The overall objective of this project is to develop a multi-modal high-resolution free-label near- real-time technology that enables research of molecular and cellular transformations at tissue level in living organisms. We propose to combine into one unique system three techniques: (i) two-photons time-resolved fluorescence spectroscopy (2p-TRFS) which provides a direct evaluation of cells and tissues composition, (ii) ultrasound backscatter microscopy (UBM) which allows a visual reconstruction of tissue microanatomy, and (iii) photoacoustic microscopy (PAM) which allows for mapping of optical absorption associated to specific morphology and physiology of tissue. Such configuration will provide complementary information about the investigated biological system and thus facilitate a more complete characterization/study of the bio-systems in question (e.g. tumor, atherosclerotic plaque, bioengineered tissue, wound healing). Specifically, this project will focus on design, engineering, and validation of instrumentation and methodologies which allow for integration of the three modalities noted above;and on demonstrating the feasibility of the integrated approach for concurrent detection of several biological-relevant parameters from living biological systems. Three aims will be addressed. Aim 1: To design and engineer an integrated hybrid diagnostic system that allows for high-resolution concurrent detection/analysis of tissue (a) autofluorescence (via 2p-TRFS), (b) micro-anatomy (via UBM), and (c) optical absorbance (via PAM). Aim 2: To validate the performance (spatial resolution, penetration depth, imaging contrast, sensitivity, signal-to-noise ratio, dynamic range, acquisition speed, data co-registration) of the hybrid 2p-TRFS - UBM - PAM system with a tissue phantom model which incorporates optical and ultrasound contrast. Aim 3: To validate the performance of the hybrid 2p-TRFS - UBM - PAM system using an in vivo hamster oral carcinoma as a model system;and to demonstrate the system feasibility for detecting and resolving concurrently multiple biological features (e.g. tissue composition, microstructures, and neo-angiogenesis) within the tumor volume. PUBLIC HEALTH RELEVANCE: The technology proposed here will make possible simultaneous detection and monitoring of compositional, morphological and functional features of biological tissues of interest. Potential applications include research in vivo of biological models of human diseases such as cancer and atherosclerotic cardiovascular disease, non-destructive evaluation of bioengineered tissues/constructs, longitudinal studies during development and wound healing mechanisms.