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A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.
PLoS Biol. 2011 Apr; 9(4):e1001041
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20 Apr 2011
Seth J Davis
F1000 Plant Biology
Max Planck Institute for Plant Breeding Research, Cologne, Germany.
F1000 Plant Biology
Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Technical Advance
DOI: 10.3410/f.9813965.10515069
Wow! The Tsien lab might have done it again with their development of a tool to examine localization of proteins via electron microscopy (EM).
Starting from a structural placement of a plant photoreceptor, the authors created miniSOG (for mini singlet oxygen generator). MiniSOG generates sufficient singlet...Continue reading
Starting from a structural placement of a plant photoreceptor, the authors created miniSOG (for mini singlet oxygen generator). MiniSOG generates sufficient singlet...Continue reading
Wow! The Tsien lab might have done it again with their development of a tool to examine localization of proteins via electron microscopy (EM).
Starting from a structural placement of a plant photoreceptor, the authors created miniSOG (for mini singlet oxygen generator). MiniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to proteins localize correctly, thus enabling ultra-structural resolution of protein localization. This technology has the chance to totally revolutionize EM.
Starting from a structural placement of a plant photoreceptor, the authors created miniSOG (for mini singlet oxygen generator). MiniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to proteins localize correctly, thus enabling ultra-structural resolution of protein localization. This technology has the chance to totally revolutionize EM.
Disclosures
None declared
Add Comment
Davis S: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 20 Apr 2011; DOI: 10.3410/f.9813965.10515069. F1000Prime.com/9813965#eval10515069
05 May 2011
Nuno Miguel MaÇarico Amorim da Costa
F1000 Neuroscience
Swiss Federal Institute of Technology, Zurich, Switzerland.
F1000 Neuroscience
Swiss Federal Institute of Technology, Zurich, Switzerland.
Technical Advance
DOI: 10.3410/f.9813965.11065054
In this study Shu and colleagues introduce mini singlet oxygen generator (miniSOG), a genetically encoded tag that is visible both at the light microscopic (LM) and electron microscopic (EM) levels. The potential of this technology for questions that require the nanometer resolution of the EM is imm since...Continue reading
In this study Shu and colleagues introduce mini singlet oxygen generator (miniSOG), a genetically encoded tag that is visible both at the light microscopic (LM) and electron microscopic (EM) levels. The potential of this technology for questions that require the nanometer resolution of the EM is immense, since it allows the identification of proteins at the ultrastructural level without the additional steps needed in conventional EM immunohistochemistry, which compromise tissue quality.
MiniSOG is based on the Arabidopsis thaliana phototropin 2, modified through mutagenesis to improve both the production of singlet oxygen and fluorescence. Through photo-oxidation the protein catalyzes the production of singlet oxygen that then reacts with diaminobenzidine (DAB) to produce an electron-dense reaction product that can be visualized in EM. As a proof of concept, the authors show several examples in which they used miniSOG to localize different intracellular proteins. The fluorescence of miniSOG has a lower quantum yield than green florescent protein (GFP) but is sufficiently strong for correlated LM and EM microcopy. The arrival of miniSOG is particularly opportune for the current renaissance of neural circuit analyses and it should enjoy a bright future.
MiniSOG is based on the Arabidopsis thaliana phototropin 2, modified through mutagenesis to improve both the production of singlet oxygen and fluorescence. Through photo-oxidation the protein catalyzes the production of singlet oxygen that then reacts with diaminobenzidine (DAB) to produce an electron-dense reaction product that can be visualized in EM. As a proof of concept, the authors show several examples in which they used miniSOG to localize different intracellular proteins. The fluorescence of miniSOG has a lower quantum yield than green florescent protein (GFP) but is sufficiently strong for correlated LM and EM microcopy. The arrival of miniSOG is particularly opportune for the current renaissance of neural circuit analyses and it should enjoy a bright future.
Disclosures
None declared
Martin K and MaÇarico Amorim da Costa N: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 05 May 2011; DOI: 10.3410/f.9813965.11065054. F1000Prime.com/9813965#eval11065054
09 May 2011
Technical Advance
DOI: 10.3410/f.9813965.11026054
This article is important as it describes a new and facile tool for both: i) correlative light and electron microscopy (EM); and ii) deeper tissue labelling with a genetically encoded probe.
EM and the ability to visualize organelles with high (often less <10nm) resolution is instrumental, even syn with...Continue reading
EM and the ability to visualize organelles with high (often less <10nm) resolution is instrumental, even syn with...Continue reading
This article is important as it describes a new and facile tool for both: i) correlative light and electron microscopy (EM); and ii) deeper tissue labelling with a genetically encoded probe.
EM and the ability to visualize organelles with high (often less <10nm) resolution is instrumental, even synonymous, with modern cell biology. But telling where specific proteins are located is not always straightforward. The most common method, immuno-EM, uses (~5-20nm) gold particles that give an easily distinguishable signal, but is limited by antibody specificity and access. Another method for producing a signal is through the generation of free oxygen radicals, usually from horseradish peroxidase (HRP), to precipitate diaminobenzidine and to generate contrast; some organic dyes can also inefficiently produce free oxygen radicals. But, in practice, HRP or HRP-tagged proteins are largely limited to exocytic and endocytic trafficking pathways. In an innovative approach, Dr Tsien and colleagues generate a genetically encoded 106 amino-acid, fluorescent protein tag from phototropin that is optimized to generate oxygen radicals: they call this new probe "mini singlet oxygen generator" ('miniSOG'). The authors show that, similar to green fluorescent protein (GFP), miniSOG-tagged proteins can be correctly targeted to multiple organelles. Upon photoconversion, correlative EM shows that the corresponding organelles show a noticeably increased labelling density. Images of connexon vesicles appear like a studded sphere and are especially appealing.
While promising, most of the examples have a relatively high local protein density. In the future, it will be important to quantitate the sensitivity of the system and its signal-to-background or degree of contrast - so that low expressing proteins targeted to an unknown location can be unambiguously identified (telling the specific signal from the general osmium tetroxide staining may not always be as obvious). Regardless of these and other potential caveats, new tools for correlative light and EM will be a welcome addition, especially for electron tomography or deep serial imaging, which often precludes post-fixation labeling with antibodies and provides little or no information about molecular localization. While clearly only the first step, one can expect that if the miniSOG labeling is sufficiently dark (by EM), then its future will be bright.
EM and the ability to visualize organelles with high (often less <10nm) resolution is instrumental, even synonymous, with modern cell biology. But telling where specific proteins are located is not always straightforward. The most common method, immuno-EM, uses (~5-20nm) gold particles that give an easily distinguishable signal, but is limited by antibody specificity and access. Another method for producing a signal is through the generation of free oxygen radicals, usually from horseradish peroxidase (HRP), to precipitate diaminobenzidine and to generate contrast; some organic dyes can also inefficiently produce free oxygen radicals. But, in practice, HRP or HRP-tagged proteins are largely limited to exocytic and endocytic trafficking pathways. In an innovative approach, Dr Tsien and colleagues generate a genetically encoded 106 amino-acid, fluorescent protein tag from phototropin that is optimized to generate oxygen radicals: they call this new probe "mini singlet oxygen generator" ('miniSOG'). The authors show that, similar to green fluorescent protein (GFP), miniSOG-tagged proteins can be correctly targeted to multiple organelles. Upon photoconversion, correlative EM shows that the corresponding organelles show a noticeably increased labelling density. Images of connexon vesicles appear like a studded sphere and are especially appealing.
While promising, most of the examples have a relatively high local protein density. In the future, it will be important to quantitate the sensitivity of the system and its signal-to-background or degree of contrast - so that low expressing proteins targeted to an unknown location can be unambiguously identified (telling the specific signal from the general osmium tetroxide staining may not always be as obvious). Regardless of these and other potential caveats, new tools for correlative light and EM will be a welcome addition, especially for electron tomography or deep serial imaging, which often precludes post-fixation labeling with antibodies and provides little or no information about molecular localization. While clearly only the first step, one can expect that if the miniSOG labeling is sufficiently dark (by EM), then its future will be bright.
Disclosures
None declared
Toomre D: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 09 May 2011; DOI: 10.3410/f.9813965.11026054. F1000Prime.com/9813965#eval11026054
15 Jul 2011
Technical Advance
DOI: 10.3410/f.9813965.13049055
The authors describe a small, genetically encoded protein tag that efficiently generates singlet oxygen, allowing sensitive detection by electron microscopy (EM).
Fluorescent proteins have revolutionized light microscopy, allowing genetic encoding of sensitive tags. Here, the authors selected for variants...Continue reading
Fluorescent proteins have revolutionized light microscopy, allowing genetic encoding of sensitive tags. Here, the authors selected for variants...Continue reading
The authors describe a small, genetically encoded protein tag that efficiently generates singlet oxygen, allowing sensitive detection by electron microscopy (EM).
Fluorescent proteins have revolutionized light microscopy, allowing genetic encoding of sensitive tags. Here, the authors selected for variants of a blue light photoreceptor protein that still fluoresce but also generate singlet oxygen, resulting in the 'miniSOG' tag, a portable 106-amino acid domain. The authors show that miniSOG generates sufficient singlet oxygen to convert diaminobenzidine (DAB) into an EM-detectable polymer that is coincident with its fluorescence localization. EM detection of multiple proteins in multiple organisms is presented. This method has the potential to revolutionize EM studies throughout biology.
Fluorescent proteins have revolutionized light microscopy, allowing genetic encoding of sensitive tags. Here, the authors selected for variants of a blue light photoreceptor protein that still fluoresce but also generate singlet oxygen, resulting in the 'miniSOG' tag, a portable 106-amino acid domain. The authors show that miniSOG generates sufficient singlet oxygen to convert diaminobenzidine (DAB) into an EM-detectable polymer that is coincident with its fluorescence localization. EM detection of multiple proteins in multiple organisms is presented. This method has the potential to revolutionize EM studies throughout biology.
Disclosures
None declared
Kaufman P: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 15 Jul 2011; DOI: 10.3410/f.9813965.13049055. F1000Prime.com/9813965#eval13049055
01 Aug 2011
Technical Advance
DOI: 10.3410/f.9813965.13542054
I fully agree with the previous evaluations (Seth J Davis, Nuno Miguel MaÇarico Amorim Da Costa and Kevan A Martin, Derek Toomre and Paul Kaufman)...Continue reading
I fully agree with the previous evaluations (Seth J Davis, Nuno Miguel MaÇarico Amorim Da Costa and Kevan A Martin, Derek Toomre and Paul Kaufman) that this new method to genetically tag proteins for electron microscopy could potentially revolutionize electron microscopy applications in many areas of biology.
Disclosures
None declared
Kehr J: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 01 Aug 2011; DOI: 10.3410/f.9813965.13542054. F1000Prime.com/9813965#eval13542054
04 Aug 2011
New Finding, Technical Advance
DOI: 10.3410/f.9813965.13624054
Despite amazing developments in confocal microscopy, the limit of point to point resolution of light microscopy remains dictated by Abbe's limit. This means that much of the information deduced by biochemistry cannot be extended to the molecular level of structures visible by transmission electron microscopy.
This...Continue reading
This...Continue reading
Despite amazing developments in confocal microscopy, the limit of point to point resolution of light microscopy remains dictated by Abbe's limit. This means that much of the information deduced by biochemistry cannot be extended to the molecular level of structures visible by transmission electron microscopy.
This exciting new method may be especially important to determine localization of structures within interphase heterochromatin, distribution between lipid rafts and primary cilia, localization at specific sites in the pathway of protein export, and distribution of Bcl-2 family proteins and capsases in different parts of the cell, among many other opportunities.
This exciting new method may be especially important to determine localization of structures within interphase heterochromatin, distribution between lipid rafts and primary cilia, localization at specific sites in the pathway of protein export, and distribution of Bcl-2 family proteins and capsases in different parts of the cell, among many other opportunities.
Disclosures
None declared
Schwartz S: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 04 Aug 2011; DOI: 10.3410/f.9813965.13624054. F1000Prime.com/9813965#eval13624054
08 Aug 2011
Technical Advance
DOI: 10.3410/f.9813965.13778054
The green fluorescent protein (GFP) revolutionized light microscopy. Localizing proteins within cells has become routine for most labs. But despite the breakthroughs that allow imaging beyond the diffraction limit (super-resolution microscopy), resolution has still lagged behind electron microscopy. this...Continue reading
The green fluorescent protein (GFP) revolutionized light microscopy. Localizing proteins within cells has become routine for most labs. But despite the breakthroughs that allow imaging beyond the diffraction limit (super-resolution microscopy), resolution has still lagged behind electron microscopy. Now, this work promises to revolutionize the use of electron microscopy by providing a genetically encoded tag for protein localization for this technology. The technology may also make transmission electron microscopy (TEM) more accessible, since it requires neither specific antibodies nor the cumbersome treatments and may be much more robust in detecting proteins since it does not rely on antigen accessibility.
Disclosures
None declared
Frommer W: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 08 Aug 2011; DOI: 10.3410/f.9813965.13778054. F1000Prime.com/9813965#eval13778054
06 Mar 2012
Technical Advance
DOI: 10.3410/f.9813965.15418087
This study represents an impressive leap forward in correlative light microscopy/electron microscopy (LM/EM). Despite recent advances in super-resolution light microscopy, the single digit nanometer resolving power and consequent exquisitely detailed ultrastructural images provided by EM are highly...Continue reading
This study represents an impressive leap forward in correlative light microscopy/electron microscopy (LM/EM). Despite recent advances in super-resolution light microscopy, the single digit nanometer resolving power and consequent exquisitely detailed ultrastructural images provided by EM are highly desired, and thus correlative LM/EM has been pursued in an effort to marry the strengths of these two techniques. Here, the authors have engineered a genetically encoded reagent, mini singlet oxygen generator (miniSOG), which produces both fluorescence (lower quantum efficiency than green fluorescent protein [GFP] but still detectable by LM) and singlet oxygen (which can be reacted with diaminobenzidine [DAB] to form an electron-dense product detectable by EM).
As proof of concept, they provide correlative LM/EM images of markers for several subcellular compartments, including chromatin, actin fibers and mitochondria.
As proof of concept, they provide correlative LM/EM images of markers for several subcellular compartments, including chromatin, actin fibers and mitochondria.
Disclosures
None declared
Trinkle-Mulcahy L: F1000Prime Recommendation of [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 06 Mar 2012; DOI: 10.3410/f.9813965.15418087. F1000Prime.com/9813965#eval15418087
F1000Prime Recommendations, Dissents and Comments for [Shu X et al., PLoS Biol 2011, 9(4):e1001041]. In F1000Prime, 19 Jun 2013; F1000Prime.com/9813965
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Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
DOI: 10.1371/journal.pbio.1001041
PMID: 21483721
