Experimental study on multicolor two-photon excited fluorescence microscopy

Two-photon excited fluorescence (TPEF) microscopy is a nonlinear optical microscopy technique. The advantages of TPEF microscopy include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. In traditional TPEF microscopy, a wavelength tunable ultrashort pulsed laser is used as an excitation source. In practical applications, sample usually contains various fluorophores or unknown components. Therefore the excitation wavelength of the ultrafast laser has to be tuned to achieve optimal excitation efficiencies of various fluorophores. In order to acquire the fluorescent signals of different fluorophores simultaneously, we develop a multicolor TPEF microscope system based on a supercontinuum laser source. In experiments, TPEF images of Lily rhizome sample slide stained by two fluorescent dyes with different excitation and emission wavelengths are obtained without tuning the wavelength. Experimental results show that the high-contrast TPEF images of the sample with various fluorophores can be obtained simultaneously by using the multicolor TPEF microscope compared with by using traditional TPEF microscopy. The system is simple in structure, easy in operation, and can provide rich information about the sample, which allows it to be widely used in life and material sciences.