WO2016075804A1 - Appareil d'observation d'organisme, dispositif d'alimentation en liquide pharmaceutique, et procédé d'observation d'organisme - Google Patents

Appareil d'observation d'organisme, dispositif d'alimentation en liquide pharmaceutique, et procédé d'observation d'organisme Download PDF

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Publication number
WO2016075804A1
WO2016075804A1 PCT/JP2014/080154 JP2014080154W WO2016075804A1 WO 2016075804 A1 WO2016075804 A1 WO 2016075804A1 JP 2014080154 W JP2014080154 W JP 2014080154W WO 2016075804 A1 WO2016075804 A1 WO 2016075804A1
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WIPO (PCT)
Prior art keywords
chemical solution
unit
refractive index
index distribution
living body
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PCT/JP2014/080154
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English (en)
Japanese (ja)
Inventor
洋平 谷川
真一 瀧本
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オリンパス株式会社
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Priority to PCT/JP2014/080154 priority Critical patent/WO2016075804A1/fr
Publication of WO2016075804A1 publication Critical patent/WO2016075804A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements

Definitions

  • the present invention relates to a living body observation apparatus, a chemical solution supply apparatus, and a living body observation method.
  • an observation method in which a scatterer that obstructs fluorescence detection is physically removed (see, for example, Patent Document 1).
  • the skull is physically excised to form an opening to expose the brain tissue to be observed.
  • the opening By using the opening as an optical window, fluorescence generated in the brain tissue can be efficiently collected.
  • Patent Document 1 The observation method disclosed in Patent Document 1 is a method in which the skull is physically excised to form an opening, so that it is highly invasive to the living body and places a burden on the living body. In addition, it may be a difficult procedure to remove the skull itself. Furthermore, since an opening is formed in the skull, the physiological state such as brain pressure changes, and the living body cannot be observed in a normal physiological state.
  • the present invention has been made in view of the above-described circumstances, and is a living body observation apparatus and a chemical solution that can easily observe a living body in a normal physiological state while reducing the burden on the living body.
  • An object of the present invention is to provide a supply device and a living body observation method.
  • One embodiment of the present invention includes a light detection unit that detects light, and a biological solution that is disposed so as to face the light detection unit so that a chemical solution penetrates and the refractive index distribution of the biological tissue is uniformized.
  • the light detection unit is disposed opposite to the outer surface of the biological tissue covering the observation target, and the chemical target is infiltrated into the biological tissue in the region facing the light detection unit to denature the observation target.
  • the biological tissue is made transparent to suppress scattering, thereby observing without removing the biological tissue. be able to. This makes it possible to easily observe a living organism in a normal physiological state while reducing the burden on the organism.
  • the determination unit determines the degree of penetration of the chemical solution in the living tissue, and adjusts the penetration rate according to the degree of penetration of the chemical solution. It is possible to prevent excessive penetration. Alternatively, the time required for penetration can be shortened by increasing the rate of penetration of the chemical solution.
  • the permeation adjustment unit determines that the refractive index distribution of the living tissue is sufficiently uniformed by the determination unit, the permeation rate of the chemical solution by the scattering suppression unit is decreased. May be. By doing in this way, it can prevent that an excess chemical
  • the said determination part may determine the grade of homogenization of refractive index distribution by the penetration time of the said chemical
  • the said determination part detects the boundary surface which optically detects the boundary surface of the area
  • a degree of homogenization of the refractive index distribution may be determined according to the distance between the boundary surface detected by the boundary surface detection unit and the outer surface of the living tissue.
  • the boundary surface detection unit can detect the boundary surface of the region where the refractive index distribution is made non-invasively from the outside of the living tissue, and more accurately make the refractive index distribution uniform. The degree can be determined.
  • the said determination part is provided with the thickness detection part which optically detects the thickness of the said biological tissue, According to the ratio of the thickness detected by this thickness detection part, and the said distance The degree of homogenization of the refractive index distribution may be determined. In this way, it is possible to more accurately determine the degree of homogenization of the refractive index distribution regardless of individual differences in the thickness of the living tissue.
  • the determination unit determines the degree of homogenization of the refractive index distribution according to the contrast of the intensity of light from the observation target detected by the light detection unit through the living tissue. You may judge. By doing so, the contrast of the intensity of light from the observation target increases as the refractive index distribution becomes more uniform, so it is possible to easily determine the degree of penetration of the chemical solution according to the contrast. it can.
  • permeation adjustment part may adjust the viscosity of the said chemical
  • permeation adjustment part may adjust the temperature of the said chemical
  • medical solution may have photocurability
  • the said penetration control part may irradiate an ultraviolet-ray to the said chemical
  • permeation adjustment part may replace
  • a chemical solution that is disposed on the outer surface of the living tissue and permeates the living tissue to denature the living tissue so that its refractive index distribution is uniform is brought into contact with the outer surface of the living tissue.
  • a chemical solution supply apparatus including a chemical solution holding unit that holds the liquid solution in a state of being held and a viscosity adjusting unit that adjusts the viscosity of the chemical solution held in the reagent holding unit.
  • the chemical solution holding unit by holding the chemical solution in contact with the outer surface of the biological tissue by the chemical solution holding unit, the chemical solution can be continuously infiltrated into the biological tissue, and the refractive index distribution of the biological tissue can be made uniform.
  • the viscosity adjusting unit is operated to increase the viscosity of the chemical solution, thereby reducing the penetration rate into the living tissue, so that the chemical solution is not excessively permeated. Can be.
  • the observation target covered with the biological tissue can be easily and non-invasively observed through the transparent biological tissue.
  • the said viscosity adjustment part may adjust the temperature of the said chemical
  • medical solution may have photocurability, and the said viscosity control part may irradiate the light for hardening to the said chemical
  • medical solution adjustment part may replace
  • a transparentizing step in which a chemical solution is applied to an outer surface of a living tissue covering an observation target and denatured so that the refractive index distribution of the living tissue is uniformed by the penetration of the chemical solution;
  • a determination step for determining the degree of homogenization of the refractive index distribution by the transparentization step; and, as a result of the determination by the determination step, if it is determined that the refractive index distribution is sufficiently uniform, the transparency
  • An osmotic adjustment step for reducing the penetration rate of the chemical solution in the step, and the biological tissue coated with the biological tissue through the biological tissue denatured in the clearing step in a state in which the penetration rate is reduced in the osmosis adjustment step.
  • a detection step for detecting light from the observed object.
  • the determination step may determine the degree of homogenization of the refractive index distribution based on the penetration time of the chemical solution in the clearing step. Further, in the above aspect, the determination step is performed by optically detecting a boundary surface of a region where the refractive index distribution is uniform formed in the living tissue due to the penetration of the chemical solution by the transparentization step. The degree of homogenization of the refractive index distribution may be determined according to the distance between the boundary surface and the outer surface of the living tissue.
  • the determination step optically detects the thickness of the living tissue, and determines a degree of homogenization of the refractive index distribution according to a ratio between the detected thickness and the distance. May be. Moreover, in the said aspect, the said determination step may determine the degree of homogenization of the said refractive index distribution according to the contrast of the intensity
  • permeation adjustment step may adjust the temperature of the said chemical
  • medical solution may have photocurability, and the said osmosis
  • permeation adjustment step may replace
  • FIG. 1 is an overall configuration diagram schematically showing a living body observation apparatus and a chemical solution supply apparatus according to an embodiment of the present invention. It is a flowchart which shows the biological observation method using the biological observation apparatus of FIG. It is a whole block diagram which shows typically the modification of the biological observation apparatus of FIG. It is a figure explaining OCT of the living body observation device of FIG. It is a figure which shows the state which the focus position of the objective lens of OCT of FIG. 4 corresponds with the outer surface of a biological tissue. It is a figure which shows the intensity signal example of the interference light detected by OCT in the state of FIG. 5A.
  • FIG. 5 is a diagram showing a state in which the focal position of the OCT objective lens in FIG.
  • the biological observation apparatus 1 is a fluorescence observation apparatus, and as shown in FIG. 1, a biological tissue at a position facing the microscope (light detection unit) 4 including the objective lens 3 and the objective lens 3.
  • medical solution supply apparatus 2 arrange
  • the chemical solution supply apparatus 2 includes a scattering suppression unit 5 and a control unit 6 that controls the scattering suppression unit 5.
  • the scattering suppression unit 5 is a cylindrical shape (for example, on a cylinder) that can be fixed in a sealed state on the outer surface of the biological tissue P so as to surround a predetermined range of the biological tissue P facing the objective lens 3 and store the drug solution X.
  • the chemical solution supply unit 8 includes a tank 10 that stores the chemical solution X, a pump 11 that supplies the chemical solution X in the tank 10 into the outer wall 7 via a pipe, and an input unit 12 that inputs an operation command for the pump 11. And.
  • the chemical solution X a high refractive index and water-soluble liquid, for example, an aqueous solution of glycerol, glucose, DMSO or the like is used.
  • an aqueous solution of glycerol, glucose, DMSO or the like is used.
  • the temperature adjusting means (permeation adjusting unit, viscosity adjusting unit) 9 is, for example, a Peltier element (hereinafter also referred to as Peltier element 9), and can cool the chemical liquid X by power supply from the driving unit 13. It has become.
  • control unit 6 receives a supply command signal from the input unit 12 to the chemical solution supply unit 8, and after the chemical solution X is supplied into the outer wall portion 7 and the penetration of the chemical solution X into the living tissue P is started.
  • a determination unit 16 that determines whether or not the time measured by the time measurement unit 14 exceeds a predetermined threshold stored in the storage unit 15. .
  • a threshold value an average time required for the chemical solution X to sufficiently permeate in the thickness direction of the living tissue P having an average thickness dimension is stored.
  • the determination part 16 outputs a drive command signal with respect to the drive part 13 of the Peltier device 9, when it determines with the time measured by the time measurement part 14 having exceeded the predetermined threshold value. . Thereby, electric power is supplied from the drive unit 13 to the Peltier element 9, and the chemical solution X stored in the outer wall 7 is cooled.
  • a living body observation method using the living body observation apparatus 1 according to the present embodiment configured as described above will be described below with reference to FIG.
  • the objective lens 3 of the microscope 4 of the living body observation apparatus 1 is disposed close to the outer surface of the living tissue P covering the observation object S, and the objective lens 3 is opposed.
  • the outer wall 7 of the chemical solution supply device 2 is fixed in a sealed state to the outer surface of the living tissue P at a position surrounding the region being formed, and the container 17 capable of storing the chemical solution X is configured (step S1).
  • step S2 When the chemical solution X is supplied to the inside of the container 17 having the outer wall portion 7 as a side wall, the chemical solution X is stored in the container 17, so that the outer surface of the living tissue P constituting the bottom surface of the container 17 is immersed in the chemical solution X. Thus, the penetration of the chemical solution X into the living tissue P is started.
  • a supply command signal is output from the input unit 12 to the control unit 6, and at this time, the time measurement unit 14 of the control unit 6 starts measuring time (step S3).
  • the drug solution X is maintained for a predetermined time in a state where the drug solution X is in contact with the outer surface of the living tissue P in this way (clearing step S4).
  • the medicinal solution X penetrates from the outer surface of the living tissue P to the inside of the living tissue P within the range surrounded by the outer wall 7, and the portion of the living tissue P in which the medicinal solution X penetrates is denatured to the inside.
  • the refractive index distribution of is uniformized.
  • the Peltier device 9 is controlled.
  • the chemical solution X in the container 17 is cooled.
  • the chemical solution X is cooled, its viscosity is increased and the penetration rate into the living tissue P is decreased (osmosis adjustment step S6).
  • observation step S7 which observes the fluorescence from the observation object S is performed after this.
  • excitation light is irradiated from the outside of the living tissue P toward the internal observation target S (irradiation step S8).
  • Irradiation of the excitation light may be performed through the living tissue P in a range where the refractive index distribution is made uniform by the penetration of the chemical solution X, or through the living tissue P in a portion where the chemical solution X does not penetrate. You may decide to irradiate the excitation light of the comparatively strong intensity
  • the fluorescent substance existing inside the observation object S is excited to generate fluorescence, and a part of the generated fluorescence is emitted from the observation object S.
  • the portion that has been made transparent in the transparentizing step S4 is transmitted through the outer surface of the living tissue P and emitted outward. Since the objective lens 3 is disposed close to the outer surface of the biological tissue P, the fluorescence transmitted through the biological tissue P from the observation target S and emitted outward from the outer surface is collected by the objective lens 3 and is then collected by the microscope 4. It is detected (detection step S9).
  • the two-dimensional or three-dimensional fluorescence image of the observation target S is acquired by repeating the detection of the fluorescence in the detection step S9 at each irradiation position while moving the irradiation position of the excitation light in the irradiation step S8. Can do.
  • the living body observation apparatus 1, the drug solution supply apparatus 2, and the living body observation method according to the present embodiment at least the living tissue P in the vicinity of the position where the objective lens 3 of the microscope 4 is arranged to be opposed is used by the drug solution X. Since the fluorescence is detected in a state where the refractive index distribution is made uniform by denaturation, the fluorescence is efficiently recovered by suppressing the scattering of the fluorescence in the living tissue P, and a clear fluorescence image of the observation target S is acquired. There is an advantage that can be.
  • the observation target S is a brain tissue and the living tissue P that covers the brain tissue is a skull
  • no opening is formed in the skull there is an advantage that it is not necessary to change physiological conditions such as brain pressure, and the living body can be observed alive in a normal physiological condition.
  • the biological observation apparatus 1, the chemical solution supply apparatus 2, and the biological observation method according to the present embodiment the degree of penetration of the chemical solution X into the living tissue P is monitored, and the excessive chemical solution X is supplied. Therefore, the observation target S can be prevented from being denatured by the chemical solution X.
  • the determination unit 16 measures the elapsed time from the start of the supply of the chemical solution X, and reduces the penetration rate of the chemical solution X when the predetermined threshold value is exceeded.
  • the following method may be adopted. First, as shown in FIG. 3, an osmotic distance measuring unit 18 that optically and non-invasively measures the osmotic distance of the drug solution X into the living tissue P is provided. The degree of penetration of the chemical solution X may be determined based on the measured penetration distance.
  • an OCT Optical Coherence Tomography, boundary surface detection unit, thickness detection unit
  • the light emitted from the broadband light source 20 is branched by the optical coupler 21 into the observation optical path and the reference optical path on the OCT probe 22 side, and reflected by the mirror 23 on the reference optical path and reflected back.
  • This is a method for accurately measuring the distance by detecting the interference light with the light by the photodetector 24.
  • reference numeral 25 denotes a single mode fiber
  • reference numeral 26 denotes a collimating lens
  • reference numeral 27 denotes an objective lens
  • reference numeral 28 denotes a mirror driving unit.
  • the penetration distance measurement unit 18 uses the property that light incident in the living tissue P returns strongly at the boundary surface of the refractive index in the living tissue P, as shown in FIGS.
  • the lens 27 is moved to focus on the outer surface of the living tissue P, and then the mirror 23 is moved by the operation of the mirror driving unit 28 to increase the intensity of the interference light detected by the photodetector 24. Record position A.
  • the mirror 23 is moved by the operation of the mirror driving unit 28, and the position B of the mirror 23 where the intensity of the interference light detected by the photodetector 24 becomes high is recorded.
  • the penetration distance of the chemical solution X can be calculated. Thereby, it is possible to more accurately determine the degree of penetration of the chemical solution X by measuring the actual penetration distance, not by the average penetration time.
  • the objective lens 27 is moved to focus on the back surface of the living tissue P, and the position C of the mirror 23 where the intensity of the interference light becomes high is recorded.
  • the thickness of the living tissue P can be calculated. As a result, it is possible to accurately determine the degree of penetration of the drug solution X based on the actual thickness of the biological tissue P, not based on the average thickness of the biological tissue P.
  • the living tissue P is a tissue containing a large amount of collagen such as a skull, SHG as shown in FIG. (Second Harmonic Generation)
  • the microscope 29 may be employed.
  • reference numeral 30 denotes a laser light source
  • reference numeral 31 denotes a beam expander
  • reference numeral 32 denotes a galvano mirror
  • reference numeral 33 denotes a relay optical system
  • reference numeral 34 denotes a dichroic mirror
  • reference numeral 35 denotes an objective lens
  • reference numeral 36 denotes a barrier filter
  • reference numeral 37 denotes A condensing lens
  • 38 is a photodetector.
  • the SHG microscope 29 utilizes the fact that the detected light intensity is high when a collagen tissue is present at the focal position.
  • the intensity of the detected light is monitored while moving the objective lens 35 in the direction of approaching the living tissue P, and the light detected when the focal position of the objective lens 35 coincides with the outer surface of the living tissue P is monitored. Since the intensity increases, the outer surface of the living tissue P is detected from this change, and the intensity of the light detected when the focal position passes the back surface of the living tissue P decreases rapidly. The back surface position of P can be accurately grasped.
  • the irradiation optical fiber 39 connected to the laser light source 30 and the emission end of the irradiation optical fiber 39 may be measured by a probe 41 including a plurality of multimode fibers 40 having incident ends arranged at different positions. That is, the laser light emitted from the emission end of the irradiation optical fiber 39 returns to the incident direction by being scattered in the living tissue P, and is incident from the incidence end of each multimode fiber 40. By suppressing the scattering in the living tissue P, the light returning to each multimode fiber 40 is reduced. Therefore, the penetration distance of the chemical solution X can be measured by monitoring the change.
  • the control unit 6 monitors the contrast of the image acquired by the microscope 4 with the contrast calculation unit 45, and when the contrast exceeds a predetermined threshold, the chemical solution X is sufficiently penetrated.
  • the determination unit 16 may determine that the process has been performed. As the refractive index distribution of the living tissue P is made uniform by the penetration of the chemical solution X, the contrast of the image of the observation target S covered with the living tissue P increases. By comparing this with the threshold value, The degree of penetration of the chemical solution X can be easily determined.
  • the penetration rate of the chemical solution X is adjusted by cooling the chemical solution X.
  • a photocurable resin such as an ultraviolet curable resin
  • a light source (penetration adjusting unit, viscosity adjusting unit) 42 that irradiates light for curing the chemical solution X above the outer wall portion 7 that stores the chemical solution X. May be arranged.
  • the chemical solution X is activated by irradiating the chemical solution X with the light source 42 by operating the light source 42. It is possible to suppress the penetration of the chemical solution X into the living tissue P.
  • two types of liquid chemicals X1 and X2 having different viscosities are prepared in different tanks 10 and 43 as the viscosity adjusting unit, and separate pipes are provided so that they can be separately supplied into the outer wall 7.
  • pumps 11 and 44 may be provided.
  • the chemical solution X1 having a low viscosity is allowed to penetrate the living tissue P, and when it is determined that the penetration of the chemical solution X1 into the living tissue P is sufficiently performed, The stored chemical solution X1 may be sucked by the reverse rotation of one pump 11 and then replaced with the chemical solution X2 having a high viscosity by the operation of the other pump 44.
  • medical solution was raised and the penetration was suppressed was demonstrated, you may use when decreasing the viscosity of a chemical

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medical Informatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

La présente invention vise à observer un organisme vivant dans un état physiologique normal de manière aisée et produisant moins de charge sur l'organisme. Un appareil d'observation d'organisme (1) selon la présente invention comprend : une unité de détection de lumière (4) qui détecte de la lumière ; une unité de suppression de dispersion (5) qui imprègne un tissu biologique P disposé à l'opposé de l'unité de détection de lumière (4) avec un liquide pharmaceutique X et dénature le tissu biologique P de telle sorte que la distribution d'indice de réfraction de ce dernier est uniforme ; une unité de détermination (16) qui détermine le niveau d'uniformité de la distribution d'indice de réfraction obtenue par l'unité de suppression de dispersion (5) ; et une unité d'ajustement d'imprégnation (9) qui ajuste le taux d'imprégnation avec le liquide pharmaceutique X par l'unité de suppression de dispersion (5) selon le résultat de détermination de l'unité de détermination (16).
PCT/JP2014/080154 2014-11-14 2014-11-14 Appareil d'observation d'organisme, dispositif d'alimentation en liquide pharmaceutique, et procédé d'observation d'organisme WO2016075804A1 (fr)

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PCT/JP2014/080154 WO2016075804A1 (fr) 2014-11-14 2014-11-14 Appareil d'observation d'organisme, dispositif d'alimentation en liquide pharmaceutique, et procédé d'observation d'organisme

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2016103425A1 (ja) * 2014-12-25 2017-10-05 オリンパス株式会社 生体観察装置および生体観察方法
WO2019244559A1 (fr) * 2018-06-21 2019-12-26 日本電信電話株式会社 Dispositif de mesure de concentration de composant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009148337A (ja) * 2007-12-19 2009-07-09 Sun Tec Kk 光断層画像表示方法
JP2013517510A (ja) * 2010-01-19 2013-05-16 ヴィジョンゲイト，インコーポレーテッド トモグラフィー光照射野顕微鏡

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009148337A (ja) * 2007-12-19 2009-07-09 Sun Tec Kk 光断層画像表示方法
JP2013517510A (ja) * 2010-01-19 2013-05-16 ヴィジョンゲイト，インコーポレーテッド トモグラフィー光照射野顕微鏡

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2016103425A1 (ja) * 2014-12-25 2017-10-05 オリンパス株式会社 生体観察装置および生体観察方法
WO2019244559A1 (fr) * 2018-06-21 2019-12-26 日本電信電話株式会社 Dispositif de mesure de concentration de composant

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