Characterization of microfluidic clamps for immobilizing and imaging of Drosophila melanogaster larva's central nervous system

Abstract

is a well-established model organism to understand biological processes and study human diseases at the molecular-genetic level. The central nervous system (CNS) of larvae is widely used as a model to study neuron development and network formation. This has been achieved by using various genetic manipulation tools such as microinjection to knock down certain genes or over-express proteins for visualizing the cellular activities. However, visualization of an intact-live neuronal response in larva's Central Nervous System (CNS) is challenging due to robust digging/burrowing behaviour that impedes neuroimaging. To address this problem, dissection is used to isolate and immobilize the CNS from the rest of the body. In order to obtain a true physiological response from the CNS, it is important to avoid dissection, while the larva should be kept immobilized. In this paper, a series of microfluidic clamps were investigated for intact immobilization of the larva. As a result, an optimized structure for rapid mechanical immobilization of larvae for CNS imaging was determined. The clamping and immobilization processes were characterized by imaging and movement measurement of the CNS through the expression of genetically encoded Calcium sensor GCaMP5 in all sensory and cholinergic interneurons. The optimal structure that included two 3D constrictions inside a narrowed channel considerably reduced the internal CNS capsule movements. It restricts the CNS movement to 10% of the motion from a glued larva and allows motion of only 10 ± 30m over 350 s immobilization which was sufficient for CNS imaging. These larva-on-a-chip platforms can be useful for studying CNS responses to sensory cues such as sound, light, chemosensory, tactile, and electric/magnetic fields.

Tools to reverse-engineer multicellular systems: case studies using the fruit fly

“…There is an additional design that pneumatically immobilizes larvae and allows for automated larva loading, immobilization and unloading. Both methods achieved significant immobilization and resulted in high-resolution imaging of neural responses [53, 54]. Mechanical restraint achieves easy immobilization but leads to reduced viability and innate response to mechanical perturbation [53, 54].…”

Single-cell Analysis with Microfluidic Devices

“…In another work, they investigated a series of microfluidic clamps for intact immobilization and imaging of Drosophila larva's central nervous system. 86…”

Microfluidics for mechanobiology of model organisms

“…Some of these devices immobilize fruit fly larvae for imaging purposes using mechanical constraints (Ghaemi, Rezai, Iyengar, & Selvaganapathy, 2015; Ghaemi, Rezai, Nejad, & Selvaganapathy, 2017), temperature (Chaudhury et al, 2017), or carbon dioxide (Ghannad-Rezaie, Wang, Mishra, Collins, & Chronis, 2012). Differences between worm and fruit fly larvae need to be considered for facile adaption of devices from one model organism to another.…”

“…Drawing inspiration from devices originally designed to study C. elegans , such as Chokshi et al (2009) and Wen et al (2012), microfluidic platforms to study mechanobiology in Drosophila larvae are starting to emerge. Some of these devices immobilize fruit fly larvae for imaging purposes using mechanical constraints (Ghaemi, Rezai, Iyengar, & Selvaganapathy, 2015; Ghaemi, Rezai, Nejad, & Selvaganapathy, 2017), temperature (Chaudhury et al, 2017), or carbon dioxide (Ghannad-Rezaie, Wang, Mishra, Collins, & Chronis, 2012). Differences between worm and fruit fly larvae need to be considered for facile adaption of devices from one model organism to another.…”