JP2836859B2 - Three-dimensional spatial and time-resolved absorption spectrum measurement device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an absorption spectrum measuring device. More specifically, the present invention relates to a confocal light-induced absorption spectrum measuring apparatus that enables three-dimensional spatial resolution and time-resolved measurement.

(Prior art and its problems) Conventionally, various methods for analyzing material phenomena and their structures using optical means have been proposed, and microscopic techniques also include new methods such as the use of laser light. Various things have been put to practical use.

However, despite the progress and development of such microscope technology, there is a limitation that the microscope technology can only obtain information on the two-dimensional structure of a substance.

For example, in a well-known transmission microscope having a configuration as shown in FIG. 2, when a thin plate-like sample having a uniform content is considered, this sample is taken in the depth direction (Z axis).
The signal obtained does not change at all, and there is a certain amount of absorption anywhere, and the depth direction, that is, the three-dimensional structure of the substance, cannot be observed.

In the case of the fluorescence microscope shown in FIG. 3, a and b in FIG.
When the positions of the sample are compared, the area where the direction of the position a is excited is small, but the concentration of the excited molecule is high.
However, the personality of the excited molecule as a whole does not change at positions a and b, and the resulting detected signal does not change at positions a and b. For this reason, even in this fluorescence microscope, information in the depth direction cannot be observed.

FIG. 4 shows a confocal laser scanning absorption microscope, which differs from the transmission microscope of FIG. 2 in that a pinhole is provided in front of a light source and in front of a detector. doing. This is realized only by using a laser beam. However, even in this laser scanning absorption microscope, since the detected light is transmitted through the sample coherently, it is not possible to obtain three-dimensional information in the depth direction.

None of the above has a three-dimensional spatial resolution, which is a weak point of the microscope technology.

On the other hand, to solve such a weak point, there is a confocal laser scanning fluorescence microscope shown in FIG. 5, for example.
This microscope differs from the fluorescence microscope of FIG. 3 in that there are pinholes in front of the light source and in front of the detector.

Although the state of the excited molecule does not change, the fluorescent light emitted incoherently from the sample at the position b in the drawing is defocused on the pinhole, so that it can hardly pass through the pinhole. On the other hand, at the position a, an image is formed on the pinhole, and most of the energy strikes the detector. For this reason, the observed signal intensity differs at the positions a and b, and from this, it is possible to recognize the difference in the depth direction. That is, information in the depth direction can be observed.

However, this laser scanning fluorescence microscope has a drawback that it has limitations as a fluorescence microscope, and cannot measure only fluorescence by laser scanning when there is absorption other than light excitation.

The present invention has been made in view of the above circumstances, and has been developed to provide a three-dimensional space-resolved measurement of photoexcitation absorption by a laser beam, and furthermore, a new method capable of observing a dynamic time change. It is an object of the present invention to provide an absorption microscope of a system and an absorption spectrum measuring apparatus having a wavelength resolution.

(Means for Solving the Problems) The present invention solves the above-mentioned problems by providing pinhole excitation light from the same direction as the optical path direction of the pinhole monitor light or from the opposite direction, thereby enabling the monitor light and the excitation light. And a three-dimensional spatially-resolved measurement of light-induced absorption of a sample using the confocal point as a confocal point.

Furthermore, the present invention also provides the above-mentioned absorption spectrum measuring apparatus for performing time-resolved measurement using pulse laser light as monitor light and excitation light.

FIG. 1 shows the configuration of an absorption spectrum measuring apparatus according to the present invention as an embodiment.

As shown in FIG. 1, the light from the pinhole monitor light source enters the sample via the condenser lens, and travels to the pinhole and the detector via the objective lens. On the other hand, light from the pinhole excitation light source is incident on the sample via a dichroic mirror and an objective lens, and optically excites the sample.

The detector via the pinhole observes the absorption due to the light excitation. At this time, although it is an absorption measurement, it has a three-dimensional spatial resolution unlike a conventional absorption microscope.

A pinhole, ie, a laser beam, is used for both the observation monitor light and the excitation light, and the monitor light and the excitation light pass through exactly the same optical path to form a confocal system.

In the spectrum measuring apparatus of the present invention having such a configuration, when comparing the positions a and b of the sample, the position a has a higher concentration of molecules excited by the excitation light but a smaller area at the position a. Since the monitor light passes only through the excited region, the signal intensity becomes smaller because a has a higher absorbance.

From such a principle, information on the sample in the depth direction can be observed. That is, three-dimensional spatially resolved absorption spectrum measurement becomes possible.

Resolution is as xy space (NA is the numerical aperture of the lens) (λ is the wavelength of light)
The axis is (in the depth direction) It is.

In the device of the present invention, even if there is absorption other than light excitation, only the light excitation absorption can be measured by the difference between when the excitation light is irradiated and when it is not irradiated, and pulse laser light is used as the excitation light and monitor light. This also enables time-resolved measurement.

Further, by changing the wavelength of the monitor light, it becomes possible to measure the absorption spectrum.

By placing a pinhole in front of the detector, it is possible to increase the contrast without the influence of scattered light and the like. Further, in the device of the present invention, since the excitation light is focused on a very small area, it is possible to supply the energy necessary for exciting the substance even if the power of the laser is not so large.

(Effect of the Invention) According to the present invention, as described in detail above, an absorption spectrum apparatus capable of three-dimensional spatial and time-resolved measurement using a laser beam, that is, a confocal laser-induced absorption microscope is realized.

[Brief description of the drawings]

FIG. 1 is an optical configuration diagram showing the configuration of the apparatus of the present invention as an embodiment. FIG. 2, FIG. 3, FIG. 4, and FIG. 5 are optical configuration diagrams each showing a conventional microscope.

Continuation of the front page (56) References JP-A-47-33662 (JP, A) JP-A-63-131116 (JP, A) JP-A-63-306414 (JP, A) JP-A-64-59018 (JP) , A) Tech Rep ISSP Ser A No. 2081 (1988) p1-16 Proc. SPIE Int. SoC. Opt. Fng. Vol 1127 (1989) p 101-108 (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 21/00-21/01 G01N 21/17-21/74 JOIS WPI / L

Claims (2)

(57) [Claims] An optical path direction of the pinhole monitor light;
Alternatively, an absorption spectrum measurement apparatus characterized in that pinhole excitation light is incident from the opposite direction to make monitor light and excitation light confocal, and three-dimensional spatially resolved measurement of light-induced absorption of a sample is performed. 2. The absorption spectrum measuring apparatus according to claim 1, wherein the time-resolved measurement is performed by using pulse laser light as the monitor light and the excitation light.