Colloidal semiconductor quantum dots are ideal fluorophores for multi-photon excitation imaging due to very large multi-photon absorption cross-sections, and are excellent energy donors in non-radiative energy transfer systems. We examine fluorescence resonance energy transfer using two-photon excited quantum dot donors and proximal dye acceptors in a variety of biologically-relevant applications including bioconjugate self-assembly, molecular sensing, and cellular imaging. We found that two-photon excitation of these bioconjugates preferentially excited the quantum dot donors such that the observed dye fluorescence was exclusively due to non-radiative energy transfer. The energy transfer mechanism itself was found to be unaffected by the excitation mode used. Multi-photon excitation is also advantageous for cellular labeling and sensing applications due to its excellent optical sectioning ability, deep tissue penetration, and limited out-of-focus excitation and photodamage. Quantum dot bioconjugates containing surface-bound peptides to promote cellular uptake were efficiently endocytosed by live cells. Two-photon confocal imaging showed that non-radiative energy transfer was efficient for conjugates comprised of favorable donor-acceptor pairs. These results can be used to develop useful in vivo sensing assemblies.
