JP2016045427A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable observation of a large specimen with high resolution without increasing the size of a scanner and relay lens.SOLUTION: A microscope device 1 includes: a scanner 2 having a galvano-mirror 2a for scanning laser light L from a light source; a relay optical system 3 for relaying the laser light L scanned by the scanner 2; an objective lens 4 that focuses the laser light L relayed by the relay optical system 3 onto a specimen A; and a diffractive optical element 8 that is disposed in a light path between the objective lens 4 and the scanner 2 to enlarge a beam diameter of the laser light L entering the objective lens 4 by means of diffraction.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microscope apparatus.

  Conventionally, there is known a microscope apparatus that observes fluorescence generated by a multiphoton excitation effect by scanning pulse laser light from a light source with a scanner, relaying it with a relay lens, and condensing it on a specimen with an objective lens ( For example, see Patent Document 1.)

  In the microscope apparatus of Patent Document 1, in order to observe a large specimen with high resolution, a low-magnification objective lens with a wide real field of view is mounted, and the numerical aperture (illumination NA) of laser light at the focal position of the objective lens Need to be increased.

JP 2010-85826 A

However, in a microscope apparatus equipped with a scanner using a galvanometer mirror, it is difficult to design the illumination field and the illumination NA independently, and the illumination field is relayed from the galvanometer mirror by the relay lens to the pupil position of the objective lens. The illumination NA is in proportion to the inverse of the magnification. Therefore, if priority is given to ensuring the illumination field, the beam diameter cannot be expanded to satisfy the pupil diameter of the objective lens, the illumination NA cannot be increased sufficiently, and the resolution is limited.
On the other hand, if priority is given to a sufficient increase in the illumination NA, there is a disadvantage that the illuminating field cannot be secured sufficiently because the swing angle of the galvano mirror is insufficient.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a microscope apparatus capable of observing a large specimen with high resolution without increasing the size of a scanner or a relay lens.

In order to achieve the above goal, the present invention provides the following means.
One embodiment of the present invention includes a scanner having a galvanometer mirror that scans laser light from a light source, a relay optical system that relays laser light scanned by the scanner, and a sample of laser light relayed by the relay optical system And a diffractive optical element that is arranged on an optical path between the objective lens and the scanner and that expands a beam diameter of laser light incident on the objective lens by diffraction. .

  According to this aspect, after the laser light from the light source is scanned by the galvanometer mirror of the scanner, it is relayed by the relay optical system and condensed on the sample by the objective lens, thereby generating fluorescence in the sample, and the generated fluorescence Is collected and detected by the objective lens, so that the fluorescence observation of the specimen can be performed. In this case, since the beam diameter of the laser beam can be enlarged by diffraction by the diffractive optical element on the optical path between the scanner and the objective lens, the pupil of the objective lens is filled without adjusting the relay magnification of the relay optical system. It can be made incident on the objective lens with a light beam diameter.

  That is, it is possible to improve the spatial resolution while securing the illumination field by setting the relay magnification that can secure the illumination field and enlarging the beam diameter by the diffractive optical element regardless of the relay magnification. Thereby, a large sample can be observed with high resolution. Further, since the relay magnification is not adjusted, it is not necessary to change the scanner, the relay optical system, and the objective lens, and the enlargement of the scanner and the relay optical system can be prevented.

In the above aspect, the relay optical system may include a pair of lenses arranged at intervals in the optical axis direction, and the diffractive optical element may be arranged at a front focal position of the lens on the objective lens side. Good.
By doing in this way, the diffusion angle of the laser beam that has focused and formed a spot at the front focal position of the objective lens side of the pair of lenses constituting the relay optical system is enlarged by the diffractive optical element, The diameter of the substantially parallel light beam output from the relay optical system can be easily increased.

Moreover, in the said aspect, the said some objective lens from which magnification differs may be provided interchangeably, and the said diffractive optical element may be provided in the said optical path so that insertion or removal is possible.
In this way, for an objective lens having a small pupil diameter, vignetting is prevented by removing the diffractive optical element from the optical path and reducing the beam diameter, and for an objective lens having a large pupil diameter. Can increase the resolution by inserting a diffractive optical element on the optical path to increase the beam diameter. Note that, instead of inserting / removing a single diffractive optical element, a plurality of diffractive optical elements having different diffraction angles can be inserted / removed to make finer adjustment in accordance with the pupil diameter of the objective lens.

In the above aspect, the diffractive optical element includes a plurality of diffractive optical elements corresponding to the wavelength of the laser light from the light source, and includes an element selection mechanism that selectively arranges these diffractive optical elements on the optical path. May be.
By doing in this way, even if the wavelength of the laser beam is changed by arranging a diffractive optical element having an appropriate diffraction angle on the optical path by an element selection mechanism in accordance with the wavelength of the laser beam from the light source, It is possible to perform high-resolution observation using laser beams having different wavelengths by setting the light beam diameter so as to fill the pupil of the objective lens without excess or deficiency.

  According to the present invention, there is an effect that a large sample can be observed with high resolution without increasing the size of a scanner or a relay lens.

1 is an overall configuration diagram showing a microscope apparatus according to an embodiment of the present invention. FIG. 2 is an overall configuration diagram illustrating a state in which laser light is scanned by an operation of a scanner in the microscope apparatus of FIG. 1. It is a figure which shows (a) the direction of diffraction by the diffractive optical element of the microscope apparatus of FIG. 1, and (b) the diffraction pattern, respectively. In the microscope apparatus of FIG. 1, (a) the diffractive optical element is removed from the optical path, (b) the cross section of the laser beam incident on the pupil of the objective lens at that time, and (c) the diffractive optical element on the optical path (D) is a diagram showing a cross section of a laser beam incident on the pupil of the objective lens at that time. (A) The intensity distribution of the laser beam when the beam diameter of the laser beam incident on the relay optical system is enlarged, and (b) the intensity distribution of the laser beam when the diffractive optical element is inserted in the microscope apparatus of FIG. FIG. FIG. 8 is a diagram illustrating a modification of the microscope apparatus of FIG. 1 and illustrating a turret in which a plurality of diffractive optical elements are alternatively arranged on an optical path. It is a figure which shows the example (a) of diffraction pattern, (b), (c).

A microscope apparatus 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 1 and 2, a microscope apparatus 1 according to the present embodiment includes a scanner 2 that scans an extremely short pulse laser beam (hereinafter simply referred to as a laser beam) L emitted from a light source (not shown), A relay optical system 3 that relays the laser light L scanned by the scanner 2 and an objective lens 4 that irradiates the sample A with the laser light L relayed by the relay optical system 3 and collects the fluorescence generated in the sample A. A dichroic mirror 5 that branches the fluorescence F collected by the objective lens 4 from the optical path of the laser light L, a condenser lens 6 that collects the fluorescence F branched by the dichroic mirror 5, and the light collection And a photodetector 7 for detecting the fluorescence F collected by the lens 6. Further, the microscope apparatus 1 according to the present embodiment includes a diffractive optical element (DOE) 8 that expands the beam diameter of the laser light L by diffraction.

  The scanner 2 includes, for example, two galvanometer mirrors 2a (only one is shown in the figure) that oscillates about non-parallel axes. The laser beam L is used as an optical axis by combining the oscillating angles of the galvanometer mirrors 2a. It is possible to scan in the intersecting two-dimensional direction.

  In the example shown in FIG. 1, the relay optical system 3 includes a pair of first lens 3 a and second lens 3 b that are spaced apart in the optical axis direction. The front focal position of the first lens 3a on the side close to the scanner 2 coincides with the rear focal position of the second lens 3b on the side far from the scanner 2, and the laser beam L incident in the form of substantially parallel light. Is once imaged and then emitted in the form of substantially parallel light.

  The dichroic mirror 5 is arranged close to the rear side of the objective lens 4 and at an angle of 45 ° with respect to the optical axis of the objective lens 4. The dichroic mirror 5 has a transmittance characteristic of transmitting the laser light L and reflecting the fluorescence F. As a result, the laser light L from the light source passes through the dichroic mirror 5 and enters the objective lens 4, while the fluorescence F collected by the objective lens 4 is deflected by 90 ° by the dichroic mirror and is reflected by the condenser lens 6. The light is condensed and detected by the photodetector 7.

  The photodetector 7 is, for example, a photomultiplier tube (PMT). By storing each scanning position of the laser light L scanned by the scanner 2 and the intensity of the fluorescence F generated by irradiating the scanning position with the laser light L, the two-dimensional fluorescent image is stored. Can be obtained.

The diffractive optical element 8 is detachably disposed on the optical path in the intermediate imaging position of the relay optical system 3, that is, in the vicinity of the rear focal position of the first lens 3a and in the vicinity of the front focal position of the second lens 3b. When inserted on the road, the diffusion angle of the laser light L diffusing from the front focal position toward the second lens 3b is expanded by diffraction.
Specifically, as shown in FIG. 3, the diffractive optical element 8 diffracts the transmitted laser light L, thereby, for example, an angle of NA 0.045 and an angle of NA 0.09 with respect to the optical axis. And diffracted in twelve directions that are equally spaced in the circumferential direction.

The operation of the microscope apparatus 1 according to the present embodiment configured as described above will be described below.
In order to perform fluorescence observation of the specimen A using the microscope apparatus 1 according to this embodiment, the laser light L from the light source is scanned two-dimensionally by the scanner 2 as shown in FIG. The laser beam L transmitted through the dichroic mirror 5 is condensed on the specimen A by the objective lens 4. As a result, the fluorescent substance existing in the specimen A at the condensing position of the laser light L is excited by the multiphoton excitation effect, and fluorescence F is generated.

  The generated fluorescence F is collected by the objective lens 4, reflected by the dichroic mirror 5, collected by the condenser lens 6, and detected by the photodetector 7. By storing the scanning position of the laser beam L by the scanner 2 and the intensity of the fluorescence F detected by the photodetector 7, a fluorescence image can be acquired.

  In this case, according to the microscope apparatus 1 according to the present embodiment, as shown in FIG. 4A, when the diffractive optical element 8 is removed from the optical path, the first lens of the relay optical system 3 is used. For example, the laser light L condensed at an angle of NA 0.03 and formed as an intermediate image is diffused by the same NA 0.03 by 3a and is converted into substantially parallel light by the second lens 3b.

  If the focal length of the first lens 3a is 50 mm and the focal length of the second lens 3b is 150 mm, the relay magnification is 3, and the luminous flux diameter of the substantially parallel light emitted from the second lens 3b is shown in FIG. As shown, for example, φ9. When the pupil diameter of the objective lens 4 is φ36, the pupil fullness ratio, which is the ratio of the laser beam L to the pupil, is 6.25%.

On the other hand, as shown in FIG. 4C, in a state where the diffractive optical element 8 is inserted on the optical path, it is condensed at an angle of NA 0.03 by the first lens 3a of the relay optical system 3 and is an intermediate image. 3 is diffracted by the diffractive optical element 8 in the angular directions of NA 0.045 and NA 0.09, as shown in FIG. 3, so that it is substantially parallel focused by the second lens 3b. The light beam diameter is, for example, φ36.
As shown in FIG. 4D, this substantially parallel light beam is a set of a plurality of light beams diffracted in a plurality of directions by the diffractive optical element 8, and the pupil filling rate is greater than 90%.

  In other words, according to the microscope apparatus 1 according to the present embodiment, the diffractive optical element 8 is simply inserted into the back focal position of the second lens 3b, and enters the objective lens 4 without changing other optical systems. The beam diameter can be expanded so that the laser beam L fills the pupil. Thereby, the NA of the laser light L irradiated to the specimen A from the objective lens 4 can be sufficiently increased, and the range in the optical axis direction in which the photon density of the laser light L condensed near the focal position can be increased. The spatial resolution in the optical axis direction can be improved by shortening.

  In particular, according to the microscope apparatus 1 according to the present embodiment, the diffractive optical element 8 is maintained regardless of the relay magnification while maintaining the relay magnification capable of ensuring a sufficiently large illumination field in relation to the galvanometer mirror 2a of the scanner 2. The spatial resolution can be improved while securing the illumination field by enlarging the beam diameter by. Thereby, the large specimen A can be observed with high resolution. Further, since the relay magnification is not adjusted, there is an advantage that the scanner 2, the relay optical system 3 and the objective lens 4 need not be changed, and the enlargement of the scanner 2 and the relay optical system 3 can be prevented.

  Further, when the enlargement of the galvanometer mirror 2a is allowed, a method of expanding the beam diameter of the laser light L incident on the objective lens 4 without changing the relay optical system 3 and without using the diffractive optical element 8. It is conceivable to increase the beam diameter of the laser beam L to be scanned. However, when such a method is adopted, as shown in FIG. 5A, the light beam of the laser light L incident on the objective lens 4 has a normal Gaussian distribution intensity distribution. Therefore, the high NA component contributing to resolution and brightness, that is, the intensity of the outer peripheral portion of the light beam is relatively low, for example, about 10% of the intensity at the center of the light beam.

  On the other hand, according to the microscope apparatus 1 according to the present embodiment, the light beam whose light beam diameter is expanded by the diffractive optical element 8 is a laser beam diffracted in a plurality of directions as shown in FIG. Since each L has an intensity distribution of Gaussian distribution, the relative intensity of the high NA component can be dramatically improved. For example, the intensity at the center of the luminous flux can be increased in the vicinity of the outer peripheral portion of the luminous flux. It can be made to have equivalent strength. As a result, there is an advantage that the illumination intensity and the spatial resolution can be further improved.

  In the present embodiment, since the diffractive optical element 8 is detachably provided on the optical path, the diffractive optical element 8 is used as a light beam when the observation is performed with the objective lens 4 having a small pupil diameter replaced. The light beam diameter of the laser light L that is extracted from the road and enters the objective lens 4 can be reduced to match the pupil diameter.

  In the microscope apparatus according to the present embodiment, the focal lengths of the lenses 3a, 3b, and 4 and the beam diameter of the laser light L are exemplified. However, the present invention is not limited to this, and arbitrary lenses 3a and 3b , 4 and a laser beam L having an arbitrary light beam diameter can be used.

In the present embodiment, the single diffractive optical element 8 is detachably provided. Instead, as shown in FIG. 6, a plurality of diffractive optical elements 8 and the diffractive optical elements 8 are provided. Alternatively, a turret (element selection mechanism) 9 that can be arranged on the optical path may be provided.
As described above, the laser light L having a light beam diameter that satisfies the pupils of the objective lenses 4 may be incident on the objective lenses 4 having different pupil diameters. Considering that the diffraction angle changes when the wavelength of the laser beam L changes, a plurality of diffractive optical elements 8 corresponding to the wavelength of the laser beam L to be used are prepared, and the wavelength of the laser beam L changes. Each time, the turret 9 may be rotated to switch the diffractive optical element 8 inserted on the optical path, and the beam diameter of the laser light L may be adjusted so that the pupil of the objective lens 4 is always filled.

  Further, as the diffractive optical element 8, a diffraction pattern in which the laser beam L is divided and diffracted in 12 directions at equal intervals in the circumferential direction in two angular directions of NA 0.045 and NA 0.09 is exemplified. Any angle direction and number, or the number of divisions of the luminous flux in the circumferential direction can be adopted.

Further, as shown in FIG. 7, as the diffraction pattern, (a) in addition to the pattern of the above embodiment, (b) a pattern that is diffracted into a ring shape continuous in the circumferential direction, and (c) a square arrangement. You may employ | adopt arbitrary patterns, such as what makes a light beam diffract.
Further, the diffractive optical element 8 is arranged in the vicinity of the front focal position of the second lens 3b. However, in addition to the case where the diffractive optical element 8 is accurately arranged at the front focal position, the diffractive optical element 8 is arranged within the focal depth range of the second lens 3b. The case where it is done is also included. By doing so, it is possible to prevent diverging light and condensed light from entering the objective lens, and to reduce the occurrence of focus shift and aberration.

A Specimen L Laser light 1 Microscope device 2 Scanner 2a Galvano mirror 3 Relay optical system 3a First lens (lens)
3b Second lens (lens)
4 Objective lens 8 Diffractive optical element 9 Turret (element selection mechanism)

Claims (4)

A scanner having a galvanometer mirror that scans laser light from a light source;
A relay optical system that relays laser light scanned by the scanner;
An objective lens for condensing the laser beam relayed by the relay optical system on a specimen;
A microscope apparatus, comprising: a diffractive optical element that is disposed on an optical path between the objective lens and the scanner and that enlarges a beam diameter of laser light incident on the objective lens by diffraction. The relay optical system includes a pair of lenses arranged at intervals in the optical axis direction,
The microscope apparatus according to claim 1, wherein the diffractive optical element is disposed in the vicinity of a front focal position of the lens on the objective lens side. A plurality of objective lenses having different magnifications are provided to be exchangeable,
The microscope apparatus according to claim 1, wherein the diffractive optical element is provided to be detachable from the optical path. A plurality of the diffractive optical elements are provided corresponding to the wavelength of the laser light from the light source,
The microscope apparatus according to any one of claims 1 to 3, further comprising an element selection mechanism that selectively arranges these diffractive optical elements on an optical path.

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