Idziak, A. | Search bioRxiv

bioRxiv2023

Fluorescent Sensors for Imaging Interstitial Calcium

Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the lack of methods that allow monitoring in tight inter-cell spaces at high spatio-temporal resolution. We engineered high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (KD) ranging from 0.8 mM to 2.9 mM, making them uniquely tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.

physiology↗

bioRxiv2023

Imaging the impact of chemical fixation on the microanatomy of mouse brain tissue

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency. We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic brain slices, allowing us to compare tissue integrity in a before-and-after experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters. While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in brain slices, unlike the situation in transcardially perfused animals where the ECS typically becomes nearly depleted. In addition to the super-resolved characterization of fixation artefacts in identified cellular and tissue compartments, our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.

neuroscience↗

bioRxiv2022

Shadow imaging for panoptical visualisation of living brain tissue

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation, do not have cellular resolution or only give a fragmented view. Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue.

neuroscience↗