JPH05223738A - Measuring device for fluorescent tomographic image - Google Patents

Abstract

PURPOSE:To observe a fluorescent source distribution image and a fluorescent tomographic image highly accurately by detecting fluorescence which is radiated in direction orthogonal to an excitation laser beam through a light-reception system with a high directivity. CONSTITUTION:For example, a certain section of a sample 61 is irradiated by a laser 60 which consists of diode arrays which are aligned in a straight line for exciting a fluorescent source, thus enabling fluorescence to be radiated in all directions. Fluorescence which is radiated in a direction crossing the excitation laser beam out of the fluorescence is received by an optical system 63 with a high directivity which takes in 0-order light of Fraunhofer's diffraction image which is laid out in a direction crossing the laser 60 for the sample 61 and is detected by a two-dimensional detector 64. Since the laser 60 is excited selectively only on a certain section, it is not excited and is not detected even if there is a fluorescent source on the other section. By sweeping the laser 60 in the direction of an arrow and applying it to each section and then exciting the fluorescent source and detecting a fluorescent image, the distribution of fluorescent image within the sample 61, namely the fluorescent tomographic image can be measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence tomographic image measuring device.

[0002]

2. Description of the Related Art Conventionally, a device for observing a fluorescent surface image of a biological sample has been developed. FIG. 14 is a diagram showing the configuration of an apparatus for observing a reflected fluorescence image on the sample surface by laser excitation. A laser beam emitted from an argon laser 1 was passed through a filter 2, a lens 3 and an aperture 4 to a sample 10 in which a small hole (1.5 cm in diameter) was exposed in the head of a rat or a gerbil to expose the cerebral cortex. 20
The X axis is set to 1 by the X scan mirror 6 and the Y scan mirror 7 which are driven by the scan driver 5 by narrowing down to about μm.
A region of 1 cm ^{2 on} the brain surface is scanned at 00 Hz and 1 Hz on the Y axis to excite NADH in the brain tissue. The reflected light and fluorescence from the brain surface are received by the lens 11 or the light guide and guided to the photomultiplier 12 through the excitation light cut filter. At this time, a part of the light is received by the photomultiplier 13 through the half mirror, and the differential amplifier 14 obtains the output difference between the two.
Correction is performed to minimize the effects of laser output fluctuation, brain tissue reflection, and blood volume fluctuation. The detection output is sampled by the analyzer 15 in synchronization with the scan of the scan driver 5, the data is processed, and the histogram display 16 is displayed.
, And the detection signal is also displayed on the monitor 17.
Observe the fluorescent surface image. Also, with He-Cd laser, 45
The distribution of the flavoprotein in the cell can also be known by exciting near 0 nm and detecting the emission at 580 nm.

The observing apparatus shown in FIG. 14 is for observing a fluorescence image on the surface of a sample, and an apparatus as shown in FIG. 15 has been proposed for observing a fluorescence image in the depth direction of a sample. FIG. 15 is a diagram showing the configuration of a laser-excited reflection fluorescence image observation apparatus by the confocal method. The beam from the laser 20 is narrowed down by a pinhole 21 and is expanded into a parallel beam by a lens 22. The scanning beam 26 is focused on an objective lens 27 while scanning with a scanning unit 25 through a filter 23 and a half mirror 24. The sample 28 is collected and irradiated on the sample 28. Only the fluorescence from the illumination spot of the sample passes through the half mirror 24 and the filter 29 and passes through the pinhole 29 placed on the back focal plane of the lens 30, and is detected by the detector 30. The detection result is processed by the computer 31. Is displayed on the CRT display 32. The excitation laser light is cut by the filter 30. Since the illumination spot of the sample and the pinhole are in a conjugate relationship, and only the depth-of-focus plane that was previously input to the computer is scanned point by point, the sample does not uniformly irradiate the excitation light and is out of focus. Signal is completely excluded and detected. Therefore, a thick sample such as an untouched tissue or a biological tissue piece can be optically sliced, and the image quality of the internal structure is hardly deteriorated.

FIG. 16 is a diagram showing the configuration of a laser-excited transmission fluorescence image observation apparatus of the confocal system. Excitation laser 4
The laser beam from 0 is focused on the focal point by the condenser lens 41 to irradiate one point of a predetermined depth in the sample, and the irradiation spot is detected by the imaging lens 44 having the front focal plane. By cutting the excitation light with the filter 43, for example, the character "A" having a predetermined depth in the sample can be observed on the observation surface 45, and the lenses 41 and 44 are interlocked to scan in the x, y, z directions. By doing so, the transmitted fluorescence image of the sample 42 can be observed.

FIG. 17 is a diagram showing a laser-excited transmission fluorescence image observation apparatus using a highly directional light receiving system. The present inventor has already proposed this observation device as Japanese Patent Application No. Hei 2-198759, and irradiates a sample 52 with a laser 50 over a predetermined region to excite it. The irradiation of the predetermined area is performed by scanning the laser beam 51, for example. The laser-excited fluorescence of the sample 52 is received by the highly directional optical system 55 via the incident light cut filter 54, the received fluorescence image is detected by the two-dimensional detector 56, and the fluorescence image distribution 53 of the sample 52 is detected. The transmission image of is detected.

Here, the high directivity optical system will be described.
The inventors of the present invention have filed Japanese Patent Application Nos. 1-62898 and 1-2.
In order to separate and extract the plane wave mixed in the scattered light in No. 50034, the 0th order spectrum of the Franforfer diffraction image (Airy disc) of the plane wave (the portion in the first dark ring of the Airy disc) Corresponds.)
It has been shown that it is only necessary to observe only the scattering component, and by doing so, almost all scattering components can be removed. As a highly directional element that realizes such observation, two pinholes P separated from each other as shown in FIG.
_An optical system consisting of ₁ and P ₂ was proposed. This optical system detects the 0th order light by the detector 55a through the pinhole P ₂ .

Further, as shown in FIG. 19, a high directivity optical system which is composed of straight and elongated hollow glass fibers 55b, and whose inner wall surface is coated with a light absorbing material, for example, an absorbing material such as carbon. Proposed element. In such an optical element, if the aperture diameter and length are appropriately set according to the measurement target, and the optical element is made sufficiently longer than the entrance aperture diameter, of the light incident on the highly directional optical element, Only plane waves parallel to the optical axis can be extracted from the exit surface. Moreover, it has been proposed that only a plane wave having a two-dimensional intensity distribution can be taken out by bundling a plurality of such high directional optical elements to form a multi-beam high directional optical system.

By the way, in the high directivity optical element as shown in FIG. 18 and FIG. 19, at the distance at which the Franforfer diffraction image can be observed, the 0th order diffraction image of the Franforfer diffraction (the airy disc (1 dark ring) is larger than the entrance-side opening diameter, and when a take-out opening having the same size as the entrance-side opening diameter is used, only part of the 0th-order diffraction image can be taken out, and the observation point is separated. The first above
It was found that the dark ring becomes larger and the energy taken out from the take-out opening becomes smaller.

Therefore, as shown in FIG. 20, the inlet opening P
_The wave diffracted by _{1 is} made incident on the convex lens L, and the pinhole P having a diameter substantially equal to the first dark ring of the diffraction image on the focal plane thereof.
It has been found that a brighter highly directional optical element can be constructed by arranging ₂ and taking out most of the 0th-order diffraction image. In this element, the diameter of the pinhole P ₂ can be made equal to or smaller than the diameter of the inlet opening. In this case, the entrance opening P ₁ may be the opening P _{0 of the} lens L itself.

FIG. 21 shows an objective lens Ob consisting of a microscope objective lens and a pinhole P arranged in the focal plane of the microscope. The pinhole P is a zero-order diffraction image of Franforfer diffraction by the objective lens Ob. It is intended to pass only.

FIG. 22 (a) shows another form of convex lens GL. This is known as the trade name “SELFOC lens” and is also called a gradient index lens.
The refractive index of this lens gradually decreases from the central axis to the periphery, and acts like a convex lens. By selecting the length appropriately, the focal plane can be made to coincide with the end face of the cylindrical body. Such a gradient index lens G
As shown in FIG. 22B, the focal plane at one end of L
It is also possible to arrange a pinhole P similar to the case of 1 so that only the 0th-order diffraction image of Franforfer diffraction is passed.

By the way, among the optical fibers, a multimode fiber, a gradient index fiber, a single mode fiber, etc. are known. Among them, the single mode fiber has an extremely small core diameter and is incident on the core end face at the incident end. It has a property of transmitting only the above-mentioned light and not transmitting the light forming a large angle with respect to the axis, and can be used in place of the pinhole P of FIGS. 21 to 22B. Moreover, since the diameter of the single-mode fiber is a value that matches the first dark ring of the Franforfer diffraction of the objective lens Ob or the gradient index lens GL, only the 0th-order diffraction image of the Franforfer diffraction is efficiently combined. Convenient for transmission. Further, since the optical fiber is used for the take-out portion, the light can be guided to any place, which is advantageous in arrangement.

FIG. 23 shows an example in which a single mode fiber SM is arranged at the focal point of the objective lens Ob to form a high directivity optical element, and FIG. 24 shows a single mode fiber SM at the focal point of one end of the gradient index lens GL. An example in which the high directional optical element is arranged is shown. In the case of a lens having a long focal length, it is possible to make the first dark ring of the Franforfer diffraction by the lens the same as the diameter of the multimode fiber. For example, put an opening in front of the lens,
The diameter of the first dark ring can be made equal to the diameter of the multimode fiber by decreasing the diameter. In such cases, multimode fibers can also be used.

FIG. 25 is a diagram in which a large number of the highly directional optical elements of FIG. 22 (b) are arranged in parallel, and a large number of similar gradient index lenses GL are regularly arranged in a bale stack in a frame, for example, from black silicon resin. Adhesive B that adheres to each other and prevents light from leaking back through the gap.
The gradient index lens array GA formed in this way
The pinhole array PA is closely attached to the rear surface of the. Each pinhole of the pinhole array PA is provided so as to coincide with the axis of each gradient index lens GL. for that reason,
A plane wave having a two-dimensional intensity distribution from the front of the gradient index lens array GA is the gradient index lens array GA.
When incident on, the intensity of light passing through each pinhole of the pinhole array PA differs according to its distribution. Therefore, the two-dimensional intensity distribution of the plane wave can be measured by disposing a separate photodetector behind each pinhole or by disposing a two-dimensional photointensity detector behind the pinhole array PA.

Further, the multi-beam high directivity optical system of FIG. 26 corresponds to a large number of high directivity optical elements of FIG. 21 arranged in parallel. In this case, instead of arranging the objective lenses in parallel,
A flat plate microlens PM is used. The flat plate microlens PM is produced by regularly arraying fine lenses on a transparent plate by using, for example, a photolithographic method, or by regularly forming the refractive index distribution lens by a method such as ion exchange or ion implantation. It was made in an array. Then, a pinhole array PA having pinholes corresponding to the focal positions of the respective microlenses is formed into a flat plate microlens P.
By disposing it on the focal plane of M, a multi-beam high directional optical system similar to the multi-beam high directional optical system of FIG. 25 can be configured.

The multi-beam high directivity optical system shown in FIG. 27 corresponds to a parallel arrangement of a large number of high directivity optical elements shown in FIG. That is, the gradient index lens array GA described with reference to FIG.
The single-mode fiber array SA formed by arranging a large number of single-mode fibers SM corresponding to the axis of each gradient index lens of the lens array GA is adhered to the rear surface of the pin array PD of FIG. A single mode fiber array SA is used in place of the above to construct an element that performs the same operation.

[0017]

By the way, the reflection fluorescence image observation apparatus shown in FIG. 14 mainly observes the fluorescence image on the surface of the sample, but it is difficult to observe the fluorescence image inside the sample. Further, according to the confocal method reflected fluorescence image and transmitted fluorescence image observation method shown in FIGS. 151 and 16, it is possible to observe the fluorescence source distribution distributed in the depth direction in the sample, but the focal plane is observed. During this time, if fluorescence or scattering occurs at other depths, they will be mixed and observed, and accurate observation will not be possible. For example, when the fluorescence image “A” is observed in FIG. 16, if the fluorescence image “B” has fluorescence or scattering, it is inevitably observed, and thus there is a limit in observation of the tomographic image. Moreover, in the transmitted fluorescence image observation apparatus shown in FIG. 17, the three-dimensional fluorescence image distribution of the sample can be detected only as a two-dimensional distribution. The present invention is intended to solve the above problems, and an object of the present invention is to provide a fluorescence tomographic image measuring apparatus capable of observing a fluorescence source distribution image and a fluorescence tomographic image with high accuracy.

[0018]

A fluorescence tomographic image measuring apparatus of the present invention is provided with an excitation laser for irradiating a sample with an excitation laser beam, and a sample arranged at a position orthogonal to the excitation laser for excitation. Of the fluorescence emitted from the fluorescence source, and a detector for detecting the fluorescence through a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image, and scans the excitation laser to generate the excitation laser. The fluorescence tomographic image is measured by detecting fluorescence emitted in a direction orthogonal to the light.

Further, the fluorescence tomographic image measuring apparatus of the present invention comprises an excitation laser for irradiating a sample with an excitation laser beam and a fluorescence source which is arranged at a position orthogonal to the excitation laser for the sample and is excited. A fluorescence tomographic image is measured by including a detector that detects a fluorescence image through an imaging lens, and scanning the excitation laser to detect fluorescence emitted in a direction orthogonal to the excitation laser light. Characterize.

Further, according to the present invention, at least the excitation laser for irradiating the sample with the excitation laser beam and at least the fluorescence from the excited fluorescence source arranged at a position orthogonal to the excitation laser with respect to the sample. 1st, 2nd which were arrange | positioned in the position which opposes on both sides of the sample which detects fluorescence via the highly directional light-receiving system which takes in the 0th-order light of a Fraunhofer diffraction image.
And a detector for scanning the excitation laser to detect fluorescence from the sample with the first and second detectors and measure a fluorescence tomographic image. An excitation laser light detector which is arranged at a position opposite to the laser and detects the excitation laser light transmitted through the sample through a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image, and from the sample Of the fluorescence wavelength laser which outputs the laser light of the same wavelength as the fluorescence of the first fluorescence, and the first or second detector and the sample are arranged between the fluorescence wavelength laser and the first laser light.
And a half mirror or sector leading to the second detector,
A multiplier for multiplying outputs of the first and second detectors by each other, the attenuation of the excitation laser light is obtained from the detection result of the excitation laser light detector, and the fluorescence wavelength laser is used as the first and second detectors. It is characterized in that the fluorescence attenuation is obtained from the output of the multiplier at the time of detection, and the measured fluorescence tomographic image is corrected.

Further, according to the present invention, an exciting laser for irradiating a sample with an exciting laser beam, and a pump arranged at a position orthogonal to the exciting laser with respect to the sample and in a circular shape centered on the sample, are excited. A plurality of fluorescence detectors for detecting fluorescence through a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image among the fluorescence from the generated fluorescence source. A device for measuring a fluorescence tomographic image by rotating and scanning, which is further arranged at a position on the opposite side of the excitation laser with the sample sandwiched therebetween, and is a highly directional light receiving device for taking in at least the 0th order light of the Fraunhofer diffraction image. An excitation laser light detector that detects excitation laser light that passes through the sample through the system, and a plurality that is arranged on the same circumference as the plurality of fluorescence detectors and that outputs laser light of the same frequency as the fluorescence from the sample Fluorescence decay measurement A laser is provided, and the attenuation of the excitation laser light is obtained from the detection result of the excitation laser light detector, and the laser light from the fluorescence attenuation measurement laser is detected by at least one of the plurality of fluorescence detectors to attenuate the fluorescence. Is obtained, and the measured fluorescence tomographic image is corrected.

Further, according to the present invention, a laser which is arranged on a circumference centered on the sample and irradiates the sample with an excitation laser beam and a laser beam for fluorescence attenuation measurement, and a sample is provided on the side opposite to the laser. And a plurality of detectors which are arranged on the circumference and which transmits at least the excitation laser light passing through the sample through the highly directional light receiving system for taking in the 0th-order light of the Fraunhofer diffraction image and the fluorescence. A device for measuring a fluorescence tomographic image by rotationally scanning the laser and a plurality of detectors,
The excitation laser light is detected by at least one of the plurality of detectors to determine the attenuation of the excitation laser light, the fluorescence attenuation measurement laser light is detected to determine the fluorescence attenuation, and the measured fluorescence tomographic image is corrected. It is characterized by doing.

Further, according to the present invention, a laser which is rotationally scanned on a circumference centered on the sample and irradiates the sample with excitation laser light and laser light for measuring fluorescence attenuation, and the laser is rotated around the sample. The excitation laser light and the fluorescence that pass through the sample are detected through the highly directional light receiving system that is arranged on the circumference having a radius larger than the radius of gyration to be scanned and that takes in at least the 0th-order light of the Fraunhofer diffraction image. An apparatus for measuring a fluorescence tomographic image by rotationally scanning the laser, comprising a plurality of detectors, wherein the plurality of detectors facing the laser detect the excitation laser light to obtain the attenuation of the excitation laser light. At the same time, the laser light for fluorescence attenuation measurement is detected to obtain the fluorescence attenuation, and the measured fluorescence tomographic image is corrected.

Further, according to the present invention, the excitation laser for irradiating the sample with the excitation laser light is arranged at a position orthogonal to the excitation laser with respect to the sample and at a position facing each other with the sample interposed therebetween. An excitation laser, comprising a second imaging lens, first and second detectors arranged at positions where fluorescent images of the sample are respectively formed by the first and second imaging lenses, Device for measuring a fluorescence tomographic image by synchronously scanning the first and second imaging lenses and the first and second detectors and detecting fluorescence from the sample by the first and second detectors Further, the excitation laser light which is arranged at a position opposite to the excitation laser with the sample interposed therebetween and which transmits the sample at least through a highly directional light receiving system for taking in the 0th-order light of the Fraunhofer diffraction image. Excitation laser photodetector that detects light and a laser with the same wavelength as the fluorescence from the sample A fluorescence wavelength laser, a half mirror or a sector arranged between the first or second detector and the sample for guiding the laser light from the fluorescence wavelength laser to the first and second detectors, and a fluorescence wavelength A condenser lens system for condensing laser light from a laser directly on one surface of the first or second detector via a half mirror or a sector and the outputs of the first and second detectors are multiplied. And the attenuation of the excitation laser light based on the detection result of the excitation laser light detector, and the attenuation of the fluorescence due to the multiplier output when the fluorescence wavelength laser is detected by the first and second detectors. It is characterized in that the obtained fluorescence fluorescence tomographic image is corrected.

Further, according to the present invention, the excitation laser for irradiating the sample with the excitation laser light is arranged at a position orthogonal to the excitation laser with respect to the sample and at a position opposed to the sample with the sample interposed therebetween. A device for measuring a fluorescence tomographic image by scanning the excitation laser and detecting fluorescence from the sample by the first and second detectors. An omnidirectional excitation laser light detector arranged at a position opposite to the excitation laser with the sample interposed therebetween, a fluorescence wavelength laser for outputting laser light having the same wavelength as the fluorescence from the sample, and the first or first Multiplying a half mirror or sector arranged between the second detector and the sample, which guides the laser light from the fluorescence wavelength laser to the first and second detectors, and the outputs of the first and second detectors. Equipped with a detector, depending on the detection result of the excitation laser light detector The attenuation of the fluorescence laser light is obtained, and the attenuation of the fluorescence is obtained by the output of the multiplier when the fluorescence wavelength laser is detected by the first and second detectors, and the measured fluorescence tomographic image is corrected. ..

[0026]

According to the present invention, a fluorescence detector is arranged at a position orthogonal to the excitation laser beam for irradiating the sample, and the influence of fluorescence or scattering other than the intended fluorescence image is reduced to measure the fluorescence tomographic image. It is a thing. In particular, a pair of detectors for detecting the fluorescence from the sample are arranged at positions facing each other across the sample to measure a fluorescence tomographic image, and further, the excitation laser light transmitted through the sample is detected to detect the excitation laser light. Calculate the damping. Also, the output from the fluorescence wavelength laser that outputs the laser light of the same wavelength as the fluorescence from the sample is detected by the pair of detectors via the half mirror or the sector, and when the detection outputs are multiplied by the multiplier, the attenuation occurs. Is generated exponentially, the optical path of the fluorescence wavelength laser light becomes the same as the fluorescence optical path at the time of fluorescence tomographic image measurement, and the attenuation of fluorescence is required. You can make corrections.

[0027]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a diagram showing the configuration of a fluorescence tomographic image measuring apparatus using a laser irradiation orthogonal direction high directional light receiving system of the present invention. In this embodiment, detection is performed using a highly directional light receiving system. For example, a sample 61 is irradiated with a laser 60 composed of a diode array linearly arranged,
A fluorescence image is received by a highly directional light receiving system 63 arranged in the direction orthogonal to the laser 60 with respect to the sample 61. That is,
When a certain cross section of the sample is irradiated by the laser 60 to excite the fluorescence source, the fluorescence is radiated in all directions. Of these, the fluorescence emitted in the direction orthogonal to the excitation laser light is two-dimensionally passed through the highly directional light receiving system 63. It is detected by the detector 64. Since the laser selectively excites only a certain cross section, even if there is a fluorescence source in another cross section, it is not excited and is not detected. It is possible to measure the fluorescence image distribution in the sample, that is, the fluorescence tomographic image, by running the laser 60 in the direction of the arrow in the figure, irradiating each section with laser light to excite the fluorescence source, and detecting the fluorescence image. it can.

FIG. 2 is a view showing the arrangement of a fluorescence tomographic image measuring apparatus using the laser irradiation orthogonal direction imaging lens light receiving system of the present invention. In the present embodiment, detection is performed using an imaging optical system, and detection is performed by a detector arranged in the direction orthogonal to the laser 60 with respect to the sample 61 as in FIG. That is, the fluorescence image 62 of a certain cross section excited by the laser light from the laser 60 is imaged and detected on the observation surface 71 by the imaging lens 70 arranged in the orthogonal direction. In the present embodiment, when the laser 60 is swept, the imaging lens 70 is also swept in synchronization with this to detect the fluorescent image in each cross section of the sample and measure the fluorescent image distribution in the sample. You can

By the way, in the method shown in FIGS. 1 and 2, the excitation light emitted from the laser is attenuated before exciting the fluorescence source, and the fluorescence emitted from the fluorescence source is attenuated before reaching the detector. .. As shown in FIG. 3A, the laser 70
A detector 72 that irradiates the sample 71 with excitation laser light of wavelength λ ₁ and that is arranged in a direction orthogonal to the laser 70
Consider the case where fluorescence of wavelength λ ₂ is detected at. The direction in which the detector arrays are arranged is the x direction, and, for example, the excitation laser light of wavelength λ ₁ is attenuated to form a curve 73 in FIG. 3B, and the fluorescence of wavelength λ ₂ is attenuated. Curve 74 of (c)
As a result, when the data obtained when neither excitation light nor fluorescence is attenuated is the curve 75 of FIG. 4D, the measured projection data is attenuated as the curve 76.

That is, the pump laser light whose intensity is Iλ ₁ as shown in FIG. 4A is exponentially attenuated as shown by a curve 73 by propagation of a predetermined distance, and as shown in FIG. 4B. Thus, the fluorescence having the intensity of Iλ ₂ is attenuated as shown by the curve 74 when it reaches the detector, and as a result, as shown in FIG.
As shown in (c), the data 75 that should be obtained when there is no attenuation is attenuated as shown by the curve 76. As described above, even if the excitation light and the detection system are orthogonally arranged with respect to the sample, a more accurate fluorescence distribution image and fluorescence tomographic image cannot be measured unless the attenuation of the excitation light and the fluorescence are considered. ..
Such attenuation is caused by absorption and scattering in the sample for both excitation light and fluorescence, and is exponentially attenuated.

FIG. 5 uses a highly directional light receiving system,
It is a figure which shows the structure of the highly directional light-receiving system fluorescence tomographic image measurement apparatus of this invention which correct | amends the attenuation of fluorescence. For example,
Wavelength λ ₁ consisting of linearly arranged diode arrays
The excitation laser 80 that emits the laser light of 1 is used as the excitation light source, and this is scanned and the sample 81 is sliced and excited by irradiation. A pair of high-directivity light receiving systems 82a, 82 sandwiching the sample in a direction orthogonal to the excitation laser 80 with respect to the sample 81.
b, the two-dimensional detectors 83a and 83b, and the fluorescence wavelength transmission filters 84a and 84b are arranged, and the fluorescence wavelength cut filter 84c, the highly directional light receiving system 82c, and the two are disposed at positions facing the excitation laser 80 with the sample 81 interposed therebetween. The dimension detector 83c is arranged.

When laser light of wavelength λ ₁ is irradiated by the excitation laser 80, the fluorescence source in the cross section irradiated with the laser is excited and fluorescence of wavelength λp is emitted in all directions, and the fluorescence wavelength transmission filters 84a and 84b. And the fluorescent images are received by the highly directional light receiving systems 82a and 82b, respectively, and are detected as fluorescent images 85a and 85b by the two-dimensional detectors 83a and 83b. On the other hand, in the highly directional light receiving system 82c, the fluorescence having the wavelength λp is cut by the fluorescence wavelength cut filter, and the light having the wavelength λ ₁ which has passed through the sample among the excitation laser light is received.
An image 85c is detected by the two-dimensional detector 83c.

As described with reference to FIGS. 3 and 4, the fluorescent image 85
Each of a, 85b and the excitation light image 85c is attenuated in its propagation optical path. For the exponential decay of the excitation light, the intensity of the emitted light in the excitation laser 80 is known, and if the position of the fluorescence source is known by the two-dimensional detectors 83a and 83b, the distance from the emission position to the fluorescence source can be known.
The amount of attenuation can be calculated from the measurement value of the two-dimensional detector 83c. Since the sample is usually an extremely thin sample, the attenuation amount in the two-dimensional detector 83c may be used as the attenuation amount up to the fluorescence source.

On the other hand, since the fluorescence is emitted in all directions, the fluorescence becomes small in inverse proportion to the square of the distance, and is attenuated exponentially by absorption and scattering. Therefore, as shown in FIG. 6, the distance between the two-dimensional detectors is L, the two-dimensional detector 3a and the fluorescent light source 81 are
the distance to a is x ₁ , the fluorescence intensity at the fluorescence source is I ₀ ,
If the attenuation coefficient is μλ _p (x), the two-dimensional detector 83
a, respectively fluorescence intensity I _1, I ₂ detected by _{83b, I 1 = (I 0} / x 1 2) exp (-∫ 0 x1 μλ p · dx) I 2 = (I 0 / (L-x ^{_{1) 2) exp (-∫ x1}} L μλ p · dx) , and the multiplication value obtained by the multiplier _{92, I 1 × I 2 = Kexp} (-∫ 0 x1 μλ p · dx) · exp (-∫ x1 ^L μλ _p · dx) = Kexp (−∫ ₀ ^L μλ _p · dx) ……………………… (1). Where K = I ₀ ² / x ₁ ² (L−x ₁ ) ²
x ₁ is a value determined by the position of the excitation laser.

Therefore, from the fluorescence wavelength laser 90 having the same wavelength λp as the fluorescence wavelength from the fluorescence source, one of the laser beams is directly transmitted to the high directivity light receiving system 82a and the two-dimensional detector 83a by the half mirror or the sector 91. The other is sample 81
The high-directivity light receiving system 82b and the two-dimensional detector 83b are irradiated through. When the position of the half mirror is set to the position of x ₂ , the detection values I ₁ ′ and I ₂ ′ at each detector are I ₁ ′ = aI ₀ exp (−∫ ₀ ^x2 μλ _p · dx) I ₂ ′ = aI ₀ becomes ^{_{exp (-∫ x2 L μλ p ·}} dx). However, a = 1/2 for a half mirror and a = 1 for a sector. Multiplication value obtained by the multiplier _{92, I 1 '×'I 2 =} (aI 0) 2 exp (-∫ 0 x2 μλ p · dx) · exp (-∫ x2 L μλ p · dx) = (aI 0 ^{_{) 2 exp (-∫ 0 L μλ}} p · dx) , and the obtained as ^{_{exp (-∫ 0 L μλ p ·}} dx) = I 1 '×'I 2 / (aI 0) 2 ...... (2). Since aI ₀ is known here,
By multiplying the detection output when the fluorescence wavelength laser 90 is used by the expression (2) by the output multiplier 92, the attenuation amount at the time of actual measurement shown by the expression (1) can be obtained.

Therefore, it is possible to accurately measure the fluorescence source distribution image and the fluorescence tomographic image by obtaining the excitation light attenuation amount and the fluorescence attenuation amount and correcting the actual measurement values.

FIG. 7 is a diagram showing an embodiment in which an excitation laser and a fluorescence detector are arranged orthogonally to each other and attenuation correction is performed to measure a fluorescence tomographic image.

A two-dimensional detector 105, in which a laser for excitation 100, a plurality of lasers for measuring fluorescence decay 101, and a plurality of detectors 102 are arranged in a circular shape around a sample 106, and a highly directional light receiving system is used, is provided. It is arranged on the side opposite to the excitation laser 100 with respect to the sample 106. In the fluorescence tomographic image, the sample 106 is excited by the excitation laser 100, the fluorescence emitted in the direction orthogonal to the excitation laser light is detected by the detector 102, and the excitation laser 100 and the detector 102 are sequentially rotated in the clockwise direction. Thus, a fluorescence tomographic image is obtained.

The attenuation of the excitation light is obtained by receiving the light with the highly directional light receiving system 103, cutting the fluorescence component with the fluorescence wavelength cut filter 104, and detecting only the excitation laser light with the two-dimensional detector 105. On the other hand, as for the fluorescence attenuation, if fluorescence of the same wavelength is emitted by the fluorescence attenuation measurement laser 101 and detected by the opposing detector, the attenuation in the optical path corresponding to the diameter of the circular arrangement is obtained, and this is the tomographic image. It is the same as the optical path of fluorescence at the time of measurement, and the measured tomographic image can be corrected from the obtained attenuation.

FIG. 8 shows an RR using a highly directional light receiving system.
It is a figure which shows the Example of the fluorescence tomographic image measuring apparatus by the (Rotation-Rotation) imaging system. In the present embodiment, the excitation / attenuation measurement laser 110 and the highly directional detector 112 provided with a filter 111 on the front surface are arranged in a circular shape with the sample 113 as the center, and the excitation / attenuation measurement laser is used. The 110 and the high directivity detector 112 are rotated clockwise, for example, to capture a tomographic image. An excitation laser (wavelength λε) is used to capture a fluorescence tomographic image.
Is irradiated to the sample, the laser and the detector are rotated, and the fluorescence (wavelength λp) from the sample is detected at each position. The excitation light attenuation is obtained by detecting the excitation laser light with the detector on the opposite side, and the fluorescence attenuation is detected by emitting the fluorescence of wavelength λp with the attenuation measurement laser and detecting with the detector on the opposite side. Is obtained in the same manner as in the case of FIG.

FIG. 9 is a diagram showing an embodiment of a fluorescence tomographic image measuring apparatus by the RR imaging system using a highly directional light receiving system whose directionality is variable. In this embodiment, a laser for excitation / attenuation measurement 120 composed of a linearly arranged diode array or the like is rotatably arranged around a sample 122, and a circular filter 123 around the sample 122 is arranged outside thereof.
Is arranged, and a highly directional light receiving system 124 whose directional direction is variable is arranged outside thereof. For example, when the parallel beam 121 is emitted from the laser 120 as shown in the figure, the light receiving element (which belongs to the range A in the figure) for receiving the parallel beam is controlled so as to direct in the direction of the parallel beam 121. , Fluorescence is emitted from the sample in all directions, so that the element that receives this is controlled so as to face the sample.

In such a structure, the fluorescence tomographic image of the sample irradiates the sample 122 with the excitation laser light while rotating the laser 120, and the fluorescence from the sample is received by the highly directional light receiving system 124 via the filter 123. It is measured by The attenuation of the excitation laser light is obtained by receiving the filter 123 as a fluorescence cut filter by the high-directional light receiving system on the opposite side, and the attenuation of the fluorescence is obtained by using the attenuation measuring laser light on the high-directional light receiving system on the opposite side. It can be obtained in the same manner as in the case of FIG.

FIG. 10 uses a lens imaging system to generate excitation light,
It is a figure which shows the structure of the lens imaging system fluorescence tomographic image measuring apparatus of this invention which correct | amends the attenuation of fluorescence. For example, a pumping laser 130 that emits laser light having a wavelength λ ₁ and is composed of linearly arranged diode arrays is used as a pumping light source, and this is scanned to slice and excite a sample 131. A pair of image forming lenses 132a and 132a sandwiching the sample in a direction orthogonal to the excitation laser 130 with respect to the sample 131.
b, and the fluorescence image is transmitted through the fluorescence wavelength transmission filter 133a,
An image is formed on the surfaces of the detectors 134a and 134b through the 133b and detected. When scanning the excitation laser, it is necessary to scan the imaging lens in synchronization with the scanning.

The excitation light is attenuated by the detectors 134a and 134b when the excitation light transmitted through the sample is received by the highly directional light receiving system 135 via the fluorescence wavelength cut filter 133c and detected by the two-dimensional detector 134c. It can be obtained because the position of the fluorescence image in the excitation light direction can be known.

On the other hand, the fluorescence is attenuated by the laser 1 for attenuation correction.
From 36, a laser beam having the same wavelength as the fluorescence from the sample is guided to the sample through the lens 137 and the half mirror 137 and the other to the detector 134a. At this time, lens 1
By making the rear focal plane of 37 conjugate with the fluorescent image position of the sample with respect to the imaging lens 132a, the laser light for attenuation measurement is focused on the fluorescent image position of the sample, and this is used as the lens 1
At 32b, an image is formed on the detector 134b and detected. Also,
The light guided to the detector 134a is detected by inserting the lens 139 indicated by the broken line in the figure so that the detection surface of the detector 134a is in a conjugate relationship with the rear focal plane of the lens 137. By doing so, it is possible to detect the laser light of the same wavelength that has passed the same optical path as the fluorescent optical path at the time of actual measurement. Therefore, by multiplying this detection output by the multiplier 140, the result in FIG. The fluorescence decay can be determined as described.

FIG. 11 is a diagram showing an embodiment of a fluorescence tomographic image measuring apparatus in which an omnidirectional light receiving system is used to perform attenuation correction of excitation light and fluorescence. Excitation laser (wavelength λ ₁ )
When the laser light from 150 is irradiated to a certain cross section of the sample 151, the fluorescence from the excited fluorescence source diffuses to the surroundings.
When this is detected through the fluorescence wavelength transmission filter by the pair of two-dimensional detectors 153a and 153b arranged in the direction orthogonal to the excitation laser with respect to the sample, for example, the fluorescence image of the character "A" is detected. By scanning the excitation laser 150, a fluorescence tomographic image at each slice level is observed.

When the excitation light is attenuated, the fluorescence wavelength cut filter 152c cuts the fluorescence from the light transmitted through the sample, and the laser light is detected by the two-dimensional detector 153c facing the excitation laser. Attenuation up to this point is obtained, and on the other hand, since the position of the fluorescence image is known by the detectors 153a and 153b, the attenuation up to the fluorescence source can be obtained.

For the attenuation of the fluorescence, one is detected by the detector 153a and the other is detected by the detector 153b through the half mirror or the sector 155 of the laser light having the same wavelength as the fluorescence emitted from the laser 154.

As shown in FIG. 12, the distance from the sample center to each detector is R, the position of the fluorescence source is x from the sample center, and the sample length is L. R is sufficiently larger than L. As shown in FIG. 13, the decay due to R starts from outside the sample and is assumed to be constant within the sample, and the fluorescence source ρ (x)
Assuming that the attenuation coefficient due to absorption and scattering in the sample of μ _p (x) and the fluorescence intensities detected by the detectors 153a and 153b are I ₁ and I ₂ , I ₁ = ρ (x) · exp (- ^{_{∫ x L / 2 μ p (}} x) · dx) / (R-x) 2 I 2 = ρ (x) · exp (-∫ -x L / 2 μ p (x) · dx) / (R + x) 2 The size L of the sample is L << R compared to the distance R of the detector, and since x ≦ L, I ₁ and I ₂ are I ₁ ≈ρ (x) · exp (−∫ _x ^{L / 2} μ _p (x) · dx) / R ² I ₂ ≈ρ (x) · exp (−∫ _-x ^{L / 2} μ _p (x) · dx) / R ² The output product of the omnidirectional detector is I here ^{_{1 × I 2 ≒ ρ 2 (}} x) · exp (-∫ -L / 2 L / 2 μ p (x) · dx) / R 4, exp (-∫ -L / 2 L / 2 μ p ( x) · dx) is determined by the size of the sample, and can be obtained from the attenuation value when the laser light from the laser 154 of which the intensity is known is detected by the detectors 153a and 153b. In this way, since the attenuation of the excitation light and the attenuation of the fluorescence are known, the fluorescence tomographic image measured in the same manner can be corrected.

[0050]

As described above, according to the present invention, the fluorescence detector is arranged at a position orthogonal to the excitation laser beam for irradiating the sample, and the influence of fluorescence or scattering other than the intended fluorescence image is reduced. It is possible to measure the fluorescence tomographic image by performing the measurement, and in particular, it is possible to measure the fluorescence tomographic image with high accuracy by correcting the fluorescence tomographic image measured by obtaining the attenuation of the excitation light and the attenuation of the fluorescence. ..

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a fluorescence tomographic image measuring apparatus using a laser beam irradiation orthogonal direction high directional light receiving system.

FIG. 2 is a diagram showing a configuration of a fluorescence tomographic image measuring apparatus using a laser irradiation orthogonal direction imaging lens light receiving system.

FIG. 3 is a diagram illustrating attenuation of measurement projection data due to attenuation of excitation light and fluorescence.

FIG. 4 is a diagram illustrating attenuation of measurement projection data due to attenuation of excitation light and fluorescence.

FIG. 5 is a diagram showing an embodiment of the present invention in which the attenuation of excitation light and fluorescence is corrected by using a highly directional light receiving system.

FIG. 6 is a diagram illustrating correction of attenuation of excitation light and fluorescence.

FIG. 7: The excitation laser and the fluorescence detector are arranged orthogonally,
It is a figure which shows the Example which has arrange | positioned the laser for fluorescence attenuation measurement on the same circumference as a fluorescence detector.

FIG. 8 is a diagram showing an embodiment of a fluorescence tomographic image measuring apparatus by an RR imaging method using a highly directional light receiving system.

FIG. 9: R using a highly directional light receiving system with variable pointing direction
It is a figure which shows the Example of the fluorescence tomographic image measuring apparatus by the -R imaging system.

FIG. 10 is a diagram showing a configuration of a fluorescence tomographic image measurement apparatus that uses a lens imaging system and corrects attenuation of excitation light and fluorescence.

FIG. 11 is a diagram showing an embodiment of a fluorescence tomographic image measuring apparatus in which an omnidirectional light receiving system is used to perform attenuation correction of excitation light and fluorescence.

FIG. 12: Excitation light when an omnidirectional light receiving system is used,
It is a figure explaining the attenuation correction of fluorescence.

FIG. 13 is a diagram illustrating attenuation characteristics depending on distance.

FIG. 14 is a diagram showing a configuration of an apparatus for observing a reflection fluorescence image of a sample surface by laser excitation.

FIG. 15 is a diagram showing a configuration of a laser-excited reflection fluorescence image observation device by a confocal method.

FIG. 16 is a diagram showing a configuration of a laser-excited transmission fluorescence image observation apparatus by a confocal method.

FIG. 17 is a diagram showing a laser-excited transmission fluorescence image observation device using a highly directional light receiving system.

FIG. 18 is a diagram showing a highly directional optical element using a pinhole.

FIG. 19 is a view showing a highly directional optical element in which a light absorbing material is applied to the inner wall surface of a hollow glass fiber.

FIG. 20 is a diagram showing a highly directional optical element using a convex lens and a pinhole.

FIG. 21 is a diagram showing a highly directional optical element using an objective lens and a pinhole.

FIG. 22 is a diagram showing a highly directional optical element using a gradient index lens and a pinhole.

FIG. 23 is a diagram showing a highly directional optical element using an objective lens and a single mode fiber.

FIG. 24 is a diagram showing a highly directional optical element using a gradient index lens and a single mode fiber.

FIG. 25 is a diagram showing a highly directional light receiving system in which a large number of highly directional optical elements of FIG. 22 (b) are arranged in parallel.

FIG. 26 is a diagram showing a highly directional light receiving system in which a large number of highly directional optical elements of FIG. 21 are arranged in parallel.

FIG. 27 is a diagram showing a highly directional light receiving system in which a large number of highly directional optical elements of FIG. 24 are arranged in parallel.

[Explanation of symbols]

80 ... Excitation laser, 81 ... Sample, 82a, 82b, 8
2c ... High-directional light receiving system, 83a, 83b, 83c ... Two-dimensional detector, 84a, 84b ... Fluorescent wavelength transmission filter, 8
4c ... Fluorescent wavelength cut filter, 85a, 85b ... Fluorescent image, 90 ... Fluorescent wavelength laser, 91 ... Half mirror or sector, 92 ... Output multiplier.

Claims (9)

[Claims] 1. A pumping laser for irradiating a sample with a pumping laser beam, and a sample arranged at a position orthogonal to the pumping laser,
Of the fluorescence from the excited fluorescence source, it is equipped with a detector that detects the fluorescence via a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image. A fluorescence tomographic image measuring device, which measures a fluorescence tomographic image by detecting fluorescence emitted in a direction orthogonal to a laser beam. 2. A pumping laser for irradiating a sample with a pumping laser beam, and a sample arranged at a position orthogonal to the pumping laser,
It is equipped with a detector that detects the fluorescence image of the excited fluorescence source through an imaging lens, and scans the excitation laser to detect the fluorescence emitted in a direction orthogonal to the excitation laser light. A fluorescence tomographic image measuring device characterized by measuring an image. 3. A pumping laser for irradiating a sample with a pumping laser beam, and a sample arranged at a position orthogonal to the pumping laser,
Of the fluorescence from the excited fluorescence source, the first is arranged at a position facing each other with a sample for detecting fluorescence being detected via a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image. A device for measuring fluorescence tomographic images by scanning the excitation laser by scanning the excitation laser with the first and second detectors, and further comprising: And an excitation laser light detector which is arranged at a position opposite to the excitation laser and which detects the excitation laser light passing through the sample through at least the highly directional light receiving system that captures the 0th-order light of the Fraunhofer diffraction image. A fluorescence wavelength laser that outputs a laser beam having the same wavelength as the fluorescence from the sample; and a first and second detection of the laser beam from the fluorescence wavelength laser, which is arranged between the first or second detector and the sample. Half mirror to guide the vessel or A sector and a multiplier for multiplying the outputs of the first and second detectors are provided, and the attenuation of the excitation laser light is obtained based on the detection result of the excitation laser light detector, and the fluorescence wavelength laser is used for the first and second fluorescence wavelength lasers. A fluorescence tomographic image measuring device, characterized in that the attenuation of fluorescence is obtained from the output of the multiplier when detected by the detector, and the measured fluorescence tomographic image is corrected. 4. A pumping laser for irradiating a sample with a pumping laser beam, and a fluorescent light which is arranged at a position orthogonal to the pumping laser with respect to the sample, and which is arranged circumferentially around the sample. A plurality of fluorescence detectors for detecting fluorescence through a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image among the fluorescence from the light source, and the plurality of fluorescence detectors are rotationally scanned. Is a device for measuring a fluorescence tomographic image, further comprising a highly directional light receiving system which is arranged at a position opposite to the excitation laser with the sample in between and which captures at least the 0th-order light of the Fraunhofer diffraction image. An excitation laser light detector that detects excitation laser light that passes through the sample, and a plurality of fluorescence attenuations that are arranged on the same circumference as the plurality of fluorescence detectors and that output laser light of the same frequency as the fluorescence from the sample Measuring laser and Comprising, the attenuation of the excitation laser light is obtained from the detection result of the excitation laser light detector, and the laser light from the fluorescence attenuation measurement laser is detected by at least one of the plurality of fluorescence detectors to obtain the fluorescence attenuation, A fluorescence tomographic image measuring apparatus, characterized in that the measured fluorescence tomographic image is corrected. 5. A laser which is arranged on a circumference centered on the sample and irradiates the sample with excitation laser light and laser light for measuring fluorescence decay, and a laser on the opposite side of the laser to the sample. An excitation laser beam that passes through a sample through a highly directional light receiving system that captures at least the 0th-order light of a Fraunhofer diffraction image, and a plurality of detectors that detect fluorescence. And a device for measuring a fluorescence tomographic image by rotationally scanning a plurality of detectors, wherein excitation laser light is detected by at least one of the plurality of detectors to obtain attenuation of the excitation laser light, and fluorescence attenuation measurement is performed. A fluorescence tomographic image measuring apparatus, characterized in that the laser light for use is detected, the attenuation of fluorescence is obtained, and the measured fluorescence tomographic image is corrected. 6. A laser which is rotatively scanned on a circumference centered on the sample and irradiates the sample with excitation laser light and laser light for measuring fluorescence decay, and the laser is rotatively scanned around the sample. A plurality of detections for detecting excitation laser light and fluorescence, which are arranged on a circumference having a radius larger than the radius of gyration and are transmitted through the sample through a highly directional light receiving system that captures at least the 0th-order light of the Fraunhofer diffraction image. And a device for measuring a fluorescence tomographic image by rotationally scanning the laser, wherein the excitation laser light is detected by a plurality of detectors facing the laser, and the attenuation of the excitation laser light is obtained. An apparatus for measuring a fluorescence tomographic image, characterized in that a laser beam for attenuation measurement is detected to determine the attenuation of fluorescence, and the measured fluorescence tomographic image is corrected. 7. The fluorescence tomographic image measuring apparatus according to claim 6, wherein the direction of the highly directional light receiving system is drive-controlled in a direction of a laser beam to be detected. 8. A pumping laser for irradiating a sample with a pumping laser beam, and first and second pumps arranged at a position orthogonal to the pumping laser with respect to the sample and facing each other across the sample. Image forming lens, and first and second detectors arranged at the positions where the fluorescence images of the sample are formed by the first and second image forming lenses, respectively. The second imaging lens and the first and second detectors are synchronously scanned to detect the fluorescence from the sample in the first and second directions.
A device for measuring a fluorescence tomographic image by detecting with a second detector, which is further arranged at a position opposite to the excitation laser with the sample interposed therebetween and at least the 0th-order light of the Fraunhofer diffraction image is An excitation laser light detector for detecting an excitation laser light transmitted through the sample via a highly directional light receiving system to be taken in, a fluorescence wavelength laser for outputting a laser light having the same wavelength as the fluorescence from the sample, and the first or second Is placed between the detector and the sample and guides the laser light from the fluorescence wavelength laser to the first and second detectors, and the laser light from the fluorescence wavelength laser directly through the half mirror or sector. A condensing lens system that condenses light on one surface of the first or second detector, and a multiplier that multiplies the outputs of the first and second detectors. Depending on the result A fluorescence tomographic image characterized in that the attenuation of fluorescence is determined by the output of the multiplier when the fluorescence wavelength laser is detected by the first and second detectors, and the measured fluorescence tomographic image is corrected. Image measuring device. 9. A pumping laser for irradiating a sample with a pumping laser beam, and first and second pumps arranged at a position orthogonal to the pumping laser with respect to the sample and facing each other across the sample. And an omnidirectional detector for scanning the excitation laser to detect fluorescence from the sample with the first and second detectors and measure a fluorescence tomographic image. An omnidirectional excitation laser light detector arranged on the opposite side of the excitation laser, a fluorescence wavelength laser that outputs laser light of the same wavelength as the fluorescence from the sample, and the first or second detection A half mirror or a sector that is arranged between the detector and the sample and guides the laser light from the fluorescence wavelength laser to the first and second detectors, and a multiplier that multiplies the outputs of the first and second detectors. Equipped with the pump laser based on the detection result of the pump laser photodetector. A fluorescence tomographic image characterized in that the attenuation of fluorescence is determined by the output of the multiplier when the fluorescence wavelength laser is detected by the first and second detectors, and the measured fluorescence tomographic image is corrected. Image measuring device.