NADH fluorescence lifetime images of hMSCs were obtained as previously reported.22,28,29 In the beginning, hMSCs were seeded onto a 24-mm diameter glass coverslip (Paul Marienfeld GmbH & Co., Lauda- Konigshofen, Germany) and osteogenesis was induced as previously described. Prior to fluorescence lifetime imaging, the coverslip was washed twice using PBS and then transferred into an imaging chamber and 1-ml aliquot of 5 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (5 mM KCl, 140 mM NaCl, 2 mM , 1 mM , 10 mM glucose, pH 7.4) was added to maintain physiological state and avoid light absorption of the culture medium. hMSCs were imaged with a two-photon laser scanning microscope and with a 1.45 PlanApochromat oil objective lens (Olympus Corp., Tokyo, Japan). NADH fluorescence was excited at 740 nm by a Verdi pumped modelocked femtosecond Ti:sapphire laser (Coherent, Inc., Santa Clara, California) at 76 MHz and the emitted fluorescent light was detected at , with the NADH fluorescence peaks detected by a bandpass filter (Semrock Inc., Rochester, New York). Fluorescence photons were detected by a photon-counting photomultiplier H7422P-40 (Hamamatsu Photonics K.K., Hamamatsu, Japan). All the images were taken at resolution with an acquisition time of 900 s. Cells were incubated at 37°C and 5% during the image acquisition.