Unveiling the Molecular Dynamics in a Living Cell to the Subcellular Organelle Level Using Second-Harmonic Generation Spectroscopy and Microscopy.

Second-harmonic generation (SHG) microscopy has been proved to be a powerful method for investigating the structures of biomaterials. SHG spectra were also generally used to probe the adsorption and cross-membrane transport of molecules on lipid bilayers in situ and in real time. In this work, we applied SHG and two-photon fluorescence (TPF) spectra to investigate the dynamics of an amphiphilic ion with an SHG and TPF chromophore, D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), on the surface of human chronic myelogenous leukemia (K562) cells and the subcellular structures inside the cells. The adsorption and cross-membrane transport of D289 into the cells and then into the organelles such as mitochondria were revealed. SHG images were also recorded and used to demonstrate their capability of probing molecular dynamics in organelles in K562 cells. This work demonstrated the first SHG investigation of the cross-membrane transport dynamics on the surface of subcellular organelles. It may also shed light on the differentiation of different types of subcellular structures in cells.