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In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, non-invasive, and multiscale experimental approach. In order to fulfill this task, a multimodal fluorescence micro-spectroscopy system was designed and built on a single platform to gain complementary molecular and cellular information at high spatial-temporal resolution. These integrated biophotonics techniques include differential interference contrast (DIC), confocal and two-photon (2P) microscopy, 2P-fluorescence lifetime imaging microscopy (FLIM), steady state fluorescence polarization anisotropy imaging, and fluorescence correlation spectroscopy (FCS). As a proof of principle, a breast cancer cell line (Hs578T) labeled with the mitochondrial marker rhodamine 123 (Rh123) was used as a model system. High resolution DIC and confocal images indicated distinct cell morphology (amorphous, polygonal shape) with a perinuclear mitochondrial distribution. In addition, the nucleus-to-cytoplasm ratio in the cancer cells appeared larger than the normal counterpart. Time-dependent and 3D confocal images revealed dynamic mitochondrial movements in the Hs578T cells. Two-photon FLIM demonstrated heterogeneous conformations of and/or environment surrounding Rh123 with an average lifetime of~ 3.1 ns that is significantly different from the free fluorophore in phosphate buffered saline at pH 7.4 (~ 3.7 ns). Steady state anisotropy imaging of Rh123 indicated a restrictive environment in mitochondria …