PROJECT SUMMARY The rapid adoption of genetically modified animals and fluorescently labeled biocompatible nanoparticles for drug delivery in biomedical science have increased demand for imaging technologies capable of fast volumetric imaging to enable longitudinal investigations of cell dynamics in their natural environment. The complexity of information required to understand tissue response to injury and treatment has also generated increased interest in multimodal imaging systems that combine complementary performance strengths. In this regard, we have developed an integrated dual-modality imaging system that combines optical coherence microscopy (OCM) and dual-channel confocal fluorescence microscopy (CFM) to enable the simultaneous measurements of fluorescence and reflectance from deep tissue layers. The combined system provides a unique opportunity to inform cells’ behavior in their natural environment by offering complementary information not available with either system alone. CFM interogates the distribution of fluorescently labeled molecules in cells. OCM is a label-free, episcopic method providing information about the cell boundaries, extracellular matrix, and thickness changes in layers of normal and pathogenic tissues. Despite its potential, the integration of these technologies is incomplete without the capability of concurrent volumetric imaging allowing the tracking of cell dynamics progressively in the same specimen. As for scanning- based systems, fast volumetric imaging in OCM and CFM requires dynamic focusing at the level of individual scanning points, which is challenging due to the limited temporal resolution of dynamic focusing devices. This project aims to establish the capability of the tunable acoustic gradient lens, the fastest dynamic focusing technology to date, in OCM and CFM to enable a fast volumetric and concurrent imaging of depth-resolved reflectance and fluorescence from deep tissue layers in vivo. This integration will have a tremendous impact on biomedical research as OCM and CFM technologies remain the most affordable, flexible, and the latter is readily available in many research facilities. We propose the following two specific aims: Aim 1: Enable fast volumetric imaging in confocal fluorescence microscopy. Aim 2: Enable extended depth-of-field in optical coherence microscopy. Significance The ability to concurrently acquire volumetric information such as reflectance and fluorescence rapidly from deep tissue layers will provide a powerful tool for longitudinal investigations into developmental and pathophysiological mechanisms in various research fields and facilitate in vivo cell tracking in the same specimen while increasing the rigor and reproducibility of studies by decreasing the inter-subject variability that can affect the outcomes of cellular processes.