Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have
been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which
maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies.
Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo
fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging
living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous
fluorophores and fluorescence labels are very different. A
multi-color ultrafast source is required for the excitation of
multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the
pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple
endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and
yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and
spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF
signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria,
microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.