WO2020150853A1 - 用于角膜神经成像的光片显微镜 - Google Patents

用于角膜神经成像的光片显微镜 Download PDF

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WO2020150853A1
WO2020150853A1 PCT/CN2019/072453 CN2019072453W WO2020150853A1 WO 2020150853 A1 WO2020150853 A1 WO 2020150853A1 CN 2019072453 W CN2019072453 W CN 2019072453W WO 2020150853 A1 WO2020150853 A1 WO 2020150853A1
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light
lens
lens group
angle
corneal
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PCT/CN2019/072453
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English (en)
French (fr)
Inventor
胡衍
杨建龙
赵一天
蒋珊珊
王浩
谢建洋
岳星宇
杨燕鹤
刘江
Original Assignee
中国科学院宁波工业技术研究院慈溪生物医学工程研究所
中国科学院宁波材料技术与工程研究所
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Priority to PCT/CN2019/072453 priority Critical patent/WO2020150853A1/zh
Publication of WO2020150853A1 publication Critical patent/WO2020150853A1/zh

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    • 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
    • A61B3/14—Arrangements specially adapted for eye photography
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

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  • the invention relates to the field of corneal nerve imaging, in particular to a corneal nerve imaging device using a light sheet microscope, which can be widely used in the field of biomedical ophthalmic imaging technology.
  • the cornea is the frontmost part of the eye, in front of the iris and pupil. It is the densest tissue innervated by nerves. Most corneal nerves are sensory nerves, which come from the thalamic branch of the trigeminal nerve. The average horizontal diameter of the cornea of an adult eye is about 11.5 mm, and the vertical diameter is 10.5 mm. The curvature of the cornea remains quite stable throughout life.
  • the optic nerve area (the anterior pupil cornea), which provides most of the refractive function of the cornea, is 4 mm in diameter. It is located in the center of the cornea and in front of the pupil. Under the condition of photopic vision, the cornea is an avascular branch of the anterior ciliary artery. Provides the curved limbus of the peripheral cornea.
  • the peripheral cornea and the central cornea are very different in physiology and pathology.
  • the corneal stroma 500 mm thick provides structural integrity to the cornea.
  • Mesenchymal keratocytes secrete collagen and proteoglycans, which are ultimately the basis for transparent cornea and hydration.
  • the cornea is rich in nerve endings and is one of the most sensitive tissues in the body. It plays an important role in maintaining the integrity of the ocular surface. Studies have shown that there are many diseases related to corneal nerves, such as keratitis, dry eye signs and diabetes.
  • the cornea is transparent, without blood vessels or lymphatic vessels, but it is rich in nerve fibers. Most of them are cholinergic nerves of parasympathetic nerves, but also adrenergic nerves of sympathetic nerves.
  • the corneal confocal microscope produced by Heidelberg is commonly used, which mainly uses Zeiss 63x, NA 0.95 water mirror, 630nm diode laser for point scanning.
  • the speed of acquiring images is 8 frames per second, so each patient generally takes 5-15 minutes to acquire the required images.
  • the confocal microscope uses the point scanning method for illumination, the image acquisition speed is slow, and it takes a long time for each patient to maintain a shooting posture, and because the objective lens and the eyeball pass through the eye when shooting the corneal nerve If the glue is in contact, long-term shooting will cause extreme discomfort to the patient's eyes, so shortening the shooting time is a problem that needs to be solved.
  • Light sheet fluorescence microscopy has been widely used in transparent tissue imaging in recent years. After the animal’s liver, kidney and other tissues are transparentized, the imaging speed of the light-sheet fluorescence microscope is significantly faster than that of the confocal fluorescence microscope, and the imaging depth is more advantageous.
  • Hong et al. proposed to use a light sheet microscope to photograph the trabecular meshwork for the diagnosis of glaucoma.
  • the device uses a 488nm Bessel Gauss laser as the illumination excitation light, and then obtains the reflected fluorescence signal. Bessel Gauss in the text The laser is obtained using a 176-degree vertex cone lens.
  • the illumination light path enters from the left side of the eyeball, and the image acquisition light path acquires the reflected image information from the trabecular meshwork.
  • this equipment may be suitable for taking out the trabecular meshwork that moves the eyeball.
  • humans have left and right eyes, and there is a bridge of nose as a partition, so this device is not suitable.
  • this kind of lighting device uses a static light sheet. During the shooting process, the image of the eyeball being shot needs to be moved to obtain information of different depths, but it is easy to cause the problem that the moving distance does not meet the required shooting depth.
  • the present invention proposes a light sheet microscope for corneal nerve imaging, which can speed up the imaging speed, reduce the shooting time, and relieve the patient's eye discomfort.
  • the light sheet microscope used for corneal neuroimaging of the present invention includes a control device, an illumination light path, and an image acquisition light path.
  • the illumination light path injects light into the cornea of the eye and forms a dynamic light sheet at an angle ⁇ to the horizontal at the cornea of the eye
  • the image signal is emitted into the image acquisition optical path at a ⁇ angle to the horizontal, and the image acquisition optical path sends the image signal to the control device for imaging; wherein the angle between the ⁇ angle and the ⁇ angle is 90 degrees, and the ⁇ angle is not equal to 90 degrees .
  • the light sheet microscope of the present invention further includes a laser light source, a first lens group, a mirror group, a second lens group, and a camera sensor; the light emitted by the laser light source sequentially passes through the first lens group and the mirror group Enter the cornea of the eye to form an illumination light path; the image signal is sequentially focused on the camera sensor through the mirror group and the second lens group to form an image acquisition light path.
  • the first lens group includes a cylindrical lens;
  • the mirror group includes a polygon mirror scanner, a lens, and an objective lens;
  • the emitted light from the laser light source is focused by the cylindrical lens and reflected by the polygon mirror scanner Enter the lens, then enter the objective lens horizontally, and finally form a dynamic light sheet at a 45 degree angle to the horizontal at the cornea of the eye;
  • the image signal is emitted back to the objective lens at an angle of 135 degrees to the horizontal, and then passes through the lens and the lens.
  • the polygon mirror scanner enters the second lens group after being reflected by the polygon mirror scanner.
  • the reflecting mirror group includes a dichroic mirror, an objective lens, and a reflecting mirror; after the laser light source emits light through the first lens group, it is reflected by the dichroic mirror and enters the objective lens, and then vertically enters the reflecting mirror and is reflected by the reflecting mirror. It then enters the cornea of the eye horizontally; the image signal is emitted back to the objective lens at a 90 degree angle with the horizontal, and enters the second lens group through the dichroic lens.
  • the first lens group includes a magnifying lens group, a cylindrical lens, a uniaxial scanning galvanometer, and a lens group.
  • the first lens group includes a magnifying lens group, a biaxial scanning galvanometer, and a lens group.
  • the reflecting mirror is fixed on the objective lens at an angle of 45 degrees to the horizontal.
  • the laser light source is a laser light source emitting near-infrared light.
  • the wavelength of light emitted by the laser light source is 400-1100 nm.
  • the light sheet microscope of the present invention further includes a conical lens, which is arranged between the laser light source and the first lens group, and the light emitted by the laser light source is passed through the conical lens to form Bessel light and enters the first lens group.
  • a conical lens which is arranged between the laser light source and the first lens group, and the light emitted by the laser light source is passed through the conical lens to form Bessel light and enters the first lens group.
  • One lens group is arranged between the laser light source and the first lens group, and the light emitted by the laser light source is passed through the conical lens to form Bessel light and enters the first lens group.
  • the light sheet microscope used for corneal nerve imaging of the present invention is a light sheet microscope used for corneal nerve imaging of the present invention.
  • the present invention uses a dynamic light sheet illumination microscope to obtain corneal nerve images. Since the illumination light does not directly hit the fundus, it can reduce the damage of the illumination light to the eyes. At the same time, the illumination laser uses near-infrared light, which not only increases the imaging depth, but also reduces the discomfort of the patient's eyes.
  • the present invention uses dynamic light sheet illumination to replace the current point scanning illumination in confocal microscopes, the imaging speed is accelerated and the shooting time is reduced.
  • the present invention adopts a lens group structure, which is easy to integrate and has good environmental adaptability, which is convenient for miniaturization and portability of equipment.
  • Figure 1 is a schematic diagram of the positional relationship between light and eyes when using an existing confocal microscope
  • FIG. 2 is a schematic diagram of the positional relationship between light and eyes under one embodiment of the light sheet microscope for corneal neuroimaging according to the present invention (refer to FIG. 4);
  • Fig. 3 is a schematic diagram of the positional relationship between light and eyes in another embodiment of the light sheet microscope for corneal neuroimaging according to the present invention (refer to Fig. 7);
  • FIG. 4 is a schematic structural diagram of an embodiment of a light sheet microscope for corneal neuroimaging according to the present invention.
  • FIG. 5 is a schematic diagram of an example structure of the first lens group in FIG. 4;
  • Fig. 6 is a schematic diagram of the position of the reflector in Fig. 4;
  • Fig. 7 is a schematic structural diagram of another embodiment of the light sheet microscope for corneal neuroimaging according to the present invention.
  • FIG. 8 is a schematic diagram of an example structure of the second lens group in FIG. 7;
  • FIG. 9 is a schematic diagram of an example of forming Bessel light by the laser light source of the light sheet microscope for corneal neuroimaging according to the present invention.
  • Figure 1 shows a schematic diagram of the positional relationship between light and eyes when a confocal microscope is used.
  • the image information 212 returns to the objective lens in its original path.
  • the illumination light path emits light into the cornea of the eye, and the emitted light is at a 90 degree angle to the horizontal at the cornea of the eye.
  • the image signal emitted light returns to the objective lens, and the image signal is at a 90 degree angle to the horizontal.
  • the image acquisition light path coincides with the cornea of the eye. In this way, because the emitted light is directed to the fundus, the patient's eyes are uncomfortable.
  • the point scanning method of the confocal microscope causes the imaging speed to be slow and aggravates eye discomfort.
  • the present invention proposes a light sheet microscope for corneal nerve imaging.
  • the light sheet microscope is only suitable for transparent tissue imaging.
  • the transparent cornea protruding from the front of the eyeball is transparent, and the light sheet microscope can be used to image the corneal nerve.
  • Traditional light sheet microscopes use two 90-degree angled objective lenses for illumination and imaging, which are not suitable for imaging corneal nerves of the eye. Therefore, the light sheet microscope designed with a single lens is suitable for corneal nerve imaging.
  • the light sheet microscope used for corneal nerve imaging includes a control device, an illumination light path, and an image acquisition light path.
  • the illumination light path injects light into the cornea of the eye, and forms a dynamic light sheet at an angle ⁇ to the horizontal at the cornea of the eye.
  • the image signal is emitted into the image acquisition optical path at a ⁇ angle with the horizontal, and the image acquisition optical path sends the image signal to the control device for imaging.
  • the angle between the angle ⁇ and the angle ⁇ is 90 degrees, and the angle ⁇ is not equal to 90 degrees.
  • the invention uses dynamic light sheet illumination to replace the point scanning illumination in the confocal microscope, which speeds up the imaging speed and reduces the shooting time.
  • the illumination light path does not directly inject light into the fundus of the eye, which can reduce the damage of the illumination light to the eyes.
  • the light sheet microscope includes a laser light source, a first lens group, a mirror group, a second lens group, and a camera sensor.
  • the light emitted by the laser light source enters the cornea of the eye through the first lens group and the reflector group in sequence to form an illumination light path.
  • the image signal is sequentially focused on the camera sensor through the mirror group and the second lens group to form an image acquisition optical path.
  • the control device includes a control computer and a control circuit.
  • the control computer provides a human-computer interaction interface for setting laser illumination wavelength, power and other parameters; controlling the laser scanning speed and angle, controlling the camera to acquire images, and sending corresponding instructions to the control circuit.
  • the function of the control circuit is to convert computer instructions into analog signals to control subsequent circuits, and receive signals from the camera, feed them back to the computer, and form real-time images on the display screen.
  • Fig. 4 shows an embodiment of the light sheet microscope for corneal nerve imaging according to the present invention.
  • the reflecting mirror group includes a bipartite mirror 36, an objective lens 38 and a reflecting mirror 39.
  • the second lens group includes a straight tube lens 37 and a filter 310.
  • the laser light source 34 emits light through the first lens group 35, it is reflected by the dichroic mirror 36 and enters the objective lens 38, then vertically enters the mirror 39, is reflected by the mirror 39 and enters the cornea of the eye horizontally, forming a horizontal light sheet.
  • the illumination light path injects light into the cornea of the eye, and forms an angle ⁇ of zero degree at the cornea of the eye.
  • the image signal is emitted back to the objective lens 38 at a 90 degree angle with the horizontal (the ⁇ angle is a 90 degree angle), enters the tube lens 37 through the dichroic lens 36, and is focused on the camera sensor 311 through the filter 310.
  • the reflector 39 is a front reflector of the lens. As shown in FIG. 6, the reflector forms a 45-degree angle with the horizontal. Generally, it can be fixed on the objective lens used after being connected with a circular ring.
  • FIG. 5 shows an example of the first lens group.
  • the first lens group includes a magnifying lens group 351, a cylindrical lens 352, a uniaxial scanning galvanometer 353, and a lens group 354.
  • the single-axis scanning galvanometer 353 can be an X-axis, Y-axis, or Z-axis scanning galvanometer. After the light emitted by the laser light source 34 is amplified and collimated by the magnifying lens group, it is focused by the cylindrical lens 352 to form a light sheet, and a single-axis scanning galvanometer 353 is used to form light sheets of different depths.
  • the first lens group includes a magnifying lens, a biaxial scanning galvanometer, and a lens group.
  • the two-axis scanning galvanometer adopts X-axis and Z-axis scanning galvanometers, and the Y-axis is defined according to the direction in which light enters the eye.
  • the magnifying lens group may adopt one or more lenses to form a lens group with magnifying function.
  • the lens group may adopt one or more lenses, which cooperate with the scanning galvanometer.
  • FIG. 2 shows the optical path in the embodiment described in FIG. 4.
  • Fig. 7 shows another embodiment of the light sheet microscope for corneal nerve imaging according to the present invention.
  • the first lens group 54 includes a cylindrical lens.
  • the mirror group includes a polygon mirror scanner 55, a lens 56, and an objective lens 57.
  • the light emitted from the laser light source 53 is focused by the first lens group 54 and then reflected by the polygon mirror scanner 55 to enter the lens 56, and then enter the objective lens 57 horizontally, and finally form a horizontal line at the cornea of the eye.
  • a dynamic light sheet at a certain angle for example, the ⁇ angle is 45 degrees).
  • the image signal is emitted back to the objective lens 57 at a certain angle with the horizontal (for example, the ⁇ angle is 135 degrees), passes through the lens 56, the polygon mirror scanner 55, and is reflected by the polygon mirror scanner 55 and enters the objective lens 57.
  • the second lens group 59 can adopt the example shown in FIG. 8, for example, two lenses 591 and 592 are used. After the image signal passes through the lens 591, the lens 592 focuses on the camera sensor.
  • FIG. 3 shows the optical path in the embodiment described in FIG. 7. After the illumination light path 233 enters the eyeball 221 at a certain angle, the image information 222 is acquired by the image acquisition light path perpendicular to the illumination light path.
  • the ⁇ angle is not limited to 45 degrees, and the ⁇ angle is not limited to 135 degrees.
  • the laser light source is a laser light source emitting near-infrared light.
  • the selected laser wavelength should be in the near-infrared wavelength range, which not only has a longer penetration depth, but also reduces the discomfort of patients because the human eye is not sensitive to light in this wavelength range.
  • the wavelength is about 700 nm to about 1100 nm.
  • the illumination laser in the 400-700 range is also suitable for this device.
  • the laser light source emits laser light through an optical fiber or directly, it enters the subsequent optical path through the first lens group.
  • the optical fiber type can be single-mode or multi-mode.
  • the commonly used Heidelberg lens is Zeiss 63X times and NA 0.95 water mirror. Also in the implementation, it is suitable for the above two design devices. Take the same Zeiss objective lens as an example to calculate the shooting time and related parameters, because The resolution of the same objective lens is the same, but the main difference lies in the imaging speed. First of all, the scanning speed of the laser scanning mirror can reach at least 500Hz. Secondly, the formed light sheet forms an illumination light sheet of corresponding size according to the lens used, and the exposure time is 5-10ms, so it can image 98-192 images per second. , So it is only limited by the current camera's speed of acquiring images. Compared with the current confocal microscope, the imaging speed of 8 frames per second is 12-24 times higher. Thus, the photographing time of the patient is greatly reduced, and the contact time between the eyeball and the objective lens is reduced. Then, it should be noted that other objective lenses with sufficient magnification to see corneal nerves are also suitable for this device.
  • the light sheet microscope for corneal neuroimaging of the present invention further includes a cone lens, which is arranged between the laser light source and the first lens group.
  • the light emitted by the laser light source can form Bessel light by using a conical lens with a certain angle.
  • the Gaussian laser 72 enters the conical mirror 71 in parallel to form Bessel light 73.
  • the linear focusing method is beneficial to produce more Large light sheet, and increase the imaging depth. This applies to the above two design options.
  • ophthalmic glue is used between the objective lens and the eyeball to avoid direct contact with the eyeball to cause damage.

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Abstract

用于角膜神经成像的光片显微镜，属于角膜神经成像技术领域。光片显微镜包括控制装置、照明光路(223,233)、图像获取光路，照明光路(223,233)将光射入眼部角膜，于眼部角膜处形成与水平成α角的动态光片；图像信号与水平成β角发射入图像获取光路，图像获取光路将图像信号送入控制装置成像；其中，α角与β角之间的夹角为90度，α角不等于90度。使用动态光片照明显微镜进行角膜神经图像的获取，加快成像速度，减少拍摄时间，避免照明光直射眼底而对眼睛造成伤害。

Description

用于角膜神经成像的光片显微镜 技术领域
本发明涉及角膜神经成像领域，具体是使用光片显微镜进行角膜神经成像设备，可广泛用于生物医学的眼科成像技术领域。
 背景技术
角膜是眼睛最前面的部分，在虹膜和瞳孔的前面。它是受神经支配的最密集的组织，大多数角膜神经是感觉神经，来自三叉神经的丘脑分支。成年人眼的角膜平均水平直径约11.5毫米，垂直直径为10.5毫米，曲率在整个生命中仍然相当稳定。视神经区（瞳孔前角膜），提供大部分角膜的折射功能，直径4毫米，位于在角膜中央、瞳孔前面，在明视条件下，角膜是无血管的前睫状动脉的分支停在它们形成了提供外周角膜的弧形的角膜缘。因此，周围角膜和中央角膜在生理学和病理学方面都非常不同。角膜层状基质（500毫米厚）为角膜提供结构完整性。间质角膜细胞分泌胶原蛋白和蛋白多糖，这最终是透明的基础角膜和水合作用。
角膜具有丰富的神经末梢，是机体中最敏感的组织之一，对于保持眼表的完整性具有重要作用。研究证明，有很多疾病与角膜神经有关，如角膜炎、干眼征和糖尿病等。角膜是透明的，无血管也无淋巴管，但却含有丰富的神经纤维。其中大多为副交感神经的胆碱能神经，也有交感神经的肾上腺素能神经。
自从1930年Sohlamm首先发现细小的白色神经从角巩膜缘进入角膜后，角膜神经的研究方法经历了70余年的发展。从经典的神经组织学方法，发展到使用光镜和电子显微镜进行研究，从运用免疫组织化学方法，发展到近来使用共聚焦显微镜进行研究。共聚焦显微镜对角膜神经是进行活体研究，成为目前临床及科研的主流的成像方式。角膜共焦显微镜（corneal confocal microscopy, CCM）以激光为光源，具有高分辨率、高清晰度的优点。通过连续激光点扫描，可以获取活体角膜各层组织和细胞的清晰图像，成为目前临床从细胞水平深入探讨疾病病理机制的重要研究手段之一。目前常用的是海德堡公司生产的角膜共焦显微镜，其主要使用蔡司63x倍，NA是0.95的水镜，630nm的二极管激光进行点扫描。其获取图像的速度是8帧每秒，所以每个病人一般都需要花费5-15分钟获取到所需要的图像。因为共聚焦显微镜是使用点扫描的方法进行照明，所以导致图像获取的速度较慢，对每个病人而言需要很长时间保持一个拍摄姿式，而且因为拍摄角膜神经时物镜与眼球通过眼用胶浆接触的，长时间的拍摄会导致患者眼睛极度不适，所以缩短拍摄时间是一个需要解决的问题。
光片荧光显微镜（light sheet fluorescence microscopy）近些年被广泛使用于透明化组织成像中。动物的肝，肾等组织经过透明化处理后，使用光片荧光显微镜成像的速度明显快于共聚焦荧光显微镜，而且成像深度更有优势。2017年Hong等人提出使用光片显微镜拍摄小梁网用于青光眼的诊断，该设备中使用488nm的贝塞尔高斯激光作为照明激发光，然后获取反射出来的荧光信号，文中的贝塞尔高斯激光是使用176度顶角锥镜得到的。这个图像获取设备中，照明光路从眼球的左侧进入，图像获取光路从小梁网获取反射回来的图像信息，这个设备对于动物实验中，可能适用于被取出的动眼球的小梁网的拍摄，但人类是有左右眼的，而且中间有鼻梁作为分隔，所以这种装置并不适用。而且这种照明装置是使用静态光片，在拍摄过程中，需要被拍摄眼球对像配合移动，从而获取不同深度的信息，但容易造成移动距离不符合所需要拍摄深度的问题。
 技术问题
本发明针对现有技术存在的问题，提出了一种用于角膜神经成像的光片显微镜，能加快成像速度，减少拍摄时间，缓解患者眼部不适。
 技术解决方案
本发明是通过以下技术方案得以实现的：
本发明用于角膜神经成像的光片显微镜，包括控制装置、照明光路、图像获取光路，所述照明光路将光射入眼部角膜，于眼部角膜处形成与水平成α角的动态光片；图像信号与水平成β角发射入图像获取光路，所述图像获取光路将图像信号送入控制装置成像；其中，α角与β角之间的夹角为90度，α角不等于90度。
作为优选，本发明光片显微镜还包括激光光源、第一透镜组、反射镜组、第二透镜组、相机传感器；所述激光光源发射光依次经所述第一透镜组、所述反射镜组进入眼部角膜，形成照明光路；图像信号依次经所述反射镜组、所述第二透镜组聚焦于所述相机传感器，形成图像获取光路。
作为优选，所述第一透镜组包括柱状透镜；所述反射镜组包括多边镜扫描仪、透镜、物镜；所述激光光源发射光经所述柱状透镜聚焦后由所述多边镜扫描仪反射光进入所述透镜，之后水平进入所述物镜，最终于眼部角膜处形成与水平成45度角的动态光片；图像信号与水平成135度角射出回到物镜，依次经过所述透镜、所述多边镜扫描仪，由所述多边镜扫描仪反射后进入所述第二透镜组。
作为优选，所述反射镜组包括二分镜、物镜、反射镜；所述激光光源发射光经所述第一透镜组后，由二分镜反射进入物镜，继而垂直射入反射镜，通过反射镜反射后水平进入眼部角膜；图像信号与水平成90度角射出回到物镜，经过二分镜进入所述第二透镜组。
作为优选，所述第一透镜组包括放大透镜组、柱状透镜、单轴扫描振镜、透镜组。
作为优选，所述第一透镜组包括放大透镜组、双轴扫描振镜、透镜组。
作为优选，所述反射镜与水平成45度角固定于所述物镜上。
作为优选，所述激光光源为发射近红外光的激光光源。
作为优选，所述激光光源发射光的波长为400-1100nm。
作为优选，本发明光片显微镜还包括锥镜，设于所述激光光源与所述第一透镜组之间，所述激光光源发射光经所述锥镜后形成贝塞尔光进入所述第一透镜组。
 有益效果
本发明用于角膜神经成像的光片显微镜：
1、由于角膜是透明组织，本发明使用动态光片照明显微镜进行角膜神经图像的获取，由于其照明光并不会直射眼底，能够减少照明光对眼睛的伤害。同时照明激光采用近红外光，不仅增加成像深度，而且减少患者眼睛的不适程度。
2、由于本发明使用动态光片照明代替目前共聚焦显微镜中的点扫描照明，加快成像速度，减少拍摄时间。
3、根据不同的需求，可以使用不同的光路进行角膜神经图像的采集。
4、本发明采用透镜组结构，易于集成并具有良好的环境适应能力，方便设备的小型化和便携化。
 附图说明
图1为采用现有共聚焦显微镜时光线与眼睛的位置关系示意图；
图2为采用本发明用于角膜神经成像的光片显微镜一实施方式（参照图4）下的光线与眼睛的位置关系示意图；
图3为采用本发明用于角膜神经成像的光片显微镜另一实施方式（参照图7）下的光线与眼睛的位置关系示意图；
图4为本发明用于角膜神经成像的光片显微镜的一实施方式结构示意图；
图5为图4中第一透镜组的某一示例结构示意图；
图6为图4中反射镜设置位置示意图；
图7为本发明用于角膜神经成像的光片显微镜的另一实施方式结构示意图；
图8为图7中第二透镜组的某一示例结构示意图；
图9为本发明用于角膜神经成像的光片显微镜的激光光源形成贝塞尔光的示例示意图。
 本发明的最佳实施方式
以下是本发明的具体实施例并结合附图，对本发明的技术方案作进一步的描述，但本发明并不限于这些实施例。
图1示出了共聚焦显微镜使用时，光线与眼睛的位置关系示意图。激光光源发射光213直射眼球211后，图像信息212原路返回物镜中。也就是说，照明光路将发光射入眼部角膜，发射光于眼部角膜处与水平成90度角，图像信号发射光回到物镜中，图像信号与水平成90度角，即照明光路与图像获取光路于眼部角膜处相重合。此方式下，由于发射光直射眼底，使得患者眼睛产生不适。并且，共聚焦显微镜的点扫描方法进行照明，导致成像速度慢，更加剧了眼睛不适感。为此，本发明提出了一种用于角膜神经成像的光片显微镜。
光片显微镜一般而言只适用于透明化组织成像，突起眼球前端的透明角膜是透明的，能够使用光片显微镜进行角膜神经的成像。传统的光片显微镜分别使用两个成90度角的物镜进行照明与成像，对于眼球角膜神经的成像并不适用，所以本发明设计使用单镜头的光片显微镜适用于角膜神经的成像。
本发明用于角膜神经成像的光片显微镜，包括控制装置、照明光路、图像获取光路。所述照明光路将光射入眼部角膜，于眼部角膜处形成与水平成α角的动态光片。图像信号与水平成β角发射入图像获取光路，所述图像获取光路将图像信号送入控制装置成像。其中，α角与β角之间的夹角为90度，α角不等于90度。本发明使用动态光片照明代替共聚焦显微镜中的点扫描照明，加快成像速度，减少拍摄时间。并且，照明光路将光不直接射入眼底，能够减少照明光对眼睛的伤害。
具体地，光片显微镜包括激光光源、第一透镜组、反射镜组、第二透镜组、相机传感器。所述激光光源发射光依次经所述第一透镜组、所述反射镜组进入眼部角膜，形成照明光路。图像信号依次经所述反射镜组、所述第二透镜组聚焦于所述相机传感器，形成图像获取光路。
所述控制装置包括控制用计算机和控制电路。所述控制用计算机提供了人机交互界面，用于设置激光照明波长，功率等参数；控制激光扫描速度与角度，控制相机获取图像，并向控制电路发出相应指令。所述控制电路作用是将计算机指令转化为模拟信号以控制后续电路，并接收由相机传回的信号，回馈给计算机，在显示屏上形成实时的图像。
图4示出了本发明用于角膜神经成像的光片显微镜的一实施方式。所述反射镜组包括二分镜36、物镜38、反射镜39。所述第二透镜组包括直筒透镜37、滤镜310。所述激光光源34发射光经第一透镜组35后，由二分镜36反射进入物镜38，继而垂直射入反射镜39，通过反射镜39反射后水平进入眼部角膜，即形成水平光片。也就是说，所述照明光路将光射入眼部角膜，于眼部角膜处形成α角为零度。图像信号与水平成90度角（β角为90度角）射出回到物镜38，经过二分镜36进入套筒透镜37，通过滤镜310聚焦到相机传感器311上。所述反射镜39为镜头前反射镜，如图6所示，所述反射镜与水平成45度角，一般通过与圆环相连接后可固定于所使用的物镜上。
图5示出了所述第一透镜组的某一示例，所述第一透镜组包括放大透镜组351、柱状透镜352、单轴扫描振镜353、透镜组354。所述单轴扫描振镜353可以为X轴、Y轴、Z轴扫描振镜。所述激光光源34发射光经放大透镜组放大、准直后，经柱状透镜352聚焦形成光片，使用单轴扫描振镜353形成不同深度的光片。在另一示例下，所述第一透镜组包括放大透镜、双轴扫描振镜、透镜组。所述双轴扫描振镜采用X轴和Z轴扫描振镜，其中Y轴根据光线射入眼部方向所定义。所述激光光源34发射光经放大透镜组放大、准直后，经双轴扫描振镜形成不同深度的光片。所述放大透镜组可采用一片或多片透镜构成具有放大功能的透镜组。所述透镜组可采用一片或多片透镜，与所述扫描振镜相配合，当扫描振镜在不同的轴上进行激光扫描后，所述透镜组在扫描振镜后将光送入二分镜。
图2示出了图4所述实施方式下的光路。照明光路223水平进入眼球221后，图像信息222由垂直方向的图像获取光路获取。此方式照明光不会直射眼底，能够减少照明光对眼睛的伤害。
图7示出了本发明用于角膜神经成像的光片显微镜的另一实施方式。所述第一透镜组54包括柱状透镜。所述反射镜组包括多边镜扫描仪55、透镜56、物镜57。所述激光光源53发射光经所述第一透镜组54聚焦后由所述多边镜扫描仪55反射光进入所述透镜56，之后水平进入所述物镜57，最终于眼部角膜处形成与水平成一定角度（如，α角为45度角）的动态光片。图像信号与水平成一定角度（如β角为135度角）射出回到物镜57，依次经过所述透镜56、所述多边镜扫描仪55，由所述多边镜扫描仪55反射后进入所述第二透镜组。所述第二透镜组59可采用图8所示示例，如采用两个透镜591、592，图像信号通过透镜591后，由透镜592聚焦到相机传感器上。
图3示出了图7所述实施方式下的光路。照明光路233成一定角度进入眼球221后，图像信息222由与照明光路垂直方向的图像获取光路获取。在此方式下，α角不限于45度角，β角不限于135度。
为了增加拍摄深度的同时减小患者眼部不适，所述激光光源为发射近红外光的激光光源。选择的激光波长，应该在近红外波长范围，不仅穿透深度更长，而且由于人眼对该波段的光不敏感，能够减少患者的不适程度，例如，波长约为700 nm到约1100 nm。然而，应该注意，400-700范围的照明激光也同样适用于该装置。所述激光光源通过光纤或者直接打出激光后，通过第一透镜组进入后续光路。其中，当通过光纤耦合输出时，光纤类型可以为单模或多模。
目前常用的海德堡所使用的镜头是蔡司63X倍，NA为0.95的水镜，同样在实施方案中，适用于以上两款设计装置，以蔡司同款物镜为例计算拍摄时间及相关参数，因为使用同款物镜，分辨率是相同的，主要区别在成像速度。首先通常激光扫描镜的扫描速度至少可以达到500Hz，其次形成的光片根据所使用的镜头形成相应大小的照明光片，以5-10ms的曝光时间计算，所以每秒能够成像98-192张图像，所以只受限于目前相机的获取图像的速度。相比于目前的共聚焦显微镜的每秒8张的成像速度高出12-24倍。从而很大程度上减少患者拍摄时间，减少其眼球与物镜的接触时间。然后，应该注意，其他的放大倍数足够看到角膜神经的物镜也适用于该装置。
在实施方案中，本发明用于角膜神经成像的光片显微镜还包括锥镜，设于所述激光光源与所述第一透镜组之间。激光光源发射光可通过使用一定角度的锥镜形成贝塞尔光，如图9所示，高斯激光72平行进入锥形镜71后形成贝塞尔光73，其线形的聚焦方式有利于产生更大的光片，且增大成像深度。这适用于以上两种设计方案。
在实施方案中，物镜与眼球之间使用眼用胶浆，避免直接接触对眼球造成伤害。
本领域的技术人员应理解，上述描述及附图中所示的本发明的实施例只作为举例而并不限制本发明。本发明的目的已经完整有效地实现。本发明的功能及结构原理已在实施例中展示和说明，在没有背离所述原理下，本发明的实施方式可以有任何变形或修改。

Claims (10)

  1. 一种用于角膜神经成像的光片显微镜，包括控制装置、照明光路、图像获取光路，其特征在于，所述照明光路将光射入眼部角膜，于眼部角膜处形成与水平成α角的动态光片；图像信号与水平成β角发射入图像获取光路，所述图像获取光路将图像信号送入控制装置成像；其中，α角与β角之间的夹角为90度，α角不等于90度。
  2. 根据权利要求1所述的用于角膜神经成像的光片显微镜，其特征在于，还包括激光光源、第一透镜组、反射镜组、第二透镜组、相机传感器；所述激光光源发射光依次经所述第一透镜组、所述反射镜组进入眼部角膜，形成照明光路；图像信号依次经所述反射镜组、所述第二透镜组聚焦于所述相机传感器，形成图像获取光路。
  3. 根据权利要求2所述的用于角膜神经成像的光片显微镜，其特征在于，所述第一透镜组包括柱状透镜；所述反射镜组包括多边镜扫描仪、透镜、物镜；所述激光光源发射光经所述柱状透镜聚焦后由所述多边镜扫描仪反射光进入所述透镜，之后水平进入所述物镜，最终于眼部角膜处形成与水平成45度角的动态光片；图像信号与水平成135度角射出回到物镜，依次经过所述透镜、所述多边镜扫描仪，由所述多边镜扫描仪反射后进入所述第二透镜组。
  4. 根据权利要求2所述的用于角膜神经成像的光片显微镜，其特征在于，所述反射镜组包括二分镜、物镜、反射镜；所述激光光源发射光经所述第一透镜组后，由二分镜反射进入物镜，继而垂直射入反射镜，通过反射镜反射后水平进入眼部角膜；图像信号与水平成90度角射出回到物镜，经过二分镜进入所述第二透镜组。
  5. 根据权利要求4所述的用于角膜神经成像的光片显微镜，其特征在于，所述第一透镜组包括放大透镜组、柱状透镜、单轴扫描振镜、透镜组。
  6. 根据权利要求4所述的用于角膜神经成像的光片显微镜，其特征在于，所述第一透镜组包括放大透镜组、双轴扫描振镜、透镜组。
  7. 根据权利要求4所述的用于角膜神经成像的光片显微镜，其特征在于，所述反射镜与水平成45度角固定于所述物镜上。
  8. 根据权利要求1所述的用于角膜神经成像的光片显微镜，其特征在于，所述激光光源为发射近红外光的激光光源。
  9. 根据权利要求8所述的用于角膜神经成像的光片显微镜，其特征在于，所述激光光源发射光的波长为400-1100nm。
  10. 根据权利要求1所述的用于角膜神经成像的光片显微镜，其特征在于，还包括锥镜，设于所述激光光源与所述第一透镜组之间，所述激光光源发射光经所述锥镜后形成贝塞尔光进入所述第一透镜。
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