A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

Shu, Xiaokun; Lev‐Ram, Varda; Deerinck, Thomas J.; Qi, Yingchuan; Ramko, Ericka B.; Davidson, Michael W.; Jin, Yishi; Ellisman, Mark H.; Tsien, Roger Y.

doi:10.1371/journal.pbio.1001041

Cited by 780 publications

(882 citation statements)

References 53 publications

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“…In addition to its conventional fluorescence readout, resorufin can also be used for singlemolecule imaging (11), chromophore-assisted light inactivation (12), and contrast generation for EM by photoxidation (13). Compared with miniSOG, a genetically encoded dual fluorescence/EM tag, resorufin is red-shifted in emission, approximately nine times brighter, yet ∼40 times smaller (by mass) (14). Resorufin therefore has suitable photophysical attributes for PRIME.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

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Section: Resultsmentioning

confidence: 99%

“…Certain fluorophores in the excited state are known to photooxidize diaminobenzidine (DAB) into a locally deposited polymer (scheme in Fig. 4B) (13,14,32). This polymer in turn recruits osmium, which is electron-dense and gives EM contrast.…”

Section: Imaging Via Stimulated Emission Depletion Microscopy and Elementioning

confidence: 99%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Correlated fluorescence and 3D EM tomography have been extended to small copy numbers of fluorescent proteins (10). Some superresolution fluorescence images were correlated with electron micrographs by using photoactivatable fluorescent proteins (PAFPs) (11), and a new protein-specific local heavy metal stain has great potential to yield high-resolution localization based on EM only (12). Nevertheless, correlative superresolution/EM requiring maximal PAFP fluorescence preservation and minimal compromise of ultrastructure has been elusive.…”

mentioning

confidence: 99%

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Kopek

Shtengel

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

227

210

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Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500-to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.correlative microscopy | photoactivated localization microscopy | electron microscopy | cryosection | mitochondrial DNA F luorescent labels can mark specific proteins directly through genetic expression of fusion proteins or by postattachment of labeled antibodies (1). Genetic expression of fusion proteins provides the best specificity and a higher labeling density, with the caveat that a fusion can disrupt the functionality and/or native localization of the protein. Alternatively, labeled antibodies allow the visualization of native proteins, but localization accuracy is dependent on labeling specificity and background, and resolution can be further degraded by a lower labeling density. When these fluorescent labels exhibit certain photophysical properties such as switching, wavelength shifting, or other nonlinear responses, a variety of new microscopy techniques can extract their position at resolutions well below the diffraction limit of approximately 200 nm, down to 20 to 100 nm (2). Some of the new microscopy techniques map molecule distributions by a patterned illumination such as saturated structured illumination microscopy (3) or stimulated emission depletion (STED) microscopy (4). Other methods, including photoactivated localization microscopy (PALM) (5, 6) or stochastic optical reconstruction microscopy (7) extract fluorescent protein positions one molecule at a time. For a single fluorophore color, those images may appear as protein distributions floating in darkness with no or little further context. The localization of fluorescently labeled proteins relative to other cellular structures, e.g., membranes, requires a second fluorescent label. Although multiple-color data are desirable, often the biologica...

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“…Last year, researchers from both groups revealed a molecular tool they had engineered called mini singlet oxygen generator (mini-SOG), which is derived from a plant photoreceptor 4 . Singlet oxygen generator molecules are easy to see because they fluoresce in light microscopy and can be stained by diaminobenzidine for EM 5 .…”

Section: Fluorescent Bridgesmentioning

confidence: 99%

Two microscopes are better than one

Smith¹

2012

Nature

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T here are many types of microscopy, each providing a unique set of benefits. Light microscopy of cells that express fluorescently labelled molecules, for example, lets scientists observe the movements of specific molecules or protein complexes in live cells in real time, or in fixed samples. Scanning electron microscopy (EM) reveals tiny details of the cell surface, and transmission EM shows the detailed cytoarchitecture of sections through fixed tissue. Other aspects of the structure of cells and tissues can be explored using techniques such as ion microscopy, total internal reflection microscopy, atomic force microscopy and super-resolution microscopy. Each type provides different information, but using two sorts of microscopy simultaneously provides even more -and molecular tools have been developed to link them together. Researchers testing the waters of this 'correlative microscopy' are beginning to discover its challenges and rewards.Correlated light-electron microscopy, for example, provides both the specificity and real-time observation of light microscopy with fluorescent labelling and the better structural resolution of EM. But such correlative microscopy has historically been tricky to use. Scientists who tried it might have needed to switch to EM halfway through an experiment, which meant moving the sample from one This image of a butterfly wing was constructed using two types of microscopy: a confocal image of the reflective eyespots shows scales (green) and wing (red); scanning electron microscopy reveals the different structure of non-reflective scales (upper left).

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A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

Cited by 780 publications

References 53 publications

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Two microscopes are better than one

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