Development of a Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM) system using a Streak Camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging

Near-Field Optical Microscopy And Spectroscopy Of One-Dimensional Metallic Photonic Crystal Slabs

We use scanning near-field optical microscopy and spectroscopy to investigate the near field of one-dimensional periodic arrays of gold nanowires on top of a slab waveguide. The Bragg resonance of these one-dimensional metallic photonic crystal slabs can coincide with the particle-plasmon resonance of the metal nanowires, giving rise to an avoided crossing. We find a rich behavior when systematically varying the polarization of the incident light, the lattice constant, and the slab thickness. Numerical simulations of the transverse magnetic component of the near field qualitatively reproduce the experiment, in agreement with the expectation from the Bethe-Bouwkamp theory. Furthermore, these calculations give insight into those electromagnetic field components which cannot easily be measured. © 2005 The American Physical Society