Imaging offers a means to draw upon the growing body of high-throughput molecular data to better understand the underlying cellular and molecular mechanisms of embryonic development; however, it is challenged by tradeoffs between speed, resolution, field of view and the photon budget. We are advancing this tradeoff by constructing two-photon light-sheet microscopes, combining the deep penetration of two-photon microscopy and the speed of light sheet microscopy, permitting 4D cell and molecular imaging with sufficient speed and resolution to generate unambiguous tracing of cells and signals in intact systems. To increase the 5 th Dimensions, we are refining a new generation of multispectral image analysis tools that exceed the performance of our previous work on Linear Unmixing by orders of magnitude in speed, error propagation and accuracy. These new analysis tools permit rapid and unambiguous analyses of multiplex-labeled specimens. Finally, to move to faster volumetric imaging, we have combined light field and light sheet approaches, offering the signal to noise needed to image thousands of neurons with high fidelity. Combined, these tools offer the multi-dimensional imaging required to follow key events in as they take place and allow us to use variance as an experimental tool rather than feeling its effects as a limitation.