The study of embryonic development has been dramatically advanced by the wealth of high-throughput molecular studies that have defined the genes and proteins involved.  This wealth of data now presents the challenge of integrating a working knowledge of how these molecular components, often present at vanishingly small concentrations, generate reliable patterns of cell migration and cell differentiation.  In typical cell biology approaches, cultures of isolated cells have been used reveal mechanism.  What is needed to understand development is to carry out studies on cells in their normal context interacting with other cells and signals in the intact embryo.  Key events of embryonic development take place over dimensions of less than 500um in less than 5 hours, making it tractable for light imaging tools to be used to answer this challenge.   

 Imaging techniques are challenged by major tradeoffs between spatial resolution, temporal resolution, and the limited photon budget.  We are attempting to advance this tradeoff by constructing light sheet microscopes that maintain subcellular resolution in thick and scattering specimens.  Our two-photon light-sheet microscope, combines the deep penetration of two-photon microscopy and the speed of light sheet microscopy to generate images with more than ten-fold improved imaging speed and sensitivity.  As with other light sheet technologies, the collection of an entire 2-D optical section in parallel offers dramatically speeds acquisition rates.  By adopting two-photon SPIM is far less subject to light scattering, permitting subcellular resolution to be maintained far better than conventional light sheet microscopes.  The combination of attributes permits cell and molecular imaging with sufficient speed and resolution to generate unambiguous tracing of cells and signals in intact systems, which presents a major challenge in data management, processing and analysis. 

Multispectral imaging offers the chance of asking multiple questions of the same embodied cells.  Multiplex analyses permit the variance and the “noise” in a system to be exploited by asking about the analytes that co-vary with a selected gene product.  Transforming fluorescent spectra to a point on a 2D-plot with sinusoidal functions (phasors) provides a powerful tool for the cumbersome tasks of visualizing and analyzing hyper-spectral imaging data.  This technique offers excellent performance in the face of biological and instrumental uncertainties. Our results on live zebrafish embryos document the accuracy of hyper-spectral phasors and demonstrates their capability to distinguish multiple spectrally overlapping fluorophores in low signal-to-noise and fast analysis time.

Combined, these tools offer the multi-dimensional imaging required to follow key events in embryos as they take place, and allow us to use variance as an experimental tool rather than a limitation. 

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EMC Abstracts - https://emc-proceedings.com/abstract/eavesdropping-on-the-molecular-signatures-of-embryonic-development/