(26e) Microfluidic Device for Life-Long High-Resolution and High-Throughput Imaging of Subtle Phenotypes in C. Elegans

San-Miguel, A., North Carolina State University
Aging produces changes in neuronal structure and function in a variety of organisms. Human beings are not an exception to this process. These age-associated functional changes can emerge in a form of diseases such as Alzheimer’s and Parkinson’s to which the growing senior population is more susceptible. Elucidating the aging process in humans is complex and poses numerous technical challenges. C. elegans, a less sophisticated model organism with a short life-span, is one of the primary systems to elucidate phenotypes associated with aging processes. These aging-induced changes encompass a wide range of phenotypes, from loss of locomotion to changes in neuronal structure and defective production of synaptic vesicles.

Conventional approaches used to study aging in C. elegans are typically labor-intensive, time-consuming, low-throughput, and incorporate drugs. Fluorodeoxyuridine (FUdR) is a chemical reagent for chemotherapeutical purposes which is frequently used in C. elegans aging studies to prevent the nematodes from developing viable progeny. FUdR treatment ensures the population is age-synchronized throughout the assay. However, C. elegans exposure to FUdR causes an extension in the population’s life-span which can hypothetically manipulate the natural aging process. In addition, Tetramisole and Levamisole are commonly used anesthetics to immobilize the nematodes prior to high-resolution microscopy which can cause side effects. Antibiotics may also be implemented to prevent any bacterial or fungal contamination during the aging studies. We have developed a microfluidic platform which enabled us to maintain a population age-synchronized throughout its life-span as well as performing high-resolution microscopy of subtle phenotypes in a drug free environment. A main chamber surrounded by hundreds of tapered channels is designed to accommodate ~800 worms while simultaneously filtering out their progeny. The main chamber is connected to a section with arrays of trapping channels with the intention of directing individual nematodes to a confined compartment. Worms are loaded into these tapered imaging channels, which restrict their motility and prepare them for imaging. The loaded population was immobilized by decreasing the temperature. A heat sink is placed on the top of the chip which extracts the heat from the PDMS and reduces its temperature. Series of high-resolution images of fluorescently tagged synaptic vesicles located at the dorsal side of the nematodes tail are acquired at different time points of the population’s life-span. The pool of images acquired is then processed to detect and quantify synapses and synaptic pattern using unbiased computer assisted techniques. The unbiased quantitative analysis performed on the data acquired suggested that the average Puncta size and intensity increases as nematodes age. In addition, the synaptic density in the dorsal side decreases. This microfluidic platform coupled with an automated quantitative analysis of data, enabled us to track the changes in the synaptic domain as nematodes age in a drug free environment