Visible-Wavelength Multiphoton Activation Confocal Microscopy

Nonlinear optical effects modify the point spread function (PSF) in laser scanning microscopy and have been utilized to enhance the spatial resolution in three dimensions. In this paper, we propose the use of visible-wavelength two-photon excitation to activate negatively switching reversibly photoswitchable fluorescent proteins (RSFPs) to introduce nonlinear relationships between the distribution of excitation light and the fluorescence emission. Single-photon excitation following the two-photon induced photoactivation provides a PSF corresponding to the cube of the excitation intensity distribution and achieves imaging properties equivalent to that using third-order nonlinear optical effects. We experimentally confirmed several species of negatively switching RSFPs can be activated by two-photon excitation in the wavelength range of 530-560 nm. We applied the cubic PSF imaging to the observation of HeLa cells stained by Skylan-NS to confirm the improvement of the spatial resolution and the image contrast in confocal scanning microscopy.