The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca+2 sensitivity (Kd ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.