JP2006055396A - Fluorescent tomogram measuring apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for accurately measuring and imaging the distribution of a fluorescent material inside a scattering medium. <P>SOLUTION: The apparatus is for measuring the tomogram of the fluorescent material present inside the light scattering medium such as a living body in vitro. The apparatus consists of an excitation laser beam, a focusing type ultrasonic transducer to modulate the intensity of the fluorescence generated by the laser beam, a photodetector with an optical filter, and a signal detector. The focusing type ultrasonic transducer is disposed so that an ultrasonic beam propagation axis orthogonally crosses or is parallel to the laser beam axis, and is structured so that the intensity of the multiple-scattered excitation light and fluorescence is locally modulated in the focal position. A modulation frequency component is detected from light signals which are obtained by three-axis scanning of the ultrasonic transducer or measuring a sample and the laser beam, and the distribution of the fluorescent material inside the scattering medium is imaged by the apparatus. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus for measuring a tomographic image of fluorescence from a fluorescent substance present in a living body from outside the living body for observation of a physiological function and gene function of a living body or pathological diagnosis.

  Conventionally, in fluorescence observation for living cells, sliced tissue, or tissue surface layers, a fluorescence observation apparatus including an excitation light source, a fluorescence observation filter, an excitation light cut filter, and a camera has been used. However, in this method, the excitation light and the fluorescence are multiple-scattered by the scattering medium of the living tissue, so that the inside of the living tissue cannot be observed when the depth is several millimeters or more.

  When observing at a cell level using a microscope, as a device capable of selectively measuring information in the depth direction to obtain a tomographic image, a confocal microscope (see Patent Document 1 below), a multiphoton microscope (US) Japanese Patent No. 55,034,613 and JP-T-10-512959) are known, but since these are effective at a thickness where light scattering does not become a problem, fluorescence tomography with a thin slice specimen is possible. It cannot be applied to living tissue with thickness.

An apparatus for imaging light absorption characteristic distribution in a scattering medium using the interaction between ultrasonic waves and laser light using differences in laser intensity and polarization characteristics due to ultrasonic modulation is disclosed in US Pat. No. 6,041. 248 (Non-Patent Document LVWang and X.Zhao: Applied Optics Vol.36, p.7277 (1997)) and the following Patent Document 2, which are known for the intensity modulation and polarization characteristics of ultrasonic waves with respect to laser light. The change is used to image the light absorption distribution of laser light.
Japanese Patent Laid-Open No. 5-60980 JP 2000-88742 A

For example, since fluorescence from a fluorescent substance in a light scattering medium such as a living body diffuses due to multiple scattering, the distribution image cannot be observed from the outside of the scattering medium. The above-mentioned known technical means is to measure the fluorescent substance distribution in a sliced specimen or to measure the light absorption distribution of laser light. Using such means, the fluorescent distribution in the living body can be accurately determined immediately. It cannot be measured.
In order to observe the distribution of the fluorescent substance in the living body from outside the living body, an imaging technique for separating and extracting only the fluorescent light originating from a specific place from the scattered fluorescence is required. Therefore, the present invention provides an apparatus for accurately measuring and imaging a fluorescent substance distribution in a scattering medium using an excitation laser beam and an ultrasonic beam that locally modulates the intensity of fluorescence generated thereby. is there.

  The present invention is a device for measuring a tomographic image of a fluorescent substance existing in a light scattering medium such as a living body from outside the living body, and at least a focusing that gives intensity modulation to an excitation laser beam and fluorescence generated thereby. Type ultrasonic transducer, a large-diameter photodetector for light detection, and a signal detection device, and a focused ultrasonic transducer arranged so as to have an ultrasonic beam propagation axis orthogonal to or parallel to the laser beam axis, The apparatus is configured to locally modulate the intensity of the multiple scattered excitation light and fluorescence at the focal position, and detects the modulation frequency component from the optical signal obtained while performing three-axis scanning with the ultrasonic transducer or the measurement sample and the laser beam. This is an apparatus for imaging the fluorescent substance distribution in the scattering medium.

[Action]
In the present invention, an ultrasonic sound field is formed in a highly scattering medium such as a living body using a focusing ultrasonic transducer. At this time, the highest sound pressure is formed at the focal point. When light propagates through this acoustic field focus, the light passing through it is intensity-modulated at the ultrasonic frequency due to light diffraction due to the refractive index distribution formed by the ultrasonic dense wave and uneven distribution of scattered molecules. . The degree of modulation at this time depends on the sound pressure.
When a fluorescent material exists in a highly scattering medium, the irradiated laser beam is multiple-scattered, but a part thereof excites the fluorescent material. Since the generated fluorescence is also scattered, the entire medium emits light almost uniformly outside the scattering medium, and it is difficult to identify the position of the fluorescent substance inside. However, at this time, if a fluorescent substance is present at the focal point of the above-described ultrasonic sound field, the generated fluorescence is intensity-modulated at the ultrasonic frequency. When only the sound wave frequency component is detected, the frequency component has information on the fluorescence intensity at the ultrasonic focus. When the focal point deviates from the position of the fluorescent material, both the modulated fluorescence and the excitation light are rapidly attenuated by scattering. Measurements can be made with a resolution of the ultrasonic focus size, and a fluorescence tomographic image can be constructed.
In the tomographic image measurement apparatus of the present invention, in the apparatus for installing the measurement sample according to the first invention in the water tank, the laser beam axis and the ultrasonic beam axis are arranged so as to be orthogonal to each other. The entire sample is scanned by moving the installed water tank with a three-axis automatic stage, and a tomographic image is obtained from fluorescence that has been intensity-modulated at an ultrasonic frequency.
In the apparatus for directly measuring a living body according to the second invention, a laser beam and an ultrasonic beam are irradiated in parallel, a detector having a large aperture is arranged on the same side as these irradiation means, and an ultrasonic wave By scanning the beam two-dimensionally or omitting the scanning action using an array type ultrasonic transducer, the fluorescence distribution in a certain depth region is detected from the surface of the living body.

According to the tomographic image measurement apparatus of the present invention, the fluorescence excited and emitted by the laser beam is detected by detecting the intensity-modulated fluorescence in the focus focal region of the ultrasonic beam and detecting the intensity-modulated fluorescence. The fluorescence from the fluorescent substance can be accurately detected, and a tomographic image can be easily measured using the scanning means.
In addition, by suspending the measurement sample in the water tank, the water tank is moved three-dimensionally to facilitate the scanning means.
Furthermore, with respect to the fluorescent substance in the living body, the fluorescent substance at the focal position of the ultrasonic beam can be accurately detected from the surface of the living body, so that the influence of scattering can be removed extremely effectively.

The embodiment of the present invention will be described with reference to FIGS. Further, FIG. 5 is a fluorescence tomographic image showing a measurement result according to one embodiment of the present invention.
FIG. 1 is a plan view showing the overall configuration of an apparatus according to an embodiment of the present invention, and FIG. 2 is a side view of the apparatus of FIG. In FIG. 1, a laser beam 11 emitted from an excitation laser light source 10 enters an optical window (not shown) installed on a side surface 31 of a water tank 30. On the water tank side surface 32 orthogonal to the optical axis of the laser beam 11 of the water tank 30, a focusing type underwater ultrasonic transducer 20 for generating the ultrasonic beam 21 is installed and fixed with the direction orthogonal to the optical axis as the propagation axis. Yes, the water tank 30 is filled with distilled water 35. The ultrasonic transducer 20 is driven by an oscillator 22 and a high frequency power amplifier 23 for driving at a resonance frequency.

The embodiment shown in FIGS. 1 and 2 is an example in which a small animal or a living tissue is used as the sample S. The sample S is mounted on a holder 41 and is mounted from a sample fixing base 40 installed in the diagonal direction of the water tank 30. It is suspended.
The ultrasonic wave absorbing plate 25 is installed on the side surface 33 of the water tank 30 facing the ultrasonic transducer installation side surface 32, and an ultrasonic traveling wave is generated between the side surfaces 32 and 33. The sample S is disposed at the focal position of the ultrasonic traveling wave. The water tank 30 is installed on an automatic stage 35 that can be moved and adjusted in three axes: a laser beam axis direction (X axis), an ultrasonic beam axis (Y axis) orthogonal thereto, and a height direction (Z axis). .

  A light detector 50 is installed on the outside of the opposite surface 34 of the laser beam incident side surface 31 of the water tank 30 with a slight offset from the optical axis of the laser beam. In the photodetector 50, an interference filter 51 that selectively transmits only the fluorescence wavelength and blocks excitation light is mounted on a large-diameter photomultiplier tube or PIN photodiode having a diameter of about 50 mm. The light detector 50 is connected to a spectrum analyzer 52, where it narrow-band detects an optical signal component that matches the ultrasonic frequency.

The spectrum analyzer 52 is connected to the personal computer 53 and scans each axis (X, Y, Z axis) of the water tank moving three-axis automatic stage 35 through the stage controller 36 under personal computer control, while Record detection data. By displaying this detection data as an image according to the signal intensity, a fluorescence tomographic image of the fluorescent substance in the sample S (for example, a living body) can be obtained.
The same effect can be obtained by simultaneously scanning the sample S and the laser beam 11 instead of scanning the ultrasonic beam 21.

  FIG. 3 and FIG. 4 show another embodiment of the present invention, which is the overall configuration of an apparatus when a human is used as a measurement target. In addition, the same code | symbol is attached | subjected and demonstrated to the part corresponding to embodiment shown in FIG. 1, FIG. In this embodiment, an ultrasonic impedance matching jelly is applied as a coupling medium directly on the surface of a living body without using a water tank, and the ultrasonic transducer is brought into contact with the body surface.

FIG. 3 shows the shape of the subject M as viewed from above. The ultrasonic transducer 20 that contacts the right chest of the subject M via the ultrasonic impedance matching jelly 36 is supported by a suitable support means 29. The ultrasonic transducer drive device 26 can be moved in two axes. And it has an adjustment mechanism (details are omitted) that can automatically adjust the height according to the height difference in the ultrasonic beam direction of the body surface and come into contact with the skin.
The laser beam 11 is guided from the excitation laser light source 10 to the ultrasonic transducer driving device 26 by the optical fiber 13 and is irradiated onto the body surface from a substantially central portion of the moving range of the ultrasonic transducer 20.

  As is clear from FIG. 4, the ultrasonic transducer driving device 26 (see FIG. 3) is provided with a large-diameter photodetector 50 so as to surround the outside of the scanning range 37 of the ultrasonic transducer, and light detection. The filter 50 is equipped with a fluorescence wavelength selection filter (not shown) as in the first embodiment. FIG. 4 is a view of a part of FIG.

  In such an embodiment, the two-dimensional scanning of the ultrasonic transducer 20 is automatically or manually scanned to measure the two-dimensional distribution of the fluorescent material at the depth determined by the focal point of the ultrasonic transducer 20. Can do. Even in such an embodiment, the configuration of peripheral electronic devices is the same as that of FIG. If a one-dimensional or two-dimensional array type is used for the ultrasonic transducer 20, a two-dimensional image can be obtained by uniaxial scanning or non-scanning.

Next, an embodiment using the apparatus according to the configuration shown in FIGS. 1 and 2 will be described.
In place of the sample S shown in FIG. 1, a rectangular solid container of 50 × 50 × 100 mm is filled with an intralipid 2% solution as a scattering medium, and a cylindrical phosphor (fluorescein) having a diameter of 2.5 mm is placed in the center of the container. ) Was installed. An argon ion laser (488 nm, 100 mW) was used as the excitation light source, and a focused ultrasonic transducer having a resonance frequency of 1 MHz and a focal length of 37 mm was used as the ultrasonic transducer.
With the apparatus configuration of FIG. 1, the water tank 30 is moved and scanned in two axes, a laser beam axis (X axis) and an ultrasonic wave propagation axis (Y axis), and the measurement result of the fluorescence two-dimensional tomographic image obtained thereby is shown in FIG. It shows.

It is a plane explanatory view of the device concerning one embodiment. It is side surface explanatory drawing of the apparatus shown in FIG. It is plane explanatory drawing of the apparatus which concerns on other embodiment. It is side surface explanatory drawing of the apparatus shown in FIG. It is a fluorescence two-dimensional tomographic image which shows the measurement result which concerns on an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Excitation laser light source 11 Laser beam 13 Optical fiber 20 Focusing type underwater ultrasonic transducer 21 Ultrasonic beam 25 Ultrasonic absorption plate 26 Ultrasonic transducer drive device 30 Water tank 35 Three-axis automatic stage for water tank movement 36 Ultrasonic impedance matching Jelly 41 Holder 40 Sample fixing table 50 Photo detector 51 Interference filter M Subject S Sample

Claims (8)

An apparatus for measuring a tomographic image of a fluorescent substance existing in a light scattering medium as a sample from outside the medium, including an excitation laser light source, a focusing ultrasonic transducer, a scanning means, and optical detection with an optical filter Detector and a signal detector, and intensity-modulates the fluorescence generated by the laser beam from the laser light source in the focal point of the ultrasonic beam, detects the output signal of the photodetector, and detects the modulation frequency component Thus, the tomographic image measuring apparatus is characterized in that only the fluorescence from the focal region is detected, and the necessary range is scanned by the scanning unit, thereby imaging the fluorescent substance distribution in the light scattering medium. The tomographic image measurement apparatus according to claim 1, further comprising a water tank for storing a sample made of a light scattering medium in which the fluorescent material is present, and an optical window for the laser beam is provided on one side of the water tank, The optical detector is provided at a position slightly shifted from the laser beam axis on the opposite side of the water tank, and the focused ultrasonic transducer is provided on the side surface of the water tank on the axis side orthogonal to the laser beam axis. A tomographic image measuring apparatus. The tomographic image measurement apparatus according to claim 2, wherein the scanning means is a three-axis automatic stage for moving the water tank. A device for measuring a tomographic image of a fluorescent substance present in a light scattering medium, which is a living body, from outside the medium, including an excitation laser light source, a focused ultrasonic transducer, scanning means, and optical detection with an optical filter Detector and a signal detector, and intensity-modulates the fluorescence generated by the laser beam from the laser light source in the focal region of the ultrasonic beam, detects the output signal of the photodetector, and detects the modulation frequency component Thus, only the fluorescence from the focal region is detected, and the fluorescent material distribution in the light scattering material is imaged by scanning the necessary range by the scanning means. The tomographic image measurement apparatus according to claim 4, wherein the laser beam and the ultrasonic beam are irradiated from the same direction, and the focused ultrasonic transducer is brought into contact with a living body surface via an ultrasonic impedance matching jelly, The tomographic image measuring apparatus according to claim 1, wherein the photodetector is arranged on the irradiation side of the laser beam and the ultrasonic beam. 6. The tomographic image measuring apparatus according to claim 5, wherein the focused ultrasonic transducer is controlled by an ultrasonic transducer driving device capable of scanning in two axial directions, and the laser beam is substantially at the center of the ultrasonic transducer driving device. A tomographic image measuring apparatus characterized by being irradiated from a portion. 7. The tomographic image measuring apparatus according to claim 5, wherein the focused ultrasonic transducer has an adjustment mechanism that can be automatically adjusted according to a difference in height of the living body surface. . An apparatus for measuring a tomographic image of a fluorescent substance existing in a light scattering medium as a living body from outside the medium, comprising an excitation laser light source, a focusing ultrasonic transducer, a photodetector with an optical filter, and signal detection An intensity modulation of the fluorescence generated by the laser beam from the laser light source in the focusing focal region of the ultrasonic beam, and signal detection of the output of the photodetector to detect the modulation frequency component, A tomographic image measuring apparatus that detects only fluorescence from a focal region and reduces a scanning action of the ultrasonic transducer by using a one-dimensional array type or a two-dimensional array type transducer as the ultrasonic transducer.

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