Fajrial, A. K. | bioRxiv Science

bioRxiv2022

Surface Acoustic Wave Integrated Microfluidics for Repetitive and Reversible Temporary Immobilization of C. elegans

Caenorhabditis elegans is an important genetic model for neuroscience studies due to its unique combination of genetics, transparency, complete synaptic connectome, and well-characterized behaviors. These factors, in turn, enable analyses of how genes control connectivity, neuronal function, and behavior. To date, however, most studies of neuronal function in C. elegans are incapable of performing microscopy imaging with subcellular resolution and behavior analysis in the same set of animals. This constraint stems from the immobilization requirement for high-resolution imaging that is incompatible with behavioral analysis. In particular, conventional immobilization methods often lead to either irreversible, partial, or slowly reversible immobilization of animals preventing a multiplexed approach. Here, we present a novel microfluidic device that uses surface acoustic waves (SAW) as a non-contact method to temporarily immobilize worms for a short period (40 seconds). This device allows non-invasive analysis of swimming behavior and high-resolution synaptic imaging in the same animal. In addition, because of the low impact of this SAW approach, the device enables fast, repeated imaging of single neurons and behavior in the same animals for three to four days. We anticipate that this device will enable longitudinal analysis of animal motility and subcellular morphological changes during development and ageing in C. elegans.

neuroscience↗

bioRxiv2020

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated in part by mechanotransduction mechanisms. However, little is known about how structure and properties of the extracellular matrix in living tissues impacts nuclear mechanics, and current experimental challenges limit the ability to detect and directly measure nuclear mechanics while cells are within the native tissue environment. Here, we hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. We aimed to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, we expanded an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue. We validated this technique by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, we found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, we demonstrated the capability for cell and nucleus elastography using the AFM needle-tip technique. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

biophysics↗