A multiphoton microscopy based on coherent anti-Stokes Raman scattering is accomplished with near-infrared ultrashort laser pulses. We demonstrate vibrational imaging of chemical and biological samples with high sensitivity, high spatial resolution, noninvasiveness, and three-dimensional sectioning capability. [S0031-9007(99)

In this work, we show how broad-bandwidth femtosecond pulses can be used to achieve high spectral resolution in nonlinear spectroscopy and microscopy. Our approach is based on chirping the excitation pulses in order to focus their entire bandwidth into a narrow spectral region. We show that spectral features which are 100 times narrower than the excitation light can be resolved with this simple spectral focusing. The gain in spectral selectivity and sensitivity makes its application to nonlinear microscopy very convenient. This is demonstrated with diffraction-limited coherent anti-Stokes Raman scattering microscopy.

The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.