Visualizing proteins by expansion microscopy
Abstract Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of<jats:underline>o</jats:underline>ne-step<jats:underline>n</jats:underline>anoscale<jats:underline>e</jats:underline>xpansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7to enable the description of 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 also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents a promising new development towards 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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Preprint| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
bioRxiv.org - (2023) vom: 14. März Zur Gesamtaufnahme - year:2023 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Shaib, Ali H. [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2022.08.03.502284 |
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XBI036795356 |
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| 520 | |a Abstract Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of<jats:underline>o</jats:underline>ne-step<jats:underline>n</jats:underline>anoscale<jats:underline>e</jats:underline>xpansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7to enable the description of 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 also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents a promising new development towards 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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