Saturated-excitation (SAX) microscopy can provide theoretically unlimited improvement of spatial resolution in laser scanning microscopy. However, in practice, the signal-to-noise ratio (SNR) limits its capability. In this research, we introduced image scanning microscopy (ISM) into SAX microscopy to improve the SNR.
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Saturated-excitation (SAX) improves the spatial resolution of laser scanning microscopy in three dimensions by inducing nonlinear fluorescence signals that localize within a focus spot. However, the spatial resolution of SAX microscopy is practically limited by the signal-to-noise ratio (SNR). In this research, we introduce image scanning microscopy (ISM) to improve the SNR of SAX microscopy. The improvement of the SNR by ISM enables the detection of weak nonlinear signal components and contributes to the improvement of the spatial resolution of SAX microscopy in practice.