JPH04350817A - Spectral confocal optical system - Google Patents

Abstract

PURPOSE:To provide the confocal optical system which provides optical slicing and spectral diffraction at the same time. CONSTITUTION:The light from a light source 12 is converged on a sample 20 through an objective 18. The fluorescent light from the sample 20 is converged through a 1st condenser lens 22. A light shield plate 24 which has a pinhole 25 is arranged behind the 1st condenser lens 22 and only fluorescent light from specific depth of the sample 20 passes through it. The light passed through the pinhole 25 is converged by a 2nd condenser lens 26 with an on-axis aberration on a point on the axis corresponding to the wavelength. A light shield plate 28 which has a pinhole 29 is arranged behind the 2nd condenser lens 26 and only light with specific wavelength passes through it. The light passed through the pinhole 29 is made incident on a photoelectric detecting element 30.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal optical system capable of performing spectroscopy.

0002

2. Description of the Related Art Confocal optical systems have superior imaging characteristics and high in-plane resolution compared to general microscopes. Furthermore, since this confocal optical system has resolution in the optical axis direction, it is possible to perform so-called optical slicing, which obtains an optical cross-sectional image of the sample at a specific position in the optical axis direction. One of the fields of application of this confocal optical system is confocal fluorescence microscopy in the biological field. In this confocal fluorescence microscopy, the spectroscopy of fluorescence from a biological specimen is important, and a method of spectroscopy of fluorescence from a specimen using a confocal optical system has been proposed. According to this method, fluorescence spectroscopy can be performed without using special optical elements such as dichroic mirrors and gratings, which were indispensable in the past. As an example of such an optical system, one shown in FIG. 3 is known. Figure 3 shows “A Proposal of Spectroscopy in Confocal Optical Systems” (Spectral Research Vol. 39, No. 6 (1990), 347-
Reprinted from page 352). In this optical system, a lens (L2) with large axial chromatic aberration is used as a detector.
) is placed in front of. Also, a pin hole is placed just in front of the detector. Sample
The fluorescence from e) is focused at different points on the optical axis depending on its wavelength due to the chromatic aberration of the lens (L2). Then, a pinhole is moved along the optical axis to allow only light of a specific wavelength to pass through, and a detector (De
sample by guiding it to the
) spectroscopy of fluorescence from

0003

However, this optical system cannot perform optical slicing, which is one of the advantages of a confocal optical system. In other words, when attempting to perform optical slicing with this optical system, wavelength information and sample depth information overlap, making it impossible to obtain an accurate optical cross-sectional image of the sample.

The present invention has been made to eliminate such inconveniences, and an object of the present invention is to provide a confocal optical system that can perform optical slicing of a specimen and spectroscopy of fluorescence from the specimen.

[0005]

[Means for Solving the Problems] The confocal optical system of the present invention includes a light source, an objective lens that focuses light from the light source onto a sample, and a first lens without chromatic aberration that focuses light from the sample. a first micro-aperture disposed at the focal point of the first lens; a second lens having axial chromatic aberration that focuses the light passing through the first micro-aperture; a second micro-aperture disposed at the focal point; a photoelectric detection element that receives light passing through the second micro-aperture; a first moving means for moving the first micro-aperture in the optical axis direction; and second moving means for moving the second minute aperture in the optical axis direction.

[0006]

[Operation] In the confocal optical system of the present invention, light from the sample is once focused by the first lens. A first micro-aperture is arranged behind the first lens, and the first micro-aperture selectively passes only light from a specific depth of the sample. The light passing through the first micro-aperture is focused by the second lens. Since the second lens has axial chromatic aberration, the light incident on the second lens is focused on different points on the optical axis depending on the wavelength. A second micro-aperture is arranged behind the second lens, and the second micro-aperture allows only light of a specific wavelength to pass through. In this optical system, by moving the first micro-aperture along the optical axis, it is possible to select only light from a specific depth of the sample, and by moving the second micro-aperture along the optical axis, it is possible to select only the light from a specific depth of the sample. The wavelength of light can be selected. That is, optical slicing is performed using the first micro-aperture, and spectroscopy is performed using the second micro-aperture.

[0007]

Embodiments Next, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 shows a confocal optical system according to an embodiment of the present invention. The light emitted from the light source 12 is converted into a parallel beam by the collimating lens 14. The parallel light beam is reflected by a dichroic mirror 16, focused by an objective lens 18, and focused onto a specimen (sample) 20. The specimen 20 is supported so as to be movable within a plane perpendicular to the optical axis, and is moved within that plane during observation. The fluorescence from specimen 20 is
It is collected by an objective lens 18 and a dichroic mirror 16.
The light passes through and is condensed by the first condensing lens 22. The first condensing lens 22 is an aberration-free lens without chromatic aberration, and the fluorescence from a certain depth of the specimen 20 is condensed to one point on the optical axis. A light shielding plate 24 having a pinhole 25 is arranged behind the first condensing lens 22 . The pinhole 25 allows only the light condensed inside the pinhole to pass through. In other words, only the fluorescence from a certain depth of the specimen 20 passes through the pinhole 25, and the fluorescence from other depths passes through the light shielding plate 25.
occluded by The depth can be changed by moving the specimen 20 or the light shielding plate 24 along the optical axis. In other words, you can perform optical slicing.

The light that has passed through the pinhole 25 is condensed again by a second condenser lens 26. The second condensing lens 26 has a relatively large axial chromatic aberration, and therefore the light is condensed at different points on the optical axis depending on the wavelength. A light shielding plate 28 having a pinhole 29 is arranged behind the second condensing lens 26 . Since the second condenser lens 26 has axial chromatic aberration, only light of a specific wavelength (λ0 in the figure) passes through the pinhole 29 and enters the photoelectric detection element 30. Light of other wavelengths (λ1 and λ2 in the figure) is blocked by the light shielding plate 2.
Blocked by 8. The light shielding plate 28 is provided movably along the optical axis, and by moving this along the optical axis, the wavelength of the light incident on the photoelectric detection element 30 can be selected. In other words, it is possible to perform spectroscopy of fluorescence from a specimen.

In the optical system of this embodiment, the first condenser lens 22 and pinhole 25 constitute a confocal optical system for optical slicing, and the second condenser lens 26 and pinhole 29 constitute a confocal optical system for optical slicing. A confocal optical system is constructed for this purpose. A cross section of the specimen to be observed is specified by a confocal optical system for optical slicing, and fluorescence from that cross section is analyzed by a confocal optical system for spectroscopy. With this configuration, optical slicing and spectroscopy can be performed simultaneously and precisely.

Next, a confocal optical system according to another embodiment of the present invention is shown in FIG. The light emitted from the light source 12 is converted into a parallel beam by a collimating lens 14, and then converted into a parallel beam by a dichroic mirror 16.
incident on and reflected. The light reflected by the dichroic mirror 16 is focused by the lens 17 and passes through the pinhole 25 of the light shielding plate 24. The light passing through the pinhole 25 is
The light enters the first condenser lens 22, becomes a parallel light beam, enters the objective lens 18, and is focused on the specimen 20. The specimen 20 is supported so as to be movable within a plane perpendicular to the optical axis, and is moved within that plane during observation. Fluorescence from the specimen 20 is collected by the objective lens 18 and then condensed by the first condensing lens 22. The first condensing lens 22 is an aberration-free lens without chromatic aberration, and the fluorescence from a certain depth of the specimen 20 is condensed to one point on the optical axis. Among them, pinhole 2
Only the fluorescence from a specific depth that is focused inside the pinhole 25 passes through the pinhole 25, and the fluorescence from other depths passes through the light shielding plate 24.
occluded by

The light passing through the pinhole 25 passes through the lens 1
7 and the dichroic mirror 16, and enters the second condensing lens 26 where it is condensed. Second condensing lens 2
Reference numeral 6 denotes a lens with large axial chromatic aberration, so that incident light is focused on different points on the optical axis depending on its wavelength. A light shielding plate 28 having a pinhole 29 is arranged behind the second condensing lens 26, and only light of a specific wavelength indicated by λ0 passes through the pinhole 29 and enters the photoelectric detection element 30. Light of other wavelengths indicated by λ1 and λ2 are blocked by the light shielding plate 28.

The optical system of this embodiment includes a light source 12, a collimating lens 14, a dichroic mirror 16, and a lens 1.
7. Optical slicing is performed by moving the light shielding plate 24, the second condensing lens 26, and the light shielding plate 28 together along the optical axis, and the light shielding plate 28 is moved relative to the second condensing lens 26. Spectroscopy is performed by moving along the optical axis. In this way, since the confocal optical system for optical slicing and the confocal optical system for spectroscopy are separately provided,
Optical slicing and spectroscopy can be performed simultaneously with high precision.

[0014]

According to the confocal optical system of the present invention, optical slicing and spectroscopy can be performed simultaneously and precisely.

[Brief explanation of the drawing]

FIG. 1 shows an embodiment of a confocal optical system according to the invention.

FIG. 2 shows another embodiment of a confocal optical system according to the invention.

FIG. 3 shows a conventional example of performing spectroscopy using a confocal optical system.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 12... Light source, 18... Objective lens, 22... First condensing lens, 26... Second condensing lens, 25, 29... Pinhole, 30... Photoelectric detection element.

Claims (1)

[Claims] Claim 1: A light source, an objective lens that focuses light from the light source onto a sample, a first lens without chromatic aberration that focuses light from the sample, and a first lens disposed at the focus point of the first lens. a first micro-aperture, a second lens having axial chromatic aberration that focuses the light that has passed through the first micro-aperture, and a second micro-aperture disposed at the focal point of the second lens; a photoelectric detection element that receives light that has passed through the second minute aperture; a first moving means that moves the first minute aperture in the optical axis direction; and a second moving means that moves the second minute aperture in the optical axis direction. A spectroscopic confocal optical system comprising a moving means.