JP3535933B2 - Fluorescence spectrometer - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence spectroscope for irradiating a subject with excitation light and for spectrally receiving the fluorescent light emitted from the subject by the excitation.

[0002]

2. Description of the Related Art Specifying the distribution of a substance in a living body itself or in a sample collected from the living body is useful for elucidating the mechanism of substance metabolism in the living body and for identifying a lesion. Obtaining fluorescence spectrum information is very effective in identifying the distribution of substances in a subject such as a living body or a biological sample.

[0003]

In order to obtain the fluorescence spectrum information, it is necessary to irradiate the subject with excitation light to excite the subject, and the excitation light is outside the wavelength band to be measured of the fluorescent light. It is necessary to select a specific wavelength. Conventionally, a laser light source, a color filter, or the like is used to obtain excitation light of a specific wavelength, but in these cases, the wavelength of the excitation light cannot be arbitrarily selected, and wavelength tuning is impossible. .

Further, a monochromator may be used in order to improve the wavelength selectivity of the excitation light and enable the tuning of the wavelength, but in that case, there is a problem that the device becomes large in size. In view of the above circumstances, the present invention is
An object of the present invention is to provide a compact fluorescence spectroscopic device capable of tuning the wavelength of excitation light at high speed and over a wide band.

[0005]

The fluorescence spectroscopic device of the present invention which achieves the above object comprises (1) an excitation light source that emits light including excitation light for exciting the subject (2) excitation from the subject by excitation light Light receiver for receiving the emitted fluorescent light (3) The light emitted from the excitation light source is made incident and the excitation light is separated from the light, and the fluorescent light emitted from the subject is made incident and the wavelength can be freely selected. Acousto-optical element (4) for extracting spectral components of different wavelengths, which is interposed between the excitation light source, the acousto-optical element, and the light receiver, and guides the light emitted from the excitation light source to the acousto-optical element, Polarization beam splitter (5) that guides the spectral components extracted by the acousto-optic element to the light receiver Excitation mode that separates the excitation light from the light emitted from the excitation light source and the spectral components from the fluorescent light emitted from the subject Spectroscopic mode It is characterized in that an acousto-optic device driver for driving the acousto-optic device by alternately switching between the mode and the mode is provided.

In the fluorescence spectroscopic device of the present invention, the driving of the acousto-optic element by the acousto-optic element driver is performed in the excitation mode by utilizing the property that the polarization direction of the output light is rotated with respect to the polarization direction of the incident light to the acousto-optic element. Since the acousto-optic device and its driver are used to separate the excitation light from the light emitted from the excitation light source and to disperse the fluorescence emitted from the subject, by alternating the time division between In addition, the wavelength selectivity of the excitation light is enhanced and a compact device is realized.

Here, in the fluorescence spectroscopic apparatus of the present invention, it is preferable that the light receiver has a plurality of light receiving elements arranged. In this case, the fluorescence spectral intensities of a plurality of points of the subject can be measured at the same time, and when a fluorescence spectral image is to be obtained, the measurement time until the fluorescence spectral image is obtained is shortened. Further, in the above-mentioned fluorescence spectroscopic device of the present invention, a pinhole plate having a pinhole that defines an optical path of light emitted from the excitation light source between the excitation light source and the polarization beam splitter and the arrangement position is freely adjusted. Is preferably provided. It is more preferable that the pinhole plate be capable of adjusting the diameter of the pinhole in addition to adjusting the arrangement position.

If a pinhole plate whose arrangement position (or, in addition to the arrangement position and the diameter of the pinhole) can be freely changed is provided, the excitation light can be irradiated only to the measurement point or measurement region targeted by the subject. Even if, for example, an autofluorescent substance is present near the measurement point or measurement region of the subject, it is possible to measure the spectral intensity of fluorescence at the measurement point or measurement region.

Further, in the fluorescence spectroscopic apparatus of the present invention, the process of irradiating the subject with excitation light of the same wavelength and receiving the spectral component of the fluorescent light emitted from the subject at a certain wavelength is repeated a plurality of times. It is preferable to provide a sequence control means for controlling the sequence as described above. If such a sequence control means is provided, it is possible to perform multiple excitation with excitation light of a certain wavelength and multiple reception of fluorescence spectroscopic light with a certain wavelength, and it is possible to perform highly sensitive and highly accurate measurement.

Further, in the fluorescence spectrometer of the present invention, the light emitted from the excitation light source is interposed between the excitation light source and the polarization beam splitter, and out of the incident light, the polarization beam splitter causes acousto-optics. It is preferable to include a polarizing element that emits the polarized component guided to the element toward the polarizing beam splitter. Further, in the fluorescence spectroscopic device of the present invention, the polarizing beam splitter emits from the excitation light source and enters the polarizing beam splitter. Of the light, the surface from which the ineffective component excluding the effective component guided to the acousto-optic element is emitted is oblique to the optical path of the ineffective component so that the ineffective component reflected by the surface is prevented from entering the light receiver. It is also a preferable embodiment that it is formed.

As described above, when the polarizing element is provided or the surface of the polarization beam splitter is formed obliquely, the ratio of the ineffective component of the light emitted from the excitation light source that is not used as the excitation light to the photodetector is reduced. , S / N can be measured well.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. FIG. 1 is a configuration diagram showing an embodiment of a fluorescence spectroscopy apparatus of the present invention. The excitation light source 1 is a white light source that emits light in a wide wavelength range, and the light 1 a emitted from the excitation light source 1
Passes through the pinhole 2a of the pinhole plate 2 and further enters the polarization beam splitter 6 via the liquid crystal shutter 3, the diaphragm 4 and the lens 5. Of the light emitted from the excitation light source 1 and incident on the polarization beam splitter 6, the P-polarized component polarized in the direction parallel to the paper surface of FIG. 1 is reflected by the reflection surface 6 a of the polarization beam splitter 6 and incident on the acousto-optic element 7. To do. Of the light emitted from the excitation light source 1 and incident on the polarization beam splitter 6, the S-polarized component polarized in the direction perpendicular to the paper surface of FIG. 1 passes through the reflecting surface 6a as it is and deviates from the effective optical path.

The acousto-optic element 7 is driven by an acousto-optic element driver 8. This acousto-optic element driver 8
The driving frequency of the personal computer 9 is switchably controlled. Here, a mode (excitation mode) for separating the excitation light for exciting the object from the light (P-polarized component) emitted from the excitation light source 1 and reflected by the reflection surface 6a of the polarization beam splitter 6, Different drive control is performed in a mode (spectral mode) in which the fluorescent light 30a emitted from the subject is dispersed to obtain the spectral light 30b.

When the acousto-optic element 7 is driven in the excitation mode, in the acousto-optic element 7, the light emitted from the excitation light source 1 and reflected by the reflection surface 6a of the polarization beam splitter 6 and incident on the acousto-optic element 7 is reflected. From inside, the excitation light 1b having a wavelength determined by the drive frequency of the acousto-optic system 7 is separated and emitted. At this time, the incident light (P
The plane of polarization of (polarized light) rotates, and S
Excitation light 1b as polarized light is emitted. The wavelength of the excitation light 1b emitted from the acousto-optic element 7 is determined by the frequency of the drive signal for driving the acousto-optic element 7. Therefore, by adjusting this frequency, the excitation light 1b is tuned to an arbitrary wavelength at high speed. can do.

Excitation light 1b emitted from the acousto-optic element 7
Irradiates a measurement region corresponding to the position of the pinhole 2a of the subject (not shown) via an objective optical system (not shown). Therefore, if the position or diameter of the pinhole 2a is changed, an arbitrary position on the subject and a region having an arbitrary area can be used as the measurement region. The subject is excited by the irradiation of the excitation light 1b and emits fluorescent light 30a. The fluorescent light 30a emitted from the subject is guided to an objective optical system (not shown) and is incident on the acoustooptic device 7, and an intermediate image 31 of the measurement region of the subject is formed inside the acoustooptic device 7. When the acousto-optic element 7 is driven in the spectroscopic mode, the spectral component 30b (S-polarized component) having a wavelength determined by the driving frequency of the acousto-optic element 7 is extracted from the fluorescent light 30a incident on the acousto-optic element 7. Then, it is incident on the polarization beam splitter 6.

Since the spectral component 30b that has entered the polarization beam splitter 6 is S-polarized, it passes through the polarization beam splitter 6 and is received by the light receiver 11 via the lens system 10. The lens system 10 has a focal length and the like so as to form an intermediate image 31 of the measurement region of the subject formed inside the acousto-optic element 7 on the light receiving surface of the light receiver 11, and also receives light. The device 11 is a CCD sensor in which a large number of light receiving elements are arranged two-dimensionally. Therefore, in the light receiving device 11, the fluorescence light 30 emitted from the measurement region of the subject is detected.
A light receiving signal representing the light amount distribution in the measurement area is obtained by the spectral component 30 of a. This received light signal is converted into a digital image signal by the AD converter 12 and is stored in the memory 1
Once stored in 3.

In the spectroscopic mode, the personal computer 9 causes the acousto-optic device driver 8 and the acousto-optic device 7 to operate.
When a command is issued to change the frequency of the drive signal of, the frequency of the drive signal of the acousto-optic element 7 is changed accordingly. Then, this time, the spectral component 30b of the fluorescent light 30a having a wavelength corresponding to the changed driving frequency is extracted and received by the light receiver 11, and the spectral component of the changed wavelength of the measurement region of the subject is detected. An image according to 30b is obtained.

The image signal obtained by each of the spectral components 30b stored in the memory 13 as described above is appropriately image-processed by the image processing device 14 and sent to the personal computer 9, and the personal computer 9 outputs the image signal. The image and the processing result thereof are displayed on the screen 9a. Figure 2
FIG. 4 is a timing chart showing an example of the timing of switching between the excitation mode and the spectroscopic mode.

As shown in (a), the excitation mode and the spectroscopic mode are alternately switched, and in the excitation mode, the excitation light of the predetermined wavelength is separated from the light emitted from the excitation light source 1 in the excitation mode. In the spectroscopic mode, the spectroscopic component is extracted from the fluorescent light emitted from the subject. Here, the acousto-optic device 7
Then, the subject is excited with excitation light of a certain wavelength over many times, and the spectral component of a certain wavelength of the fluorescence emitted from the subject by the excitation is extracted.

In the excitation mode, as shown in (b), the liquid crystal shutter 3 is opened, the light 1a from the excitation light source 1 passes through the liquid crystal shutter 3, and the acousto-optical element 7 excites the excitation light 1b.
Are separated and the subject is excited by the excitation light 1b. Further, in the light receiver (CCD sensor) 11, as shown in (c), the light receiving gate is opened only in the spectral mode, and the spectral component of fluorescence is received. Here, as mentioned above,
The spectral components of the same wavelength are repeatedly received over many times. In the light receiver (CCD sensor) 11, after the spectral components of the same wavelength are repeatedly received, as shown in (d), each element of the light receiver (CCD sensor) 11 is integrated over the many times of light reception. The received signal is the receiver (C
It is read from the CD sensor 11 and is AD-converted and sent to the memory 13. The above sequence is the fluorescent light 30a.
Of the spectral component of each wavelength. As described above, by repeatedly exciting the subject and repeatedly receiving the spectral components of the same wavelength, it is possible to perform highly sensitive and highly accurate measurement.

Although the liquid crystal shutter is used in the embodiments described with reference to FIGS. 1 and 2, it goes without saying that a mechanical shutter or the like may be provided instead of the liquid crystal shutter. FIG. 3 is a schematic view showing another part of the fluorescence spectroscopic device of the present invention, which is different from the embodiment shown in FIG.

The light emitted from the excitation light source 1 shown in FIG. 1 passes through the pinhole 2 and the like, and then enters the polarization beam splitter 61 shown in FIG. 4, which is arranged in place of the polarization beam splitter 6 shown in FIG. However, the polarizing plate 70 is arranged on the incident path in a direction in which only the P-polarized component is transmitted. Therefore, most of the incident light of the polarization beam splitter 61 (P-polarized component) is reflected at the reflection angle 61a and goes to the acousto-optic element 7 shown in FIG. However, a small amount of S-polarized light component is also included in the light transmitted through the polarizing plate 70, and most of the slightly included S-polarized light component is transmitted through the reflecting surface 61a and is emitted from the surface 61b to be effective. Although deviated from the optical path, a part of the S-polarized component is reflected by the surface 61b. At this time, if the surface 61b is perpendicular to the optical path of the S-polarized component as shown in FIG. 1, the S-polarized component reflected by this surface is reflected by the reflecting surface 61a and is incident on the light receiver 11 (see FIG. 1). However, this causes a decrease in S / N. However, the polarization beam splitter 61 shown in FIG.
, The surface 61b is formed obliquely.
The S-polarized component reflected at b deviates from the optical path toward the light receiver 11 as shown in the figure. This diverted light is blocked by the diaphragm 71 and does not enter the light receiver 11, and therefore the polarizing plate 70
With the provision of the above, it becomes possible to measure S / N well.

[0023]

As described above, according to the present invention,
Since the excitation light is separated using the acousto-optic element, the wavelength of the excitation light can be tuned at a high speed over a wide band, and the separation of the excitation light and the spectrum of the fluorescence light are the same. It is configured as a compact device because it is performed using.

[Brief description of drawings]

FIG. 1 is a configuration diagram showing an embodiment of a fluorescence spectrometer of the present invention.

FIG. 2 is a timing chart showing an example of the timing of switching between the excitation mode and the spectroscopic mode.

FIG. 3 is a schematic diagram showing another part of the fluorescence spectroscopic device of the present invention, which is different from the embodiment shown in FIG. 1.

[Explanation of symbols]

1 Excitation light source 2 pinhole plate 2a pinhole 3 LCD shutter 4 aperture 5 lenses 6 Polarizing beam splitter 7 Acousto-optic element 8 Acousto-optic element driver 9 personal computer 10 lens system 11 Light receiver 12 AD converter 13 memory 14 Image processing device 61 Polarizing beam splitter 70 Polarizer 71 Aperture

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-4-350816 (JP, A) JP-A-6-94526 (JP, A) JP-A-8-271410 (JP, A) JP-A-5- 231938 (JP, A) C.I. D. Tran and R.D. J. Furlan, "Spectrofluorometer Based on AOTF for Rapid Scanning and Multicomponent Sample Samples", Anal. Chem. , July 1, 1993, Vol. 65, No. 13, pp. 1675-1681 (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01J 3/00-3/52 G01N 21/62-21/74 JISST file (JOIS)

Claims (6)

(57) [Claims] 1. An excitation light source that emits light including excitation light that excites a subject, a light receiver that receives fluorescent light emitted from the subject by excitation by the excitation light, and the light emitted from the excitation light source is incident. Then, while separating the excitation light from the light, an acousto-optical element for entering the fluorescent light emitted from the subject and extracting the spectral component of the wavelength selected wavelength selectably, the excitation light source, and the acousto-optical element , A polarized beam which is interposed between the photoreceiver and the light guide and guides the light emitted from the excitation light source to the acousto-optic device and guides the spectral component extracted by the acousto-optic device to the photoreceiver. A splitter, and an excitation mode for separating the excitation light from the light emitted from the excitation light source, and a spectroscopic mode for extracting the spectral component from the fluorescent light emitted from the subject are alternately switched. A fluorescence spectroscopic device comprising an acousto-optic device driver for driving the acousto-optic device. 2. The fluorescence spectroscopic apparatus according to claim 1, wherein the light receiver has a plurality of light receiving elements arranged therein. 3. A pinhole plate is provided between the excitation light source and the polarization beam splitter to define an optical path of light emitted from the excitation light source, and a pinhole plate whose arrangement position is freely adjusted is provided. The fluorescence spectroscopic device according to claim 1, wherein: 4. Sequence control means for controlling the sequence so that the process of irradiating the subject with excitation light of the same wavelength and receiving the spectral component of the same wavelength of the fluorescent light emitted from the subject is repeated a plurality of times. The fluorescence spectroscope according to claim 1, further comprising: 5. The light emitted from the excitation light source is incident between the excitation light source and the polarization beam splitter, and the light is guided to the acoustooptic device by the polarization beam splitter. The fluorescence spectroscopic apparatus according to claim 1, further comprising a polarizing element that emits the polarized component to be emitted toward the polarizing beam splitter. 6. The surface of the polarization beam splitter from which the ineffective component excluding the effective component guided to the acousto-optic element of the light emitted from the excitation light source and incident on the polarization beam splitter is emitted is the surface. 2. The ineffective component reflected by the optical receiver is formed obliquely with respect to the optical path of the ineffective component so as to avoid entering the optical receiver.
The fluorescence spectroscopic device described.