WO2012075860A1 - 一种荧光内窥成像方法及*** - Google Patents

一种荧光内窥成像方法及*** Download PDF

Info

Publication number
WO2012075860A1
WO2012075860A1 PCT/CN2011/081220 CN2011081220W WO2012075860A1 WO 2012075860 A1 WO2012075860 A1 WO 2012075860A1 CN 2011081220 W CN2011081220 W CN 2011081220W WO 2012075860 A1 WO2012075860 A1 WO 2012075860A1
Authority
WO
WIPO (PCT)
Prior art keywords
sub
sample
beams
fluorescence
excitation light
Prior art date
Application number
PCT/CN2011/081220
Other languages
English (en)
French (fr)
Inventor
邵永红
屈军乐
牛憨笨
Original Assignee
深圳大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳大学 filed Critical 深圳大学
Publication of WO2012075860A1 publication Critical patent/WO2012075860A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/04—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances
    • A61B1/043—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances for fluorescence imaging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/00163—Optical arrangements
    • A61B1/00172—Optical arrangements with means for scanning

Definitions

  • the invention belongs to the field of photoelectric detection, and in particular relates to a fluorescence endoscopic imaging method and system.
  • Fluorescence microscopy has become an important tool in life sciences, especially in cell biology research. Multiphoton excited fluorescence microscopy has the advantages of small killing effect on living organisms, large penetration depth and chromatographic ability, and has become an important means of life science research. Fluorescent images provide structural and functional information for biomedical detection and analysis.
  • An object of the embodiments of the present invention is to provide a fluorescence endoscopic imaging method, which aims to solve the problem that the current fluorescence endoscopic imaging is slow and inefficient.
  • a fluorescence endoscopic imaging method includes the following steps:
  • the fluorescence emitted during the scan is acquired in real time to generate a fluorescent image.
  • Another object of embodiments of the present invention is to provide a fluorescence endoscopic imaging system, the system comprising:
  • a beam splitter configured to divide the excitation light into a plurality of sub-beams, the plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;
  • a flexible medium for adjusting the plurality of sub-beams to conduct the plurality of sub-beams into the living body
  • a focusing element for focusing each sub-beam to a sub-region of the sample
  • a scanning element for scanning the sample by using the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area
  • a dichroic mirror and an imaging medium for directing the fluorescence from the living body
  • a detector for real-time collecting fluorescence emitted during scanning to generate a fluorescent image
  • the dichroic mirror is disposed between the scanning element and the focusing element.
  • the excitation light is divided into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, and the plurality of sub-beams are transmitted to the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, which is fluorescent.
  • FIG. 1 is a flowchart of an implementation of a fluorescence endoscopic imaging method according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of a structure and an optical path diagram of a fluorescence endoscopic imaging system according to an embodiment of the present invention
  • FIG. 3 is a dot diagram of a laser array according to an embodiment of the present invention.
  • the excitation light is divided into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, and the plurality of sub-beams are transmitted to the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, which is fluorescent.
  • the two-beam scanning of the sample is performed by two sub-beams, so that the fluorescence image of the whole sample is obtained, and the time is short, the speed is fast, and the damage to the organism is small, which is beneficial to biomedical research.
  • the fluorescence emitted during the scan is acquired in real time to generate a fluorescent image.
  • a beam splitter configured to divide the excitation light into a plurality of sub-beams, the plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;
  • a flexible medium for adjusting the plurality of sub-beams to conduct the plurality of sub-beams into the living body
  • a focusing element for focusing each sub-beam to a sub-region of the sample
  • a scanning element for scanning the sample by using the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area
  • a dichroic mirror and an imaging medium for directing the fluorescence from the living body
  • a detector for real-time collecting fluorescence emitted during scanning to generate a fluorescent image
  • the dichroic mirror is disposed between the scanning element and the focusing element.
  • FIG. 1 is a flowchart showing an implementation process of a fluorescence endoscopic imaging method according to an embodiment of the present invention, which is described in detail as follows:
  • step S101 excitation light is generated
  • a femtosecond (ultra-short) pulsed laser with a working frequency of 76 MHz, a period of 120 fs, and a center wavelength of 800 nm is preferably used as the excitation light, and the excitation light can realize two-photon excitation of the fluorescent substance.
  • the pulsed laser is subjected to beam expansion collimation and its intensity distribution is adjusted to form a flat-top beam having an evenly distributed intensity.
  • step S102 the excitation light is divided into a plurality of sub-beams, and the plurality of sub-beams correspond to a plurality of sub-regions of the sample, and a fluorescent substance is distributed in the sample;
  • the excitation light uniformly distributed in intensity is divided into a plurality of sub-beams, and the plurality of sub-beams correspond one-to-one to a plurality of sub-regions of the sample having the fluorescent substance.
  • step S103 the plurality of sub-beams are adjusted such that each sub-beam is conducted into the living body and focused on a sub-region of the sample;
  • Embodiments of the present invention cause a plurality of sub-beams to be conducted in parallel to a living body, each focusing on a sub-region of the sample. Specifically, a plurality of sub-beams are first coupled into the flexible medium, and the plurality of sub-beams enter the living body in parallel via the flexible medium, and the plurality of sub-beams are focused in the living body to form the excitation light array points and respectively projected to the corresponding sub-regions.
  • the flexible medium is a photonic crystal fiber array, the input end of which is located outside the organism, and the output end is located in the living body.
  • each sub-beam is collimated such that each sub-beam becomes parallel.
  • the plurality of sub-beams are output from the flexible medium, they need to be collimated, so that each sub-beam is focused on the corresponding sample sub-area.
  • step S104 the sample is scanned by using a plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;
  • the scanning is divided into line scanning and step scanning, and the specific process is as follows:
  • the plurality of sub-beams are focused to form an excitation light array point to be projected onto the sample, and each sub-area is linearly scanned along the longitudinal direction of the sample, and the fluorescent substance in each sub-area emits fluorescence under the action of the excitation light array point. This line scans quickly and for a short time.
  • step-scanning of each sub-area in the lateral direction of the sample adjusts the position of the excitation light array point in the lateral direction of the sample.
  • the above line scan and step scan are performed cyclically until the scanning of each sub-area of the sample is completed. It should be understood that the direction of the line scan and the step scan can also be changed in the specific implementation. In addition, the sample can be scanned randomly, and the sub-areas of the sample can be scanned.
  • step S105 fluorescence emitted during scanning is acquired in real time to generate a fluorescent image.
  • the fluorescence emitted by the fluorescent substance in each sub-area is collected while scanning the sub-areas to generate a fluorescent image. Specifically, the fluorescence emitted by the fluorescent substance in each sub-area is first derived from the imaging fiber, and then the intensity information of the fluorescence and the position information of the fluorescence in the sample are acquired by the detector, and finally the fluorescence image is generated from the position and intensity information of the fluorescence.
  • FIG. 2 shows the structure of a fluorescence endoscopic imaging system according to an embodiment of the present invention. For the convenience of description, only parts related to the embodiment of the present invention are shown.
  • the fluorescence endoscopic imaging system has an excitation light path and a detection light path.
  • the excitation light path includes an excitation light source, a beam expanding collimator, a shaper, a beam splitter, a collimating lens, a first coupling lens, a photonic crystal fiber array, a first self-focusing lens, a scanning mirror, and a micro objective lens.
  • the detection optical path includes a micro objective lens, a dichroic mirror, a second autofocus lens, a imaging fiber bundle, a second coupling lens, a filter element, an imaging lens, and a detector.
  • the micro objective lens is shared by the excitation light path and the probe light path.
  • a titanium gemstone femtosecond laser 1 is preferably used as an excitation light source, which can generate a pulsed laser having a center wavelength of 800 nm, a frequency of 76 MHz, and a period of 120 fs, and the pulsed laser can realize two-photon of a fluorescent substance. excitation.
  • the pulsed laser light is converted into collimated light of a desired size via the beam expanding collimator 2.
  • the shaper is a beam shaper 3, and the collimated pulse laser is shaped by the beam shaper 3 to form a flat top beam with uniform intensity distribution.
  • the spectroscope may be a microlens array, a diffractive optical element or a beam splitter.
  • the microlens array 4 is preferred.
  • the flat top distributed pulsed laser light is divided into a plurality of sub-beams via the microlens array 4, and the plurality of sub-beams correspond to the sample 12 In the sub-region, the microlens array 4 in this embodiment is a 3 ⁇ 3 microlens array, that is, the microlens array has nine micro objective lenses.
  • the back focal plane of the collimating lens 5 coincides with the front focal plane of the microlens array 4, and the sub-beams are focused on the front focal plane of the microlens array 4, that is, on the back focal plane of the collimating lens 5, and the sub-beams are collimated through the collimating lens. 5 all become sub-beams of parallel light.
  • a plurality of sub-beams are coupled and coupled into the photonic crystal fiber array 7 via the first coupling lens 6.
  • the photonic crystal fiber array 7 is formed by equally arranging a plurality of photonic crystal fibers, and the number of photonic crystal fibers and their arrangement are the same as those of the microlens array 4.
  • a plurality of sub-beams are conducted through the photonic crystal fiber array 7 into the living body, and a plurality of sub-beams emerging from the photonic crystal fiber array 7 are projected to the scanning mirror 9 via the first autofocus lens 8.
  • the self-focusing lens is a rod lens whose refractive index is gradually gradual, and each of the sub-beams becomes parallel light via the first self-focusing lens 8.
  • Each of the sub-beams is projected by a scanning mirror 9 to a sample 12 having a fluorescent substance, and a micro-objective 11 for converging is provided between the sample 12 and the scanning mirror 9.
  • the scanning mirror 9 is preferably a MEMS (Micro-Electro-Mechanical) Systems, MEMS) scanning mirrors, MEMS scanning mirrors are two-dimensional scanning mirrors for line scanning and step scanning of samples.
  • each sub-beam is conducted through the excitation light path to form an excitation light array point and is focused on a sub-area of the sample 12, and the fluorescent substance in the excitation sample 12 emits fluorescence.
  • the fluorescence also has a plurality of sub-beams.
  • the dichroic mirror 10 is disposed between the scanning mirror 9 and the micro objective lens 11.
  • the dichroic mirror 10 is highly transparent to a pulsed laser having a center wavelength of 800 nm, and is highly reflective to a wavelength of 400 to 700 nm.
  • the dichroic mirror 10 The angle between the fluorescence and the fluorescence is 45 or 135.
  • the above fluorescence is collected by the micro objective lens 11 to form a plurality of collimated fluorescent sub-beams, and the dichroic mirror 10 reflects the fluorescence from the excitation light path and is focused by the second autofocus lens 13 on the inner end of the imaging fiber bundle 14.
  • the fluorescent sub-beam is derived from the living body by the imaging fiber bundle 14 and converted into multiple parallel light by the second coupling lens 13, and the excitation light and other stray light are filtered by the filter element 16, and focused by the imaging lens 17 to the detector 18. Sensitive face.
  • the detector 18 collects the fluorescence emitted by the scanning mirror 9 in real time, thereby obtaining the intensity information of the fluorescence and the positional information of the fluorescence in the sample 12. Finally, the fluorescence image is generated from the position and intensity information of the fluorescence.
  • the focal plane of the excitation light array point in the sample 12 and the inner end surface of the imaging fiber bundle 14 are mutually conjugated, and the outer end surface of the image fiber bundle 14 and the sensitive surface of the detector 18 are mutually conjugated surfaces, that is, Fluorescence excited from different positions in the sample 12 is focused by the micro objective lens 11 and the second self-focusing lens 13 to corresponding positions on the inner end surface of the image fiber bundle 14, and transmitted from the image fiber bundle 14 to the corresponding position on the outer end surface of the body, and then The second coupling lens 15 and the imaging lens 17 are focused to corresponding positions of the detector 18. In this way, the detector 18 can detect the fluorescence of the focal plane in the sample 12 to generate a fluorescent image.
  • the detector 18 is an area array detector for acquiring intensity and spatial information of fluorescence, and the area array detector is preferably a CCD camera or a CMOS camera.
  • the area array detector is coupled to a computer 21 which stores, processes and reads the fluorescence intensity and position information detected by the detector 18, and the computer 21 controls the exposure of the area array detector.
  • scanning mirror 9 begins scanning and detector 18 begins to expose; when scanning of sample 12 is complete, detector 18 is exposed.
  • the exposure time of the detector 18 is the same as the scanning time of the scanning mirror 9, and the scanning time may be the time when the sample is scanned once, or may be an integral multiple of the scanning time.
  • the excitation light is divided into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, and the plurality of sub-beams are transmitted to the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, which is fluorescent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Surgery (AREA)
  • Biomedical Technology (AREA)
  • Medical Informatics (AREA)
  • Optics & Photonics (AREA)
  • Pathology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Description

一种荧光内窥成像方法及*** 技术领域
本发明属于光电检测领域，尤其涉及一种荧光内窥成像方法及***。
背景技术
荧光显微技术已经成为生命科学，尤其是细胞生物学研究的重要工具。多光子激发荧光显微技术具有对生命体的杀伤作用小，穿透深度大，具有层析能力等优点，已经成为生命科学研究的重要手段。荧光图像能够为生物医学检测和分析提供结构和功能信息。
近年来, 随着新型光纤和微制造技术的迅猛发展, 光纤双光子荧光显微镜和内窥镜的研究使双光子荧光显微成像技术在活体的内部器官和活体动物中的研究成为可能。目前双光子荧光内窥显微技术已经引起了国际上的高度重视，针对这一课题做出了大量的研究成果，在内窥***设计、扫描机制、光学传导和高数值孔径的微物镜及其应用等方面取得了很多研究成果。受到活体内窥应用条件限制，成像时间不宜过长。然而目前荧光内窥成像的速度慢，效率低，耗时长，对生物体造成极大的影响。
 技术问题
本发明实施例的目的在于提供一种荧光内窥成像方法，旨在解决现有荧光内窥成像速度慢、效率低的问题。
 技术解决方案
本发明实施例是这样实现的，一种荧光内窥成像方法，包括以下步骤：
产生激发光；
将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
调整所述多个子光束，使各子光束传导至生物体内并聚焦于所述样品的子区域；
利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
实时采集扫描时发出的荧光，生成荧光图像。
本发明实施例的另一目的在于提供一种荧光内窥成像***，所述***包括：
激发光源，用于产生激发光；
分光器，用于将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
柔性介质，用于调整所述多个子光束，使所述多个子光束传导至生物体内；
聚焦元件，用于使各子光束聚焦于所述样品的子区域；
扫描元件，用于利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
双色镜及传像介质，用于将所述荧光从所述生物体内导出；
探测器，用于实时采集扫描时发出的荧光，生成荧光图像；
所述双色镜设于所述扫描元件与聚焦元件之间。
 有益效果
本发明实施例将激发光分为与样品多个子区域一一对应的多个子光束，使该多个子光束传导至生物体内，各子光束聚焦于样品的子区域，形成多点激发荧光，将荧光导出，并由多个子光束对样品进行二维扫描，从而获取整个样品的荧光图像，时间短、速度快，对生物体损伤小，有利于生物医学的研究，特别是对癌症早期诊断，具有重要意义。
 附图说明
图1是本发明实施例提供的荧光内窥成像方法的实现流程图；
图2是本发明实施例提供的荧光内窥成像***的结构及其光路图；
图3是本发明实施例提供的激光阵列点图。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例将激发光分为与样品多个子区域一一对应的多个子光束，使该多个子光束传导至生物体内，各子光束聚焦于样品的子区域，形成多点激发荧光，将荧光导出，并由多个子光束对样品进行二维扫描，从而获取整个样品的荧光图像，时间短、速度快，对生物体损伤小，有利于生物医学的研究。
本发明实施例提供的荧光内窥成像方法包括以下步骤：
产生激发光；
将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
调整所述多个子光束，使各子光束传导至生物体内并聚焦于所述样品的子区域；
利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
实时采集扫描时发出的荧光，生成荧光图像。
本发明实施例提供的荧光内窥成像***包括：
激发光源，用于产生激发光；
分光器，用于将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
柔性介质，用于调整所述多个子光束，使所述多个子光束传导至生物体内；
聚焦元件，用于使各子光束聚焦于所述样品的子区域；
扫描元件，用于利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
双色镜及传像介质，用于将所述荧光从所述生物体内导出；
探测器，用于实时采集扫描时发出的荧光，生成荧光图像；
所述双色镜设于所述扫描元件与聚焦元件之间。
以下结合具体实施例对本发明的实现进行详细描述。
图1示出了本发明实施例提供的荧光内窥成像方法的实现流程，详述如下：
在步骤S101中，产生激发光；
本发明实施例优选工作频率为76MHz，周期为120fs，中心波长为800nm的飞秒（超短）脉冲激光作为激发光，此激发光可实现荧光物质的双光子激发。通常，对脉冲激光进行扩束准直并调整其强度分布，形成强度均匀分布的平顶光束。
在步骤S102中，将激发光分为多个子光束，多个子光束对应于样品的多个子区域，样品内分布有荧光物质；
本发明实施例将强度均匀分布的激发光分为多个子光束，该多个子光束一一对应于具有荧光物质的样品的多个子区域。
在步骤S103中，调整多个子光束，使各子光束传导至生物体内并聚焦于样品的子区域；
本发明实施例使多个子光束并行传导至生物体内，各自聚焦于样品的子区域。具体地，先使多个子光束耦合进入柔性介质，多个子光束经由柔性介质并行进入生物体内，于生物体内多个子光束经聚焦形成激发光阵列点分别投射至与之对应的子区域。其中柔性介质为光子晶体光纤阵列，其输入端位于生物体外，输出端位于生物体内。
多个子光束耦合进入柔性介质之前，需对各个子光束进行准直，使各个子光束成为平行光。多个子光束从柔性介质输出之后，亦需对其进行准直，便于各子光束聚焦于与之对应的样品子区域。
在步骤S104中，利用多个子光束对样品进行扫描，使各子区域内的荧光物质发出荧光；
本发明实施例将扫描分为线扫描和步进扫描，具体过程如下：
1、线扫描
多个子光束经聚焦形成激发光阵列点投射至样品，沿样品纵向对各子区域进行线扫描，各子区域内的荧光物质在激发光阵列点的作用下发出荧光。此线扫描的速度快、时间短。
2、步进扫描
对各个子区域纵向的线扫描结束后，沿样品横向对各个子区域进行步进扫描即调整激发光阵列点在样品横向的位置。
循环执行上述线扫描和步进扫描，直至完成对样品各个子区域的扫描。应当理解，具体实施时还可以调换线扫描与步进扫描的方向。此外，还可对样品进行随机扫描，对样品各个子区域完成扫描即可。
在步骤S105中，实时采集扫描时发出的荧光，生成荧光图像。
本发明实施例对各子区域扫描的同时，采集各子区域内荧光物质发出的荧光，生成荧光图像。具体地，先由传像光纤导出各子区域内荧光物质发出的荧光，接着由探测器获取荧光的强度信息及该荧光于样品中的位置信息，最后由荧光的位置及强度信息生成荧光图像。
本领域的普通技术人员应当理解，实现上述实施例方法中的全部或部分步骤可以通过程序来指令相关的硬件完成，该程序可以存储于一计算机可读取存储介质中，如ROM/RAM、磁盘、光盘等。
图2示出了本发明实施例提供的荧光内窥成像***的结构，为了便于说明，仅示出了与本发明实施例相关的部分。
本发明实施例提供的荧光内窥成像***具有一激发光路和一探测光路。激发光路包括激发光源、扩束准直装置、整形器、分光器、准直透镜、第一耦合透镜、光子晶体光纤阵列、第一自聚焦透镜、扫描镜以及微物镜。探测光路包括微物镜、双色镜、第二自聚焦透镜、传像光纤束、第二耦合透镜、滤光元件、成像透镜以及探测器。其中微物镜为激发光路和探测光路所共用。
以下对激发光路的结构进行详细说明。
如图2所示，本发明实施例优选钛宝石飞秒激光器1作为激发光源，其可产生中心波长为800nm、频率为76MHz、周期为120fs的脉冲激光，该脉冲激光可实现荧光物质的双光子激发。脉冲激光经由扩束准直装置2变成所需尺寸的准直光。
本发明实施例中，整形器为光束整形器3，准直的脉冲激光经光束整形器3整形，形成强度均匀分布的平顶光束。分光器可为微透镜阵列、衍射光学元件或分束器，本实施例优选微透镜阵列4，平顶分布的脉冲激光经微透镜阵列4被分成多个子光束，多个子光束对应样品12的多个子区域，本实施例中微透镜阵列4为3×3微透镜阵列即微透镜阵列具有九个微物镜。
其中准直透镜5的后焦面与微透镜阵列4的前焦面重合，子光束在微透镜阵列4的前焦面即在准直透镜5的后焦面聚焦，各子光束经准直透镜5均变为平行光的子光束。
在本发明实施例中，多个子光束经第一耦合透镜6聚焦耦合进入光子晶体光纤阵列7。光子晶体光纤阵列7由多根光子晶体光纤等间距排列形成，光子晶体光纤的个数及其排列方式与微透镜阵列4的相同。多个子光束经光子晶体光纤阵列7传导至生物体内，从光子晶体光纤阵列7出射的多个子光束经第一自聚焦透镜8投射至扫描镜9。自聚焦透镜为折射率沿径向渐变的棒透镜，各子光束经第一自聚焦透镜8均变为平行光。各子光束经扫描镜9投射至具有荧光物质的样品12，样品12与扫描镜9之间设有起会聚作用的微物镜11。所述扫描镜9优选为MEMS（Micro-Electro-Mechanical Systems，微机电***）扫描镜，MEMS扫描镜为二维扫描镜，可对样品进行线扫描及步进扫描。
如图3所示，各子光束经本激发光路传导后形成激发光阵列点并聚焦于样品12的子区域，激发样品12内的荧光物质发出荧光。与此相对应地，该荧光亦具有多个子光束。
以下对探测光路的结构进行详细说明。
本发明实施例探测光路中双色镜10设于扫描镜9与微物镜11之间，双色镜10对中心波长为800nm的脉冲激光高透，对波长为400~700nm的荧光高反，双色镜10与荧光之间的夹角为45°或135°。上述荧光由微物镜11收集，形成多束准直的荧光子光束，双色镜10将此荧光从激发光路反射出来，经第二自聚焦透镜13聚焦于传像光纤束14的体内端。荧光子光束由传像光纤束14从生物体内导出，经第二耦合透镜13转化成多路平行光，经滤光元件16滤除激发光及其它杂散光，由成像透镜17聚焦到探测器18的敏感面。探测器18实时采集扫描镜9扫描时发出的荧光，从而获取荧光的强度信息及该荧光于样品12中的位置信息，最后由荧光的位置及强度信息生成荧光图像。
其中样品12内激发光阵列点的焦平面与传像光纤束14的体内端面互为共轭面，传像光纤束14的体外端面与探测器18的敏感面互为共轭面，也就是说从样品12中不同位置激发出的荧光经微物镜11和第二自聚焦透镜13聚焦到传像光纤束14体内端面的对应位置，由传像光纤束14传到体外端面的对应位置，再由第二耦合透镜15和成像透镜17聚焦到探测器18的对应位置。这样，探测器18即可对样品12内焦平面的荧光进行探测，生成荧光图像。
本发明实施例中，探测器18为用于获取荧光的强度及空间信息的面阵探测器，面阵探测器优选为CCD相机或CMOS相机。面阵探测器与计算机21连接，计算机21存储、处理和读取探测器18所探测的荧光强度及位置信息，且由计算机21控制面阵探测器的曝光。
通常，扫描镜9一开始扫描，探测器18即开始曝光；对样品12扫描完成时，探测器18曝光结束。探测器18的曝光时间与扫描镜9的扫描时间相同，扫描时间可以是对样品完成一次扫描的时间，也可以是扫描一次时间的整数倍。
本发明实施例将激发光分为与样品多个子区域一一对应的多个子光束，使该多个子光束传导至生物体内，各子光束聚焦于样品的子区域，形成多点激发荧光，将荧光导出，并由多个子光束对样品进行二维扫描，从而获取整个样品的荧光图像，时间短、速度快，对生物体损伤小，有利于生物医学的研究，特别是对癌症早期诊断，具有重要意义。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种荧光内窥成像方法，其特征在于，所述方法包括以下步骤：
    产生激发光；
    将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
    调整所述多个子光束，使各子光束传导至生物体内并聚焦于所述样品的子区域；
    利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
    实时采集扫描时发出的荧光，生成荧光图像。
  2. 如权利要求1所述的荧光内窥成像方法，其特征在于，所述扫描分为线扫描和步进扫描，所述利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光的步骤具体为：
    各子光束沿所述样品纵向线扫描对应子区域，使各子区域内的荧光物质发出荧光；
    所述线扫描结束后，沿所述样品横向对对应子区域进行步进扫描，即调整各子光束在所述样品横向的位置；
    循环执行所述线扫描和步进扫描，直至完成对各个子区域的扫描。
  3. 如权利要求1所述的荧光内窥成像方法，其特征在于，所述实时采集扫描时发出的荧光，生成荧光图像的步骤具体为：
    由传像光纤导出各子区域内荧光物质发出的荧光；
    由探测器获取荧光的强度信息及所述荧光于样品中的位置信息；
    由所述荧光的位置及强度信息生成荧光图像。
  4. 如权利要求1所述的荧光内窥成像方法，其特征在于，所述产生激发光的步骤之后还包括以下步骤：
    对所述激发光进行扩束准直；
    调整所述激发光的强度分布，使所述激发光的强度分布均匀；
    所述调整所述多个子光束，使各子光束传导至生物体内并聚焦于所述样品的子区域的步骤具体为：
    所述多个子光束耦合进入光子晶体光纤阵列，经由所述光子晶体光纤阵列进入生物体内；
    于所述生物体内多个子光束经聚焦形成激发光阵列点投射至所述样品。
  5. 一种荧光内窥成像***，其特征在于，所述***包括：
    激发光源，用于产生激发光；
    分光器，用于将所述激发光分为多个子光束，所述多个子光束对应于样品的多个子区域，所述样品内分布有荧光物质；
    柔性介质，用于调整所述多个子光束，使所述多个子光束传导至生物体内；
    聚焦元件，用于使各子光束聚焦于所述样品的子区域；
    扫描元件，用于利用所述多个子光束对所述样品进行扫描，使各子区域内的荧光物质发出荧光；
    双色镜及传像介质，用于将所述荧光从所述生物体内导出；
    探测器，用于实时采集扫描时发出的荧光，生成荧光图像；
    所述双色镜设于所述扫描元件与聚焦元件之间。
  6. 如权利要求5所述的荧光内窥成像***，其特征在于，所述激发光源与所述分光器之间还设有：
    扩束准直装置，用于调整所述激发光的尺寸并进行准直；
    整形器，用于调整所述激发光的强度分布，使所述激发光的强度分布均匀；
    所述分光器与所述柔性介质之间还设有：
    准直透镜，用于准直各子光束，使各子光束成为平行光；
    第一耦合透镜，用于使各子光束耦合进入所述柔性介质；
    所述柔性介质与所述扫描镜之间还设有：
    第一自聚焦透镜，用于准直从所述柔性介质输出的各个子光束；
    所述双色镜与所述传像介质之间还设有：
    第二自聚焦透镜，用于使所述荧光聚焦于所述传像介质的体内端；
    所述传像介质与所述探测器之间还设有：
    第二耦合透镜，用于准直从所述传像介质输出的荧光；
    成像透镜，用于将所述荧光成像于所述探测器；
    所述聚焦元件为微物镜。
  7. 如权利要求6所述的荧光内窥成像***，其特征在于，所述分光器为微透镜阵列、衍射光学元件或分束器，所述微透镜阵列的前焦面与所述准直透镜的后焦面重合；所述柔性介质为光子晶体光纤阵列，所述传像介质为传像光纤束。
  8. 如权利要求7所述的荧光内窥成像***，其特征在于，所述荧光由所述微物镜收集，并形成多束准直的荧光子光束；所述双色镜将所述荧光子光束从激发光路反射出来，经所述第二自聚焦透镜聚焦于所述传像光纤束的体内端。
  9. 如权利要求8所述的荧光内窥成像***，其特征在于，所述样品内激发光阵列点的焦平面与所述传像光纤束的体内端面互为共轭面，所述传像光纤束的体外端面与所述探测器的敏感面互为共轭面。
  10. 如权利要求5~9中任一项所述的荧光内窥成像***，其特征在于，所述探测器的曝光时间与扫描元件的扫描时间相同，所述扫描元件的扫描时间为对所述样品完成一次扫描的时间，或对所述样品完成多次扫描的时间。
PCT/CN2011/081220 2010-12-09 2011-10-25 一种荧光内窥成像方法及*** WO2012075860A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2010105808948A CN102525411A (zh) 2010-12-09 2010-12-09 一种荧光内窥成像方法及***
CN201010580894.8 2010-12-09

Publications (1)

Publication Number Publication Date
WO2012075860A1 true WO2012075860A1 (zh) 2012-06-14

Family

ID=46206600

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/081220 WO2012075860A1 (zh) 2010-12-09 2011-10-25 一种荧光内窥成像方法及***

Country Status (2)

Country Link
CN (1) CN102525411A (zh)
WO (1) WO2012075860A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107361725A (zh) * 2017-07-20 2017-11-21 无锡海斯凯尔医学技术有限公司 快速组织分子成像装置
CN108490597A (zh) * 2018-06-05 2018-09-04 张红明 一种基于光纤耦合器的共聚焦显微***
CN108937824A (zh) * 2018-08-02 2018-12-07 深圳大学 一种内窥成像装置和荧光寿命分布图像的获取方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106842232A (zh) * 2016-12-12 2017-06-13 清华大学 一种激光雷达装置及探测方法
CN109656014B (zh) * 2019-01-31 2024-03-19 北京超维景生物科技有限公司 多路荧光收集装置及三维非线性激光扫描腔体内窥镜
CN111967530A (zh) * 2020-08-28 2020-11-20 南京诺源医疗器械有限公司 一种医学荧光成像***的荧光区域识别方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050107694A1 (en) * 2003-11-17 2005-05-19 Jansen Floribertus H. Method and system for ultrasonic tagging of fluorescence
CN101339129A (zh) * 2007-09-03 2009-01-07 深圳大学 基于固定光路***的变视场扫描显微的方法及其装置
CN101375786A (zh) * 2007-09-12 2009-03-04 深圳大学 一种荧光内窥成像方法及装置
CN101361651B (zh) * 2008-09-19 2010-04-07 清华大学 荧光分子断层成像装置
CN101793829A (zh) * 2010-02-04 2010-08-04 深圳大学 一种荧光显微成像方法及***
CN101933794A (zh) * 2010-09-13 2011-01-05 深圳大学 一种荧光多参量内窥测量方法及***
CN202069570U (zh) * 2010-12-09 2011-12-14 深圳大学 一种荧光内窥成像***
CN202069569U (zh) * 2010-12-09 2011-12-14 深圳大学 一种荧光光谱内窥成像***

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101904737B (zh) * 2010-08-09 2012-07-04 华中科技大学 活体荧光内窥光谱成像装置

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050107694A1 (en) * 2003-11-17 2005-05-19 Jansen Floribertus H. Method and system for ultrasonic tagging of fluorescence
CN101339129A (zh) * 2007-09-03 2009-01-07 深圳大学 基于固定光路***的变视场扫描显微的方法及其装置
CN101375786A (zh) * 2007-09-12 2009-03-04 深圳大学 一种荧光内窥成像方法及装置
CN101361651B (zh) * 2008-09-19 2010-04-07 清华大学 荧光分子断层成像装置
CN101793829A (zh) * 2010-02-04 2010-08-04 深圳大学 一种荧光显微成像方法及***
CN101933794A (zh) * 2010-09-13 2011-01-05 深圳大学 一种荧光多参量内窥测量方法及***
CN202069570U (zh) * 2010-12-09 2011-12-14 深圳大学 一种荧光内窥成像***
CN202069569U (zh) * 2010-12-09 2011-12-14 深圳大学 一种荧光光谱内窥成像***

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107361725A (zh) * 2017-07-20 2017-11-21 无锡海斯凯尔医学技术有限公司 快速组织分子成像装置
CN107361725B (zh) * 2017-07-20 2024-02-27 无锡海斯凯尔医学技术有限公司 快速组织分子成像装置
CN108490597A (zh) * 2018-06-05 2018-09-04 张红明 一种基于光纤耦合器的共聚焦显微***
CN108937824A (zh) * 2018-08-02 2018-12-07 深圳大学 一种内窥成像装置和荧光寿命分布图像的获取方法

Also Published As

Publication number Publication date
CN102525411A (zh) 2012-07-04

Similar Documents

Publication Publication Date Title
WO2012075860A1 (zh) 一种荧光内窥成像方法及***
EP1580586B1 (en) Scanning confocal microscope
JP5142431B2 (ja) 顕微鏡用物体の検査及び操作のための装置及び方法
JP4455059B2 (ja) 特に内視鏡用の共焦点式イメージング装置
JP5547868B2 (ja) 顕微鏡系およびこれを用いた方法
JP2007501447A (ja) ダブルクラッドファイバー走査型顕微鏡
WO2019233425A1 (zh) 一种基于光纤耦合器的共聚焦显微***
US20180011309A1 (en) Device for transporting and controlling light pulses for lensless endo-microscopic imaging
Gavryusev et al. Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector
JP2012237647A (ja) 多焦点共焦点ラマン分光顕微鏡
US11703670B2 (en) Optical assembly for scanning excitation radiation and/or manipulation radiation in a laser scanning microscope, and laser scanning microscope
WO2016011611A1 (zh) 内窥式光学分子影像导航***和多光谱成像方法
WO2013036049A2 (ko) 공초점 형광 현미경
US20070109634A1 (en) Microscope apparatus
CN110208227A (zh) 一种单物镜光片显微成像***
JP3568846B2 (ja) ３次元イメージ取得方法及びその装置
CN202069570U (zh) 一种荧光内窥成像***
Lai et al. High-speed laser-scanning biological microscopy using FACED
CN108051414A (zh) 变焦光镊双光子显微成像装置和方法
CN112033538B (zh) 一种基于光谱-时间映射的超快成像装置
CN117705773A (zh) 模块化多模态显微光学分析***
CN112858250A (zh) 一种超分辨显微成像***
CN211014821U (zh) 一种显微镜
JP5302220B2 (ja) 共焦点レーザ顕微鏡
KR20110029475A (ko) 공초점 레이저 주사 현미경

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11847822

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 24.10.2013)

122 Ep: pct application non-entry in european phase

Ref document number: 11847822

Country of ref document: EP

Kind code of ref document: A1