Nanometer-scale colocalization microscopy of Streptococcus pneumoniae filaments

摘要

We present numerical simulations and experiments employing two-photon excited fluorescence in a sum-frequency mixingscheme which could be used for colocalization experiments in biophysics. By means of numerical calculations using theDebye approximation, the point spread functions (PSF) of each focused laser beam as well as the resulting PSF of the twocolortwo-photon (2C2P) excitation are calculated and discussed. Experiments are performed on Streptococcuspneumoniae and on surface filaments ("pili") of the bacteria. Two different fluorescent labels were used for staining thebacteria themselves as well as the surface filaments as structure of interest. Since one fluorophore is excited by one singlelaser and the other label only in the combination of both lasers, intrinsic colocalization of the signals on the nanometerscale is ensured. The two-color two-photon excitation is performed by an ultrashort fiber laser system with synchronized emission wavelengths at λ_1= 780nm and λ_2 = 1030 nm and pulse durations around 100 fs. The multiphoton microscopyapproach provides high resolution and allows for three-dimensional imaging of bacteria in a volume of (5 × 5 × 5) μm~3.