Label-free nonlinear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+) is widely used for high-resolution cellular redox imaging. In this work, we combined three label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: (i) TPEF optical redox ratio (ORR = FAD+/NADH + FAD+), (ii) coherent Raman scattering of cellular lipids, and (iii) second harmonic generation of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial (PME) cells and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17β-estradiol (E2) increased glycolysis in both PME and T47D ER+ breast cancer acini. However, PME cells displayed increased lipid content with no effect on ECM, while T47D cells had decreased lipid storage (P < 0.001) and significant reorganization of collagen. By measuring deuterated lipids synthesized from exogenously administered deuterium-labeled glucose, treatment of T47D cells with E2 increased both lipid synthesis and consumption rates. These results confirm that glucose is a significant source for the cellular synthesis of lipid in glycolytic breast cancer cells, and that the combination of cellular redox and lipid fraction imaging endpoints is a powerful approach with new and complementary information content.Significance: These findings provide unique insight into metabolic processes, revealing correlations between cancer metastasis and cellular redox state, lipid metabolism, and extracellular matrix. Cancer Res; 78(10); 2503-12. ©2018 AACR.