Revealing nanostructures in brain tissue via protein decrowding by iterative expansion microscopy

Sarkar, Deblina; Kang, Jinyoung; Wassie, Asmamaw T.; Schroeder, Margaret E.; Peng, Zhuyu; Tarr, Tyler B.; Tang, Aohan; Niederst, Emily; Young, Jennie Z.; Su, Hanquan; Park, Demian; Yin, Peng; Tsai, Li‐Huei; Blanpied, Thomas A.; Boyden, Edward S.

doi:10.1038/s41551-022-00912-3

Cited by 55 publications

(63 citation statements)

References 68 publications

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“…Overall, this approach indicates that single-particle averaging is indeed possible, albeit it would benefit greatly from increased resolution in the axial dimension, which can be achieved, for example, by using ExM protocols with expansion factors beyond 10x 49, 50 . This would enable us to determine better the position and tilt of each particle, thereby improving the results.…”

Section: Introductionmentioning

confidence: 95%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

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Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

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Section: Introductionmentioning

confidence: 95%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

Self Cite

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“…This strategy has been demonstrated to not only improve fluorescence brightness, increase epitope accessibility but also effectively reduce the linkage error caused by expansion. 30,75,92 For instance, as mentioned above, Zwettler et al validated and demonstrated that postexpansion immunostaining allowed the reduction of the linkage error from 17.5 nm to ∼5 nm with an expansion factor of 3.2. 75…”

Section: Pre-versus Postexpansion Immunostaining Strategiesmentioning

confidence: 91%

“…In addition, fluorescent labels also experience fluorophore destruction during radical-induced polymerization, lowering the fluorescent signal intensity postexpansion. − , To improve these issues, postexpansion immunostaining strategies have been successfully applied to visualize targets, in which biological targets first experience isotropic expansion, followed by immunostaining with antibodies (Figure ). This strategy has been demonstrated to not only improve fluorescence brightness, increase epitope accessibility but also effectively reduce the linkage error caused by expansion. ,, For instance, as mentioned above, Zwettler et al validated and demonstrated that postexpansion immunostaining allowed the reduction of the linkage error from 17.5 nm to ∼5 nm with an expansion factor of 3.2 …”

Section: Pre- Versus Postexpansion Immunostaining Strategiesmentioning

confidence: 99%

Current Progress in Expansion Microscopy: Chemical Strategies and Applications

Wen

Leen²,

Rohand

et al. 2023

Chem. Rev.

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Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre-and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development. CONTENTS R 8. Expansion Isotropy S 9. Conclusion and Outlook S

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“…Expansion microscopy has now been used in a variety of tissues and model systems to reveal nanometer-scale information ( Gallagher and Zhao, 2021 ). Some recent work has developed this approach to study synaptic structure and amyloid fibrils, and the authors terming the optimized technique “expansion revealing” ( Sarkar et al, 2022 ). This sample processing technique may help make the analysis of synaptic biology easier and more accessible, by simply making them bigger.…”

Section: Imaging Synapses In Disease Modelsmentioning

confidence: 99%

Bringing synapses into focus: Recent advances in synaptic imaging and mass-spectrometry for studying synaptopathy

Hindley

Avila

Henstridge

2023

Front. Synaptic Neurosci.

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Synapses are integral for healthy brain function and are becoming increasingly recognized as key structures in the early stages of brain disease. Understanding the pathological processes driving synaptic dysfunction will unlock new therapeutic opportunities for some of the most devastating diseases of our time. To achieve this we need a solid repertoire of imaging and molecular tools to interrogate synaptic biology at greater resolution. Synapses have historically been examined in small numbers, using highly technical imaging modalities, or in bulk, using crude molecular approaches. However, recent advances in imaging techniques are allowing us to analyze large numbers of synapses, at single-synapse resolution. Furthermore, multiplexing is now achievable with some of these approaches, meaning we can examine multiple proteins at individual synapses in intact tissue. New molecular techniques now allow accurate quantification of proteins from isolated synapses. The development of increasingly sensitive mass-spectrometry equipment means we can now scan the synaptic molecular landscape almost in totality and see how this changes in disease. As we embrace these new technical developments, synapses will be viewed with clearer focus, and the field of synaptopathy will become richer with insightful and high-quality data. Here, we will discuss some of the ways in which synaptic interrogation is being facilitated by methodological advances, focusing on imaging, and mass spectrometry.

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Revealing nanostructures in brain tissue via protein decrowding by iterative expansion microscopy

Cited by 55 publications

References 68 publications

Visualizing proteins by expansion microscopy

Visualizing proteins by expansion microscopy

Current Progress in Expansion Microscopy: Chemical Strategies and Applications

Bringing synapses into focus: Recent advances in synaptic imaging and mass-spectrometry for studying synaptopathy

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