Sensing local biomolecular environments with coherent optical nanoscopy

新型成像工具和技术的发展，包括超分辨率光学显微镜，已经彻底改变了我们对活细胞生物过程的理解。将高分辨率成像能力与活细胞中的化学传感相结合，有可能揭示现有技术几乎不可能遵循的过程，例如，定义对药物递送至关重要的内吞途径特定进入位点的局部环境。该项目的主要目的是证明和量化新型光学显微镜技术的适用性，这种技术超越了最先进的技术，可以直接在活细胞内对单个生物分子进行局部生物传感。具体来说，该方法将采用内源性生物分子的相干反斯托克斯拉曼散射（CARS）[1]显微镜和金属纳米粒子[2]的四波混合（FWM）成像的独特组合，目的是利用等离子体纳米粒子附近car的局部场增强效应，在纳米尺度上进行生物传感[3,4]。Borri的实验室中有一台独特的自制CARS/FWM显微镜[4,5]，因此本项目的主要重点将是推动其在活细胞中直接传感的适用性王晓明，王晓明，王晓明，等。非线性振动显微技术在油脂生物学研究中的应用[j] .油脂科学进展，2013,33 (4):615-632 (2013).[j]F. Masia, et ., Opt. Lett. 34, 1816（2009）。同上，物理学报，35 (4)（2012）徐海等，“电磁对表面增强拉曼散射中单分子灵敏度的贡献”物理学。中国生物医学工程学报，2003,18 (2).[10卢卡斯·佩恩等人。“单金属纳米粒子的光学微观光谱：定量消光和瞬态谐振四波混频”，法拉第学报，184,305 (2015). b[5]I Pope等人。“高光谱微分- cars、TPF和SHG同步显微技术”，光学快报21(6),7096-7106（2013）。

The development of novel imaging tools and technologies, including super-resolution optical microscopy, has revolutionised our understanding of biological processes in living cells. Combining high resolution imaging capability with chemical sensing in living cells has the potential to unravel processes virtually impossible to follow with available techniques, such as for example defining the local environment at the specific entry site of endocytic routes of key importance for drug delivery.The main aim of this project is to demonstrate and quantify the applicability of novel optical microscopy techniques beyond state-of-the-art for local biosensing of single biomolecules directly within living cells. Specifically, the method will feature a unique combination of coherent anti-Stokes Raman scattering (CARS) [1] microscopy of endogenous biomolecules and Four-Wave Mixing (FWM) imaging of metallic nanoparticles [2], with the aim to exploit the local field enhancement effect of CARS in the vicinity of a plasmonic nanoparticle for bio-sensing at the nanoscale [3,4]. A unique home-built CARS/FWM microscope is available in Borri's laboratory [4,5], therefore the main emphasis of this project will be to push its applicability for sensing directly in living cells.[1] A Zumbusch, W Langbein, P Borri "Nonlinear vibrational microscopy applied to lipid biology" Progress in lipid research 52 (4), 615-632 (2013).[2] F. Masia, et al., Opt. Lett. 34, 1816 (2009).; ibid Phys Rev B 85, 235403 (2012).[3] H Xu et al "Electromagnetic contribution to single-molecule sensitivity in surface enhanced Raman scattering" Phys. Rev. E 62, 4318 (2000).[4] Lukas Payne, et al. "Optical micro-spectroscopy of single metallic nanoparticles: quantitative extinction and transient resonant four-wave mixing", Faraday Discussions 184, 305 (2015).[5] I Pope, et al. "Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti: Sa laser" Optics express 21 (6), 7096-7106 (2013).