Hypothesis: Surface nanobubbles, which were controversially discussed in the literature, promise a number of outstanding applications, and their presence may hamper nanoscale processes at solid-aqueous interfaces. A most crucial and yet unsolved question is the rapid and conclusive identification of gas-filled (surface) nanobubbles. We hypothesize that surface nanobubbles and oil nanodroplets can be conclusively differentiated in co-localization experiments with atomic force microscopy (AFM) and time-resolved fluorescence microscopy by localizing tracer fluorophores and analyzing their fluorescence lifetimes. Experiments: Combined AFM and fluorescence lifetime imaging microscopy (FLIM) were conducted to localize the various interfaces labelled by the reporter dye rhodamine 6G (Rh6G). The dependence of the fluorescence lifetime of Rh6G on its local environment was determined for air/water, water/glass and polysiloxane/water interfaces under different conditions. Findings: In in situ co-localization experiments, surface nanobubbles labeled with Rh6G were probed by AFM with high spatial resolution and were differentiated from polysiloxane droplets as well as contamination originating from lubricant-coated syringe needles owing to the characteristic short fluorescence lifetime of Rh6G at the gas/water interface observed in FLIM. In particular, this approach lends itself to conclusively identify and rapidly differentiate these gas-filled entities from adsorbed contamination, such as siloxane-based oil nanodroplets. (C) 2019 Elsevier Inc. All rights reserved.