Dubey Vishesh, Ahmad Azeem, Singh Rajwinder, Wolfson Deanna L, Basnet Purusotam, Acharya Ganesh, Mehta Dalip Singh, Ahluwalia Balpreet Singh
Opt Express. 2018 Aug 6;26(16):19864-19876. doi: 10.1364/OE.26.019864.
Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.
全内反射荧光（TIRF）显微镜具有高灵敏度、低背景噪声、低光毒性以及对靠近膜表面的亚细胞结构进行高对比度成像等优点。尽管TIRF显微镜能提供高对比度成像，但它无法提供有关生物细胞形态特征的定量信息。在此，我们提出一种基于集成波导芯片的TIRF显微镜与无标记定量相位成像（QPI）技术。波导表面上方存在的倏逝场用于激发荧光，并用正置显微镜收集信号。将正置显微镜转换为Linnik型干涉仪，以依次提取细胞的定量相位信息和TIRF图像。基于波导芯片的TIRF显微镜具有照明光路与收集光路解耦、大视场成像以及用于多色TIRF成像的预对准配置等优点。所提出的多模态显微镜用于研究脂多糖（LPS）对大鼠巨噬细胞引起的炎症。TIRF显微镜显示，LPS炎症分子会破坏细胞膜，并导致细胞在底物上显著扩张。而QPI模块则量化了LPS刺激的巨噬细胞亚细胞内容物的变化，显示其最大相位值出现净下降。
