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

PI要求提供资金，以改善海洋微生物群落的自动化现场监测。微生物占海洋初级生产力和生物量的大部分，微生物群落的结构在很大程度上决定了更高营养水平的结构。对调节群落结构的因素的基本理解需要对浮游生物进行详细和持续的观察，这促使pi开发了原位流式细胞术和流式成像技术，以测量单个细胞的光散射和荧光特性，并利用自动图像分析来确定较大细胞的分类关系。虽然仪器设计已被证明是有效的，但两项改进将使新的研究应用成为可能。要解决的第一个挑战是，目前的仪器通常对大细胞取样不足。声波聚焦将使采样率提高10倍，从而更好地观察这些细胞，促进对不同浮游植物物种原位生长速度的研究，并有可能对微生物捕食者的活动进行物种水平的研究。要解决的第二个挑战是需要对细胞进行物理捕获以进行深入分析。乳剂微流体技术将促进基于图像的细胞分选现场样品，允许在延迟的实验室程序中进行所需的图像分析。将存储的细胞返回实验室后，将对每个存储细胞对应的图像进行分析，并通过新型微流体装置对存储细胞进行分类。更广泛的影响：如果成功，该仪器将能够对微型、纳米和微型浮游生物进行统计定量采样，水下部署时间长达半年。仪器中保存的样本将使人们对浮游生态系统的动态有前所未有的了解。如果商业化（目前正在进行）这种仪器可以成为系泊海洋观测系统生物取样的关键要素。该方案有一个重要的培训方面，包括两名研究生，以及与Zephyr基金会合作，在他们针对高中生的教育项目中开发浮游植物“单元”。全球变化的迹象是明确的，但由于我们对生态系统反应的知识有限，这些变化的后果并没有得到很好的限制。对IFCB提出的改进将提供一种手段，直接观察自然环境中初级生产者在条件变化时的反应。毫无疑问，这些知识将提高我们的建模能力，并为社会行动提供指导。

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.