Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets.

Temporal changes in macrophage metabolism are likely crucial to their role in inflammatory diseases. Label-free two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy are well suited to track dynamic changes in macrophage metabolism. We performed TPEF imaging of human macrophages following either pro- or an anti-inflammatory stimulation. Two endogenous fluorophores, NAD(P)H and FAD, coenzymes involved in key metabolic pathways, provided contrast. We used the corresponding intensity images to determine the optical redox ratio of FAD to FAD + NAD(P)H. We also analyzed the intensity fluctuation patterns within NAD(P)H TPEF images to determine mitochondrial clustering patterns. Finally, we acquired NAD(P)H TPEF lifetime images to assess the relative levels of bound NAD(P)H. Our studies indicate that the redox ratio increases, whereas mitochondrial clustering decreases in response to both pro- and anti-inflammatory stimuli; however, these changes are enhanced in pro-inflammatory macrophages. Interestingly, we did not detect any significant changes in the corresponding NAD(P)H bound fraction. A combination of optical metabolic metrics could be used to classify pro- and anti-inflammatory macrophages with high accuracy. Contributions from alterations in different metabolic pathways may explain our findings, which highlight the potential of label-free two-photon imaging to assess nondestructively macrophage functional state.