Imaging properties of saturated excitation (SAX) microscopy

Shogo Kawano; Masahito Yamanaka; Katsumasa Fujita; Nicholas I. Smith; Satoshi Kawata

doi:10.1117/12.809108

3 March 2009 Imaging properties of saturated excitation (SAX) microscopy

Shogo Kawano, Masahito Yamanaka, Katsumasa Fujita, Nicholas I. Smith, Satoshi Kawata

Proceedings Volume 7184, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI; 718415 (2009) https://doi.org/10.1117/12.809108
Event: SPIE BiOS, 2009, San Jose, California, United States

Abstract

We used nonlinear fluorescence emission under the condition of saturated excitation (SAX) of fluorescent molecules for high-resolution laser scanning microscopy. In the technique, SAX microscopy, we modulate the excitation intensity at a single frequency and demodulate the fluorescence signal at a harmonic frequency to extract a nonlinear fluorescence response that contributes to improvement of the spatial resolution. This nonlinear fluorescent response on saturated condition was analyzed by rate equations formulated from a five-level system Jablonski diagram. By calculating relationship between excitation intensity and fluorescence signal demodulated at harmonic frequencies for rhodamine-6G molecules with 532 nm excitaion, we found that the fluorescent signal exhibits high-order nonlinear dependence on the excitation intensity under conditions of saturated excitation. We also calculated effective point spread functions (ePSFs) of SAX microscopy. The result of the calculation shows that ePSFs given with the harmonic demodulation provides the spatial resolution beyond the resolution limit of conventional confocal microscopy. The optical transfer functions have also been calculated from the ePSFs. The result of the calculation shows that a higher spatial-resolution can be obtained by demodulating fluorescnece signal at a higher harmonic frequency without theoretical limitation.

Citation Download Citation

Shogo Kawano, Masahito Yamanaka, Katsumasa Fujita, Nicholas I. Smith, and Satoshi Kawata "Imaging properties of saturated excitation (SAX) microscopy", Proc. SPIE 7184, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI, 718415 (3 March 2009); https://doi.org/10.1117/12.809108

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