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.本实验采用Nanoimager-S型显微镜（Nanoimager-S microscope，ONI, 牛津纳米成像公司）开展成像，该显微镜搭载Hamamatsu ORCA-Flash4.0 V2型号的sCMOS传感器（sCMOS sensor）。使用100×、1.4数值孔径（NA）的油浸物镜（Olympus，奥林巴斯）对FluoCells™预制玻片#1（FluoCells™ Prepared Slide #1，Thermo #F36924，赛默飞）进行成像。其中，DAPI、Alexa-488与MitoTracker™ Red（MitoTracker™ Red）的激发分别由405 nm、473 nm及561 nm激光器提供。成像过程中，通过两片发射滤光片（参数分别为525/50、波段范围575-616.5）与通道分色镜561 LP（Channel Splitter dichroic 561 LP）收集光路信号。针对每个成像通道，均采集样品的3D Z堆栈图像，最终生成22帧图像，各帧在Z轴方向的间距为50 nm。