Microscopic Oxygen Imaging of Spheroids

Before OC measurement, FGM was replaced with 1.5 ml of above-mentioned imaging medium. Measurements were performed using the STOp-Q method, as described previously (36 (link)), including the VisiSens TD Mic (PreSens Precision Sensing Technology GmbH, Regensburg, Germany) readout unit for microscopic O2 imaging. Ibidi dishes containing the spheroids embedded in type I collagen and NF8 cells were inserted into 6-well plates to fit into the imaging apparatus. Microscopic images over the entire area containing the spheroids and collagen/cell matrix were acquired using the tile scan function of the LASX software and a Leica DMi8 widefield fluorescence microscope (Leica, Wetzlar, Germany). Images were acquired with a 5x objective in the TXR channel (Excitation: 560/40, Emission: 639/75) and phase contrast. After a one-hour equilibration at 37°C in the incubator containing the STOp-Q device, measurements were performed in 10 second intervals for one hour using an exposure time of 150 ms (Figure 1).