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Eight years of single-molecule localization microscopy.

Klein T, Proppert S, Sauer M - Histochem. Cell Biol. (2014)

Bottom Line: Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy.Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes.Thus, it exhibits potential to address fundamental questions of cell and developmental biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University Würzburg, Am Hubland, 97074, Würzburg, Germany, teresa.klein@uni-wuerzburg.de.

ABSTRACT
Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

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Live-cell dSTORM with SNAP tags. a Fluorescence image of histone H2B proteins in a COS-7 cell stained with SNAP-Cell TMR-Star (1 μM). Scale bar 5 μm. b Fluorescence image of the same cell but with 532 nm excitation of ~1 kW cm−2, which induced photoswitching. cdSTORM image reconstructed from 10,000 images (acquired at 50 Hz). Adapted from (Klein et al. 2011), with permission
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Fig1: Live-cell dSTORM with SNAP tags. a Fluorescence image of histone H2B proteins in a COS-7 cell stained with SNAP-Cell TMR-Star (1 μM). Scale bar 5 μm. b Fluorescence image of the same cell but with 532 nm excitation of ~1 kW cm−2, which induced photoswitching. cdSTORM image reconstructed from 10,000 images (acquired at 50 Hz). Adapted from (Klein et al. 2011), with permission

Mentions: The first demonstration of live-cell single-molecule localization microscopy with organic dyes was performed by dSTORM employing the trimethoprim (TMP) tag (Wombacher et al. 2010). TMP binds to the E. coli dihydrofolate reductase (eDHFR). The cell-permeable dye ATTO 655 was used to label the eDHFR tagged core histone protein H2B. Since the cell provides its own reductive environment, mostly due to the presence of millimolar concentration of the thiol glutathione (van de Linde et al. 2012), ATTO 655 molecules switch between a bright on- and a non-fluorescent off-state upon irradiation under physiological conditions. Applying a sliding window analysis on the localization data, the dynamic movement of histone proteins could be observed at a temporal resolution of 10 s. Slightly later, the Snap-tag technology was also successfully applied for live-cell dSTORM in various cell lines using the two rhodamine-based substrates SNAP-Cell TMR-Star and SNAP-Cell 505 (Fig. 1) (Klein et al. 2011). The results demonstrated the formation of long-lived fluorophore off-states under physiological conditions, which can be effectively and repeatedly reactivated upon irradiation at 405 nm (Klein et al. 2011; van de Linde et al. 2011a, b). Because rhodamine dyes tend to bind non-specifically on glass surfaces, coverslips were coated with 2 M glycine.Fig. 1


Eight years of single-molecule localization microscopy.

Klein T, Proppert S, Sauer M - Histochem. Cell Biol. (2014)

Live-cell dSTORM with SNAP tags. a Fluorescence image of histone H2B proteins in a COS-7 cell stained with SNAP-Cell TMR-Star (1 μM). Scale bar 5 μm. b Fluorescence image of the same cell but with 532 nm excitation of ~1 kW cm−2, which induced photoswitching. cdSTORM image reconstructed from 10,000 images (acquired at 50 Hz). Adapted from (Klein et al. 2011), with permission
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4544475&req=5

Fig1: Live-cell dSTORM with SNAP tags. a Fluorescence image of histone H2B proteins in a COS-7 cell stained with SNAP-Cell TMR-Star (1 μM). Scale bar 5 μm. b Fluorescence image of the same cell but with 532 nm excitation of ~1 kW cm−2, which induced photoswitching. cdSTORM image reconstructed from 10,000 images (acquired at 50 Hz). Adapted from (Klein et al. 2011), with permission
Mentions: The first demonstration of live-cell single-molecule localization microscopy with organic dyes was performed by dSTORM employing the trimethoprim (TMP) tag (Wombacher et al. 2010). TMP binds to the E. coli dihydrofolate reductase (eDHFR). The cell-permeable dye ATTO 655 was used to label the eDHFR tagged core histone protein H2B. Since the cell provides its own reductive environment, mostly due to the presence of millimolar concentration of the thiol glutathione (van de Linde et al. 2012), ATTO 655 molecules switch between a bright on- and a non-fluorescent off-state upon irradiation under physiological conditions. Applying a sliding window analysis on the localization data, the dynamic movement of histone proteins could be observed at a temporal resolution of 10 s. Slightly later, the Snap-tag technology was also successfully applied for live-cell dSTORM in various cell lines using the two rhodamine-based substrates SNAP-Cell TMR-Star and SNAP-Cell 505 (Fig. 1) (Klein et al. 2011). The results demonstrated the formation of long-lived fluorophore off-states under physiological conditions, which can be effectively and repeatedly reactivated upon irradiation at 405 nm (Klein et al. 2011; van de Linde et al. 2011a, b). Because rhodamine dyes tend to bind non-specifically on glass surfaces, coverslips were coated with 2 M glycine.Fig. 1

Bottom Line: Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy.Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes.Thus, it exhibits potential to address fundamental questions of cell and developmental biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University Würzburg, Am Hubland, 97074, Würzburg, Germany, teresa.klein@uni-wuerzburg.de.

ABSTRACT
Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

Show MeSH
Related in: MedlinePlus