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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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Three-dimensional PALM imaging of a vimentin network. axy (top) and xz (bottom) maximum-intensity projections of PA-mCherry1–vimentin. About 1 million unlinked localizations was rendered in each view. Insets show further magnification of white rectangles in xy (lines in xz) maximum-intensity projection, highlighting individual vimentin fibrils in 60-nm-thick z-slices (localizations are linked). Arrow region of fibril with apparent width <100 nm. b Axial extent of vimentin network with z-location indicated as a color map. For clarity, localizations corresponding to 0–1.5 μm (top) and 1.5–3 μm (bottom) are shown separately. Arrowheads in a and b indicate a fibril that persists over >2 μm axially. Only linked localizations with correlation strength >0.4 are shown. Histogram bin sizes are 60 nm for all subfigures. Scale bars 3 μm (a), 600 nm (insets), 3 μm (b). Reproduced from York et al. (2011), with permission
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Fig3: Three-dimensional PALM imaging of a vimentin network. axy (top) and xz (bottom) maximum-intensity projections of PA-mCherry1–vimentin. About 1 million unlinked localizations was rendered in each view. Insets show further magnification of white rectangles in xy (lines in xz) maximum-intensity projection, highlighting individual vimentin fibrils in 60-nm-thick z-slices (localizations are linked). Arrow region of fibril with apparent width <100 nm. b Axial extent of vimentin network with z-location indicated as a color map. For clarity, localizations corresponding to 0–1.5 μm (top) and 1.5–3 μm (bottom) are shown separately. Arrowheads in a and b indicate a fibril that persists over >2 μm axially. Only linked localizations with correlation strength >0.4 are shown. Histogram bin sizes are 60 nm for all subfigures. Scale bars 3 μm (a), 600 nm (insets), 3 μm (b). Reproduced from York et al. (2011), with permission

Mentions: In order to enable super-resolution imaging of whole living organisms, novel long-term continuous imaging methods with high spatial resolution and minimal photodamage such as selective plane illumination microscopy (SPIM) (Huisken et al. 2004) or Bessel beam plane illumination (Gao et al. 2012; Planchon et al. 2011) are urgently needed. Ideally, these methods are combined with the enhanced penetration depth and axial sectioning capabilities of two-photon microscopy. First, realizations of super-resolution imaging with confined two-photon activation (York et al. 2011) (Fig. 3) and SPIM (Cella Zanacchi et al. 2011, 2013) have recently been demonstrated. Besides the use of photoactivatable fluorescent proteins, so-called caged, non-fluorescent organic dyes that can be activated upon irradiation with UV or IR light, such as rhodamine NN dyes (Belov et al. 2010) and carborhodamine dyes masked with photolabile groups (Grimm et al. 2013), could also be used advantageously for axially confined localization microscopy.Fig. 3


Eight years of single-molecule localization microscopy.

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

Three-dimensional PALM imaging of a vimentin network. axy (top) and xz (bottom) maximum-intensity projections of PA-mCherry1–vimentin. About 1 million unlinked localizations was rendered in each view. Insets show further magnification of white rectangles in xy (lines in xz) maximum-intensity projection, highlighting individual vimentin fibrils in 60-nm-thick z-slices (localizations are linked). Arrow region of fibril with apparent width <100 nm. b Axial extent of vimentin network with z-location indicated as a color map. For clarity, localizations corresponding to 0–1.5 μm (top) and 1.5–3 μm (bottom) are shown separately. Arrowheads in a and b indicate a fibril that persists over >2 μm axially. Only linked localizations with correlation strength >0.4 are shown. Histogram bin sizes are 60 nm for all subfigures. Scale bars 3 μm (a), 600 nm (insets), 3 μm (b). Reproduced from York et al. (2011), with permission
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Three-dimensional PALM imaging of a vimentin network. axy (top) and xz (bottom) maximum-intensity projections of PA-mCherry1–vimentin. About 1 million unlinked localizations was rendered in each view. Insets show further magnification of white rectangles in xy (lines in xz) maximum-intensity projection, highlighting individual vimentin fibrils in 60-nm-thick z-slices (localizations are linked). Arrow region of fibril with apparent width <100 nm. b Axial extent of vimentin network with z-location indicated as a color map. For clarity, localizations corresponding to 0–1.5 μm (top) and 1.5–3 μm (bottom) are shown separately. Arrowheads in a and b indicate a fibril that persists over >2 μm axially. Only linked localizations with correlation strength >0.4 are shown. Histogram bin sizes are 60 nm for all subfigures. Scale bars 3 μm (a), 600 nm (insets), 3 μm (b). Reproduced from York et al. (2011), with permission
Mentions: In order to enable super-resolution imaging of whole living organisms, novel long-term continuous imaging methods with high spatial resolution and minimal photodamage such as selective plane illumination microscopy (SPIM) (Huisken et al. 2004) or Bessel beam plane illumination (Gao et al. 2012; Planchon et al. 2011) are urgently needed. Ideally, these methods are combined with the enhanced penetration depth and axial sectioning capabilities of two-photon microscopy. First, realizations of super-resolution imaging with confined two-photon activation (York et al. 2011) (Fig. 3) and SPIM (Cella Zanacchi et al. 2011, 2013) have recently been demonstrated. Besides the use of photoactivatable fluorescent proteins, so-called caged, non-fluorescent organic dyes that can be activated upon irradiation with UV or IR light, such as rhodamine NN dyes (Belov et al. 2010) and carborhodamine dyes masked with photolabile groups (Grimm et al. 2013), could also be used advantageously for axially confined localization microscopy.Fig. 3

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