High-resolution laser scanning microscopy with saturated excitation of fluorescence

摘要

We propose the use of saturated excitation to improve the spatial resolution of a laser scanning confocal fluorescence microscope. The saturated excitation induces nonlinear response in fluorescence emission that gives higher-order spatial frequency components in the point spread ruction of the microscope. To extract the nonlinear responses in fluorescence emission, we modulate the excitation intensity temporally at a single frequency (ω) and demodulate the fluorescence signal at the harmonic frequencies (2ω, 3ω, …). We found that the fluorescence signal demodulated at nth harmonic frequency is proportional to nth power of the excitation intensity, where n-fold improvement of the spatial resolution can be achieved. We experimentally confirmed that the demodulated signal at the second harmonic frequency was proportional to the square of the excitation intensity. The improvement of spatial resolution was also confirmed by observing a fluorescent sample.