Label-free multiphoton microscopy with a visible femtosecond laser

We present the design and construction of a multi-photon microscope using a home-built green femtosecond laser excitation source. Compared to typical near-infrared excitation sources used for multi-photon microscopy (MPM), visible femtosecond lasers possess substantially higher photon energy, capable of exciting numerous endogenous fluorophores present in biological samples and various materials. We show that the spatial resolution is improved by two to threefold using the shorter-wavelength visible laser, reaching 172 nm lateral resolution. We demonstrate label-free imaging performance of the microscope, highlighting the capability to detect UV two-photon fluorescence and deep-UV (DUV) second harmonic generation (SHG). Furthermore, the system is applied in high resolution label-free imaging of human cancerous pancreatic tissue and nano-micro plastic in commercial bottled water.