Probing intracellular dynamics in living cells with near-field optics.

Near-field optics (NFO) overcomes the diffraction limit of light microscopes and permits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or fluo-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions.