Probing intracellular dynamics in living cells with near-field optics

Near-field optics (NFO) overcomes the diffraction limit of light microscope s and pe;mits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of a pplications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This i s the first time a NFO probe has been inserted inside a living cell to deli ver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detect ion systems (CCD camera and avalanche photon detection) were developed to m onitor cellular responses to drug administration in two different cell type s. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) wer e penetrated with NFO probes. Intracellular Ca2+ increases post drug stimul ation were detected by NFO probes. The cells were loaded with either fura-2 /AM or flue-3/AM calcium dyes. VSMC were stimulated with angiotensin II, re sulting in a precise area of intracellular Ca2+ increase. Different respons e profiles of Ca2+ increases were observed after ionomycin and bradykinin a dministration in NG108-15 cells. Responsive heterogeneities due to ionomyci n among different cells of the same type were recorded. The results show th at 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. (C) 199 9 Elsevier Science B.V. All rights reserved.