Traps, Tensions and Transmissions: The Unionisation of Resistance Movements against External Destabilising Forces; Design, Development and Deployment of Holographic System for Active Surveillance and Targeted Manipulation
Microscopic imaging of rapid biophysical processes often relies on high-contrast, high-resolution, and high-speed acquisition. However, confocal microscopes capable of such imaging lack the capacity to manipulate the sample or its surrounding environment in real-time. As a result, the possibility to non-invasively initiate, alter or halt a biochemical process during imaging is restricted. To overcome this limitation, we have established a sui generis versatile holographic system equipped with diverse photo-perturbation techniques including photo-manipulation, photo-activation, photo-ablation, optical-trapping and optogenetics, combined with the ability for active surveillance and monitoring of biological targets through confocal imaging and potential capacity for super-resolution imaging. This hybrid system is comprised of a spinning disk confocal unit, a spatial light modulator and a digital micromirror device, and is able to elucidate the dynamics of molecules, measure local forces, and re-localise or switch molecular behaviour. Here, we present the first application of this hybrid system to the study of cell shape regulation and the role of effective membrane tension in unionising actomyosin movement to resist external deformational and destabilising forces. We demonstrate that simultaneously trapping and unfolding the cell membrane, quantitatively imaging actin network dynamics and measuring cellular forces, allowing for a multi-level understanding of how the interplay of membrane tension and actin dynamics governs cell shape. Specifically, our results show a link between the movements of myosin that lead to a surge in actin concentration and cause an increase in the effective membrane tension. These results provide direct evidence for the role of physical interactions by plasma membrane in interpreting the environment that surrounds the cell to regulate and control cell dynamics and by extension cell behaviours. Furthermore, the results strongly reiterate the role of the actomyosin cortex in regulating cell shape during the transition phase and maintaining cell shape by resisting deformation during the stationary phase. More generally, our findings have the potential to expand our understanding of how mechanical properties of the cell surface are locally and globally responsible for driving cell shape changes in physiological and disease conditions. Additionally, we demonstrate the possibility of simultaneously holographic-trapping and dynamically exciting multiple independent cellular targets each located in specific areas and having different excitation and emission wavelengths, by custom developed and optimised method which utilises a single modulator to generate multi-chromatic holograms. Our programming code uses an iterative algorithm with only ten iterations to achieve a PSNR of above 35 dB, an efficiency of 96\% and a crosstalk of less than 1\% in the results. The method retains high adaptability and customisability to prioritise the quality of the reconstructed image, or the speed of the hologram generation, or improve both quality and speed at the cost of other variables based on the application specificity.
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