Nanometer-scale colocalization microscopy of Streptococcus pneumoniae filaments

Abstract

We present numerical simulations and experiments employing two-photon excited fluorescence in a sum-frequency mixing scheme which could be used for colocalization experiments in biophysics. By means of numerical calculations using the Debye approximation, the point spread functions (PSF) of each focused laser beam as well as the resulting PSF of the two-color two-photon (2C2P) excitation are calculated and discussed. Experiments are performed on Streptococcus pneumoniae and on surface filaments (“pili”) of the bacteria. Two different fluorescent labels were used for staining the bacteria themselves as well as the surface filaments as structure of interest. Since one fluorophore is excited by one single laser and the other label only in the combination of both lasers, intrinsic colocalization of the signals on the nanometer scale is ensured. The two-color two-photon excitation is performed by an ultrashort fiber laser system with synchronized emission wavelengths at λ1 = 780 nm and λ2 = 1030 nm and pulse durations around 100 fs. The multiphoton microscopy approach provides high resolution and allows for three-dimensional imaging of bacteria in a volume of (5 x 5 x 5) μm3.