Human organoids provide a unique opportunity to model organ development in a system that is remark- ably similar to human organogenesis in vivo. Brain organoids, for example, hold great promise for modeling brain development and diseases, developing drug and neurotoxicity screens with high predictability, and studying neuro-immune interaction, among many other applications. However, current platforms to monitor organoid de- velopment, by and large, only allow end-point assays; thus, there is a significant need for in-situ functional and characterization assays during culture. This project proposes to address this unmet need using a novel label-free imaging technology, called quantitative oblique back-illumination microscopy (qOBM), which yields access to endogenous refractive index properties of cells and enables quantitative analysis of cellular and subcellular structures in 3D, non-invasively, in-situ, and in real-time. The overall goal of this R21 application is to develop a novel instrument to monitor organoid development based on qOBM, and assess the extent to which this new tool can quantify important structural and functional properties of organoids in 3D, non-invasively/in-situ, and in real-time. This project focuses on cerebral organoids, but the utility of this tool is broadly applicable for other organoid systems. The aims of this work are as follows: Aim 1 is to develop a qOBM platform that provide robust, quantitative, tomographic, multi-scale information in a scalable and easy-to-use configuration. The system will have diffraction-limited resolution, with clear cellular and subcellular contrast (depending on magnification), with a penetration depth of ~200µm into the sample. The qOBM module will be low-cost and developed as a simple add-on for any bright field microscope with a digital camera. Aim 2 is to develop pipelines for validating qOBM’s ability to follow development in human brain organ- oids. qOBM will be used to perform a daily image analysis of the development of normal cerebral organoids compared to those that have a manipulated mTOR pathway, a key pathway for regulating human cortical devel- opment. For validation, end-point whole-mount immunostaining will be used to assess qOBM’s ability to assess cell size, type, and density, as well as cortical structure. This work is innovative because it brings technological advancement for low-cost, label-free, real-time imaging of organoid development using a novel 3D microscopy technology that represents a major advance in tissue imaging. The project is significant because successful completion of this work will demonstrate a powerful and versatile new approach to performing live in-situ non-destructive monitoring of organoids that will be useful beyond brain organoids. In the future, this technology will be used to study a variety of neural developmental disorders including Autism Spectrum Disorders and fragile X syndrome, as well as neurodegenerative diseases such as Alzheim...