Pancreatic ductal adenocarcinoma (PDAC) tumors are highly fibrotic and poorly vascularized, making systemic delivery of standard chemotherapies ineffective. Targeted nanoparticles that deliver effective doses of chemotherapeutic drugs specifically to PDACs could improve chemotherapy efficacy without the toxicities. Multiphoton microscopy, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenous or exogenous fluorescent macromolecular systems and the induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter the native structure of tissue samples, can be circumvented to generate spatially and spectrally accurate 3D morphological and fine structural information. Orthotopic PDAC xenografts were established by injecting epithelioid carcinoma cell line derived from human pancreas (PANC-1 cells) into the pancreas of athymic male nude mice and were grown for 4 weeks. Near-infrared imaging agent indocyanine green (ICG) loaded calcium phosphosilicate nanoparticles (CPSNPs) including targeted CPSNPs bioconjugated with DNA Aptamer (AP1153), non-targeted CPSNPs (with no aptamer bioconjugation) and empty non-ICG containing CPSNPs were injected into the tail vein (n=4 mice/group). At 18 hours post-injection, mice were sedated and whole animal imaging was performed. To assess cellular uptake in ex vivo, whole tumor cross-sections were then imaged to spatially and spectrally localize ICG loaded nanoparticles relative to fibrotic regions (fibrillar collagen) of the tumor using MPEF and SHG imaging. Ex vivo imaging of cross-sections of whole tumors showed that cellular uptake of the targeted CPSNPs was clearly enhanced compared to untargeted CPSNPs and the 3D reconstructions showed accumulation of CPSNPs in tumor cells within the surrounding fibrotic stroma. We here conclude that multiphoton based multi-dimensional and minimally invasive imaging modalities provide spatially resolved 3D images with spectral specificities, that are sensitive enough to detect near-infrared imaging agents and to identity the bio-distributions of targeted versus untargeted drug nanoparticles in pancreatic tumor tissues.