For each pixel, the intensity of NAD(P)H was then divided by the intensity of FAD. published values. Collagen second harmonic generation images were PALLD generated using an excitation wavelength of 790 nm and an emission bandpass filter of 440/80 nm. 4.8. Image and Analysis Optical redox ratio values for all those conditions were normalized to the control condition for the same position (proximal or distal). NAD(P)H and FAD intensity and lifetime images were analyzed using SPCImage software (Becker &Hickl, Berlin, Germany) as explained previously [29,30]. The fluorescence lifetime decay curve was deconvolved with the instrument response function and fit to a two-component exponential decay model at each pixel, I(t) = 1 ? e(?t/1) + 2 ? e(?t/2) + C, where I(t) represents the fluorescence intensity at time t after the laser excitation pulse, accounts for the fractional contribution from each component, C represents the background light, and is the fluorescence lifetime of each component. Since both NAD(P)H and FAD can exist in two conformational says, bound or unbound to enzymes, a two-component model was used. The short and long lifetime components reflect the bound and Indobufen unbound conformations respectively, for FAD. While the opposite is true for NAD(P)H, the short and long lifetime components correspond with the unbound and bound conformations, respectively. The mean lifetime (m) was calculated using m = 11 + 22, for both NAD(P)H and FAD. The optical redox ratio was determined from your NAD(P)H and FAD intensity images. For each pixel, the intensity of NAD(P)H was then divided by the intensity of FAD. Using Cell Profiler, an automated cell segmentation pipeline was created . This system recognized pixels belonging to nuclear regions by using a customized threshold code. Cells were recognized by propagating out from the nuclei within the image. To refine the propagation and to prevent it from continuing into background Indobufen pixels, an Otsu Global threshold was used. The cell cytoplasm was defined as the cell borders minus the nucleus. Values for NAD(P)H m, FAD m, NAD(P)H intensity, FAD intensity, and the optical redox ratio (NAD(P)H/FAD intensity) were averaged for all those pixels within each cell cytoplasm. At least 100 cells per sample were analyzed. Cell viability microscopy images were analyzed using FIJI (https://imagej.net/Fiji/Downloads). 4.9. Statistical Analysis All experiments were repeated at least 3 impartial times. The normal distribution assumption for statistical assessments was confirmed by the KolmogorovCSmirnov test. Statistical significance was set at 0.05. For nonparametric comparisons, a KruskalCWallis test was performed followed by the MannCWhitney U test. Analysis was performed in GraphPah Prism 8 (https://www.graphpad.com/scientific-software/prism/). Acknowledgments University or college of Wisconsin Carbone Malignancy Center (AAB7173). Morgridge Research Institute. NIH grants R01 CA164492, R01 CA185747, R01 CA205101, R01 CA211082, and NSF grant CBET-1642287. Department of Defense grant W81XWH-18-PRCRP-IASF (CA181014). Supplementary Materials The following are available online at https://www.mdpi.com/1422-0067/21/23/9075/s1, Physique S1: Glucose diffusion. (A) Second harmonic generation (SHG) images showed the effect of collagen density and polymerization heat on collagen fiber business. Collagen hydrogels polymerized at 37 Indobufen C showed shorter fibers and smaller pore size compared hydrogels polymerized at 20 C. Increasing collagen concentration increased fiber density. (B) 200 M NBDG (i.e., fluorescent glucose analog) was perfused through the lumen and diffusion was analyzed after 10 min. NBDG rapidly penetrate through 1.5 mg/mL (polymerized at room temperature) and 6 mg/mL collagen hydrogels (polymerized at 37 C). Click here for additional data file.(646K,.