A microscope is described in which singlet molecular oxygen, O₂(a¹Δ_g), is produced in a femtoliter focal volume via a nonlinear two-photon photosensitized process, and the 1270 nm phosphorescence from this population of O₂(a¹Δ_g) is detected in a photon counting experiment. Although two-photon excitation of a sensitizer is less efficient than excitation by a one-photon process, nonlinear excitation has several distinct advantages with respect to the spatial resolution accessible. Pertinent aspects of this two-photon O₂(a¹Δ_g) microscope were characterized using bulk solutions of photosensitizers. These data were compared to those obtained from a single biological cell upon linear one-photon excitation of a sensitizer incorporated in the cell. On the basis of the results obtained, we outline the challenges of using nonlinear optical techniques to create O₂(a¹Δ_g) at the single cell level and to then optically detect the O₂(a¹Δ_g) thus produced in a time-resolved experiment.