JP2010072016A - Microscope device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope device that easily makes a scan with illumination rays and is suitable for observation of a deep part of a living organism. <P>SOLUTION: A separation filter 9 includes only its part (area 91) configured to transmit laser light, so that only the part is irradiated with a laser light. The laser light only passes through the separation filter to impinge on an objective 10. The objective 10 converges the laser light, and an optical scanning unit 5 scans a focal plane 20 with a convergence point thereof. A fluorescence indicator of a sample is excited with the light to emit fluorescent light. The fluorescent light is captured by the objective 10 and travels toward separation filter 9, but only the part (area 92) of the separation filter 9 makes the fluorescent light pass through. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention irradiates illumination light through an objective lens to focus the illumination light on the sample, and obtains return light from the focus through the objective lens and detects it with a detector. The present invention relates to a microscope apparatus.

  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 irradiates illumination light through an objective lens to focus the illumination light on the sample, and obtains return light from the focus through the objective lens to detect the detector. The optical path of the illumination light from the objective lens to the focal point, and the optical path from the focal point of the light detected by the detector to the objective lens among the return light, It is characterized by being separated throughout.
According to this microscope apparatus, the optical path of the illumination light from the objective lens to the focal point and the optical path from the focal point of the light detected by the detector to the objective lens among the return light are separated over the entire optical path. Therefore, the illumination light can be easily scanned and is suitable for observation of the deep part of the living body.

  The return light may be fluorescence.

  The return light may be reflected or scattered light.

  The focus of the illumination light may be scanned by the focal plane of the objective lens, and the return light from each scanned focus may be detected by the detector.

  The focus of the illumination light may be scanned by driving a mirror in the optical path of the illumination light.

  The focus of the illumination light may be scanned by driving a mask pattern member in the optical path of the illumination light.

  An area through which the illumination light from the objective lens to the focal point passes at the pupil position of the objective lens, and an area through which the light detected by the detector of the return light passes at the pupil position of the objective lens. , May be separated.

  In the pupil position of the objective lens, a region through which the illumination light passes and a region through which the return light passes are in different sections separated by at least one straight line or curve crossing the pupil position. May be present.

  In the pupil position of the objective lens, the region through which the illumination light passes and the return light are divided into different sections radially separated by at least two or more intersecting straight lines or curves crossing the pupil position. There may be a region through which

  In the pupil position of the objective lens, the region through which the illumination light passes is divided into different sections separated into the interior and the exterior by at least one closed straight line or curve existing inside the pupil position. There may be a region through which the return light passes.

  At the pupil position of the objective lens, the region through which the illumination light passes and the return light pass through different sections that are separated into the inside and the outside of at least one circle existing inside the pupil position. There may be regions.

  You may provide the optical path limiting means for isolate | separating the optical path of the said return light, and the optical path of illumination light.

  The return light may be fluorescence, and a filter or mirror having wavelength characteristics may be used as the optical path limiting means to limit the optical path of the fluorescence detected by the detector.

  As the optical path limiting means, a filter or a mirror having wavelength characteristics may be used to limit the optical path of the illumination light entering the objective lens.

  The return light may be reflected or scattered light, and a filter or mirror having polarization characteristics may be used as the optical path limiting means to limit the optical path of the return light detected by the detector.

  A part of the optical path of the illumination light or the optical path of the return light may be limited by a light shielding mask as the optical path limiting unit.

  The optical path limiting means may be provided near the pupil position of the objective lens.

  The optical path limiting means may be provided in the vicinity of the position where the pupil position of the objective lens is relayed.

  The optical path limiting means may be provided in a portion of the optical path where the illumination light or the return light forms parallel light.

  The optical path of the illumination light and the optical path of the return light may be configured by separate optical components.

  The set of focal points of the illumination light may be a line shape, and the set of detection points detected by the detector may also be a line shape that matches the set of focus points.

  The illumination light may be near infrared laser light.

  According to the microscope apparatus of the present invention, the optical path of the illumination light from the objective lens to the focal point and the optical path from the focal point of the light detected by the detector to the objective lens among the return light are separated over the entire optical path. Therefore, the illumination light can be easily scanned and is suitable for observation of the deep part of the living body.

  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, a microscope apparatus 1 according to the present embodiment includes a laser light source 2 that outputs laser light having a near-infrared wavelength, and an electric shutter 3 is disposed on the front surface thereof. Furthermore, a dichroic mirror 4 for changing the direction of the laser beam and a scanning optical unit 5 for scanning the laser beam are provided.

  The dichroic mirror 4 has a characteristic of reflecting the wavelength of the laser beam and transmitting the fluorescence excited by the laser beam. The scanning optical unit 5 includes a variable mirror 5a that can rotate about one axis, and a variable mirror 5b that can rotate about an axis substantially orthogonal to the axis of the variable mirror 5a.

  Further, the microscope apparatus 1 includes a pupil projection lens 6 that converges light, a mirror 7 that deflects a laser beam, an imaging lens 8, and an objective lens 10. A separation filter 9 is provided in the vicinity of the pupil position of the objective lens 10.

  FIG. 2 is a diagram showing the structure of the separation filter 9. As shown in FIG. 2, the separation filter 9 includes a region 91 that passes only laser light, a region 92 that passes only the above-described fluorescence, and a light shielding region 93.

The microscope apparatus 1 has a stage 11 on which a sample 12 is placed. Further, in the vicinity of the dichroic mirror 4, a fluorescent filter 13 that selectively transmits fluorescence generated from the sample 12, a lens 14 that converges a fluorescent beam, a pinhole member 15 in which a pinhole is formed, and a pinhole member 15 And a detector 16 for detecting light that has passed through the pinhole.
Furthermore, the microscope apparatus 1 has a controller 17 and a display monitor 18, and based on the control by the controller 17, the electric shutter 3 is opened and closed, the laser beam is scanned by the optical scanning unit 5, the signal is obtained from the photodetector 16 and displayed. Information output to the monitor 18 is executed.

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

  The sample 12 is placed on the stage 11 in a state in which a fluorescent indicator excited by light from the laser light source 2 is introduced.

  The laser light from the laser light source 2 passes through the electric shutter 3 when the electric shutter 3 is open, is guided to the optical scanning unit 5 by the dichroic mirror 4, and is scanned in an arbitrary direction by the optical scanning unit 5. The laser light further passes through the pupil projection lens 6, the reflection mirror 7, and the imaging lens 8, and enters the separation filter 9 near the pupil position of the objective lens 10.

  FIG. 3 is a diagram illustrating the function of the separation filter 9.

  Since the separation filter 9 is configured such that only a part (region 91) of the separation filter 9 can transmit the laser light, only the light irradiated to this part of the separation light 9 passes through the objective lens 10. The laser beam is converged by the objective lens 10, and the convergence point is scanned on the focal plane 20 by the optical scanning unit 5. This laser light excites the fluorescent indicator of the sample, and emits fluorescence. This fluorescence is captured by the objective lens 10 and travels to the separation filter 9, but the separation filter 9 allows the fluorescence to pass through only a part (region 92).

  As shown in FIG. 1, the fluorescence that has passed through the separation filter 9 travels in the opposite direction to the laser light, and passes to the dichroic mirror 4 via the imaging lens 8, the reflection mirror 9, the pupil projection lens 6, and the optical scanning unit 5. Led. The fluorescence passes through the dichroic mirror 4, a specific wavelength component is selectively transmitted by the fluorescence filter 13, and only the fluorescence from the focal plane 20 is selected by the lens 14 and the pinhole 15 and enters the detector 15. .

  The output signal from the light detector 16 is guided to the control unit 17 and converted into a digital signal in synchronization with the scanning control by the scanning optical unit 5 to create image data corresponding to the scanning position, and on the display monitor 18. Or the created data is stored in an internal memory.

  FIG. 4 is a view showing the vicinity of the convergence point of the objective lens 10. As shown in FIG. 4, the fluorescent substance is excited by the laser beam from the right side, and the fluorescence is detected from the left side. Since the pupil of the objective lens 10 is only partially used, the horizontal resolution is slightly higher. Although worse, the fluorescence in the hatched area in FIG. 4 is selectively detected, and therefore the depth resolution is excellent.

  At this time, when the optical axis of the laser beam and the optical axis of the detected fluorescence are set to approximately 90 degrees, the hatching area shown in FIG. 4 is the smallest, so that the best resolution can be obtained. Further, since the optical path of the laser beam and the optical path of the detection fluorescence are separated, the fluorescence other than the hatching region of FIG. 4 excited by the laser beam is not easily detected, and an image with good SN can be obtained during deep observation. Can shoot.

  In addition, since the fluorescence only in the hatched region returns, the confocal effect can be obtained without using the pinhole 15, but the SN is further improved by inserting the pinhole 15.

  Here, the installation position of the separation filter 9 is preferably in the vicinity of the pupil position of the objective lens 10, but may be in or near the optical scanning unit 5 on which the pupil position is projected, or between the optical scanning unit 5 and the dichroic mirror 4. You may insert in a parallel optical path. The separation filter may be installed in the vicinity of the position where the pupil position of the objective lens 10 is relayed.

  The pattern of the separation filter is not limited to this, and each region for selecting transmitted light may be provided radially or concentrically, or may be another pattern that can obtain the same effect.

  As described above, according to the microscope apparatus of the first embodiment, the optical system for projecting the laser beam and the optical system for detection are separated, and thus the resolution in the optical axis direction is excellent. In addition, since the optical system of the projection system and the optical system of the detection system are configured separately, the fluorescence that becomes noise from various places through which the optical system of the projection system passes is difficult to enter the optical path of the detection system. It has a very good S / N ratio and is suitable for deep tissue observation.

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

  FIG. 5 is a block diagram illustrating the configuration of the microscope apparatus according to the second embodiment. The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  As shown in FIG. 2, in the microscope apparatus 1A according to the second embodiment, the light beam of the laser light from the laser light source 2A is narrowed and passes through the optical path with the optical axis center removed. In addition, a light shielding mask 21 for selecting return light (fluorescence) is provided.

  In FIG. 5, the thick line indicates the luminous flux of the laser beam. The optical path of the laser light passes through the optical path indicated by a thick line, and light enters from the right side of the objective lens 10 in FIG. The fluorescence from the sample 11 returns with a thick light beam that satisfies the pupil diameter as shown by the solid line in the figure, and only a part of the light is taken out by the light shielding mask 21, and the light that has passed through the pinhole 15 is detected by the photodetector 16. .

  Since the light passing through the light shielding mask 21 is a light beam passing through the left side of the objective lens 10, the light shielding mask 21 acts substantially the same as the separation filter 9 of the first embodiment. Further, the light shielding mask may be provided in the vicinity of the pinhole member 15.

  In addition to the effects of the first embodiment, the microscope apparatus according to the present embodiment has an advantage that efficiency can be improved because most of the laser light can be used.

  Hereinafter, the microscope apparatus of Example 3 will be described with reference to FIGS. A description of the same components as those in the first embodiment is omitted. In the microscope apparatus of the third embodiment, the structure of the separation filter is different from the microscope apparatus of the first embodiment.

  FIG. 6 is a diagram showing the structure of the separation filter 9A used in the microscope apparatus of this embodiment.

  As shown in FIG. 6, in the separation filter 9A, a region 91A that passes only laser light, a light shielding region 93A, and a region 92A that passes only fluorescence are sequentially provided concentrically from the inside toward the outside.

  FIG. 7 is a diagram illustrating the function of the separation filter 9A.

  As shown in FIG. 7, in the microscope apparatus of the present embodiment, the light beam of the laser light from the laser light source is narrowed to be a light beam corresponding to the region 91A of the separation filter 9A. The laser light that has passed through the region 91A is converged by the objective lens 10. The fluorescence from the convergence point passes through the region 92A and returns through the same optical path.

  FIG. 8 is a view showing the vicinity of the convergence point of the objective lens 10. As shown in FIG. 8, in this embodiment, the fluorescent material is excited by laser light from a region close to the optical axis, and fluorescence is detected from the outside. Thereby, since the fluorescence in the hatched area in FIG. 8 is selectively detected, the resolution in the depth direction is excellent as in the first embodiment.

  Here, the installation position of the separation filter 9A is preferably in the vicinity of the pupil position of the objective lens 10, but may be in or near the optical scanning unit 5 on which the pupil position is projected, and between the optical scanning unit 5 and the dichroic mirror 4. You may insert in a parallel optical path. The separation filter may be installed in the vicinity of the position where the pupil position of the objective lens 10 is relayed.

  In addition to the effects of the first embodiment, the microscope apparatus according to the present embodiment has an advantage that efficiency can be improved because most of the laser light can be used.

  Hereinafter, the microscope apparatus according to the fourth embodiment will be described with reference to FIG. The microscope apparatus according to the fourth embodiment employs a scanning method different from that of the first embodiment.

  FIG. 9 is a block diagram illustrating a configuration of the microscope apparatus according to the fourth embodiment. The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  As shown in FIG. 9, in the microscope apparatus of this embodiment, laser light from a laser light source is incident on the scanner unit 40.

  The scanner unit 40 includes a laser light irradiation unit 41 that emits laser light from the tip of an optical fiber connected to a laser light source, and a collimator lens 42. The scanner unit 40 includes a microlens disk 43 in which microlenses are arranged in an array and a pinhole disk 44 in which pinholes are arranged in the same pattern, and these are connected by a connecting drum 45. The motor 46 can rotate integrally. A dichroic mirror 47 is disposed between the microlens disk 43 and the pinhole disk 44. The scanner unit 40 is provided with a fluorescent filter 48, a camera lens 49, and a camera 50.

  The imaging lens 8 and the objective lens 10 are disposed below the pinhole disk 44 of the scanner unit 40. In the vicinity of the pupil position of the objective lens 10, a separation filter 9 similar to that of the first embodiment is provided. Instead of the separation filter 9, the separation filter 9A of the third embodiment may be used. The camera 50 and the motor 46 are connected to the controller 17, and the synchronization of rotation and the exposure time are adjusted.

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

  Light from the tip of the optical fiber in the laser light irradiation unit 41 is converted into parallel light by the collimator lens 42. This light is applied to the microlens disk 43, passes through the dichroic mirror 47 by the action of each microlens, and then focuses on the pinhole corresponding to the microlens of the pinhole disk 44.

  The light passing through the pinhole disk 44 is converted into parallel light having an inclination corresponding to the pinhole of the passed pinhole disk 44 in the imaging lens 8. When parallel light is incident on the objective lens 10, the focal point is imaged on the focal plane in accordance with the tilt, so that the light incident on the objective lens 10 is focused at a position corresponding to the tilt.

  In FIG. 9, the light at the center of the optical axis is indicated by a solid line, and an example of a light beam shifted from the optical axis is indicated by a broken line. The fluorescence generated at the focal point is collected by the objective lens 10 and becomes parallel light. Here, due to the effect of the separation filter 9, incident light is irradiated from the right side of the objective lens 10 in FIG. 9, and light passing through the left side of the objective lens 10 passes through the filter 9 and returns (see FIG. 3). This fluorescence passes through the same optical path and again passes through the same pinhole of the pinhole disk 44. Here, the light passing through the pinhole is reflected by the dichroic mirror 47, the wavelength component is selected by the fluorescent filter 48, and an image is formed by the camera 50 by the action of the camera lens 49.

  Here, when the microlens disc 43 and the pinhole disc 44 are integrally rotated, the light passing through the pinhole scans on the corresponding focal plane, and further, the individual fluorescence passes through the same pinhole again. The image sensor of the camera 50 is scanned. By this operation, the fluorescence of the focal plane is projected onto the camera 50 and can be observed. At this time, light other than the focal plane hardly reaches the camera 50 because it hardly passes through the pinhole. As a result, the camera 50 captures a confocal image of only the light on the focal plane.

  Further, as shown in FIG. 3, excitation is performed from the right side and detection is performed from the left side, and the pupil of the objective lens 10 is only partially used. Therefore, the depth resolution is excellent.

  At this time, when the optical axis of the laser beam and the optical axis of the detected fluorescence are set to approximately 90 degrees, the hatching area in FIG. Also in this embodiment, since the optical path of the laser beam and the optical path of the detection fluorescence are separated, the fluorescence other than the hatched region of FIG. 3 excited by the laser beam is not easily detected, and deep observation is performed. Sometimes images can be taken with good SN.

  It should be noted that if the optical paths of optical systems passing through adjacent pinholes overlap each other in the vicinity of the sample, the SN is deteriorated, so that a pinhole disk having a pinhole pattern having an interval such that the optical paths in the vicinity of the sample do not overlap as much as possible. It is desirable to adopt 54 or the like.

  In the microscope apparatus of the fourth embodiment, since the scanner unit 40 that scans the laser beam by rotating the disk is used, in addition to the effects of the first embodiment, there is an advantage that the scanning speed is high.

  Hereinafter, the microscope apparatus of Example 5 will be described with reference to FIG.

  FIG. 10 is a block diagram illustrating a configuration around the objective lens in the microscope apparatus according to the fourth embodiment. FIG. 10 shows parts different from the first embodiment, and other parts are configured in the same manner as in the first embodiment.

  In the microscope apparatus of the present embodiment, the optical configuration from the imaging lens 8 to the sample 12 is different from that of the first embodiment.

  As shown in FIG. 10, in this embodiment, a dichroic mirror 51 is used to separate the optical path of illumination light from the imaging lens 8 to the sample 12 and the optical path of the detection system. That is, a dichroic mirror 51 that reflects light having a wavelength of laser light and reflects light having a wavelength longer than that of the laser light is disposed at the tip of the imaging lens 8. Further, three reflection mirrors 52, 53, and 54 are arranged in the vicinity thereof. Further, an objective lens 10A having a relatively large diameter is selected.

  In the microscope apparatus of the present embodiment configured as described above, the near-infrared laser light converted into parallel light by the imaging lens 8 is reflected by the dichroic mirror 51 and the mirror 52, and is applied to the objective lens 10A. Incident light enters the main axis and is offset from the main axis.

  As shown in FIG. 10, the light is focused on the sample 12, and fluorescence is emitted from the sample 12. A part of this fluorescence passes through another optical path which is symmetric with respect to the illumination light and the main axis of the objective lens 10A as shown in FIG. 10, and is converted again into parallel light by the objective lens 10A. Reflected. This fluorescence passes through the dichroic mirror 51 having transmission characteristics with respect to the wavelength of the fluorescence, and returns on the same optical path as the illumination light. The following returns in the same manner as in the first embodiment, passes through the dichroic mirror 4, and is detected by the detector 16 (FIG. 1).

  According to the microscope apparatus of the fourth embodiment, as in the first embodiment, since the optical system for projecting laser light and the optical system for detection are separated, the resolution in the optical axis direction is excellent. In addition, since the optical system of the projection system and the optical system of the detection system are configured separately, the fluorescence that becomes noise from various places through which the optical system of the projection system passes is difficult to enter the optical path of the detection system. It has a very good S / N ratio and is suitable for deep tissue observation.

  Hereinafter, the microscope apparatus of Example 6 will be described with reference to FIG.

  FIG. 11 is a block diagram illustrating a configuration of the microscope apparatus 1B according to the sixth embodiment. The same components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  In the microscope apparatus according to the sixth embodiment, line-shaped light is projected. The microscope apparatus of the present embodiment includes a fiber 61 that irradiates a laser beam (not shown). At the tip of the fiber 61, there are a collimating lens 62, a cylindrical lens 63, a slit member 64 having a slit extending in a direction perpendicular to the paper surface, and an imaging lens 65. The optical scanning unit 5A includes a galvanometer mirror 5a that rotates on a rotation axis perpendicular to the paper surface. Instead of the galvanometer mirror 5b (FIG. 1) in the first embodiment, a fixed mirror 68 is disposed. Further, in place of the pinhole member 15 (FIG. 1) in the first embodiment, a slit member 66 having a slit extending in a direction perpendicular to the paper surface is disposed, and a line CCD 67 is used as a photodetector.

  Hereinafter, the operation of the microscope apparatus 1B of the present embodiment will be described.

  The light from the laser light source is transmitted as a point light source through the optical fiber 61, converted into parallel light by the collimating lens 62, and condensed into a line shape by the cylindrical lens 63. The light that has passed through the slit 63 disposed at the condensing point passes through the imaging lens 64.

  Thereafter, as shown in FIG. 11, the light is imaged on the focal plane by the objective lens 10 through the same optical path as that of the first embodiment, and becomes a line shape perpendicular to the paper surface. Fluorescence from this line is collected by the objective lens 10, but each point on the line collects fluorescence only in the hatched region of FIG. 3 by the effect of the separation filter 9 as in the first embodiment. The fluorescence is imaged again on the slit of the slit member 66 by the lens system, and only the light from the focal plane that has passed through the slit is detected by the line CCD 67.

  Further, by operating the galvanometer mirror 5a, the line is scanned on the focal plane in a direction perpendicular to the line, and this information is detected by the line CCD 67 and imaged by the controller 20.

  In the microscope apparatus 1B according to the sixth embodiment, the set of focal points of the illumination light is in a line shape, and the set of detected detection points is also in a line shape that matches the set of focus points. In addition, since the line is scanned in one direction, the scanning time can be shortened.

  Hereinafter, the microscope apparatus of Example 7 will be described with reference to FIG.

  FIG. 12 is a block diagram illustrating a configuration of a microscope apparatus 1C according to the seventh embodiment. The same components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  The microscope apparatus 1C of the present embodiment has a configuration in which the light projecting optical system and the detection optical system are completely separated.

  As shown in FIG. 12, the optical system of the light projecting system does not use the dichroic mirror 4 in the optical path in the first embodiment. Further, the light from the imaging lens 8 is incident on the objective lens 10 with an offset, and the separation filter 9 of the first embodiment is removed. The fluorescence collected by the objective lens 10 is symmetrical with respect to the light of the projection system and the optical axis of the objective lens 12, and the imaging lens 71, mirror 72, pupil projection lens 73, and two galvanometer mirrors 74a and 74b. The optical scanning unit 74 having The optical path from the fluorescent filter 13 to the photodetector 16 is configured in the same manner as in the first embodiment.

  In the microscope apparatus 1C of the present embodiment, the light from the laser light source 2 enters the objective lens 10 via the electric shutter 3, the optical scanning unit 5, the pupil projection lens 6, the reflection mirror 7, and the imaging lens 8, and the objective The lens 10 is focused. The fluorescence from the focal point is collected by the objective lens 10. At this time, the light from the imaging lens 8 is incident on the objective lens 10 with an offset, and the recovered fluorescence is also recovered in the offset direction, so that only the hatched region of FIG. Fluorescence from is recovered.

  This fluorescence is converted into parallel light, reflected by the mirror 72 through the imaging lens 71, converted again into parallel light by the pupil projection lens 73, passed through the optical operation unit 74, and the wavelength component is selected by the fluorescence file 13. The Thereafter, the light is condensed into the pinhole 15 by the action of the lens 14, and only the light from the focal plane is selected by the lens 14 and the pinhole 15 and enters the detector 16.

  Further, when the optical scanning unit 5 scans the focal plane, the control unit 17 drives the optical scanning unit 74 synchronously, and the pinhole 15 does not change even if the direction of the parallel light entering the optical scanning unit 74 changes. Control is performed so that the direction of the light toward the light does not change.

  An output signal from the photodetector 16 is guided to the control unit 17 and converted into a digital signal in synchronization with the scanning control, and image data is created in correspondence with the scanning position and displayed on the display monitor 18, or The image data is stored in an internal memory.

  As described above, in the microscope apparatus 1C of the seventh embodiment, the optical path of the illumination light and the optical path of the return light are configured by separate optical parts, and the optical paths are completely separated. Since stray light such as return light of the optical system can be surely removed, there is an effect that the SN of the image is further improved.

  Hereinafter, the microscope apparatus according to the eighth embodiment will be described with reference to FIGS. 13 and 14.

  FIG. 13 is a block diagram illustrating the configuration of the microscope apparatus 1D according to the eighth embodiment, and FIG. 14 is a diagram illustrating the function of the separation filter. The same components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  The microscope apparatus of the present embodiment is an application of the present invention to a microscope apparatus in which the return light is not fluorescence but reflected or scattered light.

  As shown in FIG. 13, in the microscope apparatus 1 </ b> D, a polarizing beam splitter 81 is used instead of the dichroic mirror 4 in the first embodiment, and a polarizing filter 84 is used instead of the fluorescent filter 13. Further, instead of the separation filter 9, a separation filter 82 using polarized light is used. As shown in FIG. 14, the right side region 82a and the left side region 82b of the separation filter 82 are each configured by a polarizing filter in which the polarization direction of linearly polarized light passing therethrough differs by 90 degrees, and between the right side region 82a and the left side region 82b. A light shielding region 82c is provided. Further, a quarter wave plate 83 is inserted in front of the objective lens 10.

  In the microscope apparatus 1D configured as described above, the laser light has linearly polarized light and is reflected by the polarization beam splitter 81, and then enters the separation filter 82 through the same optical path as in the first embodiment. Here, only the right region 82a of the separation filter 82, which is configured to pass the polarized light of the laser light, is transmitted. This light is converted into circularly polarized light by the quarter-wave plate 83 and focused by the objective lens 10. Reflected and scattered light from the focal point is collected by the objective lens 10 and once again passes through the quarter-wave plate 83 to become polarized light having a polarization direction different from that of the incident laser by 90 degrees. To Penetrate. Here, the return light cannot pass through the right region 82a because the polarization direction is 90 degrees different from that of the right region 82a. The light that has passed through the left region 82 b returns in the reverse direction of the optical path of the laser light. This time, after passing through the polarization beam splitter 81, the polarization component is selected by the polarization filter 84, and the detector 19 through the pinhole 18. Is detected.

  Thus, according to the microscope apparatus of Example 8, it becomes possible to detect reflection and scattered light in the deep part of the living body. Further, since the optical system for projecting laser light and the optical system for detection are separated, the resolution in the optical axis direction is excellent. In addition, since the optical system of the light projecting system and the optical system of the detection system are configured separately, the light that becomes noise from various places through the optical system of the light projecting system is difficult to enter the optical path of the detection system. It has a very good S / N ratio and is suitable for deep tissue observation.

  Hereinafter, the microscope apparatus of Example 9 will be described with reference to FIG.

  FIG. 15 is a diagram illustrating a structure of a separation filter 9B used in place of the separation filter 9 used in the microscope apparatus 1 according to the first embodiment. Other elements are the same as those in the first embodiment.

  The separation filter 9B is configured by radially arranging areas for selecting transmitted light. As shown in FIG. 15, the separation filter 9B is composed of a region 91B that passes only laser light, a region 92B that passes only fluorescence, and a light shielding region 93B. The region 91B and the region 92B are arranged at positions facing each other with the optical axis as a target point.

  In the separation filter 9B, since the light projecting system and the detection system use positions where the pupils of the objective lens 10 face each other, the focal point is reduced and the resolution is further improved.

  The scope of application of the present invention is not limited to the above embodiment. The present invention is directed to a microscope apparatus that irradiates illumination light through an objective lens to focus the illumination light on the sample and obtains return light from the focus through the objective lens and detects it with a detector. Can be widely applied.

1 is a block diagram illustrating a configuration of a microscope apparatus according to Embodiment 1. FIG. The figure which shows the structure of a separation filter. The figure which shows the function of a separation filter. The figure which shows the convergence point vicinity of an objective lens. FIG. 6 is a block diagram illustrating a configuration of a microscope apparatus according to a second embodiment. The figure which shows the structure of a separation filter. The figure which shows the function of a separation filter. The figure which shows the convergence point vicinity of an objective lens. FIG. 6 is a block diagram illustrating a configuration of a microscope apparatus according to a fourth embodiment. The block diagram which shows the structure around an objective lens. FIG. 10 is a block diagram illustrating a configuration of a microscope apparatus according to a sixth embodiment. FIG. 9 is a block diagram illustrating a configuration of a microscope apparatus according to a seventh embodiment. FIG. 10 is a block diagram illustrating a configuration of a microscope apparatus according to an eighth embodiment. The figure which shows the function of a separation filter. The figure which shows the structure of a separation filter.

Explanation of symbols

9, 9A, 9B Separation filter (optical path limiting means)
10 Objective Lens 10A Objective Lens 16 Photodetector (Detector)
21 Shading mask (optical path limiting means)
40 Scanner unit (optical path limiting means)
51 Dichroic mirror (optical path limiting means)
67 line CCD (detector)
82 Separation filter (optical path limiting means)

Claims (22)

In the microscope apparatus that irradiates illumination light through the objective lens to tie the focus of the illumination light to the sample, and obtains return light from the focus through the objective lens and detects it with a detector,
The optical path of the illumination light from the objective lens to the focal point and the optical path from the focal point of light detected by the detector to the objective lens among the return light are separated over the entire optical path. A microscope apparatus characterized by that. The microscope apparatus according to claim 1, wherein the return light is fluorescence. The microscope apparatus according to claim 1, wherein the return light is reflected or scattered light. The microscope apparatus according to claim 1, wherein the focus of the illumination light is scanned by a focal plane of the objective lens, and the return light from each scanned focus is detected by the detector. The microscope apparatus according to claim 4, wherein the focus of the illumination light is scanned by driving a mirror in an optical path of the illumination light. The microscope apparatus according to claim 4, wherein the focus of the illumination light is scanned by driving a mask pattern member in an optical path of the illumination light. An area through which the illumination light from the objective lens to the focal point passes at the pupil position of the objective lens, and an area through which the light detected by the detector of the return light passes at the pupil position of the objective lens. The microscope apparatus according to claim 1, wherein the microscope apparatus is separated. In the pupil position of the objective lens, a region through which the illumination light passes and a region through which the return light passes are in different sections separated by at least one straight line or curve crossing the pupil position. The microscope apparatus according to claim 7, wherein the microscope apparatus is present. In the pupil position of the objective lens, the region through which the illumination light passes and the return light are divided into different sections radially separated by at least two or more intersecting straight lines or curves crossing the pupil position. The microscope apparatus according to claim 8, wherein there is a region through which to pass. In the pupil position of the objective lens, the region through which the illumination light passes is divided into different sections separated into the interior and the exterior by at least one closed straight line or curve existing inside the pupil position. The microscope apparatus according to claim 7, wherein there is a region through which the return light passes. At the pupil position of the objective lens, the region through which the illumination light passes and the return light pass through different sections that are separated into the inside and the outside of at least one circle existing inside the pupil position. The microscope apparatus according to claim 10, wherein a region exists. 2. The microscope apparatus according to claim 1, further comprising an optical path limiting unit for separating the optical path of the return light and the optical path of the illumination light. The microscope apparatus according to claim 12, wherein the return light is fluorescence, and a filter or a mirror having wavelength characteristics is used as the optical path limiting means to limit the optical path of the fluorescence detected by the detector. . 13. The microscope apparatus according to claim 12, wherein a filter or a mirror having wavelength characteristics is used as the optical path limiting means to limit an optical path of illumination light entering the objective lens. The return light is reflected or scattered light, and the optical path of the return light detected by the detector is limited by using a filter or a mirror having polarization characteristics as the optical path limiting means. The microscope apparatus described. 13. The microscope apparatus according to claim 12, wherein a part of the optical path of the illumination light or a part of the optical path of the return light is limited by a light shielding mask as the optical path limiting unit. The microscope apparatus according to claim 12, wherein the optical path limiting means is provided in the vicinity of a pupil position of the objective lens. 13. The microscope apparatus according to claim 12, wherein the optical path limiting means is provided in the vicinity of a position where the pupil position of the objective lens is relayed. 13. The microscope apparatus according to claim 12, wherein the optical path limiting means is provided in a portion of an optical path where the illumination light or the return light forms parallel light. The microscope apparatus according to claim 1, wherein the optical path of the illumination light and the optical path of the return light are configured by separate optical components. The set of the focal points of the illumination light is in a line shape, and the set of detection points detected by the detector is also a line shape that matches the set of focus points. Microscope device. The microscope apparatus according to claim 1, wherein the illumination light is near-infrared laser light.

Images