Surgical Robotics Laboratory, Department of Biomechanical Engineering, University of Twente, 7522 NB, Enschede, The Netherlands.
Surgical Robotics Laboratory, Department of Biomedical Engineering and University Medical Centre Groningen, University of Groningen, 9713 AV, Groningen, The Netherlands.
Sci Rep. 2022 Aug 4;12(1):13375. doi: 10.1038/s41598-022-17297-7.
Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 [Formula: see text] 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.
光学显微镜技术是可视化微试剂的常用选择。它们生成的图像具有相对较高的时空分辨率，但无法揭示用于区分微试剂和周围环境的编码信息。本研究提出了多色荧光显微镜，通过光谱解混实现移动微试剂和动态周围环境的彩色编码识别。我们通过可视化单个和簇状微试剂附着到用 HeLa 细胞形成的癌细胞球体来报告多色显微镜性能，作为靶向药物输送演示的概念验证。开发了微流控芯片来固定单个球体以进行附着，为多色显微镜提供稳定的环境，并创建 3D 肿瘤模型。为了确认多色显微镜能够在血管化环境中可视化微试剂，我们使用了内皮细胞形成的体外血管网络和鸡胚绒毛尿囊膜作为实验模型。通过以轮询方式顺序激发荧光团并从三个不同光谱带同步获取单个图像，实现了我们模型的全可视化。我们通过实验证明，多色显微镜利用具有良好分离光谱特性的荧光团对微试剂、有机体（癌细胞球体和脉管系统）和周围介质进行光谱分解，并允许以每秒 15 帧的速度采集 1280 [Formula: see text] 1024 像素的图像。我们的结果显示，实时多色显微镜通过彩色编码可视化提供了对微试剂跟踪、有机体形态和周围介质清晰区分的更深入理解。
