JP2000221407A - Illumination optical system for confocal laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the illumination optical system of a confocal laser scanning microscope constituted so that ring zonal luminous flux is always accurately projected on an objective pupil. SOLUTION: A ring zonal mask 18 changing circular luminous flux emitted from a laser beam source 12 to the ring zonal luminous flux is loaded on a precise XY stage 20. On the stage 20, the mask 18 is supported so that it can be continuously moved within a surface being vertical to the optical axis of the illumination luminous flux and the position thereof can be accurately and two-dimensionally adjusted. A control part 22 controlling the stage 20 is provided with a memory 24 storing the position of the mask 18 so that the mask 18 can be appearance arranged again at a proper position. Besides, the optimum position of the mask 18 with respect to the rotation amount of an image rotation prism 34 is previously stored in the memory 24 so that the mask 18 is moved to the optimum position by interlocking with the rotation of the prism 34 by the control part 22. Thus, the ring zonal luminous flux is always accurately and suitably projected on the objective pupil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an illumination optical system for a confocal laser scanning microscope, and more particularly to an illumination optical system using an annular luminous flux.

[0002]

2. Description of the Related Art Confocal laser scanning microscope (CLSM)
Can achieve high resolution as compared with a general optical microscope. The theory of CLSM is described by T. Wilson Ed.
Details are described in NFOCAL MICROSCOPY. CL
Since the point spread function (PSF) obtained by the SM is the product of the PSF of the illumination optical system (condenser lens) and the PSF of the detection optical system (objective lens), it is a basic principle that high resolution can be achieved. is there.

At present, most of the practically used CLSMs are epi-illuminated CLSMs. FIG. 2 schematically shows the configuration of the epi-illumination type CLSM. In FIG. 2, a laser beam emitted from a laser light source 12 passes through a beam expander 55 including two lenses 52 and 54, a half mirror 26, a galvanometer mirror 28, and a relay lens including two lenses 30 and 32. The sample 40 passes through the system 33 and is illuminated via the objective lens 38.

The return light from the sample 40 is transmitted to the objective lens 3
8. After passing through the relay lens system 33, the galvanometer mirror 28, and the half mirror 26, the light condensed by the confocal lens 42 and passed through the pinhole 44 is photoelectrically converted by the photodetector 46 and detected.

Although one-dimensional scanning means is shown in FIG. 2 for simplicity, two-dimensional scanning means may be provided by providing two galvanometer mirrors.

In an epi-illumination type CLSM represented by FIG.
The objective lens 38 also serves as a condenser lens. Therefore, the obtained PSF is the square of the PSF of the objective lens. FIG. 3 shows the PSF of the objective lens and its square. FIG.
It can be seen from the above that the diameter of the first dark ring does not change but the half-value width narrows due to the square effect. Further, the primary maximum is further reduced by the square effect, and both resolution and contrast can be improved as compared with a general optical microscope.

The CLSM is an excellent microscope as described above, but by using a ring-shaped light beam as an illumination light beam,
Techniques for increasing the depth of focus and further improving the resolution are disclosed in, for example, JP-A-8-334701 and JP-A-7-6350.
No. 82 and JP-A-8-15156.

In an orbicular illumination optical system using an orbicular luminous flux as an illuminating luminous flux, a diffractive optical element, a pair of axicon prisms and a ring filter are used as orbicular luminous flux forming means for converting a circular luminous flux into an orbicular luminous flux. ing.

FIG. 4 schematically shows a CLSM having an annular illumination optical system. In the optical system shown in FIG.
A pair of axicon prisms 62 and 64 and a pair of lenses 66 and 68 are provided between the half mirror 2 and the half mirror 26.
The pair of axicon prisms 62 and 64 are
A light beam forming means for converting the circular light beam from No. 2 into an annular light beam is provided. The pair of lenses 66 and 68 constitute a beam reducer for adjusting the diameter of the zonal light beam.
Other elements are the same as those of the optical system of FIG.

FIG. 5 shows the PSF of the objective lens 38 for each of the ordinary circular light beam and the annular light beam. In the figure, the broken line indicates the PSF of the objective lens 38 using a circular light beam, and the solid line indicates the PSF of the objective lens 38 using a ring light beam. From FIG. 5, it can be seen that the width of the PSF is narrower in the annular illumination, so that the resolution in the lateral direction is improved. Although not shown, it is known that the depth of focus also increases.

[0011]

As described above, in annular illumination using annular luminous flux, it is possible to improve the resolution in the horizontal direction and increase the depth of focus. However, in order to obtain this effect, it is necessary to accurately project the annular light flux onto the objective pupil. For this reason, the annular light beam forming means must be positioned with high accuracy with respect to the optical axis.

In line width measurement and the like, an image rotating mechanism using an image rotating prism or the like is used in order to increase the measurement accuracy. For a sample having directionality, such as a line width measurement, the resolution differs depending on which direction the main scanning direction is set. The image rotation mechanism has a function of rotating the main scanning direction, and the resolution is improved by rotating the main scanning direction by the image rotation mechanism so as to match the direction.

However, in general, the image rotation mechanism gives a small amount of deviation between the illumination light beam and the objective pupil due to an error of the image rotation prism and the eccentricity of the rotation mechanism. This shift amount is larger than the positional accuracy required for obtaining the above-described annular illumination effect, and there is a problem that the annular illumination effect cannot be sufficiently obtained.

The present invention has been made to solve such a problem, and an object of the present invention is to provide an illumination optical system in which an annular luminous flux is always accurately projected on an objective pupil.

[0015]

An illumination optical system of a confocal laser scanning microscope according to the present invention comprises a light source section for emitting a circular light beam as an illumination light beam, and an annular light beam forming means for converting the circular light beam into an annular light beam. And a position adjusting means for supporting the annular light flux forming means continuously movably in a direction transverse to the optical axis of the illuminating light flux and capable of adjusting the position two-dimensionally.

In one example, the illumination optical system further has a control unit for controlling the position adjusting means in conjunction with an image rotation mechanism of the confocal laser scanning microscope for rotating the main scanning direction. The image rotation mechanism includes an image rotation prism, and the control unit includes a memory that stores in advance an optimal position of the annular light beam forming unit with respect to the rotation amount of the image rotation prism,
The controller moves the orbicular zone light beam forming means to an optimum position in conjunction with the rotation of the image rotation prism based on the information stored in the memory.

[0017]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An illumination optical system of a confocal laser scanning microscope according to an embodiment of the present invention will be described with reference to FIG.

The laser beam emitted from the laser light source 12 passes through a beam expander 17 composed of two lenses 14 and 16, so that the beam diameter is enlarged.
On the optical path of the expanded light beam, an annular mask 18 constituting the annular light beam forming means is appropriately arranged. Zonal mask 18
Selectively transmits a circular light beam incident from the laser light source 12 to convert it into a ring-shaped light beam.

The annular mask 18 is mounted on a precision XY stage 20, whereby the annular mask 18 is supported so as to be continuously movable in a plane perpendicular to the optical axis of the illuminating light flux, and is two-dimensional with high precision. Can be adjusted. The precision XY stage 20 is controlled by the control unit 22. The control unit 22 includes a memory 24 for storing the position of the annular mask 18, and can be arranged at an appropriate position with good reproducibility before and after the annular mask 18 is removed.

By removing the orbicular mask 18 from the optical path by the precision XY stage 20, normal illumination with a circular luminous flux is performed. By disposing the orbicular mask 18 on the optical path,
The annular illumination is performed by the annular luminous flux.

The illumination light beam is supplied to a half mirror 26, a galvanometer mirror 28, a relay lens system 33 composed of two lenses 30 and 32, an image rotation prism 34, and an objective lens 38.
Irradiates the specimen 40 via the light source to form a spot.

The galvanomirror 28 is capable of swinging within a predetermined angle range around an axis whose reflection surface is perpendicular to the plane of the paper, and the swing scans a spot formed on the specimen 40.

In the optical system of FIG. 1, the scanning means is constituted by only one galvanometer mirror 28, but may be constituted by two galvanometer mirrors so as to realize two-dimensional scanning. In other words, two galvanometer mirrors may be preferably arranged between the half mirror 26 and the relay lens system 33 so that their axes are orthogonal to each other, so that two-dimensional scanning can be realized.

The image rotation prism 34 is rotatable around the optical axis, for example, in an angle range of 180 °. The rotation of the image rotation prism 34 rotates the main scanning direction. The main scanning direction is determined according to the sample 40. For example, in the line width measurement, the direction is preferably selected along the line width to be measured. Therefore, by adjusting the angle direction of the image rotation prism 34, the main scanning direction can be appropriately adjusted according to the specimen 40.

The error of the image rotation prism 34 and the eccentricity of the rotation mechanism change the angle and position of the illumination light beam, causing a shift between the illumination light beam and the objective pupil. This shift is
In general, even if the amount is not a problem in normal illumination, the amount is not negligible in annular illumination. That is, in annular illumination, the illumination light flux, that is, the annular light flux, needs to be projected onto the objective pupil with higher accuracy.

In the present embodiment, the displacement between the annular luminous flux and the objective pupil is determined by using the precision XY stage 20 and the annular mask 18.
Adjust the position and remove it. That is, the memory 24
Is the annular mask 18 with respect to the rotation amount of the image rotation prism 34.
The optimal position is stored in advance, and the control unit 22 moves the annular mask 18 to the optimal position in conjunction with the rotation of the image rotation prism 34. Thus, the annular luminous flux is always preferably projected onto the objective pupil with high accuracy.

The return light from the sample 40 is transmitted to the objective lens 3
8, via the image rotation prism 34, the relay lens system 33, and the galvanomirror 28, toward the half mirror 26. Part of the returning light passes through the half mirror 26 and is conveyed to the confocal lens 4.
2 collects light.

Behind the confocal lens 42, a pin pole 4
4 are provided. The opening of the pinhole 44 has a conjugate positional relationship with the spot formed on the specimen 40 by the illuminating light flux, and selectively allows only return light from the spot to pass and blocks other light. Thereby, only the return light from the spot can pass through the pinhole 44 and enter the photodetector 46 correctly.

In the illumination optical system of the confocal laser scanning microscope according to the present embodiment, by switching the normal illumination and the annular illumination by inserting and removing the annular mask 18 with respect to the optical path using the precision XY stage 20. I can do it. In addition, precision X
By adjusting the position of the annular mask 18 using the Y stage 20, the relative displacement between the annular luminous flux and the objective pupil caused by the image rotating prism 34 is corrected in real time, and thus, is always corrected. Optimal annular illumination can be obtained.

The present invention is not limited to the above-described embodiment, but includes all embodiments carried out without departing from the gist thereof.

For example, in the above-described embodiment, only the annular mask 18 is supported by the XY stage 20.
The beam expander 17 or the laser light source 12 may be supported by the XY stage 20 together with the annular mask 18. In addition, the beam expander 17 and the laser light source 1
2 may be supported by the XY stage 20.

In this configuration, the load on the XY stage 20 increases, and there is a demerit in the control aspect.
8 has the advantage that there is no relative movement between the illumination light beam from the beam expander 17 and the orbicular mask 18 and a stable orbicular light beam is formed.

Further, in the above-described embodiment, the orbicular light beam forming means is constituted by an orbicular mask which partially blocks the incident light beam, but any other known technique can be applied. It is. For example, as mentioned in the description of the related art, it may be configured by a diffractive optical element or a pair of axicon prisms.

That is, instead of the annular mask 18, a diffractive optical element or a pair of axicon prisms
0 and may be arranged at a desired position on the microscope apparatus. Furthermore, precision XY stage 2
0 is driven to insert and remove the diffractive optical element or a pair of axicon prisms supported by the precision XY stage 20 with respect to the optical path of the laser light emitted from the laser light source 12, thereby providing normal illumination and annular illumination. Can be switched.

Further, by adjusting the position of the diffractive optical element or the pair of axicon prisms supported by the precision XY stage 20 using the precision XY stage 20, the annular luminous flux caused by the image rotation prism 34 and the objective light are adjusted. The relative displacement between the pupils is corrected in real time, so that the optimal annular illumination is always obtained.

Further, not only the diffractive optical element or a pair of axicon prisms is supported by the precision XY stage 20 but also the beam expander 17 in addition to these components.
(Or a beam reducer comprising a pair of lenses 66 and 68 in FIG. 4) or the laser light source 12 is
It may be supported by the Y stage 20. The beam expander 17 (or a beam reducer including a pair of lenses 66 and 68 in FIG. 4) and the laser light source 12 may be supported by the XY stage 20.

Although this configuration has a disadvantage in the control plane for performing the alignment with respect to the objective pupil due to an increase in the load on the XY stage 20, the configuration is different from that of the above-described embodiment when the annular mask 18 is moved. Then, since the relative movement between the illumination light flux from the beam expander 17 and the annular mask 18 is reduced or eliminated, more stable annular illumination is formed, whereby the optimal annular illumination is obtained. Has advantages.

[0038]

According to the present invention, there is provided an illumination optical system of a confocal laser scanning microscope in which an annular luminous flux is always accurately projected on an objective pupil.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a confocal laser scanning microscope provided with an illumination optical system according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of the most common epi-illumination type confocal laser scanning microscope.

3 is a graph of a point spread function (PSF) function of the objective lens of FIG. 2 and its square.

FIG. 4 is a schematic diagram of a confocal laser scanning microscope provided with an annular illumination optical system.

FIG. 5 is a graph of a point spread function (PSF) function of an objective lens for a normal circular light beam and an annular light beam.

[Explanation of symbols]

 Reference Signs List 12 laser light source 18 annular mask 20 XY stage 22 control unit 24 memory

Claims (3)

[Claims] 1. An illumination optical system of a confocal laser scanning microscope, comprising: a light source for emitting a circular light beam as an illumination light beam; an annular light beam forming means for converting the circular light beam into an annular light beam; An illumination optical system for a confocal laser scanning microscope, comprising: a position adjusting means for supporting the means so as to be continuously movable in a direction transverse to the optical axis of the illumination light beam and enabling two-dimensional position adjustment. 2. The confocal laser scanning microscope has an image rotating mechanism for rotating its main scanning direction, and the illumination optical system further has a control unit for controlling position adjusting means in conjunction with the image rotating mechanism. The illumination optical system of the confocal laser scanning microscope according to claim 1, wherein: 3. The image rotation mechanism includes an image rotation prism, and the control unit includes a memory that stores an optimum position of the orbicular zone light beam forming means with respect to the amount of rotation of the image rotation prism in advance. 3. The illumination optical system of a confocal laser scanning microscope according to claim 2, wherein the annular light beam forming means is moved to an optimum position in conjunction with the rotation of the image rotation prism based on the stored information.