Collaborative Research: Enhanced imaging flow cytometry for plankton studies via acoustic focusing and emulsion microfluidics

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The PI's request funding to improve automated in situ monitoring of the marine microbial community. Microbes account for most of the primary productivity and biomass in the ocean, and the structure of the microbial community determines in large part that of higher trophic levels. A fundamental understanding of the factors that regulate community structure requires detailed and sustained observations of the plankton, which motivated the PIs to develop in situ flow cytometric and imaging-in-flow techniques that measure individual-cell light scattering and fluorescence properties, and utilize automated image analysis to determine taxonomic affiliation of the larger cells. While the instrument designs have proven and effective, two enhancements will enable new research applications. The first challenge to be addressed is that large cells are often under-sampled by current instruments. Acoustic focusing will increase sampling rate by 10-fold and thus provide a far better look at these cells, facilitating studies of in situ growth rates of different phytoplankton species, and potentially making possible species-level studies of microbial predator activity. The second challenge to be addressed is the need for physical capture of cells for in depth analysis. Emulsion microfluidics techniques will facilitate image-based cell sorting of field samples by allowing the required image analyses to be carried out in a delayed laboratory procedure. After returning the stored cells to the laboratory, analysis of the image corresponding to each stored cell will be carried out, and the stored cells sorted as desired by a novel microfluidic device.Broader Impacts: This instrument if successful will be capable of statistically quantitative sampling of pico, nano and microplankton with underwater deployment times as long as half a year. The samples archived in the instrument will permit unprecedented insights into the dynamics of the planktonic ecosystem. If made available commercially (which is in the works) instruments of this type could be key elements for biological sampling on moored ocean observing systems. The proposal has a significant training aspect with the inclusion of two graduate students as well as the collaboration with the Zephyr foundation on the development of a phytoplankton 'unit' in their educational program aimed to high school students. The signs of global change are clear, but the ramifications of these changes are not well constrained because of our limited knowledge of ecosystem responses. The proposed improvements to the IFCB will provide a means to directly observe responses of the primary producers in the natural environment as conditions change. There is no doubt that this knowledge will improve our modeling capability and provide guidance for societies actions.