Multiphoton confocal microscopy using a femtosecond Cr:Forsterite laser

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Abstract
With its output wavelength covering the in‐ frared penetrating window of most biological tissues at 1200–1250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti: sapphire laser. In this paper, we study the capability of using a femtosecond Cr: forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photolumines‐cence spectrum measurement on several commonly used bioprobes using the 1230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two‐photon or three‐photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two‐photon photoluminescence from chlorophyll, we demonstrated the two‐photon scanning paradermal and cross‐sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.