WO2020175956A1 - 플루오로퀴놀론계 항생제를 이용한 결막 내 세포영상 검사방법, 이를 이용한 안구 병변 진단방법과 안구병변 치료제 효능 검출방법 및 이를 위한 결막 내 세포영상 검사장치 - Google Patents

플루오로퀴놀론계 항생제를 이용한 결막 내 세포영상 검사방법, 이를 이용한 안구 병변 진단방법과 안구병변 치료제 효능 검출방법 및 이를 위한 결막 내 세포영상 검사장치 Download PDF

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WO2020175956A1
WO2020175956A1 PCT/KR2020/002893 KR2020002893W WO2020175956A1 WO 2020175956 A1 WO2020175956 A1 WO 2020175956A1 KR 2020002893 W KR2020002893 W KR 2020002893W WO 2020175956 A1 WO2020175956 A1 WO 2020175956A1
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Prior art keywords
conjunctiva
ocular
fluoroquinolone
light
intraconjunctival
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English (en)
French (fr)
Inventor
김성한
김기현
김명준
이승훈
장훈철
르비엣호안
박수현
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POSTECH Research and Business Development Foundation
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POSTECH Research and Business Development Foundation
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Priority to KR1020217016910A priority Critical patent/KR102559712B1/ko
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/13—Ophthalmic microscopes
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/0008—Apparatus for testing the eyes; Instruments for examining the eyes provided with illuminating means
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/13—Ophthalmic microscopes
    • A61B3/135—Slit-lamp microscopes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
    • A61B5/14551—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases
    • A61B5/14556—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases by fluorescence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4848—Monitoring or testing the effects of treatment, e.g. of medication
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • G01N21/6447—Fluorescence; Phosphorescence by visual observation
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/162—Conjunctival disorders, e.g. conjunctivitis

Definitions

  • Intraconjunctival cell imaging method using a fluoroquinolone antibiotic a method for diagnosing an ocular lesion using the same, a method for detecting the efficacy of an ocular lesion treatment agent, and an intraconjunctival cell imaging device for the same
  • the present invention relates to a method for intraconjunctival cell imaging using a fluoroquinolone antibiotic, a method for diagnosing ocular lesions using the same, a method for detecting the efficacy of an ocular lesion treatment agent, and an intraconjunctival cell imaging apparatus for the same.
  • Moxifloxacin one of the fluoroquinolone antibiotics, is stained on the goblet cells of the eye conjunctiva, and the stained goblet cells are excitation with a single photon in the near-ultraviolet area or the visible light area, and the morphological information of the living tissue is obtained from the conjunctiva.
  • Intraconjunctival cell imaging method using a fluoroquinolone antibiotic that can be obtained without damage or destruction of the body a method for diagnosing ocular lesions using it, a method for detecting the efficacy of an ocular lesion treatment agent, and an intraconjunctival cell imaging device for this.
  • Microscopy is used in biological research, and in clinical practice, it is used in ophthalmic and dermatological examinations.
  • a reflection microscope does not have a high cell contrast, so if it is necessary to improve the contrast, the living tissue is photographed using the excitation fluorescence after staining the living tissue with a fluorescent probe.
  • a fluorescence microscope is used. In one case, a fluorescence microscope is used in which the living tissue is stained with a fluorescent probe and then the living tissue is photographed using the excitation fluorescence.
  • the fluorescent material enables high-contrast and high-speed imaging by expressing a strong fluorescent signal in a specific area of interest.
  • indocyanine green and fluorescein are used as vascular staining fluorescent materials for human subjects.
  • Moxifloxacin which has no toxicity to drugs that can stain cells, is an antimicrobial agent currently used to treat or prevent bacterial infections. It has intrinsic fluorescence and is highly permeable to tissues, resulting in biotissue staining and fluorescence. It has properties that are advantageous for shooting.
  • the conjunctiva is a thin, transparent mucous membrane that covers the inner side of the eyelid and the white part of the eye (sclera).
  • the conjunctiva, together with the cornea, is on the outermost surface of the eye to protect the eye. It is composed of a superficial cell layer composed of silver epithelial cells and goblet cells and the underlying matrix. Goblet cells of the conjunctiva secrete mucus, a component of tears, and the matrix is rich in blood vessels and lymphatic tissues, and immune-related cells are distributed.
  • the imaging technology of goblet cells is helpful in diagnosing eye diseases and determining the severity of the disease, and in the case of treatment, it is useful information for evaluating the response to treatment and clinical observation of the progress.
  • Impression cytology is mainly used.
  • the method of extracting the uppermost cell layer of the conjunctiva by pressing filter paper into the limbus of the cornea, and staining it it is relatively easy and simple, but it causes slight damage. It is not recommended when testing is performed multiple times.
  • the confocal reflection microscope requires the microscope objective lens to be placed close to the eye to be tested, so it is difficult to test, the contrast is not high, and the equipment is not 2020/175956 1»(:1 ⁇ 1 ⁇ 2020/002893 There is a problem that cannot be used much because it is expensive.
  • the task to be solved in the present invention is that moxifloxacin, one of the fluoroquinolones-based antibiotics, is stained on the goblet cells of the conjunctiva of the eye, and the stained goblet cells are subjected to short-photon excitation in the near-ultraviolet region or the visible light region.
  • Intraconjunctival cell imaging method using fluoroquinolone antibiotics which can obtain morphological information of living tissues by photographing without damage or destruction of the conjunctiva of the eye, a method for diagnosing ocular lesions using the same, a method for detecting the efficacy of an ocular lesion treatment, and intraconjunctival for this It is to provide a cell imaging test device.
  • the present invention provides a conjunctival staining step of dyeing the eye conjunctiva with a fluoroquinolone antibiotic, and a light source irradiating additional light to the eye conjunctiva dyed with the fluoroquinolone antibiotic.
  • An irradiation step, and a conjunctival imaging step in which a photographing unit photographs the eye conjunctiva through the fluoroquinolone-based antibiotic fluorescence-excited by light in the light irradiation step, and the fluoroquinolone-based antibiotic in the conjunctival staining step Is staining the goblet cells of the eye conjunctiva, the light source unit outputs a single photon in the light irradiation step, and the range of the continuous wavelength output from the light source unit in the light irradiation step is a near ultraviolet region and a visible ray region
  • the photographing unit for photographing the eye conjunctiva is a high-magnification fluorescence microscope or a slit lamp microscope.
  • a method for intraconjunctival cell imaging using a fluoroquinolone antibiotic characterized in that it is a high-magnification fluorescence microscope (macroscope) or a slit
  • Fluoroquinolone antibiotics may include moxifloxacin.
  • the range of the continuous wavelength output from the light source unit may include a near ultraviolet region and a visible region.
  • the near-ultraviolet ray region and the visible ray region may be 300 nm to 476 nm.
  • the present invention relates to a method for diagnosing ocular lesions through goblet cells of the conjunctiva taken through an intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • the eye lesions are chemical burns
  • the present invention is a method for detecting the efficacy of a therapeutic agent for ocular lesions through the goblet cells of the conjunctiva taken through the intraconjunctival cell imaging method using the fluoroquinolone antibiotic according to the present invention, the eyeball Changes in the number of ocular conjunctival goblet cells injected with the ocular lesion treatment agent through the step of injecting the eye lesion treatment agent into the conjunctiva and the goblet cells of the eye conjunctiva without the injection of the eye lesion treatment agent and the step of introducing the treatment agent.
  • Provides a method of detecting the efficacy of an ocular lesion treatment agent including the step of examining the efficacy of a therapeutic agent based on a change in shape or area.
  • the present invention is an apparatus for photographing a biological tissue of an ocular conjunctiva stained with a fluoroquinolone antibiotic, a light source unit that irradiates light to the biological tissue, and fluorescence-excited by the light irradiated from the light source unit.
  • An objective lens that controls the focus of the photographing unit, a focus control part that is combined with the objective lens to change a focus position formed on the biological tissue, and an image generator that generates an image of the living tissue photographed by the photographing unit.
  • the photographing unit For each focal position changed by the focus control unit, the photographing unit generates a photographed image by photographing a plurality of retrieving biological tissues, and the image generating unit combines the images of a region in focus among the plurality of photographed images to form one living body.
  • a tissue image is created, the light source unit outputs a single photon, and the range of the continuous wavelength output from the light source unit is a near-ultraviolet ray region and a visible ray region, and the photographing unit is a high-magnification fluorescence microscope (macroscope) or a slit lamp microscope. )
  • macroscope high-magnification fluorescence microscope
  • slit lamp microscope a slit lamp microscope.
  • the near-ultraviolet ray region and the visible ray region may be 300 nm to 476 nm.
  • test method the method for diagnosing ocular lesions using the same, the method for detecting the efficacy of the treatment for ocular lesions, and the intraconjunctival cell imaging apparatus for the same have the following effects.
  • the eye conjunctiva is stained with moxifloxacin, one of the fluoroquinolones-based antibiotics, and moxifloxacin is irradiated with light in the visible light area, so you can quickly and directly take pictures without destroying the conjunctiva. There is an advantage to be able to do it.
  • an imaging device such as a high-magnification fluorescence microscope or a slit lamp microscope that does not support three-dimensional resolution can take pictures of goblet cells distributed on the surface of the conjunctiva of the eye, which has the advantage of easing the limitations of the detection equipment.
  • FIG. 1 is a view showing a flow diagram of a method for intraconjunctival cell imaging using a fluoroquinolone antibiotic according to the present invention.
  • FIG. 2 is a diagram showing a mechanism for a single photon excitation phenomenon for performing an intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • FIG. 3 is a diagram showing a short-photon excitation spectrum and a fluorescence emission spectrum of the near ultraviolet and visible light regions of moxifloxacin in the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • Figure 4 (a) is the intraconjunctival using the fluoroquinolone antibiotic according to the present invention
  • FIG. 4 Photographs of the conjunctiva of a mouse not stained with moxifloxacin in the cyto imaging method were taken with a reflex confocal microscope, and FIG. 4 (ratio is a confocal fluorescence microscope of the conjunctiva of a mouse stained with moxifloxacin. This is a picture taken.
  • Figures 5 (a) to (e) are photographs taken by staining the conjunctiva of a mouse with moxifloxacin according to the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • Figure 6 (a) to is a picture taken by staining the conjunctiva of a mouse with moxifloxacin according to the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention
  • Figure 6 (a) Is a picture taken with a confocal fluorescence microscope of the bulbous conjunctiva of a mouse stained with moxifloxacin
  • the ratio is a confocal view of the fomix con ⁇ inctiva of a mouse stained with moxifloxacin.
  • FIG. 7 is a schematic view showing the configuration of an intraconjunctival cell imaging test apparatus using a fluoroquinolone antibiotic according to the present invention.
  • FIG. 8 is a diagram showing an example of a focus area and a living tissue image changed by control of the focus control unit of the intraconjunctival cell imaging apparatus using the fluoroquinolone antibiotic according to the present invention.
  • FIG. 9 is a view showing an image of a living tissue changed by the control of the focus control unit of the intraconjunctival cell imaging apparatus using a fluoroquinolone antibiotic according to the present invention.
  • FIG. W is a diagram showing in detail the image quantification step in the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • a method for intraconjunctival cell imaging using a fluoroquinolone-based antibiotic according to the present invention to be described later is an intraconjunctival cell imaging apparatus using a fluoroquinolone-based antibiotic according to the present invention.
  • a method for intraconjunctival cell imaging using a fluoroquinolone antibiotic according to the present invention includes a conjunctival staining step (SW0), a light irradiation step (S200), and a conjunctival imaging step (S300).
  • an electron energy level in a molecule of a fluorescent substance is raised from a ground state to an excite state by using an excitation photon.
  • the activity of molecules, cells, and tissues of living organisms can be observed at high resolution through an optical fluorescence microscope when the molecules, cells, and tissues of living organisms are treated with a fluorescent material.
  • a fluorescent material This means that electrons in the fluorescent material are excitation photons. This is because it emits a unique color of fluorescent photons in the process of being excited by and returning to its place.
  • the fluorescent substance dyed on the living tissue is not toxic to the human body and fluorescence excitation is possible in the visible light region that does not negatively affect the human body, the fluorescent substance is dyed on the living tissue and the morphological information of the living tissue Will be able to provide.
  • Antibiotics include moxifloxacin, gatifloxacin, pefloxacin, difloxacin, nofloxacin, ciprofloxacin, ofloxacin and enrofloxacin, and in this specification, autofluorescence can be expressed in the visible light region. Sifloxacin was used to stain the living tissue.
  • the short-photon excitation spectrum and fluorescence emission spectrum of the near-ultraviolet and visible light regions of moxifloxacin in the intraconjunctival cell imaging method are as follows.
  • Moxifloxacin used in the test method is currently sold on the market
  • the continuous wave light source used in the intraconjunctival cell imaging method using the fluoroquinolone antibiotic according to the present invention uses a wavelength of 30011111 to 47611111, and accordingly, the fluorescence signal by using the wavelength of near-ultraviolet light end
  • the area corresponding to the near-ultraviolet region of the above-described single photon wavelength may be subject to the risk of cell damage to the internal tissues, but the single photon excitation is highly efficient, and the intensity of the light irradiated by the light source is reduced. By doing so, it can be used in living tissues.
  • moxifloxacin stains the goblet cells of the ocular conjunctiva.
  • the light source unit irradiates light, wherein the light source unit
  • the light source unit outputs continuous wave light in the near-ultraviolet and visible light ranges. Fluorescence excitation is performed.
  • a photographing unit abnormal technique to be described later is photographed through the moxifloxacin, which is fluorescently excited by light in the light irradiation step (S200), and, as will be described later, at this time, the photographing
  • the unit includes a focal transmitter and includes a general high-magnification fluorescence microscope (macroscope) or slit lamp microscope that does not support three-dimensional resolution, of course, it can also be applied to microscopes that support three-dimensional resolution.
  • It includes a change step (S320), a real-time image processing step (S330), and an image quantization step (S340).
  • the photographing unit is stained on the eyeball conjunctiva to photograph the fluorescently excited goblet cells.
  • the focus control unit to be described later moves the objective lens in the axial direction to change the focus position.
  • the cell photographing step (S310) and the focus position changing step (S320) are performed a plurality of times according to the size of the area to be photographed and the focus position.
  • the image quantification step (S340) the image of the goblet cells imaged in real time of the goblet cells is quantified, and as shown in FIG. W, image contrast adjustment is performed from the obtained image, Based on the threshold, the goblet cell image is made into a binary image to obtain quantitative information on the goblet cells.
  • the strategic information obtained through the above-described image quantification step includes the density of the goblet cells, the size distribution of the goblet cell clusters, the shape of the goblet cells, and the uniformity of the goblet cells.
  • Figure 4 (a) is a reflex confocal point before administering moxifloxacin to the conjunctiva of a rat 2020/175956 1»(:1 ⁇ 1 ⁇ 2020/002893
  • FIG. 9 This is a picture taken with a reflection confocal microscope, and FIG. 4 is a picture taken with a confocal fluorescence microscop after administration of moxifloxacin to the conjunctiva of a rat.
  • moxifloxacin exhibits fluorescence with a stronger intensity than autofluorescence while maintaining a high concentration in the goblet cells of the conjunctiva of the eye, thereby obtaining high-contrast morphological information.
  • Figure 5 (a) to (e) is a picture taken by staining the conjunctiva of a rat in a living state according to the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention with moxifloxacin.
  • Figure 5 (a) to Figure 5 (c) are the mice stained with moxifloxacin
  • FIG. 5 is a rat conjunctiva stained with moxifloxacin (fornix
  • conjunctiva is a picture taken with a high-magnification fluorescence microscope, and as shown in (d) of FIG. 5, it can be seen that the distribution of goblet cells in the distal conjunctiva is dense and the size of the cluster is large.
  • Figure 5 this is a picture taken with a high-magnification fluorescence microscope included in the imaging unit) of a rat stained with moxifloxacin (orbital or palpebral conjunctiva)
  • Figure 5 (e) also As shown, it can be seen that the distribution of goblet cells is dense and the cluster size is large in the temporal conjunctiva.
  • Fig. 5(a) to 5(e) by staining the conjunctiva of a living mouse with moxifloxacin, it is possible to check the goblet cells of the conjunctiva of the eye.
  • a high-performance microscope that supports three-dimensional resolution such as confocal microscope, but also a general high-magnification fluorescence microscope that does not support three-dimensional resolution can be used.
  • 6A to 6 are photographs taken with a confocal fluorescence microscope by staining the conjunctiva of a mouse with moxifloxacin according to the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • the imaging device may further include a confocal fluorescence microscope.
  • FIG. 6 is a photograph of the bulbar of a mouse stained with moxifloxacin, taken with a confocal fluorescence microscope, and as shown in (a) of FIG. 6, in the bulbar conjunctiva It can be seen that the distribution of goblet cells and the size of the cluster are small.
  • conjunctiva is a photograph taken with a confocal fluorescence microscope, and it can be seen that the distribution of goblet cells in the distal conjunctiva is dense and the size of the cluster is large, as shown in (non-shown in FIG. 6).
  • Figure 6 (c) shows the conjunctival conjunctival conjunctiva (bulbar) of mice stained with moxifloxacin
  • goblet cells Although there are no goblet cells, goblet cells gradually appear from the oral conjunctiva, and as the migration proceeds to the original conjunctiva, the distribution of goblet cells becomes denser and the size of the cluster increases.
  • goblet cells in the conjunctiva of the eye can be identified.
  • the method for diagnosing ocular lesions according to the present invention is as follows.
  • the lesions of the eyeball are diagnosed through the goblet cells of the conjunctiva taken through the intraconjunctival cell imaging method using a fluoroquinolone antibiotic.
  • Diagnosis is based on the standard, and the above eye lesions are chemical burns,
  • the number of goblet cells of the oral conjunctiva is damaged by 99-100%
  • diagnosis of ocular lesions can be performed not only on the basis of the change in the number of goblet cells, but also the area of goblet cells per unit area.
  • morphological information of goblet cells can be provided through the method of intraconjunctival cell imaging using a fluoroquinolone-based antibiotic according to the present invention, and such morphological information can be used for the size and distribution of goblet cells. It includes all forms, and the diagnosis of ocular lesions can be performed based on this.
  • the test method is to confirm the morphological information of the cells by staining and photographing living tissue in not only the mouse used in the above-described experiment, but also other animal (no -) models. 2020/175956 1»(:1 ⁇ 1 ⁇ 2020/002893
  • the efficacy of a therapeutic agent for ocular lesions can be detected based on the goblet cells of the conjunctiva taken using the intraconjunctival cell imaging method using a fluoroquinolone antibiotic according to the present invention.
  • the method for detecting the efficacy of an ocular lesion treatment agent according to the present invention includes a step of administering a therapeutic agent and a step of detecting the efficacy of a therapeutic agent.
  • an ocular lesion treatment agent is injected into the eye conjunctiva.
  • the goblet cells of the ocular conjunctiva to which the eye lesion treatment agent is not injected and the goblet cells of the ocular conjunctiva to which the eye lesion treatment agent is injected are photographed through the step of introducing the treatment agent.
  • the conjunctival goblet cell imaging method using a fluoroquinolone antibiotic according to the present invention can be used not only to diagnose ocular lesions, but also to verify the effectiveness in an in vivo animal model in testing the therapeutic agent performance for related lesions.
  • various types of conjunctiva included in the eye conjunctiva can be photographed, and not only the lesion of the eyeball is diagnosed based on the change in the number or area of the number or area of the goblet cells constituting the various types of conjunctiva, but also the efficacy of the treatment agent for the eye lesion is evaluated. Can be detected.
  • the intraconjunctival cell imaging apparatus using the fluoroquinolone antibiotic according to the present invention includes a light source unit 100, a photographing unit 200, a lens unit,
  • It includes a cut-off filter), a filter here), a beam splitter 3 ⁇ 4, an objective lens 300, a focus control unit 400, and an image generator 500.
  • the light source unit 100 is of the conjunctiva stained with a fluoroquinolone antibiotic.
  • the fluoroquinolone antibiotic is moxifloxacin.
  • the light source unit 100 outputs a single photon, and the range of the continuous wavelength output from the light source unit 100 is a near ultraviolet region and a visible region, and the near ultraviolet region and a visible region are 30011111 to 47611111 It is preferable to be.
  • the photographing unit 200 photographs the biological tissue that is fluorescence-excited by the light irradiated from the light source unit 100, wherein the photographing unit 200 is a high-magnification fluorescence 2020/175956 1»(:1 ⁇ 1 ⁇ 2020/002893
  • the lens unit controls the path of the light output from the light source unit 100 or the fluorescence path of the fluoroquinolone antibiotic excited by the light, and the lens unit is configured to be It includes a three-lens (L3), a description of which will be described later.
  • L3 three-lens
  • the objective lens 300 controls the focus of the photographing unit 200, and the
  • the focus control unit 400 is coupled with the objective lens 300 to move the objective lens 300 to change a focus position formed on the living tissue.
  • the photographing unit 200 photographs a plurality of reconstruction organisms to generate a photographed image, and a description thereof will be described later.
  • the image generating unit 500 is a biological tissue photographed by the photographing unit 200
  • An image is generated, and the image generation unit 500 combines images of an area in focus among the plurality of photographed images to generate a single biological tissue image.
  • the path of fluorescence excited from moxifloxacin is as follows.
  • the light source unit 100 irradiates light of a single photon, and the light is a third lens (L3), an excitation filter (EF), a beam splitter (BS), and an objective lens 300 ) To irradiate light on the living tissue of the conjunctiva of the eye.
  • L3 third lens
  • EF excitation filter
  • BS beam splitter
  • objective lens 300 objective lens
  • Fluorescent light excited by light on the living tissue is the objective lens 300, the
  • the cut-off filter (BF) and the first lens (L1) is captured by the photographing unit 200 to generate an image.
  • the image captured by the photographing unit 200 is the position of the objective lens 300 by the focus control unit 400, and more precisely, the position of the Z-axis (vertical direction) of the objective lens 300 is Depending on the position of the objective lens 300, the portion of the body tissue to focus on is different. This is because the shape of the eye conjunctiva is formed to be curved, and the area in which the focus is formed is different depending on the height difference of the eye conjunctiva. Because it loses.
  • the photographing The unit 200 generates a plurality of images II having different focal areas each time the position of the objective lens 300 is changed.
  • a single biological tissue image 12 is generated by combining the images of a region in which the focus is matched among the plurality of images.

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Abstract

본 발명은 플로오로퀴놀론계 항생제 중 하나인 목시플록사신을 안구 결막의 술잔 세포에 염색하고, 염색된 술잔 세포를 근자외선 영역 또는 가시광선 영역으로 단광자 여기하여 형광 촬영함으로써 생체 조직의 형태학적 정보를 안구 결막의 손상이나 파괴 없이 얻을 수 있는 플루오로퀴놀론계 항생제를 이용한 결막 내 세포영상 검사방법, 이를 이용한 안구 병변 진단방법과 안구병변 치료제 효능 검출방법 및 이를 위한 결막 내 세포영상 검사장치에 관한 것이다. 이를 위하여, 본 발명은 안구 결막에 플루오로퀴놀론계 항생제를 염색하는 결막 염색단계와, 상기 플루오로퀴놀론계 항생제에 염색된 상기 안구 결막에 광원부가 빛을 조사하는 광 조사단계와, 상기 광 조사단계에서 빛에 의하여 형광 여기 된 상기 플루오로퀴놀론계 항생제를 통하여 촬영유닛이 상기 안구 결막을 촬영하는 결막 촬영단계를 포함하며, 상기 결막 염색단계에서 상기 플루오로퀴놀론계 항생제는 상기 안구 결막의 술잔 세포(goblet cell)를 염색하고, 상기 광 조사단계에서 상기 광원부는 단광자를 출력하며, 상기 결막 촬영단계에서 상기 안구 결막을 촬영하는 상기 촬영유닛은 고배율 형광 현미경(macroscope) 또는 세극등 현미경(slit lamp microscope)인 것을 특징으로 하는 플루오로퀴놀론계 항생제를 이용한 결막 내 세포영상 검사방법을 제공한다.

Description

2020/175956 1»（：1/10公020/002893 명세서
발명의명칭:플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법，이를이용한안구병변진단방법과안구병변치료제 효능검출방법및이를위한결막내세포영상검사장치 기술분야
[1] 본발명은플루오로퀴놀론계항생제를이용한결막내세포영상검사방법, 이를이용한안구병변진단방법과안구병변치료제효능검출방법및이를위한 결막내세포영상검사장치에관한것으로서 ,보다상세하게는
플로오로퀴놀론계항생제중하나인목시플록사신을안구결막의술잔세포에 염색하고,염색된술잔세포를근자외선영역또는가시광선영역으로단광자 여기하여형광촬영함으로써생체조직의형태학적정보를안구결막의 손상이나파괴없이얻을수있는플루오로퀴놀론계항생제를이용한결막내 세포영상검사방법,이를이용한안구병변진단방법과안구병변치료제효능 검출방법및이를위한결막내세포영상검사장치에관한것이다.
배경기술
四 생체조직내세포를고해상도촬영할수있는광학현미경 (optical
microscopy)은생물학연구에서사용되고있으며,임상에서는안과,피부과 검사에활용되고있다.
[3] 임상에서는주로빛의반사를활용하는비침습적광학현미경을사용하며
여기에는공초점반사현미경 (confocal reflectance microscopy)등이 있다.하지만 반사현미경은세포대비도가높지않아,대비도향상이필요임상에서는주로 빛의반사를활용하는비침습적광학현미경을사용하며여기에는공초점 반사현미경 (confocal reflectance microscopy)등이 있다.
[4] 하지만,반사현미경은세포대비도가높지않아,대비도향상이필요한경우 형광물질 (fluorescent probe)로생체조직을염색한후여기 (excitation)된 형광 (fluorescence)을이용하여생체조직을촬영하는형광현미경이사용된다.한 경우형광물질 (fluorescent probe)로생체조직을염색한후여기 (excitation)된 형광 (fluorescence)을이용하여생체조직을촬영하는형광현미경이사용된다.
[5] 여기서형광물질은특정관심영역에서강한형광신호를발현함으로써고 대비도및고속영상촬영을가능하게한다.
[6] 형광물질의활용에있어서,동물대상으로는많은종류의형광물질을
사용하고있으나,인체대상으로는인도시아닌그린 (indocyanine green)과 플루오레세인 (fluorescein)등이혈관염색형광물질로써사용되고있다.
[7] 또한,혈관염색만으로는병변또는암을진단하거나,세포의형태학적정보를 얻는데어려움이있으므로,정확한진단을위해서는인체내세포염색이 필요하다. 2020/175956 1»（：1^1{2020/002893
2
[8] 이를위하여인체대상세포염색을위한형광염색약물이연구되고있으나, 현재는세포에대한독성문제등으로인하여인체에적용될수있는마땅한 형광물질이없는실정이었다.
[9] 세포에염색될수있는약물중독성이없는목시플록사신은현재임상에서 세균감염을치료하거나예방하는데사용되는향균제인데,내재적형광 (intrinsic fluorescence)을지니고있으며조직침투성이뛰어나생체조직염색및형광 촬영에유리한특성을보유하고있다.
[10] 하지만,목시늘록사신의경우여기효율 (excitation efficiency)이 280nm의
중자외선영역에서최고치로발현되고,자외선은인체에유해하게작용되므로 목시플록사신이인체의생체 (in-vivo)촬영에사용되지못하는문제가있었다.
[11] 이를해결하기위하여,최근근적외선여기파장을이용한목시플록사신의
이광자여기 (two-photon excitation)형광촬영이시연되었으며 ,이를통해 목시플록사신이생체내조직과세포를염색하고,이들의형태를고해상도로 촬영할수있음을확인하였다.
[12] 한편,안구의결막 (conjunctiva)은눈꺼풀의안쪽과안구의흰부분 (공막)을덮고 있는얇고투명한점막이다.결막은각막과함께눈의가장바깥쪽표면에있어 눈을보호한다.결막은상피세포와술잔세포로구성된표면세포층그리고 아래의기질로이루어져 있으며,결막의술잔세포는눈물구성성분인점액을 분비하고,기질은혈관과림프조직이풍부하며면역관련세포가분포되어있다.
[13] 상술한바와같이,눈물구성성분중하나인점액을만드는술잔세포의
점액분비이상이있는경우,안구건조증이안구건조에따른염증이일어날수 있다.술잔세포의점액분비이상은결막의술잔세포의밀도변화와형태 변화로부터야기되고,임상적으로술잔세포가감소하는질환은알려져 있다.
[14] 이로인하여,술잔세포의이미징기술은안구질환진단과질환의중증도를 판단하는데도움이되고치료를할경우치료에대한반응을평가하고경과를 임상관찰하는데유용한정보이다.
[15] 이러한검사방법중,현재결막내술잔세포밀도검사를위해서
압돈세포진단법 (impression cytology)이주로사용된다.압돈세포진단법은 여과지를각막윤부에압박접촉시켜결막의최상위세포층을추출하고,이를 염색해서검사하는방법인데상대적으로쉽고간단하지만약간의손상을 유발하므로검사를여러번시행하는경우에는추천되지않는다.
[16] 최근압흔세포진단법을대체할수있는방법으로공초점반사현미경 (confocal reflectance microscopy)으로비침습적으로결막을세포영상촬영을하여술잔 세포밀도를계산하는검사법이보고되었다.
[17] 이러한공초점반사현미경영상에서는술잔세포가상대적으로반사도가
높으며크고동그란형태로나타나서주변의상피세포와구별된다.그러나 공초점반사현미경은현미경대물렌즈를안구에가까이위치시켜야검사가 가능하므로,검사방법에어려움이따르고,대비도가높지않으며,장비가 2020/175956 1»（：1^1{2020/002893 고가여서 많이활용되지못하는문제점이 있다.
발명의상세한설명
기술적과제
[18] 본발명에서 해결하고자하는과제는플로오로퀴놀론계항생제중하나인 목시플록사신을안구결막의술잔세포에 염색하고,염색된술잔세포를 근자외선영역또는가시광선영역으로단광자여기하여 형광촬영함으로써 생체조직의 형태학적 정보를안구결막의손상이나파괴 없이 얻을수있는 플루오로퀴놀론계항생제를이용한결막내세포영상검사방법,이를이용한 안구병변진단방법과안구병변치료제효능검출방법 및이를위한결막내 세포영상검사장치를제공하는것이다.
과제해결수단
[19] 상술한과제를해결하기위하여,본발명은안구결막에플루오로퀴놀론계 항생제를염색하는결막염색단계와,상기플루오로퀴놀론계항생제에 염색된 상기 안구결막에 광원부가빛을조사하는광조사단계와,상기광조사단계에서 빛에 의하여 형광여기된상기플루오로퀴놀론계항생제를통하여 촬영유닛이 상기 안구결막을촬영하는결막촬영단계를포함하며,상기 결막염색단계에서 상기플루오로퀴놀론계항생제는상기 안구결막의술잔세포 (goblet cell)를 염색하고,상기 광조사단계에서상기광원부는단광자를출력하며,상기 광 조사단계에서상기광원부에서출력되는연속파파장의범위는근자외선영역 및가시광선영역이고,상기 결막촬영단계에서상기 안구결막을촬영하는상기 촬영유닛은고배율형광현미경 (macroscope)또는세극등현미경 (slit lamp microscope)인것을특징으로하는플루오로퀴놀론계항생제를이용한결막내 세포영상검사방법을제공한다.
[2이 여기서 ,상기결막염색단계에서상기 안구결막을염색하는상기
플루오로퀴놀론계항생제는목시플록사신을포함할수있다.
[21] 또한,상기광조사단계에서상기 광원부에서출력되는연속파파장의 범위는 근자외선영역 및가시광선영역을포함할수있다.
[22] 또한,상기근자외선영역 및가시광선영역은 300nm내지 476nm일수있다.
[23] 또한,본발명은본발명에 따른플루오로퀴놀론계항생제를이용한결막내 세포영상검사방법을통해촬영된안구결막의술잔세포를통해 안구의 병변을 진단하는방법에 있어서,상기술잔세포의 단위면적당개수변화,형태 변화 또는면적 변화를기준으로안구의 병변을진단하는안구병변진단방법을 제공한다.
[24] 여기서 ,상기 안구병변은화학화상 (chemical burns),
각결막염 (keratoconjunctivitis),반픈성유천포창 (Ocular Cicatricial Pemphigoid), 스티븐스존슨증후군 (Stevens-Johnson syndrome)및상윤부각결막염 (Superior limbic keratoconjunctivitis)을포함할수있다. 2020/175956 1»（：1^1{2020/002893
4
[25] 또한,본발명은본발명에 따른플루오로퀴놀론계항생제를이용한결막내 세포영상검사방법을통해촬영된안구결막의술잔세포를통해 안구병변 치료제의 효능을검출하는방법에 있어서,상기 안구결막에 안구병변치료제를 투입하는치료제투입단계와,상기 안구병변치료제를투입하지 않은안구 결막의술잔세포와상기치료제투입단계를통해상기 안구병변치료제를 투입한안구결막의술잔세포의 단위면적당개수변화,형태 변화또는면적 변화를기준으로치료제의 효능을검줄하는치료제효능검줄단계를포함하는 안구병변치료제효능검출방법을제공한다.
[26] 또한,본발명은플루오로퀴놀론계항생제로염색된안구결막의 생체조직을 촬영하는장치에 있어서 ,상기 생체조직으로빛을조사하는광원부와,상기 광원부에서조사된빛에 의하여 형광여기된상기 생체조직을촬영하는 촬영유닛과,상기 광원부로부터출력된빛또는상기빛에 의해여기된상기 플루오로퀴놀론계항생제의 형광의 경로를제어하는렌즈부와,상기
촬영유닛의초점을제어하는대물렌즈와,상기 대물렌즈와결합되어,상기 생체조직에 맺히는초점위치를변경시키는초점제어부와,상기 촬영유닛에서 촬영한생체조직의 이미지를생성하는이미지 생성부를포함하며 ,상기 초점제어부에 의하여 변경되는초점위치마다상기 촬영유닛은복수회상기 생체조직을촬영하여촬영이미지를생성하고,상기 이미지 생성부는복수개의 상기 촬영이미지중초점이맞는영역의 이미지를조합하여하나의 생체조직 이미지를생성하며 ,상기광원부는단광자를출력하며 ,상기 광원부에서 출력되는연속파파장의 범위는근자외선영역 및가시광선영역이고,상기 촬영유닛은고배율형광현미경 (macroscope)또는세극등현미경 (slit lamp microscope)인것을특징으로하는플루오로퀴놀론계항생제를이용한결막내 세포영상검사장치를제공한다.
[27] 여기서 ,상기 안구결막을염색하는상기플루오로퀴놀론계항생제는
목시플록사신을포함할수있다.
[28] 또한,상기근자외선영역 및가시광선영역은 300nm내지 476nm일수있다. 발명의효과
[29] 본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법 ,이를이용한안구병변진단방법과안구병변치료제효능검출방법 및 이를위한결막내세포영상검사장치는다음과같은효과를가진다.
[3이 첫째,안구결막을플루오로퀴놀론계항생제중하나인목시플록사신으로 염색하고,목시플록사신을가시광선영역의 빛으로조사함으로써 안구결막의 파괴 없이신속하게직접촬영할수있으므로,빠르고정확한진단을수행할수 있는이점이 있다.
[31] 둘째,목시플록사신으로염색한안구결막의술잔세포의 형광영상화에
있어서,공초점 형광현미경과같은삼차원해상도를가지는고비용,고성능 2020/175956 1»（：1^1{2020/002893
5 현미경뿐만아니라,삼차원해상도를지원하지않는고배율형광현미경또는 세극등현미경에서와같은촬영장치에서도안구결막표면에분포한술잔 세포를촬영할수있으므로검출장비의제한이완화되는이점이 있다.
[32] 셋째,안구결막의술잔세포의구성변화를통해안구의병변을진단할수있는 이점이 있다.
[33] 넷째,안구건조병을포함한결막술잔세포관련질환에대한치료제성능
검사에 있어서인비보동물모델에서의효과검증에활용되는이점이 있다. 도면의간단한설명
[34] 도 1은본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법의흐름도를도시한도면이다.
[35] 도 2는본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법을수행하기위한단광자여기현상에대한메커니즘을도시한 도면이다.
[36] 도 3은본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법에서목시플록사신의근자외선및가시광선영역의단광자여기 스펙트럼과형광방출스펙트럼을도시한도면이다.
[37] 도 4의 (a)는본발명에따른플루오로퀴놀론계항생제를이용한결막내
세포영상검사방법에서목시플록사신으로염색되지않는쥐의안구결막을 반사공초점현미경으로촬영한사진이고,도 4 (비는목시플록사신으로염색된 쥐의안구결막을공초점형광현미경으로촬영한사진이다.
[38] 도 5의 (a)내지 (e)는본발명에따른플루오로퀴놀론계항생제를이용한결막 내세포영상검사방법에따라쥐의결막을목시플록사신으로염색하여촬영한 사진으로,도 5의 (a)내지도 5의 (c)는목시플록사신으로염색된쥐의
구결막 (bulbar)을삼차원해상도를지원하지않는일반고배율형광현미경으로 촬영한사진이며 ,도 5의 (d)는목시플록사신으로염색된쥐의원개결막 (fornix conjunctiva)을고배율형광현미경으로촬영한사진이고,도 5의 (리는
목시늘록사신으로염색된쥐의검결막 (orbital또는 palpebral conjunctiva)을 고배율형광현미경으로촬영한사진이다.
[39] 도 6의 (a)내지 ( 는본발명에따른플루오로퀴놀론계항생제를이용한결막 내세포영상검사방법에따라쥐의결막을목시플록사신으로염색하여촬영한 사진으로,도 6의 (a)는목시플록사신으로염색된쥐의구결막 (bulbar)을공초점 형광현미경으로촬영한사진이고,도 6의 (비는목시플록사신으로염색된쥐의 원개결막 (fomix con如 inctiva)을공초점형광현미경으로촬영한사진이며,도 6의 ( 는목시플록사신으로염색된쥐의결막중구결막 (bulbar)과원개결막 (fornix conjunctiva)을공초점형광현미경으로이어서촬영한사진이다.
[4이 도 7은본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사장치의구성을개략적으로도시한도면이다. 2020/175956 1»（：1^1{2020/002893
6
[41] 도 8은본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사장치의초점제어부의제어에 의하여 변경되는초점영역 및생체조직 이미지의 예시를도시한도면이다.
[42] 도 9는본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사장치의초점제어부의제어에 의하여 변경되는생체조직의 이미지를도시한 도면이다.
[43] 도 W은본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법에서 이미지정량화단계를구체적으로도시한도면이다.
발명의실시를위한형태
[44] 이하,상술한해결하고자하는과제가구체적으로실현될수있는본발명의 바람직한실시 예들이 첨부된도면을참조하여설명된다.본실시 예들을 설명함에 있어서,동일구성에 대해서는동일명칭 및동일부호가사용되며, 이에 따른부가적인설명은하기에서 생략된다.
[45] 도 1내지도 6을참조하여 ,본발명에따른플루오로퀴놀론계항생제를이용한 결막내세포영상검사방법을설명하면다음과같다.
[46] 먼저 ,도 1에도시된바와같이 ,본발명에 따른플루오로퀴놀론계항생제를 이용한결막내세포영상검사방법후술하는본발명에따른플루오로퀴놀론계 항생제를이용한결막내세포영상검사장치를이용하여수행되며,본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상검사방법은결막 염색단계 (SW0),광조사단계 (S200)및결막촬영단계 (S300)를포함한다.
[47] 본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법을설명하기에 앞서,도 2를참조하여 단광자여기 현상에 대한 메커니즘을설명하면다음과같다.
[48] 도 2에도시된바와같이 ,여기광자 (excitation photon)를이용하여 형광물질 분자내전자에너지준위를바닥상태 (ground state)에서부터들뜬상태 (excite state)까지상승시킨다.
[49] 이후들뜬상태에서바닥상태로전자의 에너지준위가다시하강할때형광 광자 (fluorescence photon)가방줄되며,이때,하나의 여기 광자가톱수되고, 하나의 형광광자를방출하는현상을단광자여기 형광이라고하며，이러한 단광자여기 현광에 의하여단광자여기효율은이광자여기 효율보다높아 후술하는광원부에서조사하는빛의세기를낮추어촬영할수있게된다.
[5이 즉,생물체의분자,세포,조직의 활동모습은,생물체의분자,세포,조직을형광 물질로처리하면광학형광현미경을통해고해상도로관찰할수있다.이는 형광물질내의 전자가여기 광자에의해들뜬상태로되었다가다시 제자리로 돌아가는과정에서독특한색채의 형광광자를방줄하기 때문이다.
[51] 이러한형광물질이 생체조직에주입되고,형광물질이 생체조직의세포에 흡수되어고농도로유지되는경우,형광물질의 형광을이용하여 생체조직의고 2020/175956 1»（：1^1{2020/002893
7 대비（031 &8（；）촬영이 가능하다.
[52] 이때,생체조직에 염색되는형광물질이 인체에독성을가지지 않고,인체에 부정적 영향을주지 않는가시광선영역에서 형광여기가가능하면형광물질을 생체조직에 염색하여 생체조직의 형태학적정보를제공할수있게된다.
[53] 본발명에서 생체조직의 염색을위하여사용하는플루오로퀴놀론계
항생제에는목시플록사신,가티플록사신,페플록사신,디플록사신,노플록사신, 시프로플록사신,오플록사신및엔로플록사신등이 있으며,본명세서에서는 자가형광이가시광선영역에서발현가능한목시플록사신을사용하여 생체 조직의 염색을진행하였다.
[54] 또한,도 3을참조하여，본발명에 따른플루오로퀴놀론계항생제를이용한
결막내세포영상검사방법에서목시플록사신의근자외선및가시광선영역의 단광자여기스펙트럼과형광방출스펙트럼을설명하면다음과같다.
[55] 도 3知）와도 3（비는각각근자외선영역과가시광선영역에서목시플록사신의 여기스펙트럼과형광방출스펙트럼을나타낸다.
[56] 본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법에서사용하는목시플록사신은현재시중에서판매되고
Figure imgf000009_0001
점안액 0.5%（쇼1<:011,미국）을사용하였다.
[57] 도 3知）및도 3（비에도시된바와같이 ,목시플록사신은근자외선영역의 34011111 부근에서 여기효율이 가장높았고,그이후파장증가에따라여기효율이 점차 감소하였다.
[58] 하지만,근자외선범위밖의가시광선영역인 40511111내지 47811111파장에서도 여기가가능하며 , 40511111에서는 34011111에 비해 약 27%의 형광세기를가졌으며 , 이는 70011111여기 광기반의 이광자형광세기보다훨씬더높은것을
확인하였다.
[59] 따라서,본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법에서사용하는연속파광원은 30011111내지 47611111의 파장을사용하며 , 이에 따라근자외선내지중자외선파장을사용함으로써 형광신호가
증가되므로더빠른속도로촬영할수있게되며,가시광선에해당하는파장을 사용함으로써 생체조직에사용될수있게된다.
[6이 물론,상술한단광자파장중근자외선영역에 해당하는영역은생체내조직의 세포손상의위험이 따를수있지만,단광자여기 효율이높은특징을이용하여 광원부에서조사하는빛의 세기를낮추어 촬영함으로써 생체조직에사용될수 있게된다.
[61] 다시도 1을참조하여,본발명에따른플루오로퀴놀론계항생제를이용한결막 내세포영상검사방법을설명하면다음과같다.
[62] 상기결막염색단계（別 00）는실험하고자하는생체조직인안구결막의세포에 플루오로퀴놀론계항생제를염색하며 ,본명세서에서는플루오로퀴놀론계 항생제중하나인목시플록사신을사용한다. 2020/175956 1»（：1^1{2020/002893
8
[63] 이때,상기결막염색단계 (SW0)에서는목시플록사신이상기안구결막의술잔 세포 (goblet cell)를염색한다.
[64] 상기광조사단계 (S200)에서는목시플록사신에염색된상기안구결막에
광원부가빛을조사한다.여기서상기광원부는상기광원부는
플루오로퀴놀론계항생제형광발현을위한여기광인단광자를출력하되, 상술한근자외선과가시광선범위의연속파빛을출력하며,후술하는실험 예에서상기광원부는근자외선과가시광선범위의연속파빛을출력함으로써 형광여기를수행한다.
[65] 상기결막촬영단계 (S300)는상기광조사단계 (S200)에서빛에의하여형광 여기된상기목시플록사신을통하여후술하는촬영유닛이상기술잔세포를 촬영하며,후술하겠지만,이때,상기촬영유닛은초점이송장치를포함하고, 삼차원해상도를지원하지않는일반고배율형광현미경 (macroscope)또는 세극등현미경 (slit lamp microscope)을포함한다.물론,삼차원해상도를 지원하는현미경에도적용가능하다.
[66] 구체적으로,상기결막촬영단계는 (S300)세포촬영단계 (S310),초점위치
변경단계 (S320),실시간영상처리단계 (S330)및이미지정량화단계 (S340)를 포함한다.
[67] 상기세포촬영단계 (S310)에서는상기촬영유닛이안구결막에염색되어형광 여기된술잔세포를촬영한다.
[68] 이후,상기초점위치변경단계 (S320)에서후술하는초점제어부가대물렌즈를 축방향으로이송시켜초점위치를변경시킨다.
[69] 상기세포촬영단계 (S310)와상기초점위치변경단계 (S320)는촬영하는영역의 크기및초점위치에따라복수번수행된다.
P이 상기실시간영상처리단계 (S330)에서는촬영한술잔세포이미지를
실시간으로영상처리한다.
P 1] 상기이미지정량화단계 (S340)에서는술잔세포의실시간으로영상처리된 술잔세포의이미지를정량화하며 ,도 W에도시된바와같이 ,얻어지는 이미지로부터영상대비도조정 (contrast enhance)을수행하고,임계값을 기준으로술잔세포이미지를이진이미지 (binary image)로만들어술잔세포의 정량적정보를얻도록한다.
R2] 상술한이미지정량화단계를통해획득하는상기정략적정보는술잔세포의 밀도,술잔세포군집 (cluster)의크기분포,술잔세포의형태,술잔세포의 균일성을포함한다.
3] 상술한본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법을이용한실험예들을설명하면다음과같다.
4]
5] ＜실험예 1:목시플록사신염색여부에따른쥐의안구결막촬영＞
6] 도 4의 (a)는쥐의안구결막에목시플록사신을투여하기전에반사공초점 2020/175956 1»（：1^1{2020/002893
9 현미경 (Reflectance confocal microscope)으로촬영한사진이며 ,도 4 (비는쥐의 안구결막에목시플록사신을투여한후공초점형광현미경 (Confocal fluorescence microscop)으로촬영한사진이다.
[77] 도 4의 (a)와도 4 (비에각각도시된바와같이,쥐의안구결막의술잔세포는 자가형광이약하기때문에대비도가좋지못해목시플록사신의투여전에는 세포의촬영에어려움이따랐지만,목시플록사신투여후에는목시플록사신이 술잔세포에염색되며,목시플록사신의단광자여기형광으로인하여안구 결막의술잔세포가고대비도로촬영됨을알수있다.
8] 즉,목시플록사신이안구결막의술잔세포내에서고농도를유지하면서자가 형광보다강한세기의형광을발현한것을확인할수있으며,이에따라 고대비의형태학적정보를얻을수있게된다.
P9]
[8이 ＜실험 예 2:고배율형광현미경을사용한쥐의안구결막촬영＞
[81] 도 5의 (a)내지 (e)는본발명에따른플루오로퀴놀론계항생제를이용한결막 내세포영상검사방법에따라살아있는상태의쥐의결막을목시플록사신으로 염색하여촬영한사진이다.
[82] 여기서,도 5의 (a)내지도 5의 (c)는목시플록사신으로염색된쥐의
구결막 (bulbar)을고배율형광현미경으로촬영한사진이다.도 5의 (a)내지도 5의 ( 에도시된바와같이,구결막에서술잔세포의분포와군집의크기가작은 것을확인할수있다.
[83] 또한,도 5의예는목시플록사신으로염색된쥐의원개결막 (fornix
conjunctiva)을고배율형광현미경으로촬영한사진이며,도 5의 (d)에도시된 바와같이,원개결막에서술잔세포의분포가빽빽하고군집의크기가큰것을 확인할수있다.
[84] 또한,도 5의 (이는목시플록사신으로염색된쥐의검결막 (orbital또는 palpebral conjunctiva)을상기촬영유닛에포함되는고배율형광현미경으로촬영한 사진이며,도 5의 (e)에도시된바와같이,검결막에서술잔세포의분포가 빽빽하고군집의크기가큰것을확인할수있다.
[85] 즉,도 5의 (a)내지도 5의 (e)에도시된바와같이,살아있는상태의쥐의결막을 목시플록사신으로염색함으로써안구결막의술잔세포를확인할수있다.뿐만 아니라,안구결막의술잔세포형광영상화에는공초점현미광과같은삼차원 해상도를지원하는고성능현미경뿐만아니라,삼차원해상도를지원하지않는 일반고배율형광현미경에서촬영이가능하다.
[86] 즉,삼차원해상도를지원하지만,고가이며 ,사용이복잡하고,좁은촬영영역을 가지는공초점현미경을사용하지않더라도,삼차원해상도를지원하지않는 형광현미경에상기초점제어부를결합시킴으로써곡면의생체조직도초점을 맞춰촬영할수있다.
[87] 2020/175956 1»（：1^1{2020/002893
10
[88] ＜실험예 3:고배율형광현미경을사용한쥐의안구결막촬영＞
[89] 도 6의 (a)내지 ( 는본발명에따른플루오로퀴놀론계항생제를이용한결막 내세포영상검사방법에따라쥐의결막을목시플록사신으로염색하여공초점 형광현미경으로촬영한사진이다.
[9이 본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법에서상기촬영장치는공초점형광현미경을더포함할수있다.
[91] 여기서,도 6의 (a)는목시플록사신으로염색된쥐의구결막 (bulbar)을공초점 형광현미경으로촬영한사진이며,도 6의 (a)에도시된바와같이,구결막에서 술잔세포의분포와군집의크기가작을것을확인할수있다.
[92] 또한,도 6의 (비는목시플록사신으로염색된쥐의원개결막 (fornix
conjunctiva)을공초점형광현미경으로촬영한사진이며,도 6의 (비에도시된 바와같이,원개결막에서술잔세포의분포가빽빽하고군집의크기가큰것을 확인할수있다.
[93] 또한,도 6의 (c)는목시플록사신으로염색된쥐의결막중구결막 (bulbar)과
원개결막 (fomix conjunctiva)을공초점형광현미경으로이어서촬영한사진이며, 도 6의 ( 에도시된바와같이,각막 (cornea)와연곽 (limbus)에서는술잔
세포 (goblet cell)가없지만,구결막부터점차술잔세포가나타나원개결막으로 이동이진행될수록술잔세포의분포가빽빽하고,군집의크기가커지는것을 확인할수있다.
[94] 즉,도 6의 (a)내지 ( 에도시된바와같이,살아있는상태의쥐의결막을
목시플록사신으로염색함으로써안구결막의술잔세포를확인할수있다.
[95] 또한,본발명에따른안구병변진단방법을설명하면다음과같다.
[96] 본발명에따른안구병변진단방법은상술한본발명에따른
플루오로퀴놀론계항생제를이용한결막내세포영상검사방법을통해촬영된 안구결막의술잔세포를통해안구의병변을진단한다.
[97] 상기안구병변은단위면적당술잔세포의개수또는술잔세포의면적을
기준으로진단하며 ,상기안구병변은화학화상 (chemical burns),
각결막염 (keratoconjunctivitis),반픈성유천포창 (Ocular Cicatricial Pemphigoid), 스티븐스존슨증후군 (Stevens-Johnson syndrome)및상윤부각결막염 (Superior limbic keratoconjunctivitis)을포함하고,상기안구병변의진단을다음표 1을 기준으로수행될수있다.
[98] 2020/175956 1»(：1^1{2020/002893
11
[표 1]
Figure imgf000013_0001
[99] 상기표 1과같이 ,구결막술잔세포수가 60%손상되고,검결막술잔세포수가 40%로손상되며 ,구결막과검결막의술잔세포손상차이가 20%가될때화학 화상으로진단된다.또는,구결막술잔세포수가 70-80%손상되고,검결막술잔 세포수가 60-80%로손상되며 ,구결막과검결막의술잔세포손상차이가 0- 10%가될때각결막염으로진단된다.
[100] 또는,구결막술잔세포수가 95%손상되고,검결막술잔세포수가 90-95%로 손상되며 ,구결막과검결막의술잔세포손상차이가 0-5%가될때
반흔성유천포창으로진단된다.
[101] 또는,구결막술잔세포수가 99-100%손상되고,검결막술잔세포수가
98- 100%로손상되며,구결막과검결막의술잔세포손상차이가 0- 1%가될때 스티븐스존슨증후군으로진단된다.
[102] 마지막으로,구결막술잔세포수가 85%손상되고,검결막술잔세포수가 90%로손상되며 ,구결막과검결막의술잔세포손상차이가 5%가될때 상윤부각결막염군으로진단된다.
[103] 물론,상술한안구병변의진단은술잔세포수의 변화뿐만아니라,단위면적당 술잔세포의면적 변화를기준으로도수행될수있다.
[104] 또한,본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법을통해촬영된상기술잔세포의 형태학적인정보를제공할수있고, 이러한형태학적 정보는술잔세포의크기 및분포형태등모든형태를 포함하며,이를기준으로안구병변의진단을수행할수있게된다.
[105] 즉,본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법은상술한실험에서 활용된쥐뿐만아니라,이 외의동물생체 (노 - ) 모델에서 생체조직을염색하여촬영함으로써 세포의 형태학적 정보를확인할 2020/175956 1»（：1^1{2020/002893
12 수있으며 ,이에따라동물의 안구병변의진단을수행할수있다.
[106] 또한,본발명에 따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법을이용해촬영된안구결막의술잔세포를기반으로,안구병변 치료제의 효능을검출할수있다.
[107] 본발명에 따른안구병변치료제효능검출방법은치료제투입단계와치료제 효능검출단계를포함한다.
[108] 상기치료제투입단계에서는상기 안구결막에 안구병변치료제를투입한다.
[109] 이후,상술한본발명에따른플루오로퀴놀론계항생제를이용한결막내
세포영상검사방법을이용함으로써상기 안구병변치료제를투입하지 않은 안구결막의술잔세포와상기치료제투입단계를통해상기 안구병변치료제를 투입한안구결막의술잔세포를촬영한다.
[110] 상기치료제효능검출단계에서상기 안구병변치료제를투입하지 않은안구 결막의술잔세포와,상기치료제투입단계를통해상기 안구병변치료제를 투입한안구결막의술잔세포의 단위면적당개수변화,형태 변화또는면적 변화를기준으로치료제의 효능을검줄한다.
[111] 즉,본발명에 따른플루오로퀴놀론계항생제를이용한결막술잔세포영상화 방법은안구병변의 진단뿐만아니라,관련병변에 대한치료제성능검사에 있어서 인비보동물모델에서의효과검증에활용될수있다.
[112] 상술한바와같이,플루오로퀴놀론계항생제를이용한결막내세포영상
검사방법을통해안구결막에포함된여러종류의 결막을촬영할수있으며, 여러종류의결막을구성하는술잔세포의 개수또는면적의 변화를기준으로 안구의 병변을진단뿐만아니라,안구병변의치료제의효능을검출할수있다.
[113] 도 7내지도 9를참조하여 ,본발명에따른플루오로퀴놀론계항생제를이용한 결막내세포영상검사장치를설명하면다음과같다.
[114] 먼저,도 7을참조하여,본발명에 따른플루오로퀴놀론계항생제를이용한 결막내세포영상검사장치의구성을설명하면다음과같다.
[115] 도 7에도시된바와같이,본발명에 따른플루오로퀴놀론계항생제를이용한 결막내세포영상검사장치는광원부 (100),촬영유닛 (200),렌즈부,
차단필터여므),여기필터田므),빔스플리터여¾,대물렌즈 (300),초점제어부 (400) 및 이미지 생성부 (500)를포함한다.
[116] 상기광원부 (100)는플루오로퀴놀론계항생제로염색된안구결막의
생체조직에 빛을조사하며,여기서상기 안구결막을염색하는상기
플루오로퀴놀론계항생제는목시플록사신이다.
[117] 또한,상기광원부 (100)는단광자를출력하며,상기광원부 (100)에서출력되는 연속파파장의범위는근자외선영역 및가시광선영역이며 ,상기근자외선영역 및가시광선영역은 30011111내지 47611111인것이 바람직하다.
[118] 상기촬영유닛 (200)은상기 광원부 (100)에서조사된빛에의하여 형광여기된 상기 생체조직을촬영하며,여기서,상기 촬영유닛 (200)은고배율형광 2020/175956 1»（：1^1{2020/002893
13 현미경 (macroscope)또는세극등현미경 (slit lamp microscope)을포함한다.
[119] 상기렌즈부는상기광원부 (100)로부터출력된빛또는상기빛에의해여기된 상기플루오로퀴놀론계항생제의형광의경로를제어하며,상기렌즈부는 배치된위치및역할에따라제 1내지제 3렌즈 (L3)를포함하며,이에대한 설명은후술한다.
[12이 상기대물렌즈 (300)는상기촬영유닛 (200)의초점을제어하며 ,상기
초점제어부 (400)는상기대물렌즈 (300)와결합되어상기대물렌즈 (300)를 이동시키면서상기생체조직에맺히는초점위치를변경시킨다.
[121] 즉,상기초점제어부 (400)에의하여변경되는초점위치마다상기
촬영유닛 (200)은복수회상기생체조직을촬영하여촬영이미지를생성하며, 이에대한설명은후술한다.
[122] 상기이미지생성부 (500)는상기촬영유닛 (200)에서촬영한생체조직의
이미지를생성하며,상기이미지생성부 (500)는복수개의상기촬영이미지중 초점이맞는영역의이미지를조합하여하나의생체조직이미지를생성한다.
[123] 상기생체조직이미지를생성하는과정을설명하기이전에 ,상기
광원부 (100)에서조사되는빛의경로와,상기생체조직에염색된
목시플록사신으로부터여기된형광의경로를설명하면다음과같다.
[124] 도 7에도시된바와같이,상기광원부 (100)에서는단광자의빛을조사하며, 상기빛은제 3렌즈 (L3),여기필터 (EF),빔스플리터 (BS)및대물렌즈 (300)를거쳐 상기안구결막의생체조직에빛을조사한다.
[125] 상기생체조직에빛에의하여여기된형광은상기대물렌즈 (300),상기
차단필터 (BF)및제 1렌즈 (L1)를거쳐촬영유닛 (200)에촬영되어이미지를 생성한다.
[126] 이때 ,상기촬영유닛 (200)이촬영한이미지는상기초점제어부 (400)에의하여 상기대물렌즈 (300)의위치,정확하게는상기대물렌즈 (300)의 Z축 (수직방향) 위치가변경되며,상기대물렌즈 (300)의위치에따라,상기생체조직에서초점이 맺히는부분이달라진다.이는상기안구결막의형상이굴곡지게형성되어, 안구결막의높이차에따라초점이맺히는영역이달라지기때문이다.
[127] 즉,도 8및도 9에도시된바와같이,상기대물렌즈 (300)의위치가변경됨에 따라상기촬영유닛 (200)에서촬영한이미지마다초점이맺히는영역이 변경되며,이에따라상기촬영유닛 (200)은상기대물렌즈 (300)의위치가변경될 때마다서로다른초점영역을가지는복수개의이미지 (II)를생성한다.
[128] 이후,도 8에도시된바와같이,복수개의상기이미지중초점이맞는영역의 이미지를조합하여하나의생체조직이미지 (12)를생성한다.
[129] 이외,본발명에따른플루오로퀴놀론계항생제를이용한결막내세포영상 검사장치의구성의상세내용은상술한본발명에따른플루오로퀴놀론계 항생제를이용한결막내세포영상검사방법과동일하므로,이에대한설명은 생략한다. 2020/175956 1»（：1^1{2020/002893
14
[130] 이상설명한바와같이，본발명은상술한특정한바람직한실시 예에 한정되지 아니하며,청구범위에서 청구하는본발명의요지를벗어남없이 당해발명이 속하는기술분야에서통상의지식을가진자에의해다양한변형의실시가 가능하고이러한변형은본발명의 범위에속한다.
산업상이용가능성
[131] 본발명은안구결막을플루오로퀴놀론계항생제중하나인목시플록사신으로 염색하고,목시플록사신을가시광선영역의 빛으로조사함으로써 안구결막의 파괴 없이신속하게직접촬영할수있으므로,빠르고정확한진단을수행할수 있으며,안구의 병변을진단할수있고,치료제성능검사와관련된산업에 이용될수있다.

Claims

2020/175956 1»（：1^1{2020/002893 15 청구범위
[청구항 1] 안구결막에플루오로퀴놀론계항생제를염색하는결막염색단계 ;
상기플루오로퀴놀론계항생제에 염색된상기 안구결막에 광원부가 빛을조사하는광조사단계 ;
상기 광조사단계에서 빛에의하여 형광여기 된상기플루오로퀴놀론계 항생제를통하여촬영유닛이상기 안구결막을촬영하는결막 촬영단계;를포함하며,
상기 결막염색단계에서상기플루오로퀴놀론계항생제는상기 안구 결막의술잔세포 (goblet cell)를염색하고,
상기 광조사단계에서상기광원부는단광자를출력하며, 상기 광조사단계에서상기광원부에서출력되는연속파파장의범위는 근자외선영역 및가시광선영역이고,
상기 결막촬영단계에서상기 안구결막을촬영하는상기촬영유닛은 고배율형광현미경 (macro scope)또는세극등현미경 (slit lamp microscope)인것을특징으로하는플루오로퀴놀론계항생제를이용한 결막내세포영상검사방법 .
[청구항 2] 제 1항에 있어서,
상기 결막염색단계에서상기 안구결막을염색하는상기 플루오로퀴놀론계항생제는목시플록사신을포함하는것을특징으로 하는플루오로퀴놀론계항생제를이용한결막내세포영상검사방법.
[청구항 3] 제 1항에 있어서,
상기근자외선영역 및가시광선영역은 300nm내지 476nm인것을 특징으로하는플루오로퀴놀론계항생제를이용한결막내세포영상 검사방법.
[청구항 4] 제 1항내지제 3항중어느한항에 따른플루오로퀴놀론계항생제를 이용한결막내세포영상검사방법을통해촬영된안구결막의술잔 세포를통해안구의 병변을진단하는방법에 있어서,
상기술잔세포의 단위면적당개수변화,형태 변화또는면적 변화를 기준으로안구의 병변을진단하는안구병변진단방법.
[청구항 5] 제 4항에 있어서,
상기 안구병변은화학화상 (chemical bums),
각결막염 (keratoconjunctivitis),반픈성유천포창 (Ocular Cicatricial Pemphigoid),스티븐스존슨증후군 (Stevens-Johnson syndrome)및 상윤부각결막염 (Superior limbic keratoconjunctivitis)을포함하는것을 특징으로하는안구병변진단방법 .
[청구항 6] 제 1항내지제 3항중어느한항에 따른플루오로퀴놀론계항생제를 이용한결막내세포영상검사방법을통해촬영된안구결막의술잔 2020/175956 1»（：1^1{2020/002893
16 세포를통해안구병변치료제의효능을검출하는방법에 있어서 , 상기안구결막에안구병변치료제를투입하는치료제투입단계;및 상기안구병변치료제를투입하지않은안구결막의술잔세포와상기 치료제투입단계를통해상기안구병변치료제를투입한안구결막의 술잔세포의단위면적당개수변화,형태변화또는면적변화를 기준으로치료제의효능을검줄하는치료제효능검줄단계 ;를포함하는 안구병변치료제효능검출방법 .
[청구항 7] 플루오로퀴놀론계항생제로염색된안구결막의생체조직을촬영하는 장치에 있어서,
상기생체조직으로빛을조사하는광원부;
상기광원부에서조사된빛에의하여형광여기된상기생체조직을 촬영하는촬영유닛;
상기광원부로부터출력된빛또는상기빛에의해여기된상기
플루오로퀴놀론계항생제의형광의경로를제어하는렌즈부;
상기촬영유닛의초점을제어하는대물렌즈;
상기대물렌즈와결합되어,상기생체조직에맺히는초점위치를 변경시키는초점제어부;및
상기촬영유닛에서촬영한생체조직의이미지를생성하는이미지 생성부;를포함하며 ,
상기초점제어부에의하여변경되는초점위치마다상기촬영유닛은 복수회상기생체조직을촬영하여촬영이미지를생성하고, 상기이미지생성부는복수개의상기촬영이미지중초점이맞는영역의 이미지를조합하여하나의생체조직이미지를생성하며, 상기광원부는단광자를출력하며,상기광원부에서출력되는연속파 파장의범위는근자외선영역및가시광선영역이고,
상기촬영유닛은고배율형광현미경 (macro scope)또는세극등현미경 (slit lamp microscope)인것을특징으로하는플루오로퀴놀론계항생제를 이용한결막내세포영상검사장치 .
[청구항 8] 제 7항에 있어서,
상기안구결막을염색하는상기플루오로퀴놀론계항생제는 목시플록사신을포함하는것을특징으로하는플루오로퀴놀론계 항생제를이용한결막내세포영상검사장치.
[청구항 9] 제 7항에 있어서,
상기근자외선영역및가시광선영역은 300nm내지 476nm인것을 특징으로하는플루오로퀴놀론계항생제를이용한결막내세포영상 검사장치.
PCT/KR2020/002893 2019-02-28 2020-02-28 플루오로퀴놀론계 항생제를 이용한 결막 내 세포영상 검사방법, 이를 이용한 안구 병변 진단방법과 안구병변 치료제 효능 검출방법 및 이를 위한 결막 내 세포영상 검사장치 Ceased WO2020175956A1 (ko)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100121172A1 (en) * 2008-11-12 2010-05-13 Siemens Corporate Research, Inc. Microscopic and macroscopic data fusion for biomedical imaging
US20160077007A1 (en) * 2014-09-16 2016-03-17 Lawrence Livermore National Security, Llc System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents
KR20160037834A (ko) * 2013-03-14 2016-04-06 루미셀, 아이엔씨. 의료 이미징 장치 및 사용 방법
KR20160136738A (ko) * 2015-05-20 2016-11-30 포항공과대학교 산학협력단 플루오로퀴놀론계 항생제의 용도

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2008320965B2 (en) * 2007-11-13 2014-10-23 The Regents Of The University Of Michigan Method and apparatus for detecting diseases associated with the eye
US20130149734A1 (en) * 2010-05-28 2013-06-13 The Regents Of The University Of Colorado, A Body Corporate Multi-photon Tissue Imaging
KR101662775B1 (ko) 2014-11-26 2016-10-05 박범철 안구건조증 진단 장치 및 이를 이용한 안구건조증 진단 방법
KR101894150B1 (ko) * 2016-11-15 2018-08-31 포항공과대학교 산학협력단 다광자 특성을 이용한 안과용 진단 및 치료장치

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100121172A1 (en) * 2008-11-12 2010-05-13 Siemens Corporate Research, Inc. Microscopic and macroscopic data fusion for biomedical imaging
KR20160037834A (ko) * 2013-03-14 2016-04-06 루미셀, 아이엔씨. 의료 이미징 장치 및 사용 방법
US20160077007A1 (en) * 2014-09-16 2016-03-17 Lawrence Livermore National Security, Llc System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents
KR20160136738A (ko) * 2015-05-20 2016-11-30 포항공과대학교 산학협력단 플루오로퀴놀론계 항생제의 용도

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM , SEONGHAN AND LEE SEUNGHUN, CHANG HOONCHUL, KIM MOSES, KIM MYOUNG JOON, KIM KI HEAN: "In vivo fluorescence imaging of conjunctival goblet cells", SCIENTIFIC REPORTS, vol. 9, no. 1, 29 October 2019 (2019-10-29), pages 1 - 7, XP055734635 *

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