The activation of hypoxia-inducible factor-1 (HIF-1) has been recognized as the key event in adaptation to hypoxia. HIF-1 is composed of the O2-labile alpha- and the constitutive beta-subunit. From here the whole cascade of cellular O2 sensing was followed back ultimately leading to the elucidation of cellular O2 sensors, the prolyl and asparaginyl hydroxylases that regulate abundance and activity of oxygen-regulated alpha-subunit of HIF-1. Assembly of the HIF-1 complex then requires dimerization with constitutive nuclear beta-subunit. To localize HIF-1 subunits within the nucleus of hypoxic cells we applied 2-photon-laser microscopy (2PLM). Mobility studies of fluorescently labelled HIF-1 subunits by fluorescence recovery after photo bleaching (FRAP) revealed that HIF-1alpha migrates more slowly than HIF-1beta within the nucleus indicating that both subunits do not immediately "find" each other but may be prone to modification prior to dimerization. HIF-1 assembly was then studied in living cells in a specialized hypoxic chamber mounted on the microscopic stage which allows the in vivo analysis under a well defined oxygen tension. Finally, in vivo imaging was extended to preparations of intact mouse carotid body ex vivo. Using multifocal Nipkow disk–based imaging with oxygen-, calcium- and potential-sensitive cellular dyes we mapped oxygen-sensing properties of carotid bodies. Additionally in vivo FRET measurement revealed activation of the HIF-1 complex in vivo in carotid body preparations coinciding with changes in cellular potential. Collectively, we have successfully visualized in vitro and ex vivo oxygen sensing to further characterize cellular responses to hypoxia.