Fluorescence microscopy shadow imaging for neuroscience

Front Cell Neurosci. 2024 Feb 15:18:1330100. doi: 10.3389/fncel.2024.1330100. eCollection 2024.


Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membrane-bound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility.

Keywords: STED microscopy; SUSHI; brain extracellular space; fluorescence microscopy; neuroscience; shadow imaging; super-resolution microscopy; two-photon imaging.

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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The authors acknowledge funding for their general work from the Spanish Ministry of Science and Innovation (Grant PID2020-113894RB-I00), Project (PCI2022-135040-2) funded by the Spanish State Research Agency through PCI, as part of the AEI/NSF/NIH Collaborative Research in Computational Neuroscience program, and from the University of the Basque Country (Grant GIU21/048). This research was funded in whole or in part by Aligning Science Across Parkinson’s ASAP-020505 through the Michael J. Fox Foundation for Parkinson’s Research (MJFF). For the purpose of open access, the authors have applied a CC BY public copyright license to all authors Accepted Manuscripts arising from this submission. VP is a Marie Skłodowska Curie Fellow funded through the the European Union’s Horizon Europe research and innovation programme grant agreement (101067304, NeuroExcell).