Molecular Multicolor Multiphoton in Vivo Bioimaging Based on a Direct Diode Pumped Ti:sapphire Oscillator

Multiphoton microscopy (MPM) including two-photon excited fluorescence (TPEF), second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) is an important tool in biology and medicine to explore the dynamics of cells and to investigate tissue structure in living systems and biopsies. However, MPM still suffers from limited applicability due to the integration of highly sophisticated lasers. In this paper, we introduce our triple-modal multicolor MPM platform employing a single direct diode-pumped femtosecond Kerr-lens-mode-locked Ti:sapphire oscillator and acquire simultaneously intrinsic co-registered multicolor TPEF, TPEF, SHG and CARS signals in the backward direction. The complexity and by that the footprint as well the cost of the laser are considerably reduced by the direct diode-pumping scheme. In combination with an Yb fiber amplifier such a laser is suited for biomedical applications allowing for detailed morphologic and molecular spectroscopic in vivo investigation. The optimized emission wavelengths of our Ti:sapphire laser and Yb fiber amplifier provide simultaneous excitation at 805 nm, 1050 nm and 911 nm for multicontrast information from exogenous and endogenous fluorophores. We perform in vivo imaging of C. elegans and visualize metabolic changes in zebrafish larvae to investigate the tumor microenvironment with spectral focusing CARS and multicolor TPEF of green fluorescent protein.