title: Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae.
abstract: Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan.
authors.forename: Bibhudatta
authors.initials: B
authors.lastname: Mishra
authors.affiliation: Department of Biomedical Engineering, University of Michigan.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ghannad-Rezaie
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan.
authors.forename: Jiaxing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Life Sciences Institute, University of Michigan; Department of Cell and Developmental Biology, University of Michigan.
authors.forename: Bing
authors.initials: B
authors.lastname: Ye
authors.affiliation: Department of Biomedical Engineering, University of Michigan; Department of Mechanical Engineering, University of Michigan.
authors.forename: Nikos
authors.initials: N
authors.lastname: Chronis
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan; collinca@umich.edu.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Collins
mesh_terms: D000818:Animals; D004129:Dimethylpolysiloxanes; D004331:Drosophila melanogaster; D007814:Larva; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology*
chemical_list: D004129:Dimethylpolysiloxanes; C013830:baysilon