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.
We propose the use of visible-wavelength two-photon excitation (v2PE) for activation of reversibly photo-switchable fluorescent proteins (RSFPs) and successive confocal detection to achieve super-resolution imaging. In this method, three photons interact with the sample molecules in total, which provides imaging properties equivalent to using third-order nonlinearity in fluorescence response. Because this technique uses visible light, it can achieve higher spatial resolution than confocal microscopy. In this study, we performed experimental investigations to confirm the activation of negative RSFPs by v2PE and demonstrated super-resolution imaging of live cells.
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.
Visible-wavelength two-photon excitation (v2PE) is a powerful technique for simultaneous multicolor fluorescence imaging via simultaneous excitation of fluorescent proteins (FPs) with different emission wavelengths. We implemented v2PE into a slit-scanning confocal microscope in order to realize faster simultaneous multicolor fluorescence imaging with utilizing the capability of spectral detection. We demonstrated simultaneous multicolor imaging of living HeLa cells with expressing three types of FPs with different emission wavelengths localized at different intracellular structures. Linear un-mixing of hyperspectral images successfully separated the distribution of multiple FPs expressed in the sample.
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