Dataset Information

Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.

ABSTRACT: The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.

SUBMITTER: Biteen JS

PROVIDER: S-EPMC2655310 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.

Biteen Julie S JS Thompson Michael A MA Tselentis Nicole K NK Bowman Grant R GR Shapiro Lucy L Moerner W E WE

Nature methods 20080915 11

The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging. ...[more]

PMID: 18794860

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Project description:The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG) and can be used to label proteins sparsely, enabling single-protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

Dataset Information

Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.

Publications

Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.

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