WO2016180095A1 - 一种活体无创检测紫外光诱导皮肤损伤的方法及其检测设备 - Google Patents

一种活体无创检测紫外光诱导皮肤损伤的方法及其检测设备 Download PDF

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WO2016180095A1
WO2016180095A1 PCT/CN2016/077399 CN2016077399W WO2016180095A1 WO 2016180095 A1 WO2016180095 A1 WO 2016180095A1 CN 2016077399 W CN2016077399 W CN 2016077399W WO 2016180095 A1 WO2016180095 A1 WO 2016180095A1
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skin
autofluorescence
ultraviolet light
light source
excitation light
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PCT/CN2016/077399
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English (en)
French (fr)
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殷卫海
张铭超
贺号
张洁
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上海交通大学
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Priority to EP16791945.5A priority Critical patent/EP3295865A4/en
Priority to AU2016260153A priority patent/AU2016260153B2/en
Priority to US15/572,032 priority patent/US20180140194A1/en
Publication of WO2016180095A1 publication Critical patent/WO2016180095A1/zh

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    • 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
    • A61B5/0062—Arrangements for scanning
    • A61B5/0064—Body surface scanning
    • 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
    • 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
    • A61B5/0062—Arrangements for scanning
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A61B5/445—Evaluating skin irritation or skin trauma, e.g. rash, eczema, wound, bed sore
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2503/00—Evaluating a particular growth phase or type of persons or animals
    • A61B2503/40—Animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2503/00—Evaluating a particular growth phase or type of persons or animals
    • A61B2503/42—Evaluating a particular growth phase or type of persons or animals for laboratory research

Definitions

  • the invention relates to a method and a detection device for detecting autofluorescence of a living body. Specifically, it relates to a method for non-invasive detection of ultraviolet light-induced skin damage in a living body and a detection device thereof.
  • Ultraviolet light in sunlight is the main cause of human skin damage caused by ultraviolet radiation.
  • Ultraviolet radiation can cause various pathological effects on the skin, such as redness, peeling, inflammation, ulceration, and various skin diseases.
  • a large amount of active oxides cause DNA damage in subcutaneous cells and accelerate skin aging.
  • 90% of human skin aging is caused by ultraviolet light, and the most serious disease induced by ultraviolet light is skin cancer.
  • Skin cancer patients account for 40% of all cancer patients, and there are more than 3 million new cases of skin cancer every year worldwide, and the Skin Cancer Foundation also points out that one in five Americans will have skin at some stage in their lives. cancer.
  • the ozone layer responsible for blocking ultraviolet light in the atmosphere is continuously thinning.
  • Some experts have predicted that the thickness of the ozone layer is reduced by 1% and the intensity of ultraviolet radiation is increased by 2%. Therefore, the detection of skin ultraviolet light damage has great social and economic significance and clinical value for people to improve the skin health level and prevent the occurrence of skin diseases such as skin cancer.
  • UV skin damage has not received enough attention.
  • Many skin-injured patients will only go to the clinic after receiving particularly strong skin lesions, and in recent years, China has even The number of skin and skin cancer patients caused by UV trauma in the world is gradually increasing. Therefore, effective methods and means for detecting skin UV damage, as well as low-cost portable UV damage detection equipment, are of great significance for skin health monitoring, diagnosis and treatment of skin diseases, especially skin cancer.
  • the diagnosis of skin ultraviolet light damage is mainly based on the visual examination of dermatologists. It is highly dependent on the experience of doctors and can only evaluate the more serious skin damage, and can not achieve the accumulation of skin damage caused by ultraviolet light. It is an early diagnosis of skin cancer.
  • Existing skin lesion detection instruments can only achieve a very rough estimate of subcutaneous reflection imaging by different absorption of ultraviolet light during tissue propagation. The principle is that the ultraviolet lamp illuminates the skin and detects the light reflected back from the skin. Since the normal skin tissue and the damaged area have different absorption coefficients of light, the reflected or scattered light is imaged, so that the skin can be melanin, moisture, The lipid content is a very rough estimate.
  • the amount of photon scattering is inversely proportional to the fourth power of the wavelength.
  • the ultra-short wavelength of ultraviolet light its propagation in the skin tissue is affected.
  • the interference of scattering has been very significant, and the absorption of ultraviolet light in normal cells is also very strong. Therefore, this reflection imaging is subject to many factors, and the accuracy and repeatability are very low.
  • measurements of different muscle states in different tissues, or in the same tissue may have measured differences in results that are comparable to those caused by UV damage. Therefore, such instruments have poor clarity and sensitivity, and are unable to detect early skin damage caused by ultraviolet light.
  • the skin after ultraviolet light irradiation, the skin produces autofluorescence with a wavelength of 490-640 nm under the excitation of excitation light with a wavelength of 440-510 nm, and the autofluorescence of the wavelength is 490-640 nm.
  • Skin damage is directly proportional; at the same time X-rays are incapable of inducing an increase in autofluorescence under the endothelium of this wavelength range. Therefore, based on this new discovery, the present invention breaks through the difficulties of the prior art and designs a novel detection method and corresponding detection device which can be accurately used for predicting ultraviolet light-induced skin damage and having no damage to the human body.
  • the invention relates to a method for non-invasive detection of ultraviolet light-induced skin damage in a living body, comprising the steps of:
  • step (3) in accordance with the method of the step (2), the autofluorescence intensity of the skin of the subject not exposed to the ultraviolet light source is also detected;
  • the test is performed within 48 hours after the skin of the subject is irradiated with ultraviolet light; more preferably, the test is performed within 24 hours.
  • the method of exciting subcutaneous autofluorescence by excitation light includes at least one of a method of performing excitation using a normal continuous light output, a method of modulating excitation using electrical modulation, or a method of exciting using a pulsed laser. .
  • the wavelength of the excitation light is preferably in the range of 460 to 500 nm, and more preferably in the range of 485 to 490 nm.
  • the wavelength of the detected autofluorescence is preferably in the range of 500 to 550 nm, and more preferably in the range of 505 to 530 nm.
  • the present invention relates to a non-invasive detection device for detecting ultraviolet light-induced skin damage.
  • the detection device can be a miniaturized fluorescence detection imaging device including an excitation source, an optical conduction system, and an imaging system.
  • the excitation light source includes at least one of a single-frequency laser, a narrow-band light source, or a broadband light source capable of emitting light having a wavelength in the range of 440 to 510 nm, and optionally includes at least one filter.
  • the filter is used to filter the polychromatic light emitted by the excitation source into monochromatic light of a desired wavelength.
  • the optical conduction system is for conducting excitation light to the skin of the subject, and conducting autofluorescence emitted from the skin of the subject to the imaging system, wherein the excitation light and the autofluorescence are in optical conduction Part of the system propagates together and is separated by an optical conduction system.
  • the optical conduction system may include a two-phase color mirror for separating the excitation light and the autofluorescence, and optionally includes a pair of scanning galvanometers for adjusting the position of the spot and a pair of conjugate lenses for adjusting the depth of the excitation light.
  • excitation light and autofluorescence propagate in opposite directions on the optical path main axis, the dichroic mirror, the scanning galvanometer and the conjugate
  • the lens is arranged along the main axis; one of the excitation light source and the imaging system is located on the main axis of the optical path, and the other is located on the minor axis perpendicular to the main axis; the dichroic mirror is located at the intersection of the main axis and the sub-axis, so that the excitation light emitted by the excitation source and The collected autofluorescence is separated into a right angle relationship by a dichroic mirror, thereby allowing only autofluorescence to enter the imaging system.
  • the imaging system includes a fluorescence imaging detecting device capable of imaging light having a wavelength in the range of 490 to 640 nm and capable of calculating the intensity of the light.
  • the fluorescent imaging detecting device may be a photomultiplier tube (PMT), an avalanche diode, a photodiode, a CCD or a CMOS photodetector, or the like.
  • the invention uses ultraviolet light-induced early subcutaneous autofluorescence to directly proportional to the skin damage in the later stage, accurately predicts the degree of skin damage induced by ultraviolet light, and establishes a basis for prevention and early treatment of ultraviolet light-induced skin damage.
  • the earth reduces the prevalence of UV-induced skin diseases.
  • the detection device provided by the invention as a miniaturized fluorescence detection imaging device, can be applied in the case of medical diagnosis or research of high-end precision skin damage and fluorescence reconstruction, and can also be used in low-end skin damage medicine. It is applied in the case of diagnosis, simple skin damage in the home, etc., and has good effects such as high cost performance, accurate results, and wide application, and is very advantageous for popularization.
  • Figure 1-1 is a graph showing the increase in subcutaneous autofluorescence after 24 hours of UVC irradiation on the skin.
  • Figure 1-2 is a bar graph of subcutaneous autofluorescence after UVC irradiation of the skin for 24 hours.
  • Figure 2 is a representation of H&E staining of C57 mice after 5 days of UVC injury.
  • Figure 3 is a graph showing the increase in subcutaneous autofluorescence after 24 hours of UVC irradiation on the skin.
  • Figure 4 is a representation of H&E staining of C57 mice 5 days after UVC irradiation to the skin.
  • Fig. 5 is a bar graph showing subcutaneous autofluorescence and skin keratinization after UVC irradiation for 5 days.
  • Figure 6 is a graph showing the comparison of TUNNEL staining and epidermal apoptosis signal amount in C57 mice after UVC irradiation for 5 days.
  • Figure 7 is a graph showing the increase in subcutaneous autofluorescence after 24 hours of UVB irradiation to the skin.
  • Figure 8 is a representation of H&E staining of the C57 mouse ear region 24 hours after UVB injury.
  • Figure 9 is a histogram of H&E staining of the C57 mouse ear region after 24 hours of UVB injury.
  • Figure 10 is a representation of H&E staining of the epidermal region of C57 mice 24 hours after UVB injury.
  • Figure 11 is a histogram of H&E staining of the epidermal region of C57 mice after 24 hours of UVB injury.
  • Figure 12 is a representation of H&E staining of the C57 mouse ear region after 5 days of UVB injury.
  • Figure 13 is a histogram of H&E staining of the C57 mouse ear region after 5 days of UVB injury.
  • Figure 14 is a representation of H&E staining of the epidermal region of C57 mice after 5 days of UVB injury.
  • Figure 15 is a histogram of H&E staining of the epidermal region of C57 mice after 5 days of UVB injury.
  • Figure 16-1 is a constant graph of subcutaneous autofluorescence after 7 hours and 24 hours of synchrotron radiation X-ray irradiation on the skin.
  • Figure 16-2 is a bar graph of subcutaneous autofluorescence after 7 hours and 24 hours of synchrotron radiation X-ray irradiation on the skin.
  • Figure 17 is a graph showing subcutaneous autofluorescence of nude mice skin after UVC irradiation for 24 hours.
  • Figure 18 is a graph showing subcutaneous autofluorescence of ICR mouse skin after UVC irradiation for 24 hours.
  • Fig. 19 is a subcutaneous autofluorescence display after irradiating the skin with a solar simulator for 6 hours.
  • Figure 20 is a diagram showing the subcutaneous autofluorescence of UV-irradiated human index finger skin.
  • Fig. 21 is a schematic view showing the structure of a device for detecting non-invasive detection of ultraviolet light-induced skin damage in vivo according to the present invention.
  • ultraviolet light means electromagnetic radiation having a wavelength in the range of 100-400 nm, including naturally occurring light such as UVA, UVB, UVC, etc. in sunlight, and artificially generated wavelengths herein. Light within the range.
  • the ultraviolet light referred to in the present invention includes continuous electromagnetic radiation, pulsed electromagnetic radiation, modulated electromagnetic radiation, and the like, and the strength thereof is not particularly limited.
  • ultraviolet light-induced skin damage means damage such as skin tanning, sunburn, skin photoaging, skin allergic reaction, and skin cancer caused by ultraviolet light irradiation.
  • autofluorescence means that when a biomolecule is irradiated with excitation light of a suitable wavelength, the energy of the excitation light is absorbed into an excited state, and then exits the excited state to emit light having a longer wavelength than the excitation light. In the phenomenon, the wavelength is longer than the wavelength of the excitation light.
  • excitation light means a light capable of exciting an autofluorescence phenomenon of a biomolecule, and its wavelength should be shorter than that of autofluorescence.
  • the inventors conducted a large number of experiments to determine the relationship between the degree of autofluorescence and the degree of skin damage after UV-induced skin damage.
  • mice male C57 mice or nude mice or ICR mice are used, the UVC irradiated mice weigh between 15-25 g, and the UVB irradiated mice weigh between 18-25 g.
  • the skin of the mice was applied with a 1:1 solvent of glycerol and water, followed by corresponding ultraviolet light irradiation. After the completion of the ultraviolet radiation, the mice were housed in an animal room under the conditions of 22-24 ° C, a 12-hour light/dark cycle, and were free to eat water.
  • UV-irradiated skin was performed using a laser confocal microscope.
  • the mice were sacrificed one or five days later, and the damaged skin tissue was taken for H&E (hematoxylin-eosin staining) detection and TUNEL (in situ end labeling) detection.
  • the skin tissue of the mouse was imaged with a laser confocal microscope, wherein the excitation wavelength of the laser confocal microscope was 500-550 nm.
  • Skin tissue paraffin section soak the skin tissue in 4% paraformaldehyde solution for 24h, then According to the method for preparing paraffin sections, tap water, distilled water, gradient alcohol, xylene, and paraffin are sequentially prepared to prepare paraffin sections.
  • H&E staining of skin paraffin sections Dewaxed paraffin sections by dipping xylene, then dip in gradient alcohol, distilled water, hematoxylin staining for 10 minutes, tap water for 30 minutes, distilled water for 30s, 95% ethanol for 10s, and eosin for 30s. Wash 70% alcohol twice, sequentially dipped in gradient alcohol, xylene, and neutral resin.
  • Photographing and quantifying the staining results photographing the above two stains.
  • the indicators such as epidermal keratinization thickness and skin thickness were quantified.
  • Figure 1 After one day of UVC irradiation to the skin, the amount of subcutaneous autofluorescence increased significantly with increasing UV radiation dose.
  • Figure 1-1 in Figure 1 The autofluorescence intensity of the ear skin after 24 hours of C57 mouse ears after UVC irradiation increased with the irradiation time.
  • A, B, C, D were the control group, the irradiation dose of 0.3J / cm 2, 0.6J / cm 2 and 0.9J / cm 2.
  • Figure 1-2 in Figure 1 is a quantification of autofluorescence of the ear. * represents a P value of less than 0.05, and *** represents a P value of less than 0.001.
  • Figure 3 H&E staining after UVC irradiation for five days showed that the amount of subcutaneous autofluorescence detected after one day was correlated with the skin damage detected five days later.
  • A, B, and C were the control group, UVC irradiation for 40 minutes, and Spontaneous fluorescing of 1:1 solvent-protected skin with glycerol and water Light map.
  • Figure 4 D, E, F are the corresponding H&E staining representations; the box is the epidermis section.
  • G is a quantified map of fluorescence intensity
  • H is a quantified map of active epidermal thickness
  • I is a quantified map of keratin thickness.
  • TUNEL staining dark color is TUNNEL positive signal, indicating the presence of apoptotic cells; TUNEL staining showed subcutaneous autofluorescence and epidermal stimuli signal TUNEL signal after five days of UV light exposure (the increase is a sign of skin damage) One) is a completely positive correlation.
  • Figure 7 After one day of UVB irradiation, the subcutaneous autofluorescence increased significantly with the increase of ultraviolet radiation dose. The specific expression of fluorescence increased with the appearance of a circular fluorescent signal. The C57 mouse ears were irradiated with different doses of UVB for 24 hours. The subcutaneous autofluorescence intensity of the posterior ear skin is enhanced with the exposure dose.
  • Fig. 8 and Fig. 9 24 hours after UVB injury, H&E staining of C57 mice is 100 times magnification. After one day of UVB band irradiation, H&E staining showed no significant change in skin thickness, ie no significant damage to the skin.
  • Fig. 10 and Fig. 11 24 hours after UVB injury, H&E staining of C57 mice represents 20 times magnification. After one day of irradiation with UVB band, H&E staining showed no significant change in skin thickness, ie no significant damage to the skin.
  • FIG. 12 and Figure 13 H&E staining after UVB irradiation for five days showed that the amount of subcutaneous autofluorescence detected after one day was correlated with skin damage detected after five days; A, B, C, and D images were UVB lesions after 5 days.
  • FIG. 14 and Figure 15 H&E staining after UVB irradiation for five days showed that the amount of subcutaneous autofluorescence detected after one day was correlated with skin damage detected after five days; A, B, C, and D images were UVB lesions after 5 days.
  • FIG E picture shows the thickness of the epidermis; after UVB irradiation skin five days, H & E Staining, by analyzing the amount of subcutaneous autofluorescence detected after one day and the signs of skin damage detected after five days, showed that the amount of subcutaneous autofluorescence detected after one day was strongly proportional to the skin damage detected after five days. Relationship; subcutaneous autofluorescence is completely positively correlated with epidermal thickness; suggesting that green fluorescence enhancement can be used as an indicator for predicting future skin damage.
  • Fig. 16-1 and Fig. 16-2 The amount of subcutaneous autofluorescence did not change significantly with the change of the radiation dose after X-ray irradiation of the skin for 8 hours and one day.
  • Figure 17 UVC irradiation of nude mice skin, autofluorescence changes; as shown, after UVC irradiation, autofluorescence of nude mice skin also increased.
  • FIG. 18 UVC irradiation of ICR mouse skin, changes in autofluorescence, as shown in the figure, after After UVC irradiation, the autofluorescence of the skin of ICR mice also increases.
  • Figure 19 Changes in autofluorescence from sunlight on human skin; as shown, the autofluorescence of C57 skin is also increased after exposure to sunlight.
  • FIG. 20 UVC illuminates human skin with changes in autofluorescence; as shown, the autofluorescence of the human body increases after UVC irradiation.
  • UVB and UVC ultraviolet light
  • the skin will produce autofluorescence with a wavelength of 490-640 nm under the excitation of excitation light with a wavelength of 440-510 nm.
  • the wavelength is 490-640 nm.
  • the change in autofluorescence is directly proportional to skin damage; at the same time, X-rays have no ability to induce an increase in autofluorescence under the endothelium in this wavelength range. Therefore, subcutaneous autofluorescence can be used as a marker to predict skin damage.
  • the present invention establishes a method for non-invasive detection of ultraviolet light-induced skin lesions in vivo.
  • the method includes the following steps:
  • the skin irradiated by the subject with ultraviolet light is placed in the excitation light having a wavelength in the range of 440-510 nm within 72 hours after the skin of the subject is irradiated with a light source containing a certain dose of ultraviolet light.
  • the dose ultraviolet light power x irradiation time
  • step (3) in accordance with the method of the step (2), the autofluorescence intensity of the skin of the subject not exposed to the ultraviolet light source is also detected;
  • the means for exciting subcutaneous autofluorescence using excitation light of the present invention includes at least one of a mode of excitation using a normal continuous light output, a mode of modulation excitation using electrical modulation, or a mode of excitation using a pulsed laser.
  • the test is performed within 48 hours after the skin of the subject is irradiated with ultraviolet light; more preferably, the test is performed within 24 hours.
  • the wavelength of the excitation light is preferably in the range of 460 to 500 nm, and more preferably in the range of 485 to 490 nm.
  • the wavelength of the detected autofluorescence is preferably in the range of 500 to 550 nm, and more preferably in the range of 505 to 530 nm.
  • the method for non-invasive detection of skin damage induced by ultraviolet light in the present invention predicts the degree of skin damage of the experimental object according to the degree of change of subcutaneous autofluorescence and the degree of skin damage.
  • the present invention also provides a non-invasive detection device for detecting skin damage caused by ultraviolet light.
  • the detection device can be a miniaturized fluorescence detection imaging device including an excitation source, an optical conduction system, and an imaging system.
  • the excitation light source includes at least one of a single-frequency laser, a narrow-band light source, or a broadband light source capable of emitting light having a wavelength in the range of 440 to 510 nm, and optionally includes at least one filter.
  • the filter is used to filter the polychromatic light emitted by the excitation source into monochromatic light of a desired wavelength.
  • the optical conduction system is for conducting excitation light to the skin of the subject, and conducting autofluorescence emitted from the skin of the subject to the imaging
  • the optical conduction system may include a two-phase color mirror for separating the excitation light and the autofluorescence, and optionally includes a pair of scanning galvanometers for adjusting the position of the spot and a pair of conjugate lenses for adjusting the depth of the excitation light.
  • excitation light and autofluorescence propagate in opposite directions on the optical path main axis, the two-phase color mirror, the scanning galvanometer and the conjugate lens are arranged along the main axis; the excitation light source and the imaging system One of them is located on the main axis of the optical path, and the other is located on the minor axis perpendicular to the main axis; the dichroic mirror is located at the intersection of the main axis and the sub-axis, so that the excitation light emitted by the excitation source and the collected autofluorescence are separated by the dichroic mirror. It is in a right angle relationship so that only autofluorescence enters the imaging system.
  • the imaging system includes a fluorescence imaging detecting device capable of imaging light having a wavelength in the range of 490 to 640 nm and capable of calculating the intensity of the light.
  • the fluorescence imaging detection device can be a photomultiplier tube (PMT), an avalanche diode, a photodiode, a CCD or CMOS photodetector, or the like.
  • FIG. 1 A schematic diagram of one embodiment of the detection apparatus of the present invention is shown in FIG.
  • a detecting apparatus for non-invasive detection of ultraviolet light-induced skin damage of the present invention will be described in detail by referring to FIG.
  • main elements are arranged along the main shaft 10, and include a PMT detector 1, a dichroic mirror 3, a pair of scanning galvanometers 4, a pair of conjugate lenses 5, and an objective lens 6.
  • the counter shaft 20 is perpendicular to the main shaft 10, and some of the elements are arranged along the counter shaft 20, including a small semiconductor laser 2 and a two-phase color mirror 3 having a wavelength of 488 nm. It can be clearly seen that the dichroic mirror 3 is located at the intersection of the main shaft 10 and the counter shaft 20.
  • the laser light emitted by the laser 2 acts as excitation light, passes through the small hole, is collimated by the short focal length lens, reaches the dichroic mirror 3 and is reflected by the dichroic mirror 3, enters a pair of scanning galvanometers 4, and then passes through a For the conjugate lens 5, the objective lens 6 is entered.
  • a pair of scanning galvanometers 4 can be used to adjust the position of the spot on a plane perpendicular to the main axis 10
  • a pair of conjugate lenses 5 can be used to adjust the focus position of the excitation light on the main shaft 10, that is, the illumination of the excitation light. depth.
  • the autofluorescence signal generated by the excitation light is collected by the same objective lens 6, passes through a pair of conjugate lenses 5, returns to the scanning galvanometer 4, and then directly passes through the dichroic mirror 3, is filtered by the filter, and then focused by the lens. , and a small hole spatial filtering, and finally enter the PMT detector 1 for signal detection.
  • the positions of the detector 1 and the laser 2 can also be replaced with each other depending on the wavelength projection/reflection properties of the two-phase color mirror 3 used. Detection devices after positional interchange are also included in the scope of the present invention.
  • the scanning galvanometer 3, the PMT detector 1, and the circuit control portion of the laser 2 are integrally mounted in respective spaces.
  • the size of the entire system can be controlled within a range of 30 cm x 10 cm x 10 cm.
  • the detecting device of the present invention may further comprise other optical elements, such as light sources of other wavelengths of blue-violet light, such as 473 nm lasers, narrow-band blue LEDs, mercury lamps with color blocks, and the like; Mirror, convex lens, aspherical mirror, filter, small aperture diaphragm, CCD, CMOS, etc.
  • other optical elements such as light sources of other wavelengths of blue-violet light, such as 473 nm lasers, narrow-band blue LEDs, mercury lamps with color blocks, and the like; Mirror, convex lens, aspherical mirror, filter, small aperture diaphragm, CCD, CMOS, etc.
  • the separation of the excitation light and the autofluorescence can be achieved by simultaneously using the half mirror and the filter instead of the two-phase color mirror; for the filter used in the excitation light source, the filtering effect can also be realized by using other components such as a grating;
  • the optical path design may also differ from the above embodiments as long as the separation of the excitation light and the autofluorescence can be achieved and the autofluorescence is conducted to the imaging system.
  • the scanning galvanometer required for the point-by-point scanning imaging and the optical conduction system can be omitted in the optical conduction system for cost considerations and imaging accuracy requirements.
  • the conjugate lens required to adjust the depth position of the image is designed to design a detection device having the most basic function of detecting ultraviolet light-induced skin damage.
  • an excitation light source is used to emit excitation light, which is transmitted to the skin of the subject, and the objective lens is used to collect the subcutaneous Autofluorescence, which is then split by a dichroic mirror, and the resulting beam enters a photomultiplier tube or image sensor for detection, giving the wavelength and intensity of subcutaneous autofluorescence.
  • non-invasive detection of ultraviolet light-induced skin damage by the living body of the present invention does not depend on the detection apparatus of the present invention.
  • the method of non-invasive detection of ultraviolet light-induced skin damage of the present invention can be carried out using any apparatus capable of emitting excitation light and capable of detecting autofluorescence.

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Abstract

一种活体无创检测紫外光诱导皮肤损伤的方法及其检测设备，皮肤经过紫外光照射后，在波长为440-510nm激发光的激发下皮下会产生波长为490-640nm的自发荧光，这种波长为490-640nm的自发荧光的变化与皮肤损伤成正相关。

Description

一种活体无创检测紫外光诱导皮肤损伤的方法及其检测设备 技术领域
本发明涉及生物体自发荧光的检测方法及检测设备。具体来说，涉及一种活体无创检测紫外光诱导皮肤损伤的方法及其检测设备。
背景技术
阳光中的紫外光是人的皮肤受到紫外光辐射损伤的主要原因，紫外光辐射对于皮肤会产生多种病理作用，例如皮肤红肿，脱皮，炎症，溃烂，以及多种皮肤疾病等，并会产生大量活性氧化物，造成皮下细胞的DNA损伤，加速皮肤衰老。事实上，人体皮肤衰老90％的原因是由于紫外光照射，而紫外光诱导的最严重的疾病是皮肤癌，已有研究证明，90％以上的皮肤癌是由阳光中的紫外光照射引起的。皮肤癌患者占所有癌症患者中的40％，全世界每年有300多万新发皮肤癌病例，而皮肤癌基金会也指出，五分之一的美国人在一生中的某个阶段会得皮肤癌。随着人类工业化的进展，大气层中负责阻挡紫外光的臭氧层在不断变薄，有专家曾预测，臭氧层厚度减少1％，紫外光辐射强度增加2％。因此，对于皮肤紫外光损伤的检测对于人们提高皮肤的健康水平、防止皮肤癌等重大皮肤疾病的产生，都有着重大的社会经济意义和临床价值。
而在我国，由于人口压力巨大，诊断和就医困难，紫外皮肤损伤尚未得到足够的重视，往往很多皮肤受创患者只有受到特别强的皮损后才会门诊就医，而使得近年来，我国乃至于全世界紫外创伤导致的皮肤病和皮肤癌患者数量逐渐增多。因此，有效的皮肤紫外损伤的检测方法和手段，以及低成本便携式的紫外损伤检测设备，对于皮肤健康的监测、皮肤疾病特别是皮肤癌的诊断和治疗，意义重大。
现在临床上皮肤紫外光损伤的诊断以皮肤科医生目测为主，对医生的经验依赖性强，且只能对较严重的皮肤损伤进行评估，无法实现对紫外光造成的积累性皮肤损伤，特别是皮肤癌变的早期诊断。现有的皮肤损伤检测仪器，只能通过紫外光在组织传播过程中的吸收不同，对皮下进行反射成像实现一个非常粗略的估计。其原理是:紫外灯照射皮肤，检测皮肤反射回的光线，由于正常皮肤组织和受损区域对光线的吸收系数不同，将反射或散射回来的光线进行成像，从而可以对皮肤内黑色素、水分、脂质的含量做一个非常粗略的估计。由于激光在组织中的传播对于散射非常敏感，而且根据瑞利散射和弥散射原理，光子散射量与波长的四次方成反比，对于紫外光这种超短波长而言，其在皮肤组织的传播受到散射的干扰已经非常显著，同时紫外光在正常细胞中的吸收也非常强烈，因此，这种反射成像会受到非常多因素的干扰，准确率和重复性都非常低。对于同样一个人而言，在不同组织，或同样组织中的不同肌肉状态下的测量，可能测得的结果差异已经与紫外受损造成的差异相当。因此，这类仪器清晰度、灵敏度差，无法检测紫外光造成的早期皮肤损伤，对皮肤损伤及进一步的皮肤疾病的早期检测和预防毫无指导意义，在临床上几乎没有应用。特别地，对于皮肤科门诊，往往只能使用光实验对皮肤的紫外敏感性做一个定性评估，其方法是，在一个非见光皮肤区域，如背部的一块皮肤上进行紫外光照射，然后通过这块皮肤出现的红斑面积来评价患者的皮肤紫外敏感性，既不准确，也无法进行损伤的直接检测。
发明内容
本发明人研究发现，皮肤经过紫外光照射后，在波长为440-510nm激发光的激发下皮下会产生波长为490-640nm的自发荧光，并且这种波长为490-640nm的自发荧光的变化与皮肤损伤成正比关系；同时X射线没有能力诱导该波长范围内皮下自发荧光的增加。因此，本发明基于这一全新发现，突破了现有技术的难题设计了一种可以准确地用于预测紫外光诱导皮肤损伤并且对人体无损伤的新型检测方法和相应的检测设备。
一方面，本发明涉及一种活体无创检测紫外光诱导皮肤损伤的方法，包括以下步骤：
(1)在实验对象的皮肤受到包含一定剂量紫外光的光源照射后72小时之内，将所述实验对象经过含一定剂量紫外光的光源照射的皮肤置于波长在440-510nm范围内的激发光下，以激发出皮下自发荧光，其中所述剂量＝紫外光功率×照射时间；
(2)检测所述实验对象经过含紫外光的光源照射的皮肤的角质层以下真皮层以上部位所发出的波长在490-640nm范围内的自发荧光强度；
(3)按照所述步骤(2)的方法，同样检测所述实验对象未受到含紫外光的光源照射的皮肤所发出的自发荧光强度；
(4)将所述实验对象经过含紫外光的光源照射的皮肤的自发荧光强度和未受到含紫外光的光源照射的皮肤的自发荧光强度相对比，获得经紫外光照射诱发的自发荧光强度变化率；
(5)根据所述经紫外光照射诱发的自发荧光强度变化率，预测所述实验对象的皮肤健康状态。
前述本发明的检测方法中，优选的是，在实验对象的皮肤受到紫外光照射后的48小时之内进行检测；更优选的是，在24小时之内进行检测。
前述本发明的检测方法中，利用激发光激发皮下自发荧光的方式包括使用普通的连续光输出进行激发的方式、使用电调制进行调制激发的方式或者使用脉冲激光进行激发的方式中的至少一种。
前述本发明的检测方法中，激发光的波长优选为460-500nm的范围内，更优选为485-490nm的范围内。
前述本发明的检测方法中，所检测的自发荧光的波长优选为500-550nm的范围内，更优选为505-530nm的范围内。
另一方面，本发明涉及一种活体无创检测紫外光诱导皮肤损伤的检测设备。该检测设备可以是一种小型化的荧光探测成像设备，包括激发光源、光学传导系统和成像系统。
在前述本发明的检测设备中，所述激发光源包括能够发出波长在440-510nm范围内的光的单频激光、窄带光源或宽带光源中的至少一种，并且可选择地包括至少一个滤波片。该滤波片用于将激发光源发出的多色光过滤为所需波长的单色光。
在前述本发明的检测设备中，所述光学传导系统用于将激发光传导至实验对象的皮肤，并且将实验对象的皮肤发出的自发荧光传导至成像系统，其中激发光和自发荧光在光学传导系统的一部分中共同传播，并且被光学传导系统分离。光学传导系统可以包括用于分离激发光和自发荧光的二相色镜，并且可选择地包括用于调整光斑位置的一对扫描振镜以及用于调整激发光照射深度的一对共轭透镜。
根据本发明的某些实施例，在前述本发明的检测设备的光学传导系统中，激发光和自发荧光在光路主轴上沿相对的方向传播，所述二相色镜、扫描振镜和共轭透镜沿主轴排列；激发光源和成像系统中的一个位于光路主轴上，另一个位于与主轴垂直的副轴上；二相色镜位于主轴与副轴的交叉位置，使激发光源发出的激发光和收集的自发荧光被二相色镜分离成直角关系，从而仅使自发荧光进入成像系统。
在前述本发明的检测设备中，成像系统包括荧光成像检测器件，所述荧光成像检测器件能够成像波长在490-640nm范围内的光，并且能够计算所述光的强度。根据某些具体实施例，所述荧光成像检测器件可以是光电倍增管(PMT)、雪崩二极管、光电二极管、CCD或CMOS光电探测器等。
紫外光本发明利用紫外光诱发的早期皮下自发荧光与后期皮肤损伤成正比的关系，准确地预测紫外光诱发的皮肤损伤程度，为预防和早期治疗紫外光诱发的皮肤损伤建立了基础，可以极大地减少紫外光诱发的皮肤病的患病率。同时，本发明提供的检测设备，作为一种小型化的荧光探测成像设备，既可以在高端精密皮肤损伤与荧光重构的医学诊断或研究等情况下得到应用、也可以在低端皮肤损伤医学诊断、家庭用简易皮肤损伤等情况下得到应用，具有性价比高、结果精确并且应用广泛等良好效果，非常有利于普及。
附图说明
图1-1为UVC照射皮肤24小时后皮下自发荧光的增加图。
图1-2为UVC照射皮肤24小时后皮下自发荧光的柱状图。
图2为UVC损伤5天后，C57小鼠H&E染色代表图。
图3为UVC照射皮肤24小时后，皮下自发荧光的增加图。
图4为UVC照射皮肤5天后，C57小鼠H&E染色代表图。
图5为UVC照射皮肤5天后，皮下自发荧光与皮肤角质化柱状表示图。
图6为UVC照射皮肤5天后，C57小鼠TUNNEL染色和表皮的凋亡信号量对比图。
图7为UVB照射皮肤24小时后，皮下自发荧光增加图。
图8为UVB损伤24小时后，C57小鼠耳朵区域H&E染色代表图。
图9为UVB损伤24小时后，C57小鼠耳朵区域H&E染色代表柱状图。
图10为UVB损伤24小时后，C57小鼠表皮区域H&E染色代表图。
图11为UVB损伤24小时后，C57小鼠表皮区域H&E染色代表柱状图。
图12为UVB损伤5天后，C57小鼠耳朵区域H&E染色代表图。
图13为UVB损伤5天后，C57小鼠耳朵区域H&E染色代表柱状图。
图14为UVB损伤5天后，C57小鼠表皮区域H&E染色代表图。
图15为UVB损伤5天后，C57小鼠表皮区域H&E染色代表柱状图。
图16-1为同步辐射X-射线照射皮肤8小时、24小时后皮下自发荧光的不变图。
图16-2为同步辐射X-射线照射皮肤8小时、24小时后皮下自发荧光的柱状图。
图17为UVC照射皮肤24小时后，裸鼠皮肤皮下自发荧光显示图。
图18为UVC照射皮肤24小时后，ICR小鼠皮肤皮下自发荧光显示图。
图19为用太阳光模拟器照射皮肤6小时后，皮下自发荧光显示图。
图20为UV照射人体食指皮肤，皮下自发荧光显示图。
图21为本发明的活体无创检测紫外光诱导皮肤损伤的检测设备的设备结构示意图。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本发明中使用的术语“紫外光”，其含义为波长在100-400nm范围内的电磁辐射，包括自然产生的光，如太阳光中的UVA、UVB、UVC等，以及人工产生的波长在此范围内的光。本发明所指紫外光包括连续电磁辐射、脉冲电磁辐射和调制的电磁辐射等，并且对其强度没有特别限定。
本发明中使用的术语“紫外光诱导皮肤损伤”，其含义为由紫外光照射引起的皮肤晒黑、晒伤、皮肤光老化、皮肤过敏反应以及皮肤癌等损伤。
本发明中使用的术语“自发荧光”，其含义为生物分子在受到适当波长的激发光照射时，吸收激发光的能量进入激发态，再退出激发态从而发射出比激发光波长更长的光的现象中，所述波长比激发光波长更长的光。
本发明中使用的术语“激发光”，其含义为能够激发生物分子发生自发荧光现象的光，其波长应比自发荧光短。
紫外光诱导皮肤损伤后的自发荧光变化程度与皮肤损伤程度的关系
发明人进行了大量的实验，确定了紫外光诱导皮肤损伤后的自发荧光变化程度与皮肤损伤程度关系。
根据本发明，使用雄性C57小鼠或裸鼠或ICR小鼠，UVC照射的小鼠重量在15-25g范围之间，UVB照射的小鼠重量在18-25g范围之间。麻醉后对小鼠皮肤涂抹甘油与水的1:1溶剂，随后进行相应的紫外光照射。紫外光辐射完成后，小鼠在动物房中进行饲养，条件为22-24℃，12小时的明/暗循环，并可自由进食取水。
一天后，使用激光共聚焦显微镜对紫外光照射的皮肤进行无创成像。其后一天或五天后牺牲小鼠，取损伤皮肤组织，进行H&E(苏木精&伊红染色)检测以及TUNEL(原位末端标记)检测。小鼠的皮肤组织用激光共聚焦显微镜成像，其中激光共聚焦显微镜的激发波长为500-550nm。
皮肤组织的储存：皮肤取出后，取出适量组织浸泡于4％多聚甲醛，用于制作石蜡切片，剩余组织用铝箔纸包裹好，使用液氮冷冻，之后转移到-80℃冰箱长期保存。
皮肤组织石蜡切片：将皮肤组织浸泡4％多聚甲醛溶液24h，然后 依照石蜡切片的制作方法依次浸自来水、蒸馏水、梯度酒精、二甲苯、石蜡，制作成石蜡切片。
皮肤石蜡切片的H&E染色：将石蜡切片浸二甲苯脱蜡，然后依次浸于梯度酒精、蒸馏水，苏木素染色10分钟，自来水流水冲洗30min，蒸馏水浸泡30s，95％乙醇10s，伊红复染30s，70％酒精洗涤2次，依次浸梯度酒精、二甲苯，中性树脂封片。
对染色结果拍照、量化：对上述两种染色进行拍照。并针对表皮角质化厚度，表皮厚度等指标进行量化。
统计分析：所有数据按照平均值±标准差的方式给出，数据使用单因素方差分析进行评估，P值小于0.05认为是统计学显著的。
小鼠实验的结果如下所述：
图1：UVC照射皮肤一天后，皮下自发荧光量随紫外辐射剂量的增加而显著增加。
图1中的图1-1：C57小鼠耳部经UVC照射不同时间后，24小时后耳部皮肤的自发荧光强度随照射时间的延长而增强。A、B、C、D分别为对照组，照射剂量为0.3J/cm2，0.6J/cm2和0.9J/cm2。
图1中的图1-2：为对耳部自发荧光的量化。*代表P值小于0.05，***代表P值小于0.001。
图2：紫外光照射皮肤一天后，H&E染色，发现皮肤没有明显损伤。
图3：UVC照射皮肤五天后，H&E染色，显示一天后检测出的皮下自发荧光量和五天后检测出的皮肤损伤有相关关系，A、B、C分别为对照组、UVC照射40分钟以及具有甘油与水1:1溶剂保护的皮肤自发荧 光图。
图4：D、E、F为相应的H&E染色代表图；方框中为表皮部分。
图5：G为荧光强度的量化图；H为活性表皮厚度的量化图，I为角质厚度的量化图。
由图5可知UVC照射皮肤五天后，H&E染色，通过分析一天后检测出的皮下自发荧光和五天后检测出的皮肤损伤的标志，显示出一天后检测出的皮下自发荧光和五天后检测出的皮肤损伤有很强的正相关关系：皮下自发荧光和皮肤活性表皮除角质层以外的表皮层(其减少是皮肤损伤的标志之一)成完全负相关关系；而皮下自发荧光和表皮角质化(其增加是皮肤损伤的标志之一)成完全成正相关关系；因此，本发明首次公开了皮下自发荧光的增强可以作为预测未来皮肤损伤的指标。
图6：TUNEL染色，图中暗色为TUNNEL阳性信号，代表存在凋亡细胞；紫外光照射皮肤五天后，TUNEL染色显示皮下自发荧光和表皮的凋亡信号量TUNEL信号(其增加是皮肤损伤的标志之一)成完全正相关关系。
图7：UVB照射皮肤一天后，皮下自发荧光随紫外辐射剂量的增加而显著增加，其荧光增加具体表现形式为出现圆形的荧光信号，C57小鼠耳部经UVB照射不同剂量后，24小时后耳部皮肤的皮下自发荧光强度随照射剂量的延长而增强。
图8和图9：UVB损伤后24小时，C57小鼠H&E染色代表图100倍放大，紫外光UVB波段照射皮肤一天后，H&E染色，发现皮肤厚度没有明显变化，即皮肤没有明显损伤。
图10和图11：UVB损伤后24小时，C57小鼠H&E染色代表图20倍放大，紫外光UVB波段照射皮肤一天后，H&E染色，发现表皮厚度没有明显变化，即皮肤没有明显损伤。
图12和图13：UVB照射皮肤五天后，H&E染色，显示一天后检测出的皮下自发荧光量和五天后检测出的皮肤损伤有相关关系；A，B，C，D图为UVB损伤5天后H&E染色代表图，分别对应UVB损伤剂量为0J/cm2，2.5J/cm2，5.0J/cm2，7.5J/cm2；E图为耳朵厚度的量化图；UVB照射皮肤五天后，H&E染色，分析一天后检测出的皮下自发荧光量和和五天后检测出的皮肤损伤的标志，显示出一天后检测出的皮下自发荧光量和五天后检测出的皮肤损伤有很强的正比关系；皮下自发荧光量和耳朵厚度呈完全正相关。
图14和图15：UVB照射皮肤五天后，H&E染色，显示一天后检测出的皮下自发荧光量和五天后检测出的皮肤损伤有相关关系；A，B，C，D图为UVB损伤5天后H&E染色代表图，分别对应UVB损伤剂量为0J/cm2，2.5J/cm2，5.0J/cm2，7.5J/cm2；E图为表皮厚度的量化图；UVB照射皮肤五天后，H&E染色，通过分析一天后检测出的皮下自发荧光量和和五天后检测出的皮肤损伤的标志，显示出一天后检测出的皮下自发荧光量和和五天后检测出的皮肤损伤有很强的正比关系；皮下自发荧光量和表皮厚度呈完全正相关；提示，绿色荧光的增强可以作为预测未来皮肤损伤的指标。
图16-1和图16-2：X射线照射皮肤8小时以及一天后，皮下自发荧光量随辐射剂量的改变没有而显著变化。
图17：UVC照射裸鼠皮肤，自发荧光的变化；如图显示，经过UVC照射之后，裸鼠皮肤的自发荧光也会增加。
图18：UVC照射ICR小鼠皮肤，自发荧光的变化，如图显示，经过 UVC照射之后，ICR小鼠皮肤的自发荧光也会增加。
图19：太阳光照射人体皮肤，自发荧光的变化；如图显示，用太阳光照射之后，C57皮肤的自发荧光也会增加。
图20：UVC照射人体皮肤，自发荧光的变化；如图显示，经过UVC照射之后，人体的自发荧光也会增加。
由以上实验可以得出，皮肤经过紫外光(UVB和UVC)照射后，在波长为440-510nm激发光的激发下皮下会产生波长为490-640nm的自发荧光，这种波长为490-640nm的自发荧光的变化与皮肤损伤成正比关系；同时X射线没有能力诱导该波长范围内皮下自发荧光的增加。因此，皮下自发荧光能够作为预测皮肤损伤的标志物。
本发明基于这一发现，建立了一种活体无创检测紫外光诱导皮肤损伤的方法。该方法包括以下步骤：
(1)在实验对象的皮肤受到包含一定剂量紫外光的光源照射后72小时之内，将所述实验对象经过含紫外光的光源照射的皮肤置于波长在440-510nm范围内的激发光下，以激发出皮下自发荧光，其中所述剂量＝紫外光功率×照射时间；
(2)检测所述实验对象经过含紫外光的光源照射的皮肤的角质层以下真皮层以上部位所发出的波长在490-640nm范围内的自发荧光强度；
(3)按照所述步骤(2)的方法，同样检测所述实验对象未受到含紫外光的光源照射的皮肤的自发荧光强度；
(4)将所述实验对象经过含紫外光的光源照射的皮肤的自发荧光强度和未受到含紫外光的光源照射的皮肤的自发荧光强度相对比，获得经紫外光照射诱发的自发荧光强度变化率；
(5)根据所述经紫外光照射诱发的自发荧光强度变化率，预测所述实验对象的皮肤健康状态。
本发明的利用激发光激发皮下自发荧光的方式包括使用普通的连续光输出进行激发的方式、使用电调制进行调制激发的方式或者使用脉冲激光进行激发的方式中的至少一种。
在本发明的检测方法中，优选的是，在实验对象的皮肤受到紫外光照射后的48小时之内进行检测；更优选的是，在24小时之内进行检测。
在本发明的检测方法中，激发光的波长优选为460-500nm的范围内，更优选为485-490nm的范围内。
在本发明的检测方法中，所检测的自发荧光的波长优选为500-550nm的范围内，更优选为505-530nm的范围内。
本发明的活体无创检测紫外光诱导皮肤损伤的方法，根据皮下自发荧光的变化程度与皮肤损伤程度呈正比的规律，预测实验对象的皮肤损伤程度。
此外，本发明还提供了一种活体无创检测紫外光诱导皮肤损伤的检测设备。该检测设备可以是一种小型化的荧光探测成像设备，包括激发光源、光学传导系统和成像系统。
在本发明的检测设备中，所述激发光源包括能够发出波长在440-510nm范围内的光的单频激光、窄带光源或宽带光源中的至少一种，并且可选择地包括至少一个滤波片。该滤波片用于将激发光源发出的多色光过滤为所需波长的单色光。
在本发明的检测设备中，所述光学传导系统用于将激发光传导至实验对象的皮肤，并且将实验对象的皮肤发出的自发荧光传导至成像 系统，其中激发光和自发荧光在光学传导系统的一部分中共同传播，并且被光学传导系统分离。光学传导系统可以包括用于分离激发光和自发荧光的二相色镜，并且可选择地包括用于调整光斑位置的一对扫描振镜以及用于调整激发光照射深度的一对共轭透镜。
在本发明的检测设备的光学传导系统中，激发光和自发荧光在光路主轴上沿相对的方向传播，所述二相色镜、扫描振镜和共轭透镜沿主轴排列；激发光源和成像系统中的一个位于光路主轴上，另一个位于与主轴垂直的副轴上；二相色镜位于主轴与副轴的交叉位置，使激发光源发出的激发光和收集的自发荧光被二相色镜分离成直角关系，从而仅使自发荧光进入成像系统。
在本发明的检测设备中，成像系统包括荧光成像检测器件，所述荧光成像检测器件能够成像波长在490-640nm范围内的光，并且能够计算所述光的强度。根据某些具体实施例，荧光成像检测器件可以是光电倍增管(PMT)、雪崩二极管、光电二极管、CCD或CMOS光电探测器等。
本发明的检测设备的一个具体实施例的示意图如图21所示。以下将通过参考图21，详细描述本发明的活体无创检测紫外光诱导皮肤损伤的检测设备。
在图21所示的检测设备中，主要元件沿主轴10排列，包括PMT探测器1、二相色镜3、一对扫描振镜4、一对共轭透镜5和物镜6。副轴20与主轴10垂直，部分元件沿副轴20排列，包括波长488nm的小型半导体激光器2和二相色镜3。明显可以看出，二相色镜3位于主轴10和副轴20的交叉位置。
激光器2发出的激光作为激发光，经过小孔后由短焦距透镜准直，到达二相色镜3并被二相色镜3反射，进入一对扫描振镜4，之后经过一 对共轭透镜5，进入物镜6。其中，一对扫描振镜4可以用于调整光斑在垂直于主轴10的平面上的位置，并且一对共轭透镜5可以用于调整激发光在主轴10上的聚焦位置，即激发光的照射深度。
经激发光激发产生的自发荧光信号由同一个物镜6收集，经过一对共轭透镜5后，返回扫描振镜4，然后直接穿过二相色镜3，经过滤波片滤波，而后由透镜聚焦，和一个小孔空间滤波，最后进入PMT探测器1进行信号探测。
需要注意的是，根据所使用的二相色镜3的波长投射/反射性质，探测器1和激光器2的位置也可以相互替换。位置互换后的检测设备，也包含在本发明的范围内。
作为小型化的荧光探测成像设备，扫描振镜3、PMT探测器1和激光器2的电路控制部分集成安装于相应的空间。整个系统的大小可以控制在30cm×10cm×10cm的范围内。
除上述元件之外，本发明的检测设备中还可以包含其他光学元件，例如其它波长的蓝紫光波段的光源，如473nm激光器，窄带的蓝光LED，具有滤色块的汞灯等等；以及银镜，凸透镜，非球面镜，滤波片，小孔光阑，CCD，CMOS等等。
本领域的技术人员应当理解，基于本发明的检测设备的原理，可以采用其他方法和元件实现本发明的检测设备。例如，也可以通过同时使用半透半反镜和滤波片代替二相色镜，实现激发光和自发荧光的分离；对于激发光源中使用的滤波片，也可以用光栅等其它元件实现滤波效果；光路设计也可以不同于上述实施例，只要能够实现激发光和自发荧光的分离、并且把自发荧光传导至成像系统即可。另外，在本发明的检测设备中，出于成本的考虑和成像精度的要求，可以在光学传导系统中省略逐点扫描成像所需的扫描振镜，以及光学传导系统 中调整成像深度位置所需的共轭透镜，从而设计具有最基本的检测紫外光诱导皮肤损伤的功能的检测设备。这些修改和/或简化的检测设备，也在本发明的范围之内。
在使用本发明的活体无创检测紫外光诱导皮肤损伤的检测设备进行本发明的活体无创检测紫外光诱导皮肤损伤的方法时，使用激发光源发出激发光，传导到实验对象的皮肤，采用物镜收集皮下自发荧光，然后由二相色镜分光，所分得的光束进入光电倍增管或图像传感器中进行检测，得出皮下自发荧光的波长和强度。
应该理解的是，本发明的活体无创检测紫外光诱导皮肤损伤的方法的实施并不依赖于本发明的检测设备。可以使用任何能够发出激发光并且能够对自发荧光进行检测的设备来实施本发明的活体无创检测紫外光诱导皮肤损伤的方法。
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求的限制。本申请中引用的所有文献均完整地并入本文作为参考。

Claims (12)

  1. 一种活体无创检测紫外光诱导皮肤损伤的方法，包括以下步骤：
    (1)在实验对象的皮肤受到包含一定剂量紫外光的光源照射后72小时之内，将所述实验对象经过含紫外光的光源照射的皮肤置于波长在440-510nm范围内的激发光下，以激发出皮下自发荧光；
    (2)检测所述实验对象经过含紫外光的光源照射的皮肤的角质层以下真皮层以上部位所发出的波长在490-640nm范围内的自发荧光强度；
    (3)按照所述步骤(2)的方法，同样检测所述实验对象未受到含紫外光的光源照射的皮肤所发出的自发荧光强度；
    (4)将所述实验对象经过含紫外光的光源照射的皮肤的自发荧光强度和未受到含紫外光的光源照射的皮肤的自发荧光强度相对比，获得经紫外光照射诱发的自发荧光强度变化率；
    (5)根据所述经紫外光照射诱发的自发荧光强度变化率，预测所述实验对象的皮肤健康状态。
  2. 如权利要求1所述的方法，其特征在于：
    所述利用激发光激发皮下自发荧光的方式包括使用普通的连续光输出进行激发的方式、使用电调制进行调制激发的方式或者使用脉冲激光进行激发的方式中的至少一种。
  3. 如权利要求1所述的方法，其特征在于：
    在所述光源照射后的48小时之内进行检测。
  4. 如权利要求3所述的方法，其特征在于：
    在所述光源照射后的24小时之内进行检测。
  5. 如权利要求1所述的方法，其特征在于：
    所述激发光的波长在460-500nm范围内。
  6. 如权利要求5所述的方法，其特征在于：
    所述激发光的波长在485-490nm范围内。
  7. 如权利要求1所述的方法，其特征在于：
    所述自发荧光的波长在500-550nm范围内。
  8. 如权利要求7所述的方法，其特征在于：
    所述自发荧光的波长在505-530nm范围内。
  9. 一种活体无创检测紫外光诱导皮肤损伤的检测设备，该检测设备包括激发光源、光学传导系统和成像系统，其特征在于：
    所述激发光源包括能够发出波长在440-510nm范围内的光的单频激光、窄带光源或宽带光源中的至少一种；
    所述光学传导系统用于将所述激发光传导至实验对象的皮肤，并且将所述实验对象的皮肤发出的自发荧光传导至所述成像系统，其中所述激发光和所述自发荧光在所述光学传导系统的一部分中共同传播，并且被所述光学传导系统分离；
    所述成像系统包括荧光成像检测器件，所述荧光成像检测器件能够成像波长在490-640nm范围内的光，并且能够计算所述光的强度。
  10. 如权利要求9所述的检测设备，其特征在于：
    所述激发光源还包括至少一个滤波片；
    所述光学传导系统包括用于分离所述激发光和所述自发荧光的二相色镜，用于调整光斑位置的一对扫描振镜以及用于调整所述激发光照射深度的一对共轭透镜。
  11. 如权利要求10所述的检测设备，其特征在于：
    所述激发光和所述自发荧光在所述光学传导系统的光路主轴上沿 相对的方向传播，所述二相色镜、所述扫描振镜和所述共轭透镜沿所述主轴排列；
    所述激发光源和所述成像系统中的一个位于所述主轴上，另一个位于与所述主轴垂直的副轴上；
    所述二相色镜位于所述主轴与所述副轴的交叉位置，使所述激发光和所述自发荧光被所述二相色镜分离成直角关系，从而仅使所述自发荧光进入所述成像系统。
  12. 如权利要求9所述的检测设备，其特征在于：
    所述成像系统包括光电倍增管、雪崩二极管、光电二极管、CCD或CMOS光电探测器中的至少一种。
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