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WO2019059379A1 - Dispositif d'examen d'un corps biologique et procédé afférent - Google Patents

Dispositif d'examen d'un corps biologique et procédé afférent Download PDF

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Publication number
WO2019059379A1
WO2019059379A1 PCT/JP2018/035198 JP2018035198W WO2019059379A1 WO 2019059379 A1 WO2019059379 A1 WO 2019059379A1 JP 2018035198 W JP2018035198 W JP 2018035198W WO 2019059379 A1 WO2019059379 A1 WO 2019059379A1
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Prior art keywords
light
fluorescence
time
living body
excitation
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English (en)
Japanese (ja)
Inventor
吾朗 西村
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Hokkaido University NUC
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Hokkaido University NUC
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Priority to JP2019543744A priority Critical patent/JPWO2019059379A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to a biopsy apparatus and a biopsy method.
  • the lymph node detection device described in Patent Document 1 is one example thereof, in which excitation light is irradiated to a living body including a lymph node in the vicinity of a tumor in which a fluorescent dye has been injected in advance, and fluorescence from the living body is thereby obtained. An image for detecting a lymph node is acquired.
  • An object of the present invention is to provide a living body inspection apparatus and a living body inspection method capable of grasping the intensity of fluorescence from a fluorescent dye embedded in a living body with high sensitivity.
  • the present inventors focused on the following points in order to effectively improve the sensitivity for detecting fluorescence from a fluorescent dye. That is, the fluorescence from the fluorescent dye from deep to the tissue surface tends to diffuse, and the intensity per unit area decreases. Then, background light due to fluorescence or the like from the tissue itself becomes relatively larger than the fluorescence from the fluorescent dye. This makes fluorescence indistinguishable from background light.
  • the present inventors focused on the fact that even if the background light is relatively large relative to fluorescence, the difference in the time change mode (waveform) of these intensities tends to appear in the measurement results.
  • the temporal change mode of the background light intensity can be obtained, for example, by measurement in advance using a sample, measurement at a position sufficiently away from the position where the fluorescent dye is embedded, or the like.
  • the living body inspection apparatus of the present invention obtained by focusing on the above points comprises: a light source for irradiating excitation light for exciting the fluorescent dye to the living body in which the fluorescent dye is embedded; and light emitted from the living body
  • the light detection means for detecting the intensity, the time change aspect of the intensity of the emitted light including the fluorescence generated by the fluorescent dye due to the irradiation of the excitation light to the living body by the light source, and the background for the fluorescence
  • a fluorescence evaluation means for evaluating the degree of difference between light and at least one of the intensities of light with time change mode of the intensity at a wavelength of the fluorescence, and an evaluation result for perceptually outputting the degree evaluated by the fluorescence evaluation means And output means.
  • the present inventor decided to provide a means for evaluating the degree of difference between the time change aspect of the intensity of the light emitted from the living body and the time change aspect of the intensity of the background light as one means for evaluating the fluorescence. . Then, the degree of the difference is perceptually output.
  • the above difference in the time change mode is caused by the generation of fluorescence from the fluorescent dye.
  • the extent of the difference serves as an index showing the intensity of fluorescence from the fluorescent dye. Therefore, the user can recognize the intensity of the fluorescence from the fluorescent dye based on the output result according to the degree of the difference.
  • the present invention includes, as another means related to the fluorescence evaluation means, a means for evaluating the degree of difference between the time change aspect of the intensity of the light emitted from the living body and the time change aspect of the intensity of the fluorescence from the fluorescent dye. It may be This means corresponds to the above-described means for evaluating the degree of difference between the time change aspect of the intensity of the light emitted from the living body and the time change aspect of the intensity of the background light.
  • the difference between the temporal change of the intensity of the light emitted from the living body and the temporal change of the intensity of the background light is the time change of the intensity of the emitted light from the living and the intensity of the fluorescence from the fluorescent dye.
  • the difference with the time change aspect of is small (the similarity between the two is high).
  • standard of evaluation can be acquired by prior measurement, for example.
  • “evaluating at the wavelength of fluorescence” corresponds to evaluating the component of the fluorescence wavelength in the light to be evaluated.
  • the component of the fluorescence wavelength is physically extracted from the light to be evaluated using, for example, an optical filter.
  • the component of the fluorescence wavelength may be extracted by calculation from the time response function of the light to be evaluated.
  • the background light decays faster than the fluorescence decay, and the fluorescence evaluation means generates the fluorescence during a period after the background light becomes maximum. It is preferable to evaluate the degree based on the emitted light within a predetermined period which is a period.
  • the predetermined period is set to a period after the timing when the background light is maximum. Background light decays faster than fluorescence, so the period after the timing when the background light is maximum tends to be a period where the difference between the waveform of the fluorescence and the waveform of the background light is large. Therefore, based on the emitted light from the living body during this period, the intensity of the fluorescence from the fluorescent dye is likely to be appropriately evaluated.
  • the predetermined period includes a timing at which the ratio of the intensity of the emitted light including the fluorescence to the intensity of the background light is maximum. According to this, based on the emitted light from the living body in this period, the intensity of the fluorescence from the fluorescent dye can be more appropriately evaluated.
  • the light detection means can detect the incidence of photons, and the fluorescence evaluation means is based on the timing at which the light source irradiates the living body with the excitation light as a reference.
  • the excitation / detection probe has a contact surface in contact with the surface of the living body, and the irradiation portion of the excitation light and the light receiving portion of the emission light from the living body are formed on the surface of the contact surface. It is preferable to further comprise According to this, while being able to irradiate excitation light appropriately towards a living body, it is possible to appropriately receive light emitted from a range irradiated with excitation light in the living body.
  • the contact surface is curved so as to protrude toward the living body. According to this, the adhesion of the excitation / detection probe to the living body is improved. Therefore, it is difficult for the excitation light emitted from the irradiation unit to leak to the light receiving unit.
  • a switch is provided on the excitation / detection probe, and switching between a state in which the excitation light is emitted and a state in which the excitation light is not emitted from the irradiation portion of the excitation light Is preferred. According to this, it is easy to switch on / off of the irradiation of the excitation light by the operation.
  • a contact surface is in contact with the surface of the living body, the irradiation portion of the excitation light and the light receiving portion of the emission light from the living body are formed on the surface of the contact surface, and a switch is provided. It is preferable to further include an excitation / detection probe, and to change the predetermined period based on the operation status of the switch.
  • the setting of the predetermined period affects the fluctuation (contrast) of change with respect to the measurement position in the photon counting result. For this reason, when grasping the position of the fluorescent dye based on the relationship between the measurement position and the photon counting result, it is possible to adjust the ease of the grasping by the switch operation.
  • a switch for changing conditions other than a predetermined period may be provided instead of or in addition to the above switch.
  • the evaluation result output means outputs at least one of a character, an image and a sound expressing the degree. According to this, the evaluation result is perceptible to the user by at least one of characters, images and sounds.
  • the fluorescence evaluation unit evaluates the degree based on the correlation between the time change aspect of the intensity of the emitted light and the time change aspect serving as a reference.
  • the time change aspect of background light is used as a reference time change aspect
  • the time change aspect of the fluorescence from a fluorescent dye is used as a time change aspect used as a reference
  • standard it turns out that the fluorescence from a fluorescent dye is strongly appeared by the emitted light from a biological body, so that correlation is high.
  • the correlation is used to obtain an appropriate evaluation result.
  • a living body inspection method is a method of inspecting a living body by evaluating light emitted from a living body in which a fluorescent dye is embedded, which is caused by irradiating the living body with excitation light.
  • the degree of difference between the time change aspect of the intensity of the emitted light including the fluorescence generated by the fluorescent dye and the time change aspect of the intensity of at least one of the background light and the fluorescence with respect to the fluorescence is the fluorescence Evaluate at the wavelength of
  • the degree of difference between the time variation aspect of the intensity of the emitted light from the living body and the time variation aspect of the fluorescence from the fluorescent dye and / or the background light is evaluated. did.
  • the above difference in the time change mode is caused by the generation of fluorescence from the fluorescent dye.
  • the extent of the difference serves as an index showing the intensity of fluorescence from the fluorescent dye. Therefore, based on the degree of the difference, the intensity of the fluorescence from the fluorescent dye can be appropriately evaluated.
  • the intensity of fluorescence from a fluorescent dye can be properly grasped.
  • FIG. 6 (a) is an image showing the change with respect to the position of the total numerical value of photons during the measurement period of 90 seconds.
  • FIG. 6 (b) is an image showing the change with respect to the position of the photon count value limited within the period corresponding to the time gate in the measurement period of 90 seconds.
  • FIG. 8A is a graph showing the result of counting the number of times of detection of photons by the photon detector with respect to the elapsed time from the incident timing of the excitation light (pulsed light).
  • FIG. 8 (b) is a graph showing the background ratio. It is a graph which shows the change of the count value with respect to a measurement position at the time of changing a time gate variously based on the simulation result of FIG. FIG.
  • FIGS. 14 (a) and 14 (b) is a front view of a modification of the excitation / detection probe according to the first to third embodiments.
  • the detection principle of the fluorescent dye that is the basis of each embodiment according to the present invention will be described, and then the specific configuration of the present embodiment based on the principle will be described.
  • the basic idea for improving the detection sensitivity of the fluorescence from the fluorescent dye in each embodiment according to the present invention is the time response (light of light) emitted from the living body after the excitation light for the fluorescent dye is incident on the living body
  • the time-varying aspect of the intensity is to utilize the difference between the fluorescence from the fluorescent dye and the background light.
  • the waveform means a waveform showing a time response (for example, a waveform showing a time change of the number of detected photons). 1) Quantitatively measure the difference between the shape of the time response of the background light and the shape of the time response of the light emitted from the subject.
  • fmeas (ti) is a function of the measurement value of the number of photons emitted from the subject after the excitation light for the fluorescent dye is incident on the subject.
  • the waveform indicated by fmeas (ti) is taken as a measurement waveform.
  • fref (ti) is a function of the reference waveform value.
  • w (ti) is a weighting function and ⁇ is a scaling factor. [Equation 1]
  • ⁇ is a coefficient value for causing D to be small (ideally to be 0) when the waveform shapes match even if the values themselves differ between the measured value and the reference waveform value. .
  • the absolute value may vary.
  • ⁇ is determined so as to minimize D.
  • the value of D ⁇ 2 increases as the measured waveform differs from the shape of the reference waveform.
  • fback indicating the waveform shape of background light is used as fref.
  • D 2 According to the distance of the measurement position from the fluorescent dye, it is understood by D 2 that fmeas deviates from fback.
  • the method using D ⁇ 2 can be said to be a method of effectively subtracting the background light contribution from the measurement value.
  • the method based on D2 satisfies the above requirements 1) and 3) simultaneously.
  • w (ti) used in the determination of ⁇ different from w (ti) used in the final evaluation of D of the measured values In this case, D ⁇ 2 in the final evaluation of the measured values is not minimized, but it is effective to determine w (ti) so that background light can be efficiently suppressed.
  • Equation 2 In the detection using Equation 1, one of the simplest methods is to set fref to 0, and set w to the following weight w0 for extracting the time domain [tmin, tmax] where the difference in waveform appears most It is. [Equation 2]
  • this D is used to detect fluorescence by comparing the difference between the measured value and the background light value.
  • the fmeas natural sum may be used. That is, a value obtained by adding fmeas from tmin to tmax may be used. That is, the time gating method itself may be used. Also, in these cases, instead of using fmeas respectively, the ratio fmeas / fback of the measurement value to the value of the background light may be used.
  • D ⁇ 2 represents a squared deviation from the reference waveform
  • D represents an average difference in intensity.
  • D approaches 0 as the shape of the measurement waveform approaches the background light waveform shape.
  • the background light waveform needs to be known in advance, but in general, the time to the incidence of the excitation light to the subject before administering the fluorescent dye to the subject and the tissue opposite to the affected area to be measured It is possible to know by measuring the response.
  • D can not be used as it is because the similarity is seen.
  • a correlation coefficient may be used as an evaluation criterion different from Equation 1.
  • similarities or differences can be assessed. Since the value of the correlation coefficient is limited to [-1, 1], using the correlation coefficient only satisfies the above requirements 1) or 2). In order to satisfy the above requirement 3), it is necessary to combine the correlation coefficient with Equation 1.
  • ⁇ 0 is an upper limit value or an ideal upper limit value 1 estimated from data substantially matching background light.
  • rho fmeas, ffluo
  • L changes the rho 0-rho of the right side of equation 4 to rho-rho 0 and then It can be used for evaluation as well.
  • L may be rephrased to be the intensity of the average difference between the waveform of background light or fluorescence from fluorescent dye and the measured waveform in consideration of the waveform shape of background light or fluorescence from fluorescent dye.
  • the first embodiment relates to a method using the time gating method described above.
  • the biological inspection apparatus 1 includes a fluorescent probe 2, a light source 3, a photon detector 4 (light detection means), an excitation / detection probe 10, a time-resolved measurement unit 20 (detection time acquisition means), an analysis unit 30 (counting means), control A unit 40, a sound generation unit 7, and a display unit 8 (evaluation result output means) are provided.
  • the function of the fluorescence evaluation means in the present invention is realized by the functions of both the time-resolved measurement unit 20 and the analysis unit 30 in the present embodiment.
  • fluorescent dye of the fluorescent probe 2 for example, indocyanine green is used. Indocyanine green is used for fluorescence angiography of sentinel lymph nodes.
  • the fluorescent probe 2 is embedded in the human body of a subject (subject to be examined).
  • the light source 3 is an emitter of pulse laser light having a pulse width of picosecond or femtosecond scale.
  • the pulsed laser light includes a light pulse having an excitation wavelength of the fluorescent dye of the fluorescent probe 2.
  • the pulsed laser light is composed of an optical pulse train in which light pulses are continuously arranged at predetermined pulse intervals. For example, a fiber laser is used as the light source 3 and the light source 3 is caused to generate picosecond pulse light with a wavelength of 785 nm continuous at a pulse interval corresponding to 10 MHz.
  • the pulse interval is sufficiently longer than the fluorescence lifetime of the fluorescent dye (indocyanine green).
  • a timing signal indicating the timing of emitting each light pulse is output from the light source 3 to the time-resolved measurement unit 20.
  • an optical fiber 14 is connected to the light source 3 via an optical filter 5.
  • the other end of the optical fiber 14 is connected to the excitation and detection probe 10.
  • the pulse laser light emitted from the light source 3 enters the optical fiber 14 through the optical filter 5.
  • the optical filter 5 transmits light having the excitation wavelength of the fluorescent dye of the fluorescent probe 2 while blocking light of wavelengths other than the excitation wavelength. Thus, only excitation light for exciting the fluorescent dye of the fluorescent probe 2 is transmitted to the excitation / detection probe 10 through the optical fiber 14.
  • indocyanine green When indocyanine green is used as a fluorescent dye, its excitation light is light having a near infrared wavelength around 700 nm.
  • the excitation / detection probe 10 has a cylindrical holder 11 having an outer diameter of about 1 cm, and an irradiating unit 12 and a light receiving unit 13 provided on the tip surface 11 a (contact surface) of the holder 11. , Switches 17 and 18.
  • the excitation / detection probe 10 is used in contact with human tissue and in a state where the tip surface 11a is in close contact with the tissue.
  • the holder 11 has a shape suitable for holding the entire probe by hand to scan a subject.
  • the irradiation unit 12 emits the excitation light transmitted from the light source 3 through the optical fiber 14. When the emitted excitation light is irradiated to the fluorescent probe 2 embedded in the human body, the fluorescent dye of the fluorescent probe 2 generates fluorescence.
  • the light receiving unit 13 receives outgoing light emitted from the subject.
  • the light received by the light receiving unit 13 is the fluorescence generated by the fluorescent dye of the fluorescent probe 2 and the background serving as the background for the fluorescence It will contain the light.
  • Background light is component light having a fluorescence wavelength of a fluorescent dye that is derived from other than the fluorescent dye of the fluorescent probe 2. Background light includes, for example, fluorescence from the subject's tissue itself. In the following, “fluorescence” means fluorescence generated by the fluorescent dye of the fluorescent probe 2 unless otherwise noted.
  • optical fiber 16 One end of an optical fiber 16 is connected to the excitation / detection probe 10.
  • the other end of the optical fiber 16 is connected to the photon detector 4 via an optical filter 6 (see FIG. 1).
  • the optical filter 6 transmits light having the fluorescence wavelength of the fluorescent dye of the fluorescent probe 2 while blocking light having a wavelength other than the fluorescence wavelength. Of the light received by the light receiving unit 13, only component light having a fluorescence wavelength reaches the photon detector 4 through the optical fiber 16.
  • the switch 17 is a switch that switches on / off of the function of generating pulse laser light in the light source 3.
  • a signal indicating the state of the switch 17 is output to the light source 3.
  • the light source 3 generates pulse laser light or stops the generation based on a signal indicating the state of the switch 17. Since the switch 17 can be operated by the user, it is easy to perform the on / off switching operation of the pulse laser beam.
  • the switch 18 is a switch for adjusting a time gate, which will be described later.
  • a signal indicating the state of the switch 18 is output to the control unit 40.
  • the control unit 40 controls the analysis unit 30 based on a signal indicating the state of the switch 18 as described later.
  • the photon detector 4 is a photon counting detector that detects the incidence of photons from the optical fiber 16 on a photon basis. The detection result of the incidence of photons is output to the time-resolved measurement unit 20 for each detection of one photon.
  • the time-resolved measurement unit 20 is a time-digital converter.
  • the timing signal from the light source 3 is input to the time-resolved measurement unit 20.
  • the time-resolved measurement unit 20 generates a digital value indicating the time when the detection result is output from the photon detector 4 (the elapsed time from the latest reference timing), using each timing indicated by the timing signal as a reference timing.
  • the digital value is output to the analysis unit 30. This digital value is output from the photon detector 4 as the detection result indicating the incidence of photons from the timing when the light pulse is emitted from the light source 3, that is, the timing when the excitation light is irradiated to the human body from the excitation / detection probe 10. Timing, that is, the time until the photon is detected in the photon detector 4.
  • the analysis unit 30 includes digital comparators 32 and 33, a gate circuit 34, a counter 35, and a time reference generator 36, as shown in FIG.
  • Digital values from the time-resolved measurement unit 20 are input to digital comparators 32 and 33 respectively.
  • the digital comparator 32 outputs to the gate circuit 34 a signal indicating the result of comparing the digital value from the time-resolved measurement unit 20 with the upper limit value.
  • the digital comparator 33 outputs to the gate circuit 34 a signal indicating the result of comparing the digital value from the time-resolved measurement unit 20 with the lower limit value.
  • the upper limit value and the lower limit value are input from the control unit 40 as described later.
  • the gate circuit 34 outputs a signal indicating the logical product of the signal from the digital comparator 32 and the signal from the digital comparator 33 to the counter 35. This logical product indicates whether the digital value from the time-resolved measurement unit 20 is in the range from the lower limit value to the upper limit value.
  • the time reference generator 36 generates a reference signal every unit time (for example, 100 milliseconds) and outputs the reference signal to the counter 35.
  • the counter 35 counts the number of times the digital value from the time-resolved measurement unit 20 falls within the range from the lower limit value to the upper limit value based on the signal from the gate circuit 34.
  • the counting is performed for each period having the length of the unit time based on the reference signal from the time reference generator 36.
  • the counting result is output to the control unit 40 each time a unit time passes. Each counting result indicates the number of times per unit time that the digital value from the time-resolved measurement unit 20 is in the range from the lower limit value to the upper limit value.
  • the control unit 40 is constructed by a combination of a computer and software.
  • the computer is recorded in a memory device such as hardware including a memory device such as a central processing unit (CPU), a read-only memory (ROM) and a random access memory (RAM), and various interfaces such as an input / output interface.
  • a memory device such as a central processing unit (CPU), a read-only memory (ROM) and a random access memory (RAM), and various interfaces such as an input / output interface.
  • software comprising program data and the like.
  • hardware executes various information processing such as arithmetic processing and input / output processing according to software, thereby realizing various functions in the control unit 40 described below.
  • the control unit 40 outputs the upper limit value and the lower limit value to the digital comparators 32 and 33 of the analysis unit 30.
  • the lower limit corresponds to the start timing of the time gate
  • the upper limit corresponds to the end timing of the time gate.
  • This time gate is set to a period in which the difference between the waveform of fluorescence and the waveform of background light is large, as shown in the first experimental example described later.
  • the time gate is set at a wavelength of fluorescence, which is a period after the timing at which background light is maximized and in which fluorescence is generated.
  • the time gate is a period including the timing at which the incident light to the photon detector 4 is maximized relative to the background light (the timing at which the ratio to the background described later is maximized), and the background light is It is preferable to set to the period which does not include the timing which becomes the maximum. Alternatively, the time gate may be set to a period in which the fluorescence is maximum but not including the timing in which the background light is maximum. Further, the control unit 40 adjusts the time gate according to the state of the switch 18 based on the signal output from the excitation / detection probe 10. For example, the control unit 40 changes the start timing (that is, the lower limit value) of the time gate according to the state of the switch 18.
  • control unit 40 records the counting result output from the counter 35 of the analyzing unit 30 in a recording device such as a hard disk, and controls the sound generating unit 7 and the display unit 8 according to the counting result. Specifically, the control unit 40 causes the sound generation unit 7 to generate a sound having a size corresponding to the counting result, and causes the display unit 8 to perform display corresponding to the counting result.
  • the sound generation unit 7 generates a sound of the size and timbre according to the instruction of the control unit 40, and outputs the sound from the speaker. For example, the control unit 40 instructs the sound generation unit 7 to output a larger sound as the counting result from the counter 35 is larger.
  • the display unit 8 displays the counting result from the counter 35 on the display using various expressions such as numerical values and graph images according to the instruction of the control unit 40.
  • the counting result from the counter 35 indicates the number of times per unit time that the digital value from the time-resolved measurement unit 20 is in the range from the lower limit value to the upper limit value. That is, the counting result indicates the number of times per unit time that the photon detector 4 has detected photons in the time gate.
  • the time gate is a period in which the difference between the waveform of the fluorescence and the waveform of the background light is largely generated. Therefore, the counting result from the counter 35 strongly reflects the intensity of the fluorescence.
  • the user can recognize the intensity of the fluorescence by perceiving the size and timbre of the sound generated by the sound generation unit 7.
  • the user can search for the position of the fluorescent probe 2 by following the change of the sound from the sound generator 7 while moving the excitation / detection probe 10. For example, when the sound from the sound generation unit 7 is getting louder, it is known that the excitation / detection probe 10 is approaching the fluorescent probe 2. In addition, when the sound from the sound generation unit 7 becomes smaller, it is understood that the excitation / detection probe 10 is moved away from the fluorescent probe 2. Note that, instead of or in addition to the sound from the sound generation unit 7, the display content of the display unit 8 may be used for searching for the position of the fluorescent probe 2.
  • the user can change the time gate by operating the switch 18 of the excitation and detection probe 10.
  • the setting of the time gate affects the behavior for the measurement position in the photon counting result as shown in the first experimental example described later.
  • the variation degree of the counting result with respect to the change of the measurement position changes due to the difference of the time gate. Therefore, when the position of the fluorescent probe 2 is grasped by following the change of the sound from the sound generation unit 7 while moving the excitation / detection probe 10, the ease of the grasping is adjusted by the operation of the switch 18. It is possible.
  • the voice or the like is output based on the result of counting the number of times the photon incident is detected in the time gate by the photon detector 4.
  • the time gate is set as a period in which the difference between the waveform of fluorescence and the waveform of background light is large, as shown in a first experimental example described later. Therefore, the intensity of fluorescence is likely to be reflected in the above-mentioned counting result.
  • the emitted light from the subject and the background light are evaluated by evaluating the intensity of the emitted light from the subject during the gate period, which is a period during which the difference between the waveforms of the fluorescence and the background light is large.
  • the user can recognize the intensity of the fluorescence generated from the fluorescent probe 2 in the subject's body from the output result. If the intensities of fluorescence at a plurality of positions in the subject are known, as described above, the relationship between those positions and the position of the fluorescent probe 2 can be grasped. Thus, according to the present embodiment, it is possible to detect fluorescence from a relatively deep position from the tissue surface and to allow the user to recognize the position of the fluorescent probe 2 from the intensity.
  • the probe P1 shown in FIG. 4 was used for irradiation of excitation light and detection of fluorescence.
  • the probe P1 has outlets A and B for emitting laser light, and inlets CH1 and CH2 for receiving light from a sample. The distance between these exit and entrance is 10 mm.
  • laser light to be excitation light for a fluorescent target picosecond pulse light generated at a pulse interval corresponding to 10 MHz and a wavelength of 785 nm generated from a fiber laser was used. This laser beam is transmitted to the emission ports A and B formed in the probe P1 through an optical fiber.
  • Excitation light was irradiated to the sample by emitting laser light from the emission ports A and B while bringing the emission ports A and B into contact with the sample.
  • One end of a bundle fiber of 3 mm in diameter was connected to the entrances CH1 and CH2.
  • the other end of the bundle fiber was connected to the photon detector 4 via the optical filter 6.
  • the light incident on the entrances CH 1 and CH 2 is transmitted through the bundle fiber, passes through the optical filter 6, and is incident on the photon detector 4.
  • emitted from a sample and a fluorescence target was measured using the time correlation single photon detection system. This was repeated while changing the measurement position with respect to the sample. Specifically, the measurement was repeated while translating the probe P1 in parallel by 5 mm in each of the x direction and the y direction of the xy plane set for the sample. The measurement at each measurement position was performed by holding the probe P1 at that position for 90 seconds.
  • Each graph in FIG. 5 counts the number of times a photon is detected by the photon detector 4 with respect to the elapsed time from the incident timing of each pulse for all the pulses of the laser light irradiated to the sample during the 90 second period. The results are shown.
  • the horizontal axis in FIG. 5 indicates the elapsed time from the incident timing of each pulse, and the vertical axis in FIG. 5 indicates the number of photons per bin.
  • the graph g1 shows the counting result of the background light (counting result at the measurement position farthest from the fluorescent target).
  • Graphs g2 to g4 show three counting results at different measurement positions closer to the fluorescent target than graph g1.
  • the graph g0 shows the time response function of the measuring device itself. As shown in FIG.
  • the graph g1 showing the time response of background light has a faster decay than the graphs g2 to g4 showing the time response of light containing fluorescence. That is, background light decays faster than fluorescence.
  • “Fast decay” indicates, for example, that the time from the timing when the intensity peaks to the time when the intensity becomes half or an appropriate ratio with respect to the peak time is short.
  • 6 (a) and 6 (b) are gray scale images showing the counting results for each position in the xy plane. Gray scale indicates that the higher the tonal value, the larger the photon count.
  • FIG. 6 (a) corresponds to the total numerical value of photons during the entire 90 seconds
  • FIG. 6 (b) shows the time gate indicated by the line segment G in FIG.
  • the tone values are relatively large over the entire range of the gray scale image of FIG. 6 (a). That is, even at the measurement position away from the target, the counting result does not decrease much as compared with the measurement position close to the target. Thus, the total number of photons during the entire 90 second period results in low contrast.
  • FIGS. 7 (a) and 7 (b) The results of determining the background ratio in this manner are shown in FIGS. 7 (a) and 7 (b).
  • the square points are the ratio of background to background obtained from the total number of photons during the entire period of 90 seconds at every 5 mm position.
  • the round dots correspond to the contrast obtained from the photon counts limited within the time gate G during the 90 second period.
  • Graphs g5 and g7 are the results of fitting square points.
  • Graphs g6 and g8 are results of fitting round points. As shown in FIGS.
  • the use of the time gate G increased the ratio to background by about six times. This indicates that when a time gate is used, the presence of a fluorescent target can be clearly distinguished from background light when the measurement position is brought close to the target while scanning the tissue surface.
  • the time gate G is set to be a period after the timing when the background light is maximum at the wavelength of fluorescence and a period in which fluorescence is generated. More specifically, time gate G is a period including the timing at which light incident on photon detector 4 is at a maximum relative to background light, and includes the timing at which background light is at a maximum. There is no period set.
  • the other gray solid lines show three time responses at different measurement locations. Also, based on these time response function data, the result of similarly obtaining the background ratio at each measurement position is shown in FIG. 8 (b).
  • the horizontal axis of FIG. 8 (b) shows the elapsed time from the incident timing of each pulse in the laser light, and the vertical axis of FIG. 8 (b) shows the ratio to background.
  • the background ratio here is the time response function at each side fixed position divided by the time response function of background light.
  • the graph which used the measurement data of measurement time 90 second as it is is displayed on the upper right of each of FIG. 8 (a) and FIG.8 (b) for comparison.
  • the ratio to the background is maximum around 2 nanoseconds.
  • the 100 ms data lacks statistical properties around 2 nanoseconds, and the ratio to background is maximum at a position slightly faster than 2 nanoseconds. From this, it is considered that a section having a relatively large ratio to the background may be set as the time gate, including around 2 nanoseconds where the ratio to the background is maximum.
  • candidate segments of the time gate are shown in FIG. 8 G1: [0.797 nanoseconds: 3.2972 nanoseconds] (2.5 nanoseconds wide), G2: [1.497 nanoseconds: 2.497 nanoseconds] (1 nanosecond width), G3: [1.747 nanoseconds: 2.247 nanoseconds] (0.5 nanosecond width).
  • FIG. 9 (a) and 9 (b) show the change to the position of the photon counts limited within these time gates and the change to the position of the photon counts without time gates.
  • Inverted triangle points, circle points, square points and triangle points correspond to no gate, gate G1, gate G2 and gate G3, respectively.
  • Each solid line is the result of fitting these by Formula 5, respectively. It can be seen that the number of photons is maximized when the gate is not used and becomes smaller as the gate width becomes narrower. Therefore, the statistical variation of the signal becomes larger as the gate width becomes narrower.
  • FIG. 10 (a) and 10 (b) show the change to the position in the background to background ratio based on the count of photons limited within the time gates G1 to G3 and the back to the count of photons not using the time gate.
  • the change to the position in the ground ratio is shown.
  • the triangle point, the inverted triangle point, the round point and the square point in FIG. 10A correspond to the gate G3, the gate G2, the gate G1 and no gate, respectively.
  • the triangle points, the square points, the round points, and the inverse triangle points in FIG. 10B correspond to the gate G3, the gate G2, the gate G1, and no gate, respectively. Contrary to the case of FIG. 9, higher contrast was obtained when the time gate was applied. In addition, even if the gate width is 0.5 nanosecond wide, the contrast is not improved as compared with the 1.0 nanosecond width. From the above results, it is concluded that the width of the time gate may be greater than 1.0 nanosecond. Furthermore, in practice, it is preferable to use the time gate determined as described above as an initial setting value and to optimize the time gate in clinical settings such that the change in contrast with position is the largest.
  • the second embodiment relates to a method of using the Euclidean distance D shown in Formula 1 instead of using the time gate of the first embodiment.
  • the biological examination apparatus according to the second embodiment is mainly different from the biological examination apparatus according to the first embodiment in the configuration of the analysis unit 30.
  • the analysis unit according to the second embodiment will be mainly described.
  • the analysis unit of the second embodiment calculates D in accordance with Equation 1 based on the digital value from the time-resolved measurement unit 20.
  • Fmeas in Equation 1 indicates the result of counting (counting) the number of times of photon detection by the photon detector 4 during a predetermined period and the elapsed time from the timing when each light pulse is emitted by the light source 3.
  • fmeas corresponds to, for example, the graphs g2 to g4 in FIG. 5 and the gray solid line graph in FIG. 8 (a).
  • fref, w and ⁇ have values determined according to the method described above in the description of the principle.
  • the analysis unit outputs the calculated D to the control unit 40.
  • the control unit 40 causes the sound generation unit 7 to generate a sound corresponding to the size of D, and causes the display unit 8 to perform display corresponding to the size of D.
  • the waveform data fmeas used for the evaluation is data on a waveform (waveform shown in FIG. 8A) corresponding to the measurement time of 100 milliseconds generated from the measurement data in the first experimental example.
  • the evaluation section was a time section covering the entire range of the waveform.
  • the measurement result at the position farthest from the fluorescent target was used for the waveform fback of the background light which is the reference fref.
  • w is a constant 1 / N (N is the number of time data).
  • ⁇ was decided to minimize D ⁇ 2.
  • FIG. 11 shows the change with respect to the position at D calculated in this manner.
  • the result in FIG. 11 shows the fluorescent target position with a little contrast as compared with the steady light, that is, the simple integral value of the time response function (the value indicated by the square point in FIG. 10). This situation will be further improved if the measurement time is longer than 100 milliseconds.
  • the method using Equation 1 and the method using a time gate may be used in combination.
  • the third embodiment relates to a method using a correlation function shown in Formula 4 in place of the time gate of the first embodiment.
  • the analysis unit according to the third embodiment calculates L in accordance with Equation 4 based on the digital value from the time-resolved measurement unit 20 and outputs the calculated value to the control unit 40.
  • the calculation of D is based on the same method as the calculation method in the second embodiment.
  • the control unit 40 causes the sound generation unit 7 to generate a sound corresponding to the size of L and causes the display unit 8 to display according to the size of L as in the first embodiment.
  • the waveform data fmeas used for the evaluation is data on a waveform (waveform shown in FIG. 8A) corresponding to the measurement time of 100 milliseconds created from the measurement data in the first experimental example.
  • the evaluation section was a time section covering the entire range of the waveform.
  • the measurement result at the position farthest from the fluorescent target was used for the waveform fback of the background light which is the reference fref.
  • w is a constant 1 / N (N is the number of time data).
  • ⁇ 1.
  • the correlation ⁇ in Equation 4 was calculated using Pearson's correlation coefficient.
  • FIG. 12 shows the change with respect to the position at L calculated in this manner.
  • the result in FIG. 12 shows the position of the fluorescent target with good contrast, as in the first experimental example using a time gate.
  • the value of the position where the measurement waveform is close to the background light waveform is almost 0, and the contrast is very good.
  • the distribution width of L is narrow. Therefore, the method using Equation 4 is considered to be effective for detection of a fluorescent probe.
  • the similarity of the time response function between human tissue and the sample used in the first experiment was confirmed as follows.
  • the graph g10 of FIG. 13 shows the time response function obtained by measuring the time response in the same manner as the first experimental example without embedding the fluorescent dye in the human upper arm.
  • Graph g11 is the result of using beef.
  • the graphs g10 and g11 are normalized so that the waveforms match each other near the peaks. It can be seen that although the decay of the time response function was somewhat faster in humans compared to beef, both show approximately the same decay. As shown in FIG. 13, it is convenient in terms of separating background light and fluorescence that the background light decays in the case of humans faster than in the case of beef.
  • the fluorescence time response decays more slowly than the background light time response.
  • the excitation / detection probe 110 shown in FIG. 14A or the excitation / detection probe 120 shown in FIG. 14B may be used instead of the excitation / detection probe 10 in the above-described embodiment.
  • the excitation / detection probe 110 has a holder 111 from which a tip surface 111 a protrudes.
  • the tip surface 111a is smoothly curved.
  • the irradiation unit 12 and the light receiving unit 13 are provided on the front end surface 111 a.
  • optical filters 5 and 6 are provided on the tip surface 111 a of the excitation / detection probe 110.
  • the laser light emitted from the irradiation unit 12 is irradiated onto the living body through the optical filter 5.
  • the optical filters 5 and 6 are provided on the tip surface 121 a of the holder 121.
  • a laser diode 103 and a photon detector 104 are provided in the holder 121. If the source of excitation light and the photon detector can be miniaturized, they may be thus provided in the excitation / detection probe.
  • the laser light emitted from the laser diode 103 is irradiated to the subject through the irradiation unit 12 and the optical filter 5.
  • Light emitted from the living body enters the photon detector 104 through the optical filter 6 and the light receiving unit 13.
  • the analysis unit 30 is constructed using a digital circuit such as the digital comparator 32 or the like.
  • the analysis unit may be constructed by a combination of computer and software.
  • the software may cause the computer to function so as to play the same role as the analysis unit 30.
  • the result of counting by the counter 35 that is, the output of voice or numerical value corresponding to the number of times of detection of photons per unit time is performed.
  • output may be made according to the background ratio.
  • the control unit 40 calculates the to-background ratio based on the counting result of the counter 35.
  • the sound generation unit 7 generates a sound having a magnitude corresponding to the calculated background ratio.
  • the ratio to the background is calculated by dividing the counting result of the counter 35 by the background level b.
  • a value calculated in advance using Equation 5 is used based on the measurement value measured in advance according to the time gate.
  • G G1 to G3 Time gate 1 Biological examination device 2 Fluorescent probe 3 Light source 4 Photon detector 5, 6 Optical filter 7 Audio generation unit 8 Display unit 10, 110, 120 Excitation / detection probe 12 Irradiation unit 13 Light receiving unit 17 , 18 switches 20 time-resolved measurement unit 30 analysis unit 40 control unit

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Abstract

Un objet de la présente invention est de déterminer, à une sensibilité élevée, l'intensité de la lumière fluorescente émanant d'un fluorochrome incorporé dans un corps biologique. Une source de lumière 3 émet, par l'intermédiaire d'un filtre optique 5 et d'une sonde d'excitation/détection 10, une lumière d'excitation dirigée sur un sujet incorporé dans une sonde fluorescente 2. La lumière sortant du sujet pénètre dans un détecteur de photons 4 par l'intermédiaire de la sonde d'excitation/détection 10 et d'un filtre optique 6. Quand un photon est détecté par le détecteur de photons 4, une unité de mesure à résolution temporelle 20 délivre un signal de synchronisation de détection à une unité d'analyse 30. L'unité d'analyse 30 compte le nombre de détections de photons effectuées par le détecteur de photons 4 pendant une période équivalant à une fenêtre temporelle prescrite, en fonction de la sortie de l'unité de mesure à résolution temporelle 20. Une unité de commande 40 amène une unité de génération de son 7 à émettre un son à un volume qui correspond au compte effectué par l'unité d'analyse 30.
PCT/JP2018/035198 2017-09-21 2018-09-21 Dispositif d'examen d'un corps biologique et procédé afférent Ceased WO2019059379A1 (fr)

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