CN111323389A - 高光谱成像显微镜及成像方法 - Google Patents
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Abstract
本发明提出高光谱成像显微镜，包括激发光源、载物台、物镜组、科勒光学透镜组、分光镜以及会聚透镜；激发光源发出光依次透过科勒光学透镜组、分光镜、物镜组并照射在载物台上，分光镜背离载物台的一侧依次设置有会聚透镜、液晶可调谐滤波器、面阵光电探测器，载物台将部分激发光源发出光反射并依次透过物镜组、分光镜、会聚透镜、液晶可调谐滤波器、面阵光电探测器。该显微镜采用液晶可调谐滤波器，适应不同波长下的样品的反射光以及荧光，拥有较高分辨率的反射成像以及荧光成像，无需制作多个窄宽滤波片；并且，该显微镜采用单波长激光器作为激发光源，具有较多的光子，使样品具有较为明显的反射光。
Description
技术领域
本发明涉及生物医学领域，具体涉及高光谱成像显微镜及成像方法。
背景技术
光谱成像技术在观察细胞活动领域是研究生命机制的强有力手段，尤其应用于细胞检测，可以起到鉴别不同细胞、区分细胞的不同生长阶段、分析细胞中某元素的作用以及检测细胞给药后的药物疗效等。随着光谱成像技术的不断发展与改进，其将在在生物医学领域发挥更大的作用，在显微成像方面，为深入研究微观组织的各种结构和特性提供了有力的支持。
反射成像可以检测组织散射和吸收性质的局部变化，荧光成像可以探测到组织化学成分的变化。
生物组织吸收的光一部分转换为热能，另外一部用以激发荧光和磷光。在紫外或者近红外波段的入射光会激发组织分子并诱导其发射荧光，大部分荧光团与组织结构基质以及细胞代谢途径有关。生物组织基质中最常见的荧光团是胶原蛋白和弹性蛋白，而涉及细胞代谢的主要荧光团则是烟酰胺腺嘌呤二核苷酸(NADH)，黄素腺嘌呤二核苷酸(FAD)和脂质体。这些荧光团在不同的光谱范围里有着不同的荧光强度。例如，胶原蛋白或者弹性蛋白在400-1700nm的光波照射下产生的荧光的带宽高于400-600nm的光，根据这个差异可以用来区分不同类型的组织，例如上皮组织和结缔组织。不同病变程度的细胞有着不同的结构和代谢速率，因此它们的发射荧光光谱也有差异。因此，在不使用荧光剂的情况下，可以通过荧光成像实时地检测组织并进行疾病的诊断。
传统的成像例如：荧光光谱成像，拉曼光谱成像，具有相当的光谱检测能力，但荧光光谱成像只能够实现某些离散波长(特定的部分波长下)下的光谱成像，信息量不足，不利于对样品进行深入的研究分析，拉曼光谱成像作为一种非线性光学现象，拉曼散射的效率非常低，通常在105个光子中，只有一个光子能够通过拉曼效应而产生频率漂移，在普通光照中很难被观测到，需要采用高光子密度的激光作为激发光源。
发明内容
为了克服现有技术中的不足，本发明提出高光谱成像显微镜，包括激发光源、载物台、物镜组、科勒光学透镜组、分光镜以及会聚透镜，其特征在于，
为了实现上述目的，所述激发光源发出光依次透过科勒光学透镜组、分光镜、物镜组并照射在载物台上，所述分光镜背离载物台的一侧依次设置有会聚透镜、液晶可调谐滤波器、面阵光电探测器，所述载物台将部分激发光源发出光反射并依次透过物镜组、分光镜、会聚透镜、液晶可调谐滤波器、面阵光电探测器。
进一步的，所述液晶可调谐滤波器的透光波段连续可调。
进一步的，所述面阵光电探测器包括EMCCD相机、CCD相机、coms相机、scoms相机、InGaAs相机。
进一步的，所述物镜组的分辨率可调。
进一步的，所述激发光源包括一个或者多个不同波长的激光器。
本发明还提出一种用于实施上述高光谱成像显微镜而设计的高光谱成像方法，包括以下步骤：S1：利用不同波长的光源照射载物台上的样品，样品在光源的刺激下发出荧光和反射部分激发光源，荧光和反射光依次通过物镜组、分光镜、会聚透镜到达液晶可调谐滤波器；
S2：调整液晶可调谐滤波器的透光波段，使不同波长下的光源照射下样品产生的荧光和反射光透过，传递到面阵光电探测器表面；
S3：被激发出的荧光或反射光会聚在面阵光电探测器上，可扫描出样品的反射高光谱图像和荧光高光谱图像；
S4：面阵光电探测器将接收到的光信号转换为电信号并传递给计算机，计算机处理接收到的数据并显示样品的反射高光谱图像和荧光高光谱图像。
有益效果：1、本发明采用液晶可调谐滤波器，其透光波段可连续调节，可以适应不同波长下的样品的反射光以及荧光，特别是在近红外和可见光波长范围内样品荧光，从而拥有较高分辨率的反射成像以及荧光成像，无需制作多个窄宽滤波片；
2、本发明采用单个或多个激光器组成激发光源，可激发荧光范围广，具有较多的光子，可以使样品具有较为明显的反射光。
附图说明
下面结合附图对本发明作进一步描写和阐述。
图1是本发明的整体的结构示意图；
图2是本发明的流程图。
附图标记：1、激发光源；2、科勒光学透镜；3、分光镜；4、物镜组；5、载物台；6、样品；7、会聚透镜；8、液晶可调谐滤波器；9、面阵光电探测器；10、计算机。
具体实施方式
下面将结合附图、通过对本发明的优选实施方式的描述，更加清楚、完整地阐述本发明的技术方案。
实施例
本发明提出的高光谱成像显微镜，包括载物台5、物镜组4、分光镜3以及会聚透镜7、激发光源1、科勒光学透镜2组、液晶可调谐滤波器8以及面阵光电探测器9。
如图1所示，在载物台5上放置样品6(例如细胞玻片)，激发光源1同时发出某个波长的光，该光经过科勒光学透镜2组照在分光镜3上，分光镜3将光反射，反射后的光依次透过物镜组4、载物台5并照在样品6上。样品6在激发光源1的光照下激发出荧光，同时样品6也会反射部分单波长激发光源1的光(以下简称反射光)，荧光以及反射光共同依次透过载物台5、物镜组4、分光镜3、会聚透镜7以及液晶可调谐滤波器8。液晶可调谐滤波器8通过透光波段调整，使荧光以及反射光进行二维空间分光。
设置在液晶可调谐滤波器8光路末端的面阵光电探测器9对二维空间分光后的荧光或者反射光分步接收并显示，展示的分别为反射高光谱图像以及荧光高光谱图像。
面阵光电探测器9采集一次荧光和反射光后，关闭并重启激发光源1，激发光源1使用与前述波长不同的另一个波长的光重新照射样品6，样品6再次经过上述过程在面阵光电探测器9上二次呈现反射高光谱图像和荧光高光谱图像。
如图1所示，激光光源包含一个或多个不同波长的激光器，每个波长的激光器有一个或多个，例如型号为BOT980-5000D的激光器，激发光源的发射光子的波长范围包括部分可见光和部分红外光，输出的激光波长包括405nm、488nm、561nm、640nm、785nm、808nm、980nm、1064nm等。激发光源的发射光具有多个光子；
本发明采用上述激光光源，通过发出激光的波长较多，克服了现有技术中采用离散波长(特定的部分波长下)样品6成像的信息量不足的缺点，激发光源1具有较多的波长选择，可以获得不同波长下反射高光谱图像和荧光高光谱图像，获得的样品6成像信息量更多更完整。同时，采用激发光源1，具有较多的光子，可以使样品6具有较为明显的反射光。
液晶可调谐滤波器8采用型号为KURIOS_WL1的可调谐滤光器，液晶可调谐滤波器是根据液晶电控双折射效应而制成的器件，具有透光波段连续可调的功能，用以滤除激发光源及其他激发荧光波段外的干扰光，样品6发出的荧光经过液晶可调谐滤波器后，被成像物镜成像在高灵敏面阵光电探测器9上。由于激光器发出的激光波长的范围较宽，采用连续可调的液晶可调谐滤波器8可以适应不同波长的光的滤光。
如图1所示，高灵敏面阵光电探测器9主要是由EMCCD(电子倍增CCD)相机、CCD相机、coms(互补金属氧化物导体器件)相机、scoms相机、InGaAs相机等相机组成的一个相机组。高灵敏面阵光电探测器9用于在不同的激发光源1的波长下接收的液晶可调谐滤波器8传递的样品6的多个成像，再在多个成像中人为选出高分辨率的样品6的成像。为了搭配高灵敏面阵光电探测器9，以及同样适应不同的单波长激发光源1的波长下样品6成像高分辨率，所以物镜同样具有分辨率可调的功能，使物镜具有高透光率。
激发光源1、液晶可调谐滤波器8以及面阵光电探测器9均通过通信接口(例如USB3.0接口)与计算机10电性连接，计算机10既可以对激发光源1的启停控制，计算机10也可以实现液晶可调谐滤波器8的透光波段调节，又可以将面阵光电探测器9检测的荧光高光谱图像或者反射高光谱图像进行显示。
如图2所示，本发明还提出一种用于上述高光谱成像显微镜所设计的高光谱成像方法，包括如下步骤：
S1：利用不同波长的光源照射载物台上的样品，样品在光源的刺激下发出荧光和反射部分激发光源，荧光和反射光依次通过物镜组、分光镜、会聚透镜到达液晶可调谐滤波器；
S2：调整液晶可调滤光片的透光波段，使不同波长下的光源照射下样品产生的荧光和反射光透过，传递到面阵光电探测器表面；
S3：被激发出的荧光或反射光会聚在面阵列光电探测器上，可扫描出样品的反射高光谱图像和荧光高光谱图像；
S4：面阵光电探测器将接收到的光信号转换为电信号并传递给计算机，计算机处理接收到的数据并显示样品的反射高光谱图像和荧光高光谱图像。
本发明还具备的好处有：
1、采用液晶可调谐滤波器，液晶可调谐滤波器的透过波段范围切换速度快、光谱扫描速度快、操作简单、选通波长带宽窄、光谱分辨率高、无移动部件、没有机械抖动、孔径大、视场角大、体积小、并具有良好的光学特性；
2、采用高光谱成像技术，通过窄带滤光片液晶可调谐滤波器将交叠的荧光光谱区域通过算法分离，实现荧光光谱分离，对荧光标记物精准的定量分析；
3、显微物镜具有高透光率、高NA值、高成像分辨率的4X\10X\20X\40X\50X\60X，同时需匹配相应的面阵光电探测器，物镜可以切换倍率；
4、液晶可调制波段宽且连续可调，该系统适用多种生物荧光、荧光探针、纳米材料。另外，对于样品体内的多种荧光标记，快速切换激发光源，配合液晶可调谐滤波器透光波段的调节，被激发出的荧光在即可通过液晶可调谐滤波器被成像物镜会聚在面阵光电探测器上，可扫描出样品的高光谱图像；
5、显微镜镜体采用正置荧光显微镜或者倒置荧光显微镜镜头。上述具体实施方式仅仅对本发明的优选实施方式进行描述，而并非对本发明的保护范围进行限定。在不脱离本发明设计构思和精神范畴的前提下，本领域的普通技术人员根据本发明所提供的文字描述、附图对本发明的技术方案所作出的各种变形、替代和改进，均应属于本发明的保护范畴。本发明的保护范围由权利要求确定。
Claims (7)
1.高光谱成像显微镜，包括激发光源、载物台、物镜组、科勒光学透镜组、分光镜以及会聚透镜，其特征在于，
所述激发光源发出光依次透过科勒光学透镜组、分光镜、物镜组并照射在载物台上，所述分光镜背离载物台的一侧依次设置有会聚透镜、液晶可调谐滤波器、面阵光电探测器，所述载物台将部分激发光源发出光反射并依次透过物镜组、分光镜、会聚透镜、液晶可调谐滤波器、面阵光电探测器。
2.根据权利要求1所述的高光谱成像显微镜，其特征在于，所述液晶可调谐滤波器的透光波段连续可调。
3.根据权利要求1所述的高光谱成像显微镜，其特征在于，所述面阵光电探测器包括EMCCD相机、CCD相机、coms相机、scoms相机、InGaAs相机。
4.根据权利要求1所述的高光谱成像显微镜，其特征在于，所述物镜组的分辨率可调。
5.根据权利要求1所述的高光谱成像显微镜，其特征在于，所述激发光源包括一个或者多个不同波长的激光器。
6.权利要求1所述的高光谱成像显微镜，其特征在于，所述激光器的发射光子的波长范围包括部分可见光和部分红外光，进一步的，所述激发光源发出的光的波长包括405nm、488nm、561nm、640nm、785nm、808nm、980nm、1064nm。
7.用于实施权利要求1-5任意一项所述的高光谱成像显微镜而设计的高光谱成像方法，其特征在于，包括以下步骤：
S1：利用不同波长的光源照射载物台上的样品，样品在光源的刺激下发出荧光和反射部分激发光源，荧光和反射光依次通过物镜组、分光镜、会聚透镜到达液晶可调谐滤波器；
S2：调整液晶可调谐滤波器的透光波段，使不同波长下的光源照射下样品产生的荧光和反射光透过，传递到面阵光电探测器表面；
S3：被激发出的荧光或反射光会聚在面阵光电探测器上，可扫描出样品的反射高光谱图像和荧光高光谱图像；
S4：面阵光电探测器将接收到的光信号转换为电信号并传递给计算机，计算机处理接收到的数据并显示样品的反射高光谱图像和荧光高光谱图像。
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