Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Analyst. 2021 May 4;146(9):3062-3072. doi: 10.1039/d1an00129a.
Nonlinear optical microscopy that leverages an objective based total internal reflection (TIR) excitation scheme is an attractive means for rapid, wide-field imaging with enhanced surface sensitivity. Through select combinations of distinct modalities, one can, in principle, access complementary chemical and structural information for various chemical species near interfaces. Here, we report a successful implementation of such a wide-field nonlinear optical microscope system, which combines coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and sum frequency generation (SFG) modalities on the same platform. The intense optical fields needed to drive these high order nonlinear optical processes are achieved through the use of femtosecond pulsed light in combination with the intrinsic field confinement induced by TIR over a large field of view. The performance of our multimodal microscope was first assessed through the experimental determination of its chemical fidelity, intensity and polarization dependences, and spatial resolution using a set of well-defined model systems. Subsequently, its unique capabilities were validated through imaging complex biological systems, including Hydrangea quercifolia pollen grains and Pantoea sp. YR343 bacterial cells. Specifically, the spatial distribution of different molecular groups in the former was visualized via vibrational contrast mechanisms of CARS, whereas co-registered TPF imaging allowed the identification of spatially localized intrinsic fluorophores. We further demonstrate the feasibility of our microscope for wide-field CARS imaging on live cells through independent characterization of cell viability using spatially co-registered TPF imaging. This approach to TIR enabled wide-field imaging is expected to provide new insights into bacterial strains and their interactions with other species in the rhizosphere in a time-resolved and chemically selective manner.
利用基于物镜的全内反射 (TIR) 激发方案的非线性光学显微镜是一种快速、宽场成像的有吸引力的方法，具有增强的表面灵敏度。通过选择不同模式的独特组合，人们可以原则上获取界面附近各种化学物质的互补化学和结构信息。在这里，我们报告了一种成功实现的宽场非线性光学显微镜系统，该系统将相干反斯托克斯拉曼散射 (CARS)、双光子荧光 (TPF)、二次谐波产生 (SHG) 和和频产生 (SFG) 模式结合在同一平台上。这些高阶非线性光学过程所需的强光学场是通过使用飞秒脉冲光结合 TIR 诱导的固有场限制在大视场中实现的。我们通过使用一组定义明确的模型系统，通过实验确定其化学保真度、强度和偏振依赖性以及空间分辨率来评估多模态显微镜的性能。随后，通过对复杂生物系统进行成像，包括绣球花粉粒和 Pantoea sp. YR343 细菌细胞，验证了其独特的功能。具体来说，通过 CARS 的振动对比机制可视化了前者中不同分子基团的空间分布，而共注册的 TPF 成像允许识别空间局部固有荧光团。我们通过使用共注册的 TPF 成像对细胞活力进行独立表征，进一步证明了我们的显微镜在活细胞上进行宽场 CARS 成像的可行性。这种基于 TIR 的宽场成像方法有望以时间分辨和化学选择性的方式为研究根际中的细菌菌株及其与其他物种的相互作用提供新的见解。
