title: Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions.
abstract: We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.
authors: Department of Applied Physics , Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871 , Japan.
authors: Masahito
authors: M
authors: Yamanaka
authors: The Institute of Scientific and Industrial Research , Osaka University , 8-1 Mihogaoka, Ibaraki, Osaka 567-0047 , Japan.
authors: Kenta
authors: K
authors: Saito
authors: Immunology Frontier Research Center , Osaka University , 3-1 Yamadaoka, Suita, Osaka 565-0871 , Japan.
authors: Nicholas I
authors: NI
authors: Smith
authors: Department of Applied Physics , Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871 , Japan.
authors: Satoshi
authors: S
authors: Kawata
authors: The Institute of Scientific and Industrial Research , Osaka University , 8-1 Mihogaoka, Ibaraki, Osaka 567-0047 , Japan.
authors: Takeharu
authors: T
authors: Nagai
authors: Department of Applied Physics , Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871 , Japan.
authors: Katsumasa
authors: K
authors: Fujita
keywords: confocal microscopy; fluorescence microscopy; high resolution; live-cell imaging; saturated excitation