
@article {109935,
	title = {A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans.},
	journal = {J Vis Exp},
	year = {2022},
	month = {2022 Apr 11},
	abstract = {<p>Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.</p>
},
	keywords = {Animals, Biological Phenomena, Caenorhabditis elegans, Diagnostic Imaging, Microfluidics},
	issn = {1940-087X},
	doi = {10.3791/63136},
	author = {Dubey, Jyoti and Mondal, Sudip and Koushika, Sandhya P}
}
@article {94156,
	title = {Tracking mitochondrial density and positioning along a growing neuronal process in individual  neuron using a long-term growth and imaging microfluidic device.},
	journal = {eNeuro},
	year = {2021},
	month = {2021 May 21},
	abstract = {<p>The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and sub-cellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show inter-animal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in sub-cellular events such as organelle positions, we developed a microfluidic device to feed and immobilize for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process Sub-cellular imaging of an individual neuron in multiple animals, over 36 hours in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles at synapses. Long-term imaging of individual touch receptor neurons shows that the addition of new mitochondria takes place along the entire neuronal process length at a rate of \~{}0.6 mitochondria per hour. The threshold for the addition of a new mitochondrion occurs when the average separation between the two pre-existing mitochondria exceeds 24 {\textmu}m. Our assay provides a new opportunity to move beyond simple observations obtained from assays to allow the discovery of genes that regulate positioning of mitochondria in neurons.Axonal transport of mitochondria is required for the normal function and health of a developing animal with continuously growing axonal processes. Existing technologies are unable to monitor the addition of a new mitochondrion in a growing axon , as it requires continuous or intermittent tracking of the same individual neuron over several hours to days. We have developed a microfluidic device that enables long-term high-resolution imaging of individual in an anesthetic-free setting. Using this device, we observe that the addition of a new mitochondrion can occur anywhere along the entire neuronal process, likely mediated by actively transported mitochondria, and at docking sites that occur with high probability when the separation between adjacent mitochondria crosses 24 {\textmu}m threshold.</p>
},
	issn = {2373-2822},
	doi = {10.1523/ENEURO.0360-20.2021},
	author = {Mondal, Sudip and Dubey, Jyoti and Awasthi, Anjali and Sure, Guruprasad Reddy and Vasudevan, Amruta and Koushika, Sandhya P}
}
