Abstract
The tissue-specific etiology of aging and stress has been elusive due to limitations in data processing of current techniques. Despite that many techniques are high-throughput, they usually use singular features of the data (e.g. whole fluorescence). One technology at the nexus of fluorescence-based screens is large particle flow cytometry (“biosorter”), capable of recording positional fluorescence and object granularity information from many individual live animals. Current processing of biosorter data, however, do not integrate positional information into their analysis and data visualization. Here, we present a bioanalytical platform for the quantification of positional information (“longitudinal profiling”) of C. elegans, which we posit embodies the benefits of both high-throughput screening and high-resolution microscopy. We show the use of these techniques in (1) characterizing distinct responses of a transcriptional reporter to various stresses in defined anatomical regions, (2) identifying regions of high mitochondrial membrane potential in live animals, (3) monitoring regional mitochondrial activity in aging models and during development, and (4) screening for regulators of muscle mitochondrial dynamics in a high-throughput format. This platform offers a significant improvement in the quality of high-throughput biosorter data analysis and visualization, opening new options for region-specific phenotypic screening of complex physiological phenomena and mitochondrial biology.
Highlights
Methods to reliably identify causative factors of cellular phenomena at the organismal level have been laborious and time consuming due to limitations in current imaging techniques
While LAMPro can be used for a variety of model organisms, here we demonstrate, with applications to C. elegans biology, i.e. stress response, 1Department of Molecular & Cellular Biology, University of California, Berkeley, Berkeley, CA, 94720-3370, USA. 2Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA
Individual organism measurements done by large particle flow cytometry represent the total response per organism1–3
Summary
Methods to reliably identify causative factors of cellular phenomena at the organismal level have been laborious and time consuming due to limitations in current imaging techniques. With the intent to improve the quality and visualization of high-throughput, whole-organism, biosorter data we developed a methodology and freely available software (which we have called the “Longitudinal Alignment Metabolic Profiler” or “LAMPro”) to exploit previously under-used positional information and define key regions of interest in various celluar phenomena (e.g. mitochondrial biology). We posit that this platform embodies advantages of both high-throughput screening and high-resolution microscopy, while substantially removing artefactual data. Bioenergetics, and mitochondrial morphology/dynamics, how LAMPro’s streamlined data analysis and unprecedented data visualization capability can improve the quality and sensitivity of conventional biosorter data
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