KAUST Research Workshop on Innovative Technologies to Study Brain Energy Metabolism
Cervo Brain Research Center, Université Laval, Canada
Pierre MarquetCERVO, Brain Research Center in Mental Health, Québec, Québec, Canada; Department of Psychiatry and Neuroscience, Université Laval, Québec, Québec, Canada. International Joint Research Unit in Neurodevelopment and Child Psychiatry, Psychiatry Department, Lausanne University Hospital, University of Lausanne, Switzerland, Université Laval, Quebec, Quebec, CanadaQuantitative Phase Imaging (QPM) has recently emerged as a powerful new imaging modality to non-invasively visualize transparent specimens, including living cell culture. Among these different QPI techniques, Quantitative Phase Digital Holography Microscopy (QP-DHM) is particularly well suited to explore, with a nanometric axial sensitivity, cell structure and dynamics. Concretely, accurate interferometric measurements of the phase retardation of a light wave when transmitted through living cells are performed. This phase retardation, namely the Quantitative Phase Signal (QPS) depends on both the thickness of the observed cells as well as the difference between its refractive index and that of the surrounding medium. The refractive index difference is generated by the presence of organic molecules, including proteins, DNA, organelles, nuclei present in cells. QPS provides thus information about both cell morphology and cell contents. Furthermore, thanks to the development of different experimental procedures, allowing to separately measure cell thickness and intracellular refractive index from the QPS, relevant biophysical cell parameters were successfully calculated, including cell shape, absolute volume, intracellular protein concentration, nanoscale membrane fluctuations, membrane mechanical properties and water permeability, transmembrane water movements.Furthermore, membranes of animal cells are highly permeable to water; movements of water across membranes are therefore dictated in large part by osmotic pressure gradients. Any imbalance in intracellular and extracellular osmolarity is thus paralleled by water movements across cell membranes affecting cell volume along with the concentration of intracellular compounds. However, even at constant extracellular osmolarity, both the volume constancy and the concentration of intracellular compounds of any mammalian cell are permanently challenged by the normal cell activity, including transport of osmotically active substances, cell metabolism, enzyme activity, which all induce osmotic water movements. A large variety of cellular processes are all extremely dependent on a homeostatic intracellular environment. Consequently, the ability to control cell volume while maintaining ion and water homeostasis is pivotal for cell function. Accordingly, volume regulatory mechanisms involve multiple ion transport processes, including channels, cotransporters, and exchangers, as well as transmembrane water movements.Thus, simultaneous dynamic imaging of intracellular ion and transmembrane water movements is likely to reveal new insights about the volume regulatory processes. A multimodal microscopy approach, combining epifluorescene and QP-DHM, permitted to study how glutamate challenges the volume and ion balance of neurons and astrocytes. We also explored whether the dynamics of cell volume regulation can represent an early indicator of cell capacity to maintain homeostasis. A paradigm involving L-Lactate stressed that neuronal volume regulation is an early sign of the L-Lactate protection against excitotoxicity mediated by glutamate.