CN102551661B - Fluorescence spectrum endoscopic imaging method and system - Google Patents
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Abstract
本发明适用于光电检测领域，提供了一种荧光光谱内窥成像方法及系统，所述荧光光谱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，生成光谱分辨的荧光图像。本发明由多个子光束对样品进行二维扫描，从而获取整个样品光谱分辨的荧光图像，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。
The present invention is applicable to the field of photoelectric detection, and provides a fluorescence spectrum endoscopic imaging method and system. The fluorescence spectrum endoscopic imaging method includes the following steps: generating excitation light; dividing the excitation light into multiple sub-beams, and the The multiple sub-beams correspond to multiple sub-regions of the sample, and fluorescent substances are distributed in the sample; the multiple sub-beams are adjusted so that each sub-beam is transmitted into the living body and focused on the sub-regions of the sample; using the multiple sub-beams The light beam scans the sample so that the fluorescent substances in each sub-area emit fluorescence; the fluorescence emitted during scanning is collected in real time to generate a spectrally resolved fluorescence image. The present invention scans the sample two-dimensionally by a plurality of sub-beams to obtain a spectrally resolved fluorescence image of the entire sample, with short time, high speed, and little damage to organisms, which is beneficial to biomedical research, especially for early diagnosis of cancer. Significance.
Description
技术领域 technical field
本发明属于光电检测领域，尤其涉及一种荧光光谱内窥成像方法及系统。The invention belongs to the field of photoelectric detection, in particular to a fluorescent spectrum endoscopic imaging method and system.
背景技术 Background technique
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。荧光光谱图像能够为生物医学检测和分析提供结构和功能信息。Fluorescence microscopy has become an important tool in life sciences, especially cell biology research. Multi-photon excitation 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. Fluorescence spectroscopy images can provide structural and functional information for biomedical detection and analysis.
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。目前双光子荧光光谱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，在内窥系统设计、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。然而目前荧光光谱内窥成像的速度慢，效率低，耗时长，对生物体造成极大的影响。In recent years, with the rapid development of new optical fiber and micro-manufacturing technology, the research of fiber-optic two-photon fluorescence microscope and endoscope has made it possible to study two-photon fluorescence microscopy imaging technology in living internal organs and living animals. At present, two-photon fluorescence spectroscopy endoscopic microscopy technology has attracted great attention in the world, and a large number of research results have been made on this subject, including endoscopic system design, scanning mechanism, optical transmission and high numerical aperture micro-objective lens Many research results have been obtained in its application and other aspects. Limited by the application conditions of in vivo endoscopy, the imaging time should not be too long. However, the current endoscopic imaging of fluorescence spectroscopy is slow, inefficient, and time-consuming, which has a great impact on organisms.
发明内容 Contents of the invention
本发明实施例的目的在于提供一种荧光光谱内窥成像方法，旨在解决现有荧光光谱内窥成像速度慢、效率低的问题。The purpose of the embodiments of the present invention is to provide a fluorescence spectrum endoscopic imaging method, aiming at solving the problems of slow speed and low efficiency of the existing fluorescence spectrum endoscopic imaging.
本发明实施例是这样实现的，一种荧光光谱内窥成像方法，包括以下步骤：The embodiment of the present invention is achieved in this way, a fluorescent spectrum endoscopic imaging method, comprising the following steps:
产生激发光；generate excitation light;
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；Dividing the excitation light into a plurality of sub-beams, the plurality of sub-beams correspond to a plurality of sub-regions of the sample, and fluorescent substances are distributed in the sample;
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；adjusting the plurality of sub-beams so that each sub-beam is transmitted into the living body and focused on a sub-region of the sample;
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；Scanning the sample by using the plurality of sub-beams, so that the fluorescent substances in each sub-area emit fluorescence;
实时采集扫描时发出的荧光，生成光谱分辨的荧光图像。Fluorescence emitted during scanning is collected in real time, generating spectrally resolved fluorescence images.
本发明实施例的另一目的在于提供一种荧光光谱内窥成像系统，所述系统包括：Another object of the embodiments of the present invention is to provide a fluorescence spectrum endoscopic imaging system, the system comprising:
激发光源，用于产生激发光；an excitation light source for generating excitation light;
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；a beam splitter, configured to divide the excitation light into multiple sub-beams, the multiple sub-beams correspond to multiple sub-regions of the sample, and fluorescent substances are distributed in the sample;
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；A flexible medium, used to adjust the multiple sub-beams so that the multiple sub-beams are transmitted into the living body;
聚焦元件，用于使各子光束聚焦于所述样品的子区域；a focusing element for focusing each sub-beam on a sub-area of the sample;
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；a scanning element, configured to scan the sample with the plurality of sub-beams, so that the fluorescent substances in each sub-area emit fluorescence;
双色镜及传像介质，用于将所述荧光从所述生物体内导出；a dichroic mirror and an imaging medium for deriving said fluorescence from said organism;
色散元件，用于使所述荧光沿光谱方向展开；a dispersive element for spreading the fluorescence along a spectral direction;
探测器，用于实时采集扫描时发出的荧光，生成光谱分辨的荧光图像；The detector is used to collect the fluorescence emitted during scanning in real time to generate a spectrally resolved fluorescence image;
所述双色镜设于所述扫描元件与聚焦元件之间。The dichroic mirror is arranged between the scanning element and the focusing element.
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，导出荧光并将其沿光谱方向展开，由多个子光束对样品进行二维扫描，从而获取整个样品光谱分辨的荧光图像，时间短、速度快，对生物体损伤小，有利于活体和在体研究，特别是对癌症早期诊断，具有重要意义。In the embodiment of the present invention, the excitation light is divided into multiple sub-beams corresponding to multiple sub-regions of the sample, so that the multiple sub-beams are transmitted into the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, and the fluorescence is derived And spread it along the spectral direction, and scan the sample two-dimensionally by multiple sub-beams to obtain a spectrally resolved fluorescence image of the entire sample. The time is short, the speed is fast, and the damage to the organism is small, which is beneficial to living and in vivo research, especially It is of great significance to the early diagnosis of cancer.
附图说明 Description of drawings
图1是本发明实施例提供的荧光光谱内窥成像方法的实现流程图；Fig. 1 is a flow chart of the implementation of the fluorescence spectrum endoscopic imaging method provided by the embodiment of the present invention;
图2是本发明实施例提供的荧光光谱内窥成像系统的结构及其光路图；Fig. 2 is the structure and optical path diagram of the fluorescent spectrum endoscopic imaging system provided by the embodiment of the present invention;
图3a是扫描元件对样品进行线扫描的位置图；Figure 3a is a position diagram of the scanning element performing line scanning on the sample;
图3b是与图3a对应的光谱分辨的荧光图像；Figure 3b is a spectrally resolved fluorescence image corresponding to Figure 3a;
图4a是扫描元件对样品进行线扫描的另一位置图；Figure 4a is another positional diagram of the scanning element performing line scanning on the sample;
图4b是与图4a对应的光谱分辨的荧光图像。Figure 4b is a spectrally resolved fluorescence image corresponding to Figure 4a.
具体实施方式 Detailed ways
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。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
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，导出荧光并将其沿光谱方向展开，由多个子光束对样品进行二维扫描，从而获取整个样品光谱分辨的荧光图像，时间短、速度快，对生物体损伤小，有利于活体和在体研究。In the embodiment of the present invention, the excitation light is divided into multiple sub-beams corresponding to multiple sub-regions of the sample, so that the multiple sub-beams are transmitted into the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, and the fluorescence is derived And spread it along the spectral direction, and scan the sample two-dimensionally with multiple sub-beams to obtain a spectrally resolved fluorescence image of the entire sample, with short time, fast speed, and little damage to organisms, which is conducive to in vivo and in vivo research.
本发明实施例提供的荧光光谱内窥成像方法包括以下步骤：The fluorescence spectrum endoscopic imaging method provided by the embodiment of the present invention includes the following steps:
产生激发光；generate excitation light;
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；Dividing the excitation light into a plurality of sub-beams, the plurality of sub-beams correspond to a plurality of sub-regions of the sample, and fluorescent substances are distributed in the sample;
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；adjusting the plurality of sub-beams so that each sub-beam is transmitted into the living body and focused on a sub-region of the sample;
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；Scanning the sample by using the plurality of sub-beams, so that the fluorescent substances in each sub-area emit fluorescence;
实时采集扫描时发出的荧光，生成光谱分辨的荧光图像。Fluorescence emitted during scanning is collected in real time, generating spectrally resolved fluorescence images.
本发明实施例提供的荧光光谱内窥成像系统包括：The fluorescence spectrum endoscopic imaging system provided by the embodiment of the present invention includes:
激发光源，用于产生激发光；an excitation light source for generating excitation light;
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；a beam splitter, configured to divide the excitation light into multiple sub-beams, the multiple sub-beams correspond to multiple sub-regions of the sample, and fluorescent substances are distributed in the sample;
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；A flexible medium, used to adjust the multiple sub-beams so that the multiple sub-beams are transmitted into the living body;
聚焦元件，用于使各子光束聚焦于所述样品的子区域；a focusing element for focusing each sub-beam on a sub-area of the sample;
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；a scanning element, configured to scan the sample with the plurality of sub-beams, so that the fluorescent substances in each sub-area emit fluorescence;
双色镜及传像介质，用于将所述荧光从所述生物体内导出；a dichroic mirror and an imaging medium for deriving said fluorescence from said organism;
色散元件，用于使所述荧光沿光谱方向展开；a dispersive element for spreading the fluorescence along a spectral direction;
探测器，用于实时采集扫描时发出的荧光，生成光谱分辨的荧光图像；The detector is used to collect the fluorescence emitted during scanning in real time to generate a spectrally resolved fluorescence image;
所述双色镜设于所述扫描元件与聚焦元件之间。The dichroic mirror is arranged between the scanning element and the focusing element.
以下结合具体实施例对本发明的实现进行详细描述。The implementation of the present invention will be described in detail below in conjunction with specific embodiments.
图1示出了本发明实施例提供的荧光光谱内窥成像方法的实现流程，详述如下：Figure 1 shows the implementation process of the fluorescence spectrum endoscopic imaging method provided by the embodiment of the present invention, which is described in detail as follows:
在步骤S101中，产生激发光；In step S101, generating excitation light;
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。In the embodiment of the present invention, a femtosecond (ultrashort) pulse laser with a working frequency of 76 MHz, a period of 120 fs, and a center wavelength of 800 nm is preferably used as excitation light, which can realize two-photon excitation of fluorescent substances. Usually, the pulsed laser is expanded and collimated and its intensity distribution is adjusted to form a flat-hat beam with uniform intensity distribution.
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；In step S102, the excitation light is divided into multiple sub-beams, the multiple sub-beams correspond to multiple sub-regions of the sample, and fluorescent substances are distributed in the sample;
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。In the embodiment of the present invention, the excitation light with uniform intensity distribution is divided into multiple sub-beams, and the multiple sub-beams correspond to multiple sub-regions of the sample with fluorescent substances.
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；In step S103, adjusting multiple sub-beams, so that each sub-beam is transmitted into the living body and focused on a sub-region of the sample;
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。In the embodiment of the present invention, multiple sub-beams are transmitted into the living body in parallel, each focusing on a sub-region of the sample. Specifically, multiple sub-beams are first coupled into the flexible medium, and the multiple sub-beams enter the living body in parallel through the flexible medium, and the multiple sub-beams are focused in the living body to form excitation light array points and projected to corresponding sub-regions respectively. Wherein the flexible medium is a photonic crystal fiber array, the input end of which is located outside the living body, and the output end is located inside the living body.
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。Before multiple sub-beams are coupled into the flexible medium, each sub-beam needs to be collimated so that each sub-beam becomes parallel light. After multiple sub-beams are output from the flexible medium, they also need to be collimated so that each sub-beam can be focused on the corresponding sample sub-area.
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；In step S104, the sample is scanned with multiple sub-beams, so that the fluorescent substances in each sub-area emit fluorescence;
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：In the embodiment of the present invention, scanning is divided into line scanning and step scanning, and the specific process is as follows:
1、线扫描1. Line scan
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。A plurality of sub-beams are focused to form an excitation light array point and projected onto the sample, and each sub-area is scanned along the longitudinal direction of the sample, and the fluorescent substances in each sub-area emit fluorescence under the action of the excitation light array point. The scanning speed of this line is fast and the time is short.
2、步进扫描2. Step scan
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。After the longitudinal line scanning of each sub-region is completed, each sub-region is step-scanned along the lateral direction of the sample to adjust the position of the excitation light array point in the lateral direction of the sample.
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。The above-mentioned line scanning and step scanning are performed cyclically until the scanning of each sub-area of the sample is completed. It should be understood that the directions of the line scan and the step scan may also be exchanged during specific implementation.
在步骤S105中，实时采集扫描时发出的荧光，生成光谱分辨的荧光图像。In step S105, the fluorescence emitted during scanning is collected in real time to generate a spectrally resolved fluorescence image.
本发明实施例对样品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，生成光谱分辨的荧光图像。具体地，先导出各子区域内荧光物质发出的荧光，接着将荧光沿其光谱方向展开，然后采集光谱分辨的荧光信息，生成荧光图像。In the embodiment of the present invention, while scanning each sub-area of the sample, the fluorescence emitted by the fluorescent substance in each sub-area is collected to generate a spectrally resolved fluorescence image. Specifically, the fluorescence emitted by the fluorescent substances in each sub-region is firstly derived, and then the fluorescence is spread along its spectral direction, and then spectrally resolved fluorescence information is collected to generate a fluorescence image.
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。Those of ordinary skill in the art should understand that all or part of the steps in the methods of the above-mentioned embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, such as ROM/RAM, disk , CD, etc.
图2示出了本发明实施例提供的荧光光谱内窥成像系统的结构，为了便于说明，仅示出了与本发明实施例相关的部分。FIG. 2 shows the structure of the fluorescence spectrum endoscopic imaging system provided by the embodiment of the present invention. For the convenience of description, only the parts related to the embodiment of the present invention are shown.
本发明实施例提供的荧光光谱内窥成像系统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、扫描元件以及微物镜。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、纠偏元件、色散元件、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。The fluorescence spectrum endoscopic imaging system provided by the embodiment of the present invention has an excitation light path and a detection light path. The excitation optical path includes an excitation light source, a beam expander and collimator, a shaper, a beam splitter, a collimator lens, a first coupling lens, a photonic crystal fiber array, a first self-focusing lens, a scanning element and a micro-objective lens. The detection optical path includes a micro-objective lens, a dichroic mirror, a second self-focusing lens, an image transmission fiber bundle, a second coupling lens, a filter element, a deflection correction element, a dispersion element, an imaging lens and a detector. The micro-objective lens is shared by the excitation optical path and the detection optical path.
以下对激发光路的结构进行详细说明。The structure of the excitation light path will be described in detail below.
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。As shown in Figure 2, the embodiment of the present invention preferably uses a Ti:Sapphire femtosecond laser 1 as an excitation light source, which can generate a pulsed laser with 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 generation of fluorescent substances excitation. The pulsed laser light is transformed into collimated light of required size through the beam expanding and collimating device 2 .
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。In the embodiment of the present invention, the shaper is the beam shaper 3, and the collimated pulsed laser is shaped by the beam shaper 3 to form a flat-hat beam with uniform intensity distribution. The beam splitter can be a microlens array, a diffractive optical element or a beam splitter. In this embodiment, the microlens array 4 is preferred, and the flat-top distributed pulsed laser light is divided into multiple sub-beams through the microlens array 4. The multiple sub-beams correspond to the multiple beams of the sample 12. In this embodiment, the microlens array 4 is a 3×3 microlens array, that is, the microlens array has nine microobjective lenses.
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。Wherein the rear focal plane of collimator lens 5 coincides with the front focal plane of microlens array 4, and the sub-beams focus at the front focal plane of microlens array 4, namely at the back focal plane of collimator lens 5, and each sub-beam is through collimating lens 5 become sub-beams of parallel light.
上述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投射至扫描元件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。各子光束经扫描元件9投射至具有荧光物质的样品12，样品12与扫描元件9之间设有起会聚作用的微物镜11。所述扫描元件9优选为MEMS(Micro-Electro-Mechanical Systems，微机电系统)扫描镜，MEMS扫描镜为二维扫描镜，即可对样品进行线扫描及步进扫描。多个子光束经扫描元件9对样品进行二维扫描，具体过程如下：The above multiple sub-beams are focused and coupled into the photonic crystal fiber array 7 through the first coupling lens 6 . The photonic crystal fiber array 7 is formed by a plurality of photonic crystal fibers arranged at equal intervals, and the number and arrangement of the photonic crystal fibers are the same as those of the microlens array 4 . The multiple sub-beams are transmitted into the living body through the photonic crystal fiber array 7 , and the multiple sub-beams emitted from the photonic crystal fiber array 7 are projected to the scanning element 9 through the first self-focusing lens 8 . The self-focusing lens is a rod lens whose refractive index gradually changes along the radial direction, and each sub-beam becomes parallel light through the first self-focusing lens 8 . Each sub-beam is projected to a sample 12 with fluorescent substances through the scanning element 9 , and a micro-objective lens 11 for converging is arranged between the sample 12 and the scanning element 9 . The scanning element 9 is preferably a MEMS (Micro-Electro-Mechanical Systems, Micro-Electro-Mechanical Systems) scanning mirror, and the MEMS scanning mirror is a two-dimensional scanning mirror, which can perform line scanning and step scanning on the sample. A plurality of sub-beams scan the sample two-dimensionally through the scanning element 9, and the specific process is as follows:
1、线扫描1. Line scan
多个子光束经微物镜11聚焦形成激发光阵列点投射至样品，沿样品12纵向对各子区域进行线扫描，各子区域内的荧光物质于激发光阵列点的作用下发出荧光形成荧光点阵，经扫描元件9线扫描即形成荧光线阵。此线扫描的速度快、时间短。Multiple sub-beams are focused by the micro-objective lens 11 to form excitation light array points and projected onto the sample, and each sub-area is line-scanned along the longitudinal direction of the sample 12, and the fluorescent substances in each sub-area emit fluorescence under the action of the excitation light array points to form a fluorescent lattice , the fluorescent linear array is formed by scanning the scanning element 9 lines. The scanning speed of this line is fast and the time is short.
2、步进扫描2. Step scan
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于样品横向的位置。After the vertical line scanning of each sub-region is completed, each sub-region is step-scanned along the lateral direction of the sample, that is, the position of the excitation light array point in the lateral direction of the sample is adjusted.
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。The above-mentioned line scanning and step scanning are performed cyclically until the scanning of each sub-area of the sample is completed. It should be understood that the directions of the line scan and the step scan may also be exchanged during specific implementation.
各子光束经本激发光路传导后形成激发光阵列点投射于样品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。扫描元件9进行线扫描的同时，由探测器20采集光谱分辨的荧光信息，生成荧光图像。该光谱分辨的荧光信息包括荧光的强度信息、光谱信息及其于样品中的位置信息。Each sub-beam is transmitted through the excitation optical path to form an excitation light array point projected on the sub-area of the sample 12 to excite the fluorescent substance in the sample 12 to emit fluorescence. Correspondingly, the fluorescence also has a plurality of sub-beams. While the scanning element 9 is performing line scanning, the detector 20 collects spectrally resolved fluorescence information to generate a fluorescence image. The spectrally resolved fluorescence information includes fluorescence intensity information, spectral information and position information in the sample.
以下对探测光路的结构进行详细说明。The structure of the detection light path will be described in detail below.
本发明实施例探测光路中双色镜10设于扫描元件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将各荧光子光束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。各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，由滤光元件16滤除激发光及其它杂散光，经纠偏元件17(如扫描镜)投射至色散元件18，色散元件18将多束荧光子光束沿光谱方向展开形成光谱分辨的多线阵列，再由成像透镜19聚焦于探测器20的敏感面，记录光谱分辨的多线阵列的荧光强度信息，生成光谱分辨的荧光图像。In the detection light path of the embodiment of the present invention, the dichromatic mirror 10 is arranged between the scanning element 9 and the micro-objective lens 11. The dichromatic mirror 10 is highly transparent to the pulsed laser with a center wavelength of 800nm, and highly reflective to the fluorescence with a wavelength of 400-700nm. The dichromatic mirror 10 The included angle with fluorescence is 45° or 135°. The above-mentioned fluorescence is collected by the micro-objective lens 11 to form multiple collimated fluorescent sub-beams. The dichroic mirror 10 reflects each fluorescent sub-beam from the excitation optical path, and focuses on the end of the body of the imaging fiber bundle 14 through the second self-focusing lens 13 . Each fluorescent sub-beam is derived from the living body by the image fiber bundle 14, converted into multi-path parallel light through the second coupling lens 13, the excitation light and other stray light are filtered out by the filter element 16, and the deflection correction element 17 (such as a scanning mirror) ) to the dispersive element 18, the dispersive element 18 expands the multi-beam fluorescent sub-beams along the spectral direction to form a spectrally resolved multi-line array, and then the imaging lens 19 is focused on the sensitive surface of the detector 20 to record the spectrally resolved multi-line array Fluorescence intensity information to generate spectrally resolved fluorescence images.
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20的敏感面互为共轭面，也就是说从样品12不同位置激发出的荧光点阵经微物镜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，经传像光纤束14传至其体外端面的对应位置，由色散元件18将荧光点阵沿光谱方向展开形成光谱分辨的荧光强度分布，再由成像透镜19聚焦到探测器20的对应位置。这样探测器20即可对样品12发出的荧光进行光谱探测，生成光谱分辨的荧光图像。Wherein the focal plane of the excitation light array point in the sample 12 and the internal end face of the image transmission fiber bundle 14 are mutually conjugate surfaces, and the in vitro end surface of the image transmission fiber bundle 14 and the sensitive surface of the detector 20 are mutually conjugate surfaces, that is to say The fluorescent lattices excited from different positions of the sample 12 are focused to the corresponding position of the internal end surface of the image-transmitting fiber bundle 14 through the micro-objective lens 11 and the second self-focusing lens 13, and transmitted to the corresponding position of the external end surface of the image-transmitting fiber bundle 14, by The dispersion element 18 expands the fluorescent dot matrix along the spectral direction to form a spectrally resolved fluorescent intensity distribution, which is then focused to the corresponding position of the detector 20 by the imaging lens 19 . In this way, the detector 20 can perform spectral detection on the fluorescence emitted by the sample 12 to generate a spectrally resolved fluorescence image.
本发明实施例中，探测器20为用于生成光谱分辨的荧光图像的面阵探测器，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用于存储、处理和读取探测器20所探测的光谱分辨的荧光图像，且由计算机21控制面阵探测器的曝光。In the embodiment of the present invention, the detector 20 is an area array detector for generating spectrally resolved fluorescence images, and the area array detector is preferably a CCD camera or a CMOS camera. The area array detector is connected to a computer 21, and the computer 21 is used to store, process and read the spectrally resolved fluorescence images detected by the detector 20, and the exposure of the area array detector is controlled by the computer 21.
上述光谱方向与扫描元件9的扫描方向垂直，即如果扫描方向为X方向，则光谱方向应为Y方向。扫描元件9一开始线扫描，探测器20即开始曝光；扫描元件9线扫描使样品12产生了荧光线阵；扫描元件9对样品12完成一次线扫描时，探测器20一次曝光结束，此时记录一幅光谱分辨的荧光图像，该荧光图像对应于样品多条线位置，探测器20由此记录了样品12多条荧光线的信息，如图3a和图3b所示。之后，扫描元件(MEMS扫描镜)9沿另一方向Y+步进一个像素，纠偏元件17相应地沿Y-方向步进一个像素，使荧光线阵于探测器20的位置不变。然后，扫描元件(MEMS扫描镜)9开始重复上述线扫描过程，探测器20亦开始重复上述曝光过程，扫描元件(MEMS扫描镜)9对样品12线扫描完成，同时探测器20曝光结束，记录另一幅光谱分辨的荧光图像，该图像对应于样品12内另一组多条线位置，如图4a和图4b所示。重复上述过程，直至激发光阵列点扫描完整个样品12为止。至此，获取了样品12内所有点光谱分辨的荧光图像，通过算法重构即可获得样品12光谱分辨的荧光信息。The above spectral direction is perpendicular to the scanning direction of the scanning element 9 , that is, if the scanning direction is the X direction, then the spectral direction should be the Y direction. As soon as the scanning element 9 starts line scanning, the detector 20 starts to expose; the scanning element 9 lines scanning makes the sample 12 produce a fluorescent line array; A spectrally resolved fluorescence image is recorded, the fluorescence image corresponds to the positions of the multiple lines of the sample, and the detector 20 thus records the information of the multiple fluorescent lines of the sample 12, as shown in Fig. 3a and Fig. 3b. Afterwards, the scanning element (MEMS scanning mirror) 9 steps one pixel along the Y+ direction, and the correction element 17 steps one pixel along the Y- direction accordingly, so that the position of the fluorescent line array on the detector 20 remains unchanged. Then, the scanning element (MEMS scanning mirror) 9 begins to repeat the above-mentioned line scanning process, and the detector 20 also begins to repeat the above-mentioned exposure process, and the scanning element (MEMS scanning mirror) 9 completes the line scanning of the sample 12, while the exposure of the detector 20 ends, and the recording Another spectrally resolved fluorescence image corresponding to another set of multiple line locations within the sample 12 is shown in Figures 4a and 4b. The above process is repeated until the excitation light array spots scan the entire sample 12 . So far, the spectrally resolved fluorescence images of all points in the sample 12 have been obtained, and the spectrally resolved fluorescence information of the sample 12 can be obtained through algorithm reconstruction.
当扫描元件(MEMS扫描镜)9沿另一方向Y+步进一个像素时，可使探测器20相应地沿Y-方向步进一个像素，以使荧光线阵于探测器20的位置不变，即可省却纠偏元件17，简化荧光光谱内窥成像系统，提高精度，节约成本。When the scanning element (MEMS scanning mirror) 9 steps one pixel along the other direction Y+, the detector 20 can be made to step one pixel correspondingly along the Y-direction, so that the position of the fluorescent linear array on the detector 20 remains unchanged, The deviation correcting element 17 can be omitted, the fluorescence spectrum endoscopic imaging system can be simplified, the precision can be improved, and the cost can be saved.
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，导出荧光并将其沿光谱方向展开，由多个子光束对样品进行二维扫描，从而获取整个样品光谱分辨的荧光图像，时间短、速度快，对生物体损伤小，有利于活体和在体研究，特别是对癌症早期诊断，具有重要意义。In the embodiment of the present invention, the excitation light is divided into multiple sub-beams corresponding to multiple sub-regions of the sample, so that the multiple sub-beams are transmitted into the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, and the fluorescence is derived And spread it along the spectral direction, and scan the sample two-dimensionally by multiple sub-beams to obtain a spectrally resolved fluorescence image of the entire sample. The time is short, the speed is fast, and the damage to the organism is small, which is beneficial to living and in vivo research, especially It is of great significance to the early diagnosis of cancer.
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。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.
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| CN202069569U (en) * | 2010-12-09 | 2011-12-14 | 深圳大学 | Fluorescent spectrum endoscope system |
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