SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

Moeyaert, Benjamien; Vandenberg, Wim; Dedecker, Peter

doi:10.1101/802199

Cited by 11 publications

(19 citation statements)

References 42 publications

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“…However, redFAST did show single-molecule intensity fluctuations suitable for SOFI imaging. Two-color SOFI imaging could be achieved using live mammalian cells co-expressing microtubule-targeted redFAST (stained with HBR-3,5DOM) and membrane-targeted Skylan-S, 28 a green fluorescent protein that was showed to be robustly well-performing in pcSOFI 29 (Figure 2b). Second-order pcSOFI analysis showed the background rejection and two-fold gain in spatial information intrinsic to the method.…”

Section: Two Color Superresolution Microscopymentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

et al. 2020

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Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development, and the development of split complementation systems capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

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Section: Two Color Superresolution Microscopymentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

et al. 2020

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“…However, redFAST did show single-molecule intensity fluctuations suitable for SOFI imaging. Two-color SOFI imaging could be achieved using live mammalian cells co-expressing microtubule-targeted redFAST (stained with HBR-3,5DOM) and membrane-targeted Skylan-S, 27 a green fluorescent protein that was showed to be robustly well-performing in pcSOFI 28 (Figure 2g). Second-order pcSOFI analysis showed the background rejection and two-fold gain in spatial information intrinsic to the method.…”

Section: Resultsmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

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Fluorescence microscopy is an indispensable tool in biological research, allowing subsecond and sub-micrometer mapping of molecules or processes inside living cells.Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one-and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy. 3

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“…To construct the SOFI image using the Localizer package in IgorPro (35), 600 frames where used, dropping the initial 15 frames to exclude non-stationary signal. The images produced where checked for artifacts using SOFIevaluator (36). Since the SNR of the images was limited, the image was subsequently convolved with a gaussian (FWHM 112,5nm) to further smooth out the background noise and bring out the labelled features.…”

Section: Methodsmentioning

confidence: 99%

“…It is The copyright holder for this preprint this version posted March 1, 2021. ; https://doi.org/10.1101/2021.02.26.433138 doi: bioRxiv preprint stationary signal. The images produced where checked for artifacts using SOFIevaluator (36). Since the SNR of the images was limited, the image was subsequently convolved with a gaussian (FWHM 112,5nm) to further smooth out the background noise and bring out the labelled features.…”

Section: Gcn5 Imaging and Pcsofi Processingmentioning

confidence: 99%

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

Genechten

Demuyser

Duwe

et al. 2021

Preprint

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Fluorescence microscopy is a standard research tool in many fields, though collecting reliable images can be difficult in systems characterized by low expressions levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly- or endogenously-expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungals agents in the fungal pathogen C. albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by a high background fluorescence.ImportanceUnderstanding the spatial and temporal organization of proteins-of-interest is key to unravel cellular processes and identify novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans and resistance mechanisms against these therapeutic agents is rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable imaging of lowly-expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems allowing the visualization of intricate processes.

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SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

Cited by 11 publications

References 42 publications

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

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