Hyperspectral fluorescence imaging by using visible-wavelength two-photon excitation

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.