JP2013156467A - Scanning type observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that suppresses vignetting of off-axis light due to a housing accommodating a detection element.SOLUTION: A two-photon excitation microscope 1 is a scanning type observation device having a scanner 3 that scans a sample S by using illumination light emitted from a laser 2. Observation light generated in the sample S irradiated with illumination light is condensed by an object lens 6 and enters a detection element 8a via a pupil relay optical system 7 that makes a pupil PP of the object lens 6 almost conjugate to a light receiving surface 8b of a detection element 8a and is detected. In the two-photon excitation microscope 1, the detection element 8a is disposed such that the most off-axis principal ray of the observation light emitted from the pupil relay optical system 7 and the optical axis of the pupil relay optical system 7 intersect with each other between the light receiving surface 8b and the pupil relay optical system 7.

Description

  The present invention relates to a scanning observation apparatus provided with scanning means for scanning a specimen.

  In the field of observation devices, as a configuration for efficiently detecting observation light generated from a certain region on a specimen, an objective lens or a condenser lens (hereinafter referred to as an objective lens) on which the observation light is incident on the light receiving surface of the detection element is used. And the condensing lens are known to be arranged on the pupil conjugate plane of the condensing lens). Such a configuration is disclosed in Patent Document 1, for example.

  The scanning microscope disclosed in Patent Document 1 is configured such that the pupil of the condenser lens is projected onto the light receiving surface of the photodetector. The principal ray of the observation light incident on the condenser lens from the specimen passes through the pupil position of the condenser lens regardless of the position on the specimen where the observation light is generated. For this reason, according to the scanning microscope disclosed in Patent Document 1 in which the light receiving surface is arranged on the pupil conjugate plane, it is possible to efficiently detect the observation light from the specimen regardless of the scanning position.

JP 2008-2775763 A

  By the way, the detection element is housed in a housing that plays various roles such as shielding of an electric field and magnetic field from the outside and cooling of the detection element, and is usually arranged at the bottom of the opening formed in the housing and parallel to the optical axis. Has been.

  The principal ray of observation light generated from a position deviated from the optical axis of the condenser lens, that is, the principal ray of off-axis light is incident on the pupil position while being inclined with respect to the optical axis. The housing may become an obstacle and vignetting may occur.

In order to suppress such vignetting due to the housing, a method of increasing the pupil projection magnification and reducing the incident angle can be considered. However, when the pupil projection magnification increases, the luminous flux incident on the light receiving surface of the detection element becomes thicker. For this reason, if the projection magnification is increased too much, the phenomenon that the observation light protrudes from the light receiving surface and the phenomenon that the peripheral portion of the thickened light flux is blocked by the housing occur, and the efficiency is lowered.
Based on the above situation, an object of the present invention is to provide a technique for suppressing vignetting of off-axis light by a housing that houses a detection element.

  The first aspect of the present invention includes a scanning unit that scans a specimen with illumination light emitted from a light source, a condensing lens that collects observation light generated by the specimen irradiated with the illumination light, and the observation A detection element that detects light, and a pupil relay optical system that conjugates a pupil of the condenser lens with the light receiving surface of the detection element, wherein the detection element is emitted from the pupil relay optical system There is provided a scanning observation apparatus arranged such that a principal ray outside the most axis of the observation light and an optical axis of the pupil relay optical system intersect between the light receiving surface and the pupil relay optical system.

  According to a second aspect of the present invention, in the scanning observation apparatus according to the first aspect, the detection element has a pupil conjugate position of the condenser lens located between the light receiving surface and the pupil relay optical system. Thus, a scanning observation apparatus is provided.

  According to a third aspect of the present invention, in the scanning observation apparatus according to the first aspect, the pupil relay optical system is configured such that the off-axis principal ray and the optical axis of the pupil relay optical system are the light receiving surfaces. And a pupil observation optical system having a pupil aberration that intersects the pupil relay optical system.

  According to a fourth aspect of the present invention, in the scanning observation apparatus according to the first aspect, the detection element has a pupil conjugate position of the condenser lens located between the light receiving surface and the pupil relay optical system. The pupil relay optical system is arranged such that the off-axis principal ray and the optical axis of the pupil relay optical system intersect between the light receiving surface and the pupil relay optical system. A scanning observation apparatus having pupil aberration is provided.

  According to a fifth aspect of the present invention, in the scanning observation apparatus according to any one of the second to fourth aspects, the scanning unit is on an illumination optical path and the pupil of the condenser lens. Provided is a scanning observation apparatus arranged at a conjugate position.

  According to a sixth aspect of the present invention, there is provided the scanning observation apparatus according to any one of the first to fifth aspects, wherein the scanning observation apparatus is a nonlinear optical microscope. To do.

  ADVANTAGE OF THE INVENTION According to this invention, the technique which suppresses the vignetting of the off-axis light by the housing which accommodates a detection element can be provided.

It is the figure which illustrated the structure of the scanning observation apparatus which concerns on Example 1 of this invention. It is a figure for demonstrating the difference between the scanning observation apparatus which concerns on a prior art, and the scanning observation apparatus which concerns on Example 1. FIG. It is the figure illustrated about the relationship between arrangement | positioning of a detection element, and aperture efficiency. FIG. 2 is an enlarged view of a detector having a typical aperture shape illustrated in FIG. 1. It is a figure for demonstrating the difference between the scanning observation apparatus which concerns on a prior art, and the scanning observation apparatus which concerns on Example 2 of this invention. It is the figure which illustrated the mode of the fluorescence inject | emitted from each of the pupil relay optical system which has a different pupil aberration. FIG. 7 is a spherical aberration diagram of each of the pupil relay optical systems exemplified in FIG. 6. FIG. 7 is a diagram illustrating the relationship between the arrangement of detection elements and the aperture efficiency when each of the pupil relay optical systems illustrated in FIG. 6 is used. It is the figure which illustrated the structure of the scanning observation apparatus which concerns on Example 3 of this invention.

  FIG. 1 is a diagram illustrating the configuration of a two-photon excitation microscope according to the present embodiment. A two-photon excitation microscope 1 illustrated in FIG. 1 is a scanning observation apparatus including a scanner 3 that observes a specimen S by detecting fluorescence that is observation light generated from the specimen S.

  The two-photon excitation microscope 1 includes a laser 2 that emits laser light for exciting the specimen S, a scanner 3 that is a scanning unit that scans the specimen S with laser light emitted from the laser 2, and a pupil relay optical system 4 A dichroic mirror 5 that reflects laser light and transmits fluorescence generated from the specimen S, an objective lens 6, a pupil relay optical system 7, and a detector 8 that includes a detection element 8a that detects fluorescence. Contains.

  The scanner 3 is, for example, a galvanometer mirror, a polygon mirror, an acoustooptic deflection element, and the like, and is disposed on the illumination optical path between the dichroic mirror 5 and the laser 2 and at the pupil conjugate position of the objective lens 6. The pupil relay optical system 4 includes a lens 4a and a lens 4b, and is configured so that the pupil PP of the objective lens 6 is substantially conjugate with the scanner 3.

  The dichroic mirror 5 is an optical path branching unit that branches the illumination optical path and the detection optical path. The objective lens 6 is a condenser lens that irradiates the sample S with the laser light and collects the fluorescence generated in the sample S irradiated with the laser light. The pupil relay optical system 7 includes a lens 7a and a lens 7b, and is configured such that the pupil PP of the objective lens 6 is substantially conjugate with the light receiving surface 8b of the detection element 8a.

The detector 8 includes a housing 8c that houses the detection element 8a in addition to the detection element 8a. An opening parallel to the optical axis through which the fluorescence incident on the detection element 8a passes is formed in the housing 8c provided for shielding an electric field and a magnetic field from the outside, cooling the detection element 8a, and the like.
The detector 8 is, for example, a cooled photomultiplier tube (photomultiplier), and GaAsP or the like is adopted as the material of the detection element 8a.

  The detection element 8a is disposed at the bottom of the opening so that the normal line of the light receiving surface 8b of the detection element 8a matches the optical axis of the pupil relay optical system 7. In addition, the detection element 8a is configured such that the principal ray off the most axis of fluorescence emitted from the pupil relay optical system 7 and the optical axis of the pupil relay optical system 7 intersect between the pupil relay optical system 7 and the light receiving surface 8b. To be arranged.

  FIG. 2 is a diagram for explaining the difference between the scanning observation apparatus according to the prior art and the scanning observation apparatus according to the present embodiment. FIG. 3 is a diagram illustrating the relationship between the arrangement of the detection elements and the aperture efficiency. The vertical axis in FIG. 3 shows the aperture efficiency, which is the ratio of the area of the off-axis light beam to the area of the light beam on the axis at the pupil position of the fluorescence emitted from the pupil relay optical system, and the horizontal axis in FIG. Indicates the shift amount of the light receiving surface 8b from the pupil conjugate position P0 of the objective lens. The shift amount is defined so as to be positive when the light receiving surface 8b is further away from the pupil relay optical system than the pupil conjugate position P0.

  Hereinafter, with reference to FIGS. 2 and 3, the arrangement of the detection element 8a of the two-photon excitation microscope, which is a scanning observation apparatus, and the influence thereof will be described in more detail.

  FIG. 2A shows the arrangement of the detection elements 8a included in the two-photon excitation microscope 100, which is a scanning observation apparatus according to the prior art. FIG. 2B shows the arrangement of the detection elements 8a included in the two-photon excitation microscope 1 that is the scanning observation apparatus according to the present embodiment. In the two-photon excitation microscope 100 and the two-photon excitation microscope 1, only the distance between the lens 7b and the detector 8 is different.

As illustrated in FIG. 2A, in the two-photon excitation microscope 100 according to the related art, the detection element 8a is disposed on the optical axis so that the light receiving surface 8b coincides with the pupil conjugate plane CP. In this case, if there is no housing 8c, all the principal rays (for example, the principal ray L0 of the on-axis light, the principal ray L1 of the off-axis light, and the principal ray L2 of the off-axis light) are located at the center of the light receiving surface 8b. Since the light enters the position P0, it is possible to detect the light generated from each position of the sample S represented by the principal ray as long as the light beam diameter does not exceed the size of the detection element 8a. However, since the housing 8c actually exists, vignetting by the housing 8c occurs in off-axis light whose principal ray is inclined with respect to the optical axis.
A cover glass (parallel plate) called a window lens is usually disposed at the tip of the opening of the housing 8c for dust prevention and protection. Here, only the lines are described, but when the thickness affects, the air equivalent length may be considered.

Specifically, in the two-photon excitation microscope 100, since the shift amount of the light receiving surface 8b from the pupil conjugate position P0 is 0, as shown in FIG. 3, the axial light represented by the principal ray L0 has an aperture. The efficiency is 100%, whereas the off-axis light represented by the principal ray L1 has an aperture efficiency of about 55%, and the off-axis light represented by the off-axis light represented by the principal ray L2 has an aperture efficiency of 0%. It becomes.
3, the opening shape of the housing 8c is cylindrical, the diameter b is φ6 mm, the opening depth c is 8.9 mm, the diameter a of the light receiving surface 8b is φ5 mm, and the focal point of the lens 7b. Calculation is made assuming that the distance f is 35.3 mm, the principal ray height of the most off-axis light beam on the entrance surface side of the lens 7b is 11.8 mm, and the light beam diameter of the on-axis light beam on the exit surface side of the lens 7b is φ3.2 mm. did.

  On the other hand, as illustrated in FIG. 2B, in the two-photon excitation microscope 1 according to the present embodiment, the detection element 8a has the pupil conjugate position P0 of the objective lens 6 such that the light receiving surface 8b and the lens 7b (pupil relay optics). It is arranged on the optical axis so as to be located between the system 7). For this reason, in the two-photon excitation microscope 1, the principal ray (principal ray L1, principal ray L2) of off-axis light is generated at the end of the opening formed in the housing 8c on the specimen side (hereinafter referred to as the opening end). Compared to the case of the scanning observation apparatus 100, it passes through a position closer to the optical axis. As a result, vignetting in the housing 8c can be suppressed, and as shown in FIG. 3, the aperture efficiency of off-axis light is improved by shifting the light receiving surface 8c from the pupil conjugate position P0.

Note that the principal rays (principal ray L1 and principal ray L2) of off-axis light after passing through the pupil conjugate position P0 spread from the optical axis as the distance from the pupil conjugate position P0 increases. If it is too close to the aperture end, the chief ray spreads greatly after passing through the pupil conjugate position P0, and vignetting occurs in the aperture. As shown in FIG. 3, in the case of the two-photon excitation microscope 1, the shift amount from the pupil conjugate position P0 of the light receiving surface 8c is preferably about 4 mm to 6 mm, and once the shift amount is increased, it is improved once. The aperture efficiency of the off-axis light that has been generated is reduced again. For this reason, the light receiving surface of the detection element 8a should not be disposed at a position far away from the pupil conjugate position P0, but is desirably disposed in the vicinity of the pupil conjugate position P0.
In particular, when the ratio c / b of the depth c to the diameter b of the housing opening is about 1.3 to 1.6, the shift amount z with respect to the focal length f of the lens system at the rear stage of the intermediate image of the pupil relay optical system The ratio z / f is preferably about 0.1 to 0.18.

As described above, the shift amount z of the light receiving surface 8c from the pupil conjugate position P0 has an optimum range in which the occurrence of vignetting can be effectively suppressed. FIG. 4 is an enlarged view of a detector having the typical aperture shape illustrated in FIG. In the case of the detector 8 illustrated in FIG. 4, the optimum range of the shift amount z that can effectively suppress the occurrence of vignetting is expressed by the following equation (1).

Here, the parameter a is the light receiving surface diameter of the detection element 8a (the diameter of the light receiving surface 8b), the parameter b is the mechanical frame diameter (the diameter of the opening of the housing 8c), and the parameter c is the mechanical frame length (of the housing 8c). The depth of the opening). The parameter _hu is the upper ray width of the most off-axis light (the interval between the principal ray of the most off-axis light and the upper marginal ray), and the parameter _hl is the lower ray width of the most off-axis light ( The distance between the principal ray of the most off-axis light and the lower marginal ray), and the parameter θ is the ray incidence angle of the most off-axis light (when the principal ray of the most off-axis light is incident on the pupil conjugate position) , The angle between the principal ray and the optical axis). Note that the parameter a and the parameter b have a relationship of a ≦ b, and the parameter c is represented by an air conversion length when a window lens is disposed in the opening.

Equation (1) can be calculated on condition that the amount of vignetting calculated by the following equations (2) to (5) is minimized.

Here, the shading amount u _a is the upper vignetting of the light receiving surface side (distance between the upper end of the light-receiving surface and the upper marginal ray on the light-receiving surface in the same plane), the shading amount u _b is at the mechanical frame side amount upper vignetting a (distance between the upper marginal ray and the upper open end at the open end flush), vignetting amount l _a is lower marginal in the lower vignetting amount (light receiving surface flush with the light receiving surface side the spacing) between the lower end of the beam and the light receiving surface, the distance between the eclipse amount l _b is lower marginal ray and a lower opening end in the lower vignetting amount (opening end flush at the mechanical frame side ). Incidentally, vignetting amount u _a, vignetting amount u _b, vignetting amount l _a, vignetting amount l _b, respectively, as positive value when the shading occurs, is calculated as a negative value when the eclipse does not occur .

  According to the two-photon excitation microscope 1 according to the present embodiment configured as described above, the pupil conjugate position P0 of the objective lens 6 is in the vicinity of the light receiving surface 8b and between the light receiving surface 8b and the pupil relay optical system 7. By disposing the detection element 8a so as to be positioned between them, vignetting of off-axis light by the housing 8c can be suppressed.

  FIG. 5 is a diagram for explaining the difference between the scanning observation apparatus according to the prior art and the scanning observation apparatus according to the present embodiment. FIG. 5A shows the state of light emitted from the pupil relay optical system of the two-photon excitation microscope 100, which is a scanning observation apparatus according to the prior art, toward the detector 8. FIG. FIG. 5B shows the state of light emitted toward the detector 8 from the pupil relay optical system of the two-photon excitation microscope 10 which is the scanning observation apparatus according to the present embodiment.

  In the two-photon excitation microscope 10 according to the present embodiment, the detection element 8a is arranged so that the lens 17b is included instead of the lens 7b constituting the pupil relay optical system, and the light receiving surface 8b coincides with the pupil conjugate plane CP. This is different from the two-photon excitation microscope 1 illustrated in FIG. The detection element 8a is arranged so that the light receiving surface 8b coincides with the pupil conjugate plane CP, which is the same as the two-photon excitation microscope 100 according to the related art.

  In the pupil relay optical system including the lens 17b illustrated in FIG. 5B and the lens 7a (not shown), the principal ray outside the most axis and the optical axis of the pupil relay optical system are the light receiving surface 8b and the pupil relay. It has pupil aberration that intersects with the optical system. Further, the pupil aberration is the largest spherical aberration off the most axis, and the spherical aberration becomes smaller as it approaches the optical axis.

  For this reason, in the two-photon excitation microscope 10 having a pupil relay optical system having pupil aberration, the position at which the principal ray intersects the optical axis differs for each principal ray, and the principal light of off-axis light that is further away from the optical axis. The light beam intersects the optical axis at a position away from the pupil conjugate position P0. Note that the pupil position and the pupil conjugate position are generally defined by paraxial rays.

  FIG. 6 is a diagram illustrating the state of fluorescence emitted from each of the pupil relay optical systems having different pupil aberrations. FIG. 7 is a spherical aberration diagram of each of the pupil relay optical systems illustrated in FIG. FIG. 8 is a diagram illustrating the relationship between the arrangement of the detection elements and the aperture efficiency when each of the pupil relay optical systems illustrated in FIG. 6 is used. The vertical axis in FIG. 8 indicates the aperture efficiency, which is the ratio of the fluorescence detected by the detection element 8a to the fluorescence emitted from the pupil relay optical system, and the horizontal axis in FIG. 8 indicates the pupil conjugate position P0 of the objective lens. The shift amount of the light-receiving surface 8b from is shown. The shift amount is defined so as to be positive when the light receiving surface 8b is further away from the pupil relay optical system than the pupil conjugate position P0.

The pupil relay optical system including the lens 18b illustrated in FIG. 6A has a pupil aberration (spherical aberration) of about 2.5 mm off the most axis as shown in FIG. 7A. FIG. 8A shows the aperture efficiency when the pupil relay optical system including the lens 18b is used. The pupil relay optical system including the lens 17b illustrated in FIG. 6B is a pupil relay optical system having a pupil aberration (spherical aberration) of about 5 mm off the most axis as shown in FIG. 7B. FIG. 8B shows the aperture efficiency when a pupil relay optical system including the lens 17b is used. The pupil relay optical system including the lens 19b illustrated in FIG. 6C is a pupil relay optical system having a pupil aberration (spherical aberration) of about 8 mm off the most axis as shown in FIG. 7C. FIG. 8C shows the aperture efficiency when a pupil relay optical system including the lens 19b is used.
Here, in deriving the aperture efficiency of FIG. 8, the aperture of the housing has the same shape as in FIG. 3, and the lens 18b in FIG. 6 (a), the lens 17b in FIG. 6 (b), and the lens in FIG. 6 (c). The ratio (R1: R2) of the curvature radius R1 of the incident side surface 19b and the curvature radius R2 of the exit side surface of 19b was calculated as 26.9: ∞, 50: 54.6, and 100: 35.6, respectively.

  As shown in FIG. 8B, when a pupil relay optical system including the lens 17b is used, vignetting of off-axis light by the housing 8c is performed with the light receiving surface 8b aligned with the pupil conjugate plane CP. Suppressing and realizing high aperture efficiency for off-axis light. On the other hand, as shown in FIGS. 8A and 8C, when the pupil relay optical system including the lens 18b and the lens 19b is used, the pupil aberration is insufficient and excessive, respectively. Therefore, in the state where the light receiving surface 8b coincides with the pupil conjugate plane CP, the vignetting of off-axis light represented by the principal ray L2 is particularly large.

  As described above, by using the pupil aberration of the pupil relay optical system, it is possible to obtain the same effect as the case where the pupil conjugate position CP is shifted with respect to the light receiving surface 8b. For this reason, by using pupil aberration within a certain range, vignetting in the housing 8c is suppressed and the aperture efficiency of off-axis light is improved even when the light-receiving surface 8b is aligned with the pupil conjugate position CP. Can be improved.

  According to the two-photon excitation microscope 10 according to the present embodiment configured as described above, the pupil conjugate position P0 of the objective lens 6 is disposed in the vicinity of the light receiving surface 8b (including a state in which it coincides with the light receiving surface 8b), and The pupil relay optical system has a pupil aberration such that the off-axis principal ray and the optical axis of the pupil relay optical system intersect between the light receiving surface 8b and the pupil relay optical system. Similar to the photon excitation microscope 1, vignetting of off-axis light by the housing 8c can be suppressed.

  Note that the fluctuation of 1 mm of pupil aberration of the pupil relay optical system acts similarly to the fluctuation of 1 mm of the shift amount of the light receiving surface 8b. For this reason, the optimal range of pupil aberration that can effectively suppress the occurrence of vignetting can be calculated using the equation (1), as in the first embodiment.

  In the first embodiment, the pupil conjugate position CP is shifted with respect to the light receiving surface 8b. In the second embodiment, the pupil relay optical system has pupil aberration, thereby suppressing vignetting in the housing 8c. However, these may be used in combination. For example, when vignetting can be most effectively suppressed with the light receiving surface 8b shifted by 5 mm with respect to the pupil conjugate position CP, for example, a pupil relay optical system having an off-axis pupil aberration of 2 mm is used. In addition, vignetting may be suppressed by shifting the light receiving surface 8b by 3 mm.

  FIG. 9 is a diagram illustrating the configuration of a two-photon excitation microscope according to the present embodiment. A two-photon excitation microscope 20 illustrated in FIG. 9 is a scanning observation apparatus including a scanner 22 that observes the specimen S by detecting fluorescence generated from the specimen S as observation light.

  The two-photon excitation microscope 20 includes a laser 21 that emits laser light for exciting the specimen S, a scanner 22 that is a scanning unit that scans the specimen S with the laser light emitted from the laser 21, and a pupil relay optical system 23. A lens 24 that irradiates the sample S with laser light, a lens 25 that is disposed facing the lens 24 across the sample S, a pupil relay optical system 26, and a barrier filter that blocks the laser light and transmits fluorescence. 27 and a detector 28 having a detection element 28a for detecting fluorescence.

  The scanner 22 is, for example, a galvano mirror, a polygon mirror, an acousto-optic deflection element, and the like, and is disposed on the illumination optical path between the sample S and the laser 21 and at the pupil conjugate position of the lens 25. The pupil relay optical system 23 includes a lens 23 a and a lens 23 b, and is configured such that the pupil of the lens 24 formed at a position optically conjugate with the pupil PP of the lens 25 is conjugate with the scanner 22.

  The lens 25 is a condensing lens that condenses the fluorescence generated in the sample S irradiated with the laser light. The pupil relay optical system 26 includes a lens 26a and a lens 26b, and is configured such that the pupil PP of the lens 25 is substantially conjugate with the light receiving surface 28b of the detection element 28a.

  The detector 28 includes a housing 28c that houses the detection element 28a in addition to the detection element 28a. An opening parallel to the optical axis through which the fluorescence incident on the detection element 28a passes is formed in the housing 28c provided for shielding an electric field and a magnetic field from the outside and cooling the detection element 28a.

  The detection element 28a is arranged at the bottom of the opening so that the normal line of the light receiving surface 28b of the detection element 28a and the optical axis of the pupil relay optical system 26 coincide. Further, in the detection element 28a, the principal ray off the most axis of fluorescence emitted from the pupil relay optical system 26 and the optical axis of the pupil relay optical system 26 intersect between the pupil relay optical system 26 and the light receiving surface 28b. To be arranged.

  More specifically, similarly to the first embodiment, the detection element 28a may be arranged so that the pupil conjugate position of the lens 25 is located between the light receiving surface 28b and the lens 26b. The pupil relay optical system 26 may have a pupil aberration such that the off-axis principal ray and the optical axis of the pupil relay optical system intersect between the light receiving surface 8b and the pupil relay optical system.

  The two-photon excitation microscope 20 according to the present embodiment configured as described above can also suppress vignetting of off-axis light by the housing 28c, similarly to the two-photon excitation microscope according to the first and second embodiments. it can.

  In the first to third embodiments, the two-photon excitation microscope is illustrated as the scanning observation apparatus. However, the scanning observation apparatus to which the present invention is applied is not limited to the two-photon excitation microscope. Any scanning observation apparatus that is required to efficiently detect off-axis light, for example, a microscope other than a fluorescence microscope such as a second harmonic (SHG) microscope may be used.

  In the case of a non-linear optical microscope using a non-linear optical phenomenon, light is generated only from one point, but is spread out due to scattering in the sample and emitted from the sample. For this reason, the nonlinear optical microscope which needs to take in these scattered light without waste is especially suitable as an application object of this invention.

1, 10, 20, 100... 2 photon excitation microscope 2, 21... Laser 3, 22, scanner 4, 23, pupil relay optical system 4 a, 4 b, 7 a, 7 b, 17 b, 18 b, 19b, 23a, 23b, 24, 25, 26a, 26b ... lens 5 ... dichroic mirror 6 ... objective lens 7, 26 ... pupil relay optical system 8, 28 ... detectors 8a, 28a ... detecting elements 8b, 28b ... light receiving surfaces 8c, 28c ... housing 27 ... barrier filter P0 ... pupil conjugate position PP ... pupil plane CP ... pupil conjugate planes L0, L1, L2 ... chief ray S ... specimen

Claims (6)

Scanning means for scanning the specimen with illumination light emitted from a light source;
A condensing lens that condenses the observation light generated in the specimen irradiated with the illumination light;
A detection element for detecting the observation light;
A pupil relay optical system that makes the pupil of the condenser lens substantially conjugate with the light receiving surface of the detection element;
In the detection element, the principal ray off the most axis of the observation light emitted from the pupil relay optical system and the optical axis of the pupil relay optical system intersect between the light receiving surface and the pupil relay optical system. A scanning observation apparatus, which is arranged as described above. The scanning observation apparatus according to claim 1,
The scanning type observation apparatus, wherein the detection element is arranged so that a pupil conjugate position of the condenser lens is located between the light receiving surface and the pupil relay optical system. The scanning observation apparatus according to claim 1,
The pupil relay optical system has pupil aberration such that the off-axis principal ray and the optical axis of the pupil relay optical system intersect between the light receiving surface and the pupil relay optical system.
A scanning observation apparatus characterized by that. The scanning observation apparatus according to claim 1,
The detection element is arranged such that a pupil conjugate position of the condenser lens is located between the light receiving surface and the pupil relay optical system,
The pupil relay optical system has pupil aberration such that the off-axis principal ray and the optical axis of the pupil relay optical system intersect between the light receiving surface and the pupil relay optical system.
A scanning observation apparatus characterized by that. The scanning observation apparatus according to any one of claims 2 to 4,
The scanning observation apparatus, wherein the scanning unit is arranged on an illumination optical path and at a pupil conjugate position of the condenser lens. The scanning observation apparatus according to any one of claims 1 to 5,
The scanning observation apparatus is a nonlinear optical microscope.