Simultaneous label-free two-photon fluorescence and second-harmonic generation microscopy for visualization of mouse pulmonary alveoli

We demonstrate a label-free imaging system capable of generating two-photon fluorescence (TPF) and second-harmonic generation (SHG) signals simultaneously, using a wavelength-tunable mode-locked Ti:Sapphire laser. TPF and SHG images are acquired at two wavelength ranges. One is between 415 nm and 455 nm, and the other is between 495 nm and 635 nm. The capability of the imaging system is demonstrated by performing simultaneous TPF and SHG imaging of freshly excised mouse lung lobes. At 870 nm excitation, the microenvironment of pulmonary alveoli is revealed by TPF from elastin fibers and SHG from collagen fibers. Macrophages residing inside apparent alveolar lumens are also identified by autofluorescence.