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Proceedings Paper

Imaging properties of saturated excitation (SAX) microscopy
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Paper 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.

Paper Details

Date Published: 3 March 2009
PDF: 10 pages
Proc. SPIE 7184, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI, 718415 (3 March 2009); doi: 10.1117/12.809108
Show Author Affiliations
Shogo Kawano, Osaka Univ. (Japan)
Masahito Yamanaka, Osaka Univ. (Japan)
Katsumasa Fujita, Osaka Univ. (Japan)
Nicholas I. Smith, Osaka Univ. (Japan)
Satoshi Kawata, Osaka Univ. (Japan)
RIKEN (Japan)

Published in SPIE Proceedings Vol. 7184:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson, Editor(s)

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