JP4461250B2 - Scanning microscope capable of acquiring confocal image and evanescence illumination image information - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention includes a confocal optical system that gives an image obtained by optically cutting a specimen, and an evanescence microscope optical system that uses illumination by evanescence light formed at a glass-water (or air) interface. The present invention relates to a scanning microscope that can simultaneously acquire both the image information and the evanescence illumination image information.
[0002]
[Prior art]
A confocal microscope obtains a microscopic image obtained by cutting along a plane perpendicular to a single point on the z-axis of a target specimen, and is used in fields such as medical biology and semiconductor engineering.
The confocal microscope is realized by using a Nipkow disk with a lens in which a plate having an illumination lens and a pinhole plate that transmits a signal from an image are combined.
[0003]
On the other hand, the inventor illuminates the specimen with extremely thin light using evanescence light generated in the total reflection region of the interface according to the invention of “Ultra-thin illumination light generator for microscope and observation method using the device” described below. Alternatively, an ultra-thin illumination light generator for a microscope that obtains information generated as a result of excitation is proposed. The evanescence illumination method can generate ultra-thin illumination light that can be cut thinner than 1 μm.
[Patent Document 1]
Japanese Patent Application No. 2003-186769 By using this apparatus, it is possible to acquire three-dimensional information such as cells from the tomographic information of a cell as a specimen and information of two or more tomographic cells.
[0004]
[Problems to be solved by the invention]
When the confocal scanning microscope and the microscope apparatus using the evanescence light are combined, information on cells and the like can be acquired simultaneously, but such an apparatus has not been proposed.
The objective of this invention is providing the scanning microscope which can acquire a confocal image and evanescence illumination image information.
[0005]
[Means for Solving the Problems]
In order to achieve the object, a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to claim 1 according to the present invention,
A transparent plate having a first surface and a second surface and supporting the specimen on the first surface;
A confocal scanning microscope optical system disposed on the first surface side;
The evanescent illumination light is formed by making the second surface incident on the second surface at an angle larger than the total reflection angle so that the thickness of the light component that passes from the second surface to the first surface becomes a unit of micron or less. and evanescence illumination light source for supplying illumination light for cutting specimens by the evanescence illumination light,
A scanning microscope optical system for an evanescence illumination image disposed on the second surface side ,
Illuminate a specific surface of the specimen with illumination from the confocal scanning microscope optical system, receive a signal light generated by the illumination, and image the specimen in units of microns or less with the evanescence illumination light. It is configured to simultaneously observe an evanescent illumination image that is imaged by receiving signal light from that portion after being cut by light of thickness, and from the illumination point by the evanescent illumination light and the confocal scanning microscope optical system The illumination points are configured so that they do not overlap on the specimen.
[0006]
The scanning microscope capable of acquiring the confocal image and the evanescence illumination image information according to claim 2 according to the present invention is the scanning microscope according to claim 1, wherein the confocal scanning microscope optical system is a lens-equipped disk, Nipkou disk assembly consisting of corresponding pinhole disks, dichroic mirrors arranged between the disks, confocal scanning microscope optical system illuminating a specific surface of the specimen, and signals generated by the illumination The light is reflected by the pinhole disk and the dichroic mirror and imaged.
[0007]
A scanning microscope capable of acquiring confocal image and evanescence illumination image information according to claim 3 according to the present invention is the scanning microscope according to claim 1,
Scanning microscope optics for evanescence illumination images
The signal light generated by the evanescence illumination is imaged through a Nipkow disk having a pinhole corresponding to the signal light generation point through the objective lens and the converging lens.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of an apparatus according to the present invention will be described below with reference to the drawings.
FIG. 1 is a view showing a first embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention, and FIG. 2 is an enlarged view showing an objective lens and a sample portion of the microscope. .
[0009]
In the first embodiment, a confocal optical system is formed by a Nipkou disk assembly 2 including a lens disk 2 a with a microlens and a pinhole disk 2 b and an objective lens 5. At the same time, a laser beam scanning system is formed using the other area of the Nipkou disk assembly 2 and irradiated from the opposite side of the specimen to the specimen 10 through the periphery of another objective lens 11. This creates evanescence illumination light. The light emitted from the fluorescent material placed in the evanescence field is collected by the objective lens 11.
By shifting the focal position of the fluorescence image of the evanescence illumination light from that of the confocal system, the two optical systems can be operated independently (without being influenced by other optical systems). It is.
[0010]
A first embodiment according to the above principle will be further described with reference to FIGS. As shown in each figure, the specimen 10 is arranged on the upper side which is the first surface of the transparent plate 9 made of a cover glass or the like. A confocal scanning microscope optical system is disposed on the first surface side.
[0011]
The confocal scanning microscope optical system includes a Nipkou disk assembly 2. The Nipkou disk assembly 2 includes a lens disk 2a and a pinhole disk 2b having a pinhole corresponding to each lens, and a dichroic mirror 6 is provided between the disks.
The light from the window 1a of the light source 1 that has passed through the pinhole illuminates a specific surface (plane P _{1 in} FIG. 2) of the specimen through the objective lens 5 supported by the lens barrel 4 of the confocal scanning microscope optical system. To do.
The signal light (excited fluorescence, scattered light, etc.) generated by the illumination is reflected by the pinhole of the pinhole disk 2b and the dichroic mirror 6 and imaged by a camera comprising a lens 7 and an image sensor.
[0012]
The evanescence illumination light source supplies illumination light for cutting the specimen with evanescence illumination light from the second surface side of the transparent plate 9. The scanning microscope optical system for the evanescence illumination image is disposed on the second surface side. The light beam indicated by L ₁ in FIG. 2 is light for forming the evanescence illumination light L ₂ and is supplied via the objective lens 11 of the scanning microscope optical system for the evanescence illumination image.
[0013]
The evanescence illumination light beam is formed by the light from the window 1b of the light source. The light transmitted through the micro lens of the lens disk 2a of the Nipkou disk assembly 2 by the light from the window 1b of the light source passes through the corresponding pinhole of the pinhole disk 2b and is collimated by the lens 3. The collimated light is bent by the prism 13, reflected by the dichroic mirror 14 disposed in the optical axis of the scanning microscope optical system for the evanescence illumination image, and the transparent plate 9 by the objective lens 11 supported by the lens barrel 12. Is incident on.
[0014]
Although it is described in detail in the above-mentioned Patent Document 1 of the present applicant, an interface between a water phase and a transparent plate 9 such as a cover glass is assumed as shown in FIG. Consider the case where it exists. When the incident angle of the incident light L _{1 at} the interface is set to be slightly larger than the critical angle, total reflection occurs , and a light component that escapes to the water side is generated. This missing light L ₂ is nearly parallel to the interface and becomes light having a thickness of about 1 μm. By cutting a specimen such as a cell with this light, signal light (scattered light, fluorescence, etc.) is generated from the portion of the specimen.
[0015]
The signal light is imaged through the objective lens 11 through the dichroic mirror 14 and the lens 15 at the pinhole position of the Nipkou disk assembly 16 in the confocal plane with the signal light generation point, and the light passing through the pinhole is The light is reflected by the mirror 19 and picked up by the image pickup means including the lens 17 and the image pickup device 18.
[0016]
In the first embodiment described above, the illumination of the confocal scanning microscope and the total reflection evanescence optical system is realized by using a single Nipkou disk assembly 16. In order to improve the illumination power of the total reflection evanescence optical system, another Nipkou disk assembly that rotates in synchronization with the Nipkou disk assembly 2 can be used for illumination of the total reflection evanescence optical system.
In the second embodiment shown in FIG. 3, a light beam for evanescence excitation is introduced from an obliquely downward direction of the second Nipkou disk assembly 16 that is interlocked with the first Nipkou disk assembly 2.
The third embodiment shown in FIG. 4 is a pinhole on the opposite side of the portion used for imaging (for fluorescence observation) of the second Nipkou disk assembly 16 linked to the first Nipkou disk assembly 2. Is to be used.
In the fourth embodiment shown in FIG. 5, a pinhole in the vicinity of a pinhole used for imaging (for fluorescence observation) of the second Nipkou disk assembly 16 interlocked with the first Nipkou disk assembly 2 is used. It is what you use. Each example will be briefly described below.
[0017]
FIG. 3 is a diagram showing a second embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention. Components having the same functions as those in the above embodiment are given the same numerals. The configuration of the confocal scanning microscope optical system disposed on the first surface side in this embodiment is the same as that of the above-described embodiment apparatus. The light source 20 for the evanescence microscope is arranged on the lower side so that illumination light is supplied to the Nipkou disk assembly 16 via the lens disk 16a supporting the microlens and the pinhole disk 16b. . The light from the light source 20 is transmitted through the lens of the lens disk 16a that supports the microlenses of the Nipkou disk assembly 16, and the lens disk 16a is transmitted through the dichroic mirror 24 disposed between 16b and is a pinhole disk. Pass the corresponding pinhole of 16b. The light passing through the pinhole passes through the lenses 15 and 11 and is incident on the interface between the specimen 10 and the transparent plate 9 at an angle slightly smaller than the total reflection angle, and emits evanescence light. The signal light generated on the cut specimen surface by the evanescence light passes through the objective lens 11 and the lens 15 through a pinhole at a position conjugate with the signal light generation point. The signal light transmitted through the pinhole is reflected by the dichroic mirror 24 and picked up by the image pickup means including the lens 17 image pickup device 18.
[0018]
FIG. 4 is a diagram showing a third embodiment of a scanning microscope capable of acquiring confocal images and evanescence illumination image information according to the present invention. Components having the same functions as those in the other embodiments are given the same numerals. The configuration of the confocal scanning microscope optical system disposed on the first surface side in this embodiment is the same as that of the above-described embodiment apparatus. The light source 21 for the evanescence microscope is disposed on the lower side, and illumination light is supplied to the Nipkou disk assembly 16 through the lens disk 16b and the pinhole disk 16a that support the microlenses. The light transmitted through the pinhole of the pinhole disk 16 a is collimated by the lens 22, reflected by the prism 13, reflected by the dichroic mirror 14, and incident on the periphery of the objective lens 11. This light forms the aforementioned evanescence light for illumination, and signal light is generated by the evanescence light. This signal light passes through the pinhole located at a conjugate position with the signal light generation point via the objective lens 11, the dichroic mirror 14, and the lens 15.
The signal light that has passed through the pinhole is reflected by the mirror 19 and picked up by the image pickup means comprising the lens 17 image pickup device 18.
[0019]
FIG. 5 is a diagram showing a fourth embodiment of the scanning microscope capable of acquiring the confocal image and the evanescence illumination image information according to the present invention.
Components having the same functions as those in the other embodiments are given the same numerals.
The configuration of the confocal scanning microscope optical system disposed on the first surface side in this embodiment is the same as that of the above-described embodiment apparatus.
The light source 21 for the evanescence microscope is disposed on the lower side, and illumination light is supplied to the Nipkou disk assembly 16 via a lens disk 16a for supporting the microlens and a pinhole disk 16b.
The light transmitted through the pinhole of the pinhole disk 16b is collimated by the lens 23 and supplied to the peripheral portion of the objective lens 11, and the evanescence light for illumination is generated as described above.
[0020]
【The invention's effect】
As described above, the present invention includes a confocal optical system that gives an image obtained by optically cutting a specimen, and an evanescence microscope optical system that uses illumination by evanescence light that can be formed at the glass-water (or air) interface. Both confocal images and evanescence illumination image information can be acquired simultaneously for the same specimen.
In order to obtain a two-dimensional surface image, it is necessary to scan each point. By linking the scanning means, the light points in both illumination modes do not overlap each other. Two two-dimensional images obtained by the methods do not affect each other, that is, the two images are not superimposed, and two two-dimensional images according to the respective methods can be obtained separately .
[Brief description of the drawings]
FIG. 1 is a diagram showing a first embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention.
FIG. 2 is an enlarged view showing a sample portion and an objective lens portion in the embodiment.
FIG. 3 is a diagram showing a second embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention.
FIG. 4 is a diagram showing a third embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention.
FIG. 5 is a diagram showing a fourth embodiment of a scanning microscope capable of acquiring confocal image and evanescence illumination image information according to the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Light source 2 Nipkou disk assembly 3 Lens 4 Confocal scanning microscope barrel 5 Confocal scanning microscope objective lens 6 Dichroic mirror 7 Imaging lens 8 Imaging device 9 Transparent plate 10 Sample 11 Evanescence microscope objective lens 12 Evanescence microscope mirror Tube 13 Prism 14 Dichroic mirror 15 Lens 16 Nipkou disk assembly 17 Imaging lens 18 Imaging device 19 Mirror 20, 21 Evanescence microscope light source 23 Collimator lens 24 Dichroic mirror

Claims (3)

A transparent plate having a first surface and a second surface and supporting the specimen on the first surface;
A confocal scanning microscope optical system disposed on the first surface side;
The evanescent illumination light is formed by making the second surface incident on the second surface at an angle larger than the total reflection angle so that the thickness of the light component that passes from the second surface to the first surface becomes a unit of micron or less. and evanescence illumination light source for supplying illumination light for cutting specimens by the evanescence illumination light,
A scanning microscope optical system for an evanescence illumination image disposed on the second surface side ,
Illuminate a specific surface of the specimen with illumination from the confocal scanning microscope optical system, receive a signal light generated by the illumination, and image the specimen in units of microns or less with the evanescence illumination light. It is configured to simultaneously observe an evanescent illumination image that is imaged by receiving signal light from that portion after being cut by light of thickness, and from the illumination point by the evanescent illumination light and the confocal scanning microscope optical system The illumination point is structured so that it does not overlap on the specimen.
Scanning microscope that can acquire confocal image and evanescence illumination image information.   2. The scanning microscope according to claim 1, wherein the confocal scanning microscope optical system comprises: a disk with a lens for light from a light source; a Nipkou disk assembly comprising a corresponding pinhole disk; and a dichroic disposed between the disks. A specific surface of the specimen is illuminated via a mirror and a confocal scanning microscope optical system objective lens, and the signal light generated by the illumination is reflected by the pinhole disk and the dichroic mirror and imaged. Scanning microscope that can acquire confocal image and evanescence illumination image information. The scanning microscope according to claim 1, wherein
Scanning microscope optics for evanescence illumination images
Confocal image and evanescence illumination image information configured to image signal light generated by evanescence illumination through a objective lens and a converging lens through a Nipkou disk having a pinhole corresponding to the signal light generation point. Scanning microscope that can be acquired.

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