1.2 na objective

HT22 cells were seeded at a density of 20,000 cells/mL and cultured on cell culture dishes (35mm) with a cover glass in high glucose DMEM supplemented with pyruvate (1mM), L-glutamine (4 mM), and 10% FBS. For the fluorescence life time imaging microscopy (FLTIM), cells were incubated in tris (2, 2′-bipyridyl) dichlororuthenium (II) hexahydrate (120 μM), an oxygen sensing dye, for 2 h. Washed Cells were incubated in Dextrose (10 mM)-supplemented sterile Dulbecco's phosphate buffered saline. MB (10 μM) and glucose oxidase (GO), as a positive control, were added during microscopy. Time resolved images were obtained on a confocal MicroTime 200 system (PicoQuant GmbH, Berlin). Excitation was provided from 470 nm pulsed diode laser operating at a 320 kHz repetition rate and it was reflected off of a 490 nm dichroic plate into an Olympus IX71 inverted microscope. The light passed through an Olympus 60X 1.2 NA objective, and the collected fluorescence was filtered by a 488 nm long wave pass, interference filter before passing through a 50μm confocal pinhole. The signal from the detector was routed into time correlated single photon counting module (PicoHarp 300). Fluorescence decay curves were analyzed by the software SymPhoTime, v. 5.3.2.