illustrating the positional relationship of the objective lens.

  As shown in FIG. 5, the focal plane 28A of the objective lens 28 and the focal plane 30A of the objective lens 30 intersect each other, and the focal points of the objective lens 28 and the objective lens 30 are positioned on a straight line formed by the intersecting portion. The shaped image 29 is also located on this straight line.

  When an object such as a living body is present at the position of the image 29 and scattered light or fluorescence is emitted, the light is converted into parallel light by the action of the objective lens 30. The light converted into parallel light is reflected by the mirror 31 and the mirror 32 and is condensed on the slit 40 by the action of the imaging lens 39.

  The light that has passed through the slit 40 is imaged on the pixels of the high sensitivity camera 43 by the action of the relay lens 41 and the relay lens 42.

  FIG. 6 is an enlarged view of the line-shaped image 29.

  FIG. 6 shows the beam spread by the point spread function (PSF) of the objective lens 28 and the beam by the point spread function (PSF) when parallel light is introduced into the objective lens 30 from the opposite direction along the optical axis. The spread of is shown. Since only the light in the region where the beam shapes overlap by the two objective lenses shown by hatching in FIG. 6 is projected onto the high-sensitivity camera 43, the line-shaped information (collection with high resolution in the optical axis direction) is collected. The intensity distribution on the line of light 7) is detected.

  When obtaining scattered light from the imaging 29, the configuration shown in FIG. 4 may be used. However, when obtaining fluorescence information from the object, a fluorescent filter (not shown) is appropriately provided near the front surface of the high-sensitivity camera 43. It is desirable to insert.

  Here, by driving the X-axis stage 34 in the X direction by the controller 46, the line-shaped imaging 29 is scanned in the X direction.

  At this time, since the optical axis between the mirror 26 and the mirror 27 and the optical axis between the mirror 31 and the mirror 32 are parallel light and these optical axes are in the X direction, the X drive unit 33 can be moved. The optical system before and after that is not affected.

  Simultaneously with the driving by the X-axis stage 34, the X-axis stage 44 is also moved in synchronization with the corresponding position. For example, when the magnification in the optical system is 10, when moving the X drive unit 33 by 1 μm, the moving stage 44 is simultaneously moved by 10 μm. Thus, the controller 46 synchronizes the two stages and adjusts the exposure time of the high sensitivity camera 43 in synchronization with these scans. As a result, the high-sensitivity camera 43 shoots an image on the XY plane obtained by scanning the line-shaped image 29 in the X direction with uniform brightness, and transfers the image to the controller 46 for observation on the monitor 47. Or recording to a recording device inside the controller 46.

  Further, by driving the Z-axis stage 37 in the Z direction by the controller 46, the line-shaped imaging 29 is scanned in the Z direction. Further, since the optical axis between the mirror 26 and the imaging lens 25 and the optical axis between the mirror 32 and the imaging lens 39 are parallel lights, and these optical axes are in the Z direction, the Z drive unit 36 is Even if it is moved, the optical system before and after that is not affected.

  Further, the X-axis stage 44 is also moved in synchronization with the corresponding position simultaneously with the driving of the Z drive unit 36. For example, when the magnification in the optical system is 10, when the Z drive unit 36 is moved by 1 μm, the moving stage 44 is simultaneously moved by 10 μm. Thus, the controller 46 synchronizes the two stages and adjusts the exposure time of the high sensitivity camera 43 in synchronization with these scans. As a result, the high-sensitivity camera 43 shoots an image on the YZ plane obtained by scanning the line-shaped image 29 in the Z direction with uniform brightness, and transfers the image to the controller 46 for observation on the monitor 47. Or recording to a recording device inside the controller 46.

  In this embodiment, two objective lenses are used for image formation. However, the present invention is not limited to this, and other lens systems having similar functions may be used. A reflective optical system such as a concave mirror may be used. As a simpler configuration, the two slits 24 and the slits 40 may be omitted.

  As described above, according to the microscope apparatus of the present embodiment, the two objective lenses are arranged at an angle with respect to the optical axis so that the focal positions thereof coincide with each other. Is obtained.

  In addition, since the light projecting light path and the light collecting light path are separate, the light other than the light collecting position is difficult to return, and an image with a good SN ratio can be obtained.

  Further, since a lens having a small NA is used as the objective lens, WD can be taken and aberrations are small, which is suitable for deep observation.

  Furthermore, since the image is obtained by scanning the above-described lines, the scanning method is easy and high-speed scanning is possible.

  Furthermore, since near infrared light is used as the light source, it is suitable for deep observation.

  In addition, since only the necessary optical unit is scanned, the speed can be further increased.

  In addition, since a confocal optical system is formed by inserting slits on the light projecting side and the light receiving side, information on only the focal plane is obtained and SN is improved.

  Furthermore, since a high-sensitivity camera is used for light reception, and the high-sensitivity camera is configured to shoot while moving relatively, an image of the target surface can be easily obtained.

  As described above, according to the microscope apparatus of the present invention, the main axis of the objective lens of the light projecting optical system and the main axis of the objective lens of the detection optical system intersect at a constant angle, and the detector has a linear shape. Since the intensity distribution on the line of the light generated by the illumination light is detected, the illumination light can be easily scanned, and a microscope apparatus suitable for observation of the deep part of the living body can be obtained.

  The scope of application of the present invention is not limited to the above embodiment. The present invention relates to a projection optical system that projects line-shaped illumination light onto an object, a detection optical system that receives light generated by the line-shaped illumination light and forms an image on a detector, and line-shaped illumination. A scanning unit that scans light, and widely applied to a microscope apparatus that acquires image information on a plane obtained by scanning a line-shaped illumination light by the scanning unit based on a signal from a detector. be able to.

1 is a block diagram illustrating a configuration of a microscope apparatus according to Embodiment 1. FIG. The figure which shows a condensing point typically. The enlarged view of line-shaped condensing. FIG. 6 is a block diagram illustrating a configuration of a microscope apparatus according to a second embodiment. The figure which shows the positional relationship of an objective lens. The enlarged view of line-shaped image formation.

Explanation of symbols

5 Cylindrical lens (objective lens)
7 Condensation (illumination light)
8 Objective lens 8A Focal plane 11 Line CCD (detector, line sensor)
12 2-axis stage (scanning means)
15 Controller (scanning means)
24 slit 34 X drive stage (scanning means)
37 Z drive stage (scanning means)
40 slit 28 objective lens 28A focal plane 30 objective lens 30A focal plane 43 high sensitivity camera (detector, two-dimensional sensor)

Claims (15)

A projection optical system that projects line-shaped illumination light onto an object, a detection optical system that receives light generated by the line-shaped illumination light and forms an image on a detector, and the line-shaped illumination light And a scanning device that scans, and obtains image information on a plane obtained by scanning the line-shaped illumination light by the scanning device based on a signal from the detector,
The principal axis of the objective lens of the projection optical system and the principal axis of the objective lens of the detection optical system intersect at a constant angle,
The said detector detects the intensity distribution on the said line of the said light produced by the said line-shaped illumination light, The microscope apparatus characterized by the above-mentioned. The microscope apparatus according to claim 1, wherein the light generated by the line-shaped illumination light is fluorescence from the object. The microscope apparatus according to claim 1, wherein the light generated by the line-shaped illumination light is reflected light or scattered light from the object. The microscope apparatus according to claim 1, wherein the angle is 90 degrees. The microscope apparatus according to claim 1, wherein the angle is in a range of 30 degrees to 180 degrees. The microscope apparatus according to claim 1, wherein the light projecting optical system irradiates near infrared laser light. The microscope apparatus according to claim 1, wherein the light projecting optical system projects the linear illumination light by causing parallel light to enter a cylindrical lens. The microscope apparatus according to claim 7, wherein the line-shaped illumination light is on a focal plane of the detection optical system. The microscope apparatus according to claim 1, wherein the light projecting optical system projects the linear illumination light by forming an image of a slit on the symmetrical object. The microscope apparatus according to claim 9, wherein the line-shaped illumination light is irradiated onto a line where a focal plane of the light projecting optical system intersects with a focal plane of the detection optical system. The microscope apparatus according to claim 1, wherein the scanning unit scans the line-shaped illumination light in a direction perpendicular to the line. The microscope according to claim 1, wherein the scanning unit scans the illumination light by moving all or part of the light projecting optical system and the detection optical system. apparatus. The microscope apparatus according to any one of claims 1 to 12, wherein a slit is disposed on an imaging surface of the detection optical system. The microscope device according to claim 1, wherein the detector is a line sensor. The microscope according to claim 1, wherein the detector is a two-dimensional sensor, and the two-dimensional sensor is moved in accordance with detection light in synchronization with scanning by the scanning unit. apparatus.

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