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Advanced STORM super-resolution techniques for astrocyte-specific molecular imaging

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Zöldi Miklós

János Szentágothai Neurosciences Division

Dr. Dr. Bereczki Dániel

HUN-REN Kísérleti Orvostudományi Kutatóintézet, tanterem

2026-12-08 14:00:00

Neuromorphology and cell biology

Dr. Alpár Alán

Dr. Katona István

Dr. Adorján István

Dr. Hegyi Zoltán

Dr. Kellermayer Miklós

Dr. Gulyás-Onódi Zsófia

Dr. Rácz Bence

Although astrocytes are essential to nearly every aspect of brain function, the relationship between the number and distribution of key molecules within astrocytes and their extensive roles remains poorly understood. In this thesis, I provide a brief overview of the key concepts of super-resolution imaging and present a methodology for visualizing astrocytic proteins at the nanoscale level using STORM imaging. I demonstrate the effectiveness of this method by differentiating between astrocytic and non-astrocytic pools of MAGL, a key enzyme in the endocannabinoid signaling pathway with major clinical relevance. We employed postnatal electroporation to label astrocytes selectively and sparsely in the mouse hippocampus and to reconstruct their architecture with nanoscale precision using STORM. We showed that this level of resolution is a prerequisite to reliably visualize the astrocytic arbor. Moreover, shifting the timing of electroporation enabled the targeting of other cell types such as radial glia. As an added benefit, no transgenic mouse lines or cell type-specific promoters were needed for the sparse labeling that provides low fluorescent background for high-quality super-resolution imaging in tissue. Then, we demonstrated that dual-color STORM imaging can readily differentiate between synaptic and astrocytic proteins. We performed various control measurements to exclude the possibility of severe optical aberrations, and we presented an analysis workflow based on colocalization and nearest neighbor distance measurements to assign molecules to cells of the tripartite synapse. As a proof of concept, we distinguished between neuronal and astrocytic pools of the endocannabinoid-degrading enzyme monoacylglycerol lipase (MAGL). Our results revealed that a sparse, but specific signal of MAGL can be attributed to hippocampal astrocytes, and the enzyme in glia cells does not exhibit any preferential localization toward synapses. This suggests a synapse-independent role, supporting previous findings on astrocytic MAGL’s involvement in regulating inflammation rather than the time course of endocannabinoid signaling. With MAGL inhibitors already in clinical trials, our research could have potential clinical implications. Overall, our methodology offers a versatile approach for studying various nanoscale protein distributions in astrocytes in brain tissue preparations.