High-resolution laser scanning microscopy with saturated excitation of fluorescence

Katsumasa Fujita; Shogo Kawano; Minoru Kobayashi; Satoshi Kawata

doi:10.1117/12.700660

14 February 2007 High-resolution laser scanning microscopy with saturated excitation of fluorescence

Katsumasa Fujita, Shogo Kawano, Minoru Kobayashi, Satoshi Kawata

Proceedings Volume 6443, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIV; 64430Y (2007) https://doi.org/10.1117/12.700660
Event: SPIE BiOS, 2007, San Jose, California, United States

Abstract

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 fuction 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.

Citation Download Citation

Katsumasa Fujita, Shogo Kawano, Minoru Kobayashi, and Satoshi Kawata "High-resolution laser scanning microscopy with saturated excitation of fluorescence", Proc. SPIE 6443, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIV, 64430Y (14 February 2007); https://doi.org/10.1117/12.700660

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