Volumetric imaging of fluorescently labeled BPAE cells

Using the Nanoimager-S microscope (ONI, Oxford Nanoimaging) with a sCMOS sensor (Hamamatsu, ORCA-Flash4.0 V2), FluoCells™ Prepared Slide #1 (Thermo, #F36924) were imaged with a 100X, 1.4 NA, oil-immersion objective (Olympus). DAPI, Alexa-488 and MitoTracker™ Red excitation was delivered by 405 nm, 473 nm and 561 nm lasers, respectively. Light was collected while using two emission filters (1: 525/50; 2: Band 1 575-616.5) and a Channel Splitter dichroic 561 LP. A 3D Z-stack of the sample was acquired for each channel. 22 frames were generated, each separated 50 nm from each other in Z.