Diffraction‐Unlimited Fluorescence Microscopy of Living Biological Samples Using pcSOFI

Duwé, Sam; Moeyaert, Benjamien; Dedecker, Peter

doi:10.1002/9780470559277.ch140025

Cited by 8 publications

(8 citation statements)

References 34 publications

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“…We evaluated the use of the developed RSFPs for sub-diffraction microscopy by employing these labels in pcSOFI 8,65 and RESOLFT 26,61 experiments. pcSOFI experiments were successful in TIRF mode using the Lyn tag described above (Figure 8 and Figure S17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 23…”

Section: Sub-diffraction Microscopymentioning

confidence: 99%

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Duwé¹,

Zitter²,

Gielen³

et al. 2015

ACS Nano

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Smart fluorophores", such as reversibly switchable fluorescent proteins (RSFPs), are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available and many display reduced biological performance compared to more classical variants.We present the development of robustly photoswitchable variants of EGFP, named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37°C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multi-modal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ~70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"-and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance, and provides a practical strategy for developing maturation-and stability-enhanced photochromic fluorescent proteins.KEYWORDS: fluorescent proteins, reversible photoswitching, super-resolution fluorescence microscopy, SOFI, RESOLFT, crystal structure determination, rsEGFP, superfolder Fluorescent proteins (FPs) enable the minimally-invasive labeling of intracellular structures in live systems. 1 The discovery and development of "smart photoactive FPs", 2,3 with features such as irreversible photoactivation and photoconversion, or reversible photoswitching, allowed the development of diffraction-unlimited imaging techniques such as (f)PALM 4,5 ((fluorescence) photoactivated localization microscopy), RESOLFT 6 (reversible saturable optical fluorescence transitions) and (pc)SOFI 7,8 ((photochromic) stochastic optical fluctuation imaging). These techniques strongly rely on the performance of the fluorophores and considerable efforts have therefore been dedicated to create optimized "smart labels". 9 This is exemplified by the continuous optimization and diversification of the EosFP family, 10-15 or the development of Dronpa 16 mutants with different or added photophysical properties. [17][18][19][20][21][22] Probes that combine multiple "smart" behaviors have also been engineered. [23][24][25] On the whole, however, the general acceptance of the FP-based "smart labels" has not quite risen up to the high expectations set by the many applications they enable. In some cases this is due to concerns surrounding the biological compatibility of the labels, meaning that the label may interfere with the functioning of the syst...

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Section: Sub-diffraction Microscopymentioning

confidence: 99%

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Duwé¹,

Zitter²,

Gielen³

et al. 2015

ACS Nano

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“…There have been select applications, where single-color SMLM achieved <1 s time resolution, but usually tens of seconds acquisitions are necessary 28,31 . We previously demonstrated live-cell SOFI with acquisition times~1 s (~325 frames 17 ) using optimized, fast switching of Dreiklang and others achieved live-cell SOFI for several fluorescent proteins by analyzing~500 frames 32 .…”

Section: Resultsmentioning

confidence: 99%

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

et al. 2020

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Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using n th order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-toimplement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

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“…Apart from the capability for RESOLFT nanoscopy, we also found GMars-T displays obvious stochastic photoswitching under wide-field illumination scheme at single pixel level. By taking advantage of such fluorescence properties, 53,54 pcSOFI analysis could also be performed which enables long-term time-lapse live cell fluorescence imaging at superresolution level. Besides multimodal super-resolution structural imaging which reveals fine subcellular structures beyond diffraction limit, we also developed GMars-T-based BiFC sensor reporting PPIs with high specificity in live cells.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells

et al. 2018

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Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

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Diffraction‐Unlimited Fluorescence Microscopy of Living Biological Samples Using pcSOFI

Cited by 8 publications

References 34 publications

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells

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