Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.
Transition metal complexes such as ruthenium complexes, having metal-to-ligand charge transfer (MLCT) states, are extensively used in solar energy conversion and electron transfer in biological systems and at interfaces. The dynamics of metal-to-ligand charge transfer and subsequent intermolecular, intramolecular, and interfacial electron transfer processes can be highly complex and inhomogeneous, especially when molecules are involved in interactions and
perturbations from heterogeneous local environments and gated by conformation fluctuations. We have employed single-molecule
spectroscopy, a powerful approach for studying inhomogeneous systems, to study the electron transfer dynamics of ruthenium complexes. We have applied a range of statistical analysis methods to reveal nonclassical photon emission behaviors of single ruthenium complexes, e.g., photon antibunching and photophysical ground-state recovering dynamics on a microsecond time-scale. The use of photon antibunching to measure phosphorescence lifetimes and single-molecule electron transfer dynamics at room temperature is demonstrated, which is a novel way of probing ground state regeneration in back electron transfer processes.
Overexpression of HER2 alters the cellular behavior of EGF receptor (EGFR) and itself, with great implications on cell fate. To understand the molecular interactions underlying these alterations, we quantified the association between the two receptors by looking at efficiency changes in fluorescence resonance energy transfer (FRET) between a small number of molecules at the membrane of living cells. Human mammary epithelial (HME) cells expressing varying degrees of HER2 were studied, to identify and compare the degree of receptors interactions as a function of HER2 overexpression. A high resolution wide-field laser microscope combined with a high sensitivity cooled CCD camera was used to capture simultaneously donor and acceptor emissions. Alternating between green and red lasers every 80 msec, donor, FRET, and acceptor images were acquired and were used to calculate FRET efficiency. Automated image analysis was developed to create FRET efficiency maps from overlapping donor, acceptor and FRET images, and derive FRET efficiency histograms to quantify receptor-receptor interactions pixel by pixel. This approach enabled us to detect subtle changes in the average distance between EGFR molecules, and between EGFR and HER2. We found pre-existing EGFR homoassociations, and EGFR-HER2 heteroassociations in cells overexpressing HER2, and identified the changes in these interactions with ligand stimulation. These observations demonstrate the power of FRET measurements between small numbers of molecules in identifying subtle changes in molecular interactions in living cell.