Time‐ and wavelength‐resolved spectroscopy in two‐photon excited fluorescence microscopy

Two‐photon excited fluorescence spectroscopy has been performed at a microscopic scale in combination with normal, white‐light microscopy. This gave simultaneously a spectral resolution of 20 nm and a temporal resolution of 20 ps, from a volume element less than 5 μm in all three dimensions. The sample was excited with the light from a continuously mode‐locked Ti: sapphire laser that was focused on the sample in a fluorescence microscope. A polychromator and a streak‐camera were used for detection. The method has been used on tissue, plant and paper samples. It has also been demonstrated how substances naturally occurring in the samples can be identified from their spectroscopic properties and the spatial distribution of these substances can be observed. 1994 Blackwell Science Ltd