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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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a Nucleus of a Xenopus laevis A6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x–z-cross section b is taken; c and d show the respective x–y- and y–z-views of the distal appendage protein CEP152 of centrioles from a U2OS cell; e represents another pair of centrioles in a COS-7 cell. All stainings were performed with Alexa Fluor 647. Scale bara, b 1 μm; c, d 200 nm; e 500 nm; color-code (blue to red) a, b 0–4.6 μm; c, d 0–400 nm
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Fig2: a Nucleus of a Xenopus laevis A6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x–z-cross section b is taken; c and d show the respective x–y- and y–z-views of the distal appendage protein CEP152 of centrioles from a U2OS cell; e represents another pair of centrioles in a COS-7 cell. All stainings were performed with Alexa Fluor 647. Scale bara, b 1 μm; c, d 200 nm; e 500 nm; color-code (blue to red) a, b 0–4.6 μm; c, d 0–400 nm

Mentions: The astigmatism approach introduces a cylindrical lens into the detection path of the microscope system. This leads to a stretching of the PSF because only one spatial direction is tightly focused while the other is defocused (Fig. 2). In practice, there is one point of equal PSF widths (which can be set to be z = 0 nm), and PSFs originating from above or below are spread to a horizontal or vertical line, respectively. The rough axial position can be extracted from the orientation of the PSF and the widths in x and y can be calibrated to yield exact z-coordinates. After the initial use in single-particle tracking (Holtzer et al. 2007; Kao and Verkman 1994), the method has been broadly applied to single-molecule localization microscopy (Dani et al. 2010; Huang et al. 2008a, b; Xu et al. 2012). For calibration of the defocusing behavior, usually single fluorophores, quantum dots or small fluorescent beads are adsorbed on a bare coverslip and moved in z while recording their PSF. The obtained PSF widths in x and y are fitted with a polynomial of second order, which represents a physically derived model (Holtzer et al. 2007) or a fourth-order polynomial to account for imperfections in the optical system (Huang et al. 2008a). To avoid fitting of a more or less physically derived function to the calibration data, a lookup table can be created for the extraction of the actual axial position. In the open-source QuickPALM plugin for ImageJ (Henriques et al. 2010), the standard deviations of the calibration PSF in x and y are determined and the known z-position is plotted against σx − σy. The obtained straight line serves to look up the z-coordinates during the course of a measurement.Fig. 2


Eight years of single-molecule localization microscopy.

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

a Nucleus of a Xenopus laevis A6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x–z-cross section b is taken; c and d show the respective x–y- and y–z-views of the distal appendage protein CEP152 of centrioles from a U2OS cell; e represents another pair of centrioles in a COS-7 cell. All stainings were performed with Alexa Fluor 647. Scale bara, b 1 μm; c, d 200 nm; e 500 nm; color-code (blue to red) a, b 0–4.6 μm; c, d 0–400 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: a Nucleus of a Xenopus laevis A6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x–z-cross section b is taken; c and d show the respective x–y- and y–z-views of the distal appendage protein CEP152 of centrioles from a U2OS cell; e represents another pair of centrioles in a COS-7 cell. All stainings were performed with Alexa Fluor 647. Scale bara, b 1 μm; c, d 200 nm; e 500 nm; color-code (blue to red) a, b 0–4.6 μm; c, d 0–400 nm
Mentions: The astigmatism approach introduces a cylindrical lens into the detection path of the microscope system. This leads to a stretching of the PSF because only one spatial direction is tightly focused while the other is defocused (Fig. 2). In practice, there is one point of equal PSF widths (which can be set to be z = 0 nm), and PSFs originating from above or below are spread to a horizontal or vertical line, respectively. The rough axial position can be extracted from the orientation of the PSF and the widths in x and y can be calibrated to yield exact z-coordinates. After the initial use in single-particle tracking (Holtzer et al. 2007; Kao and Verkman 1994), the method has been broadly applied to single-molecule localization microscopy (Dani et al. 2010; Huang et al. 2008a, b; Xu et al. 2012). For calibration of the defocusing behavior, usually single fluorophores, quantum dots or small fluorescent beads are adsorbed on a bare coverslip and moved in z while recording their PSF. The obtained PSF widths in x and y are fitted with a polynomial of second order, which represents a physically derived model (Holtzer et al. 2007) or a fourth-order polynomial to account for imperfections in the optical system (Huang et al. 2008a). To avoid fitting of a more or less physically derived function to the calibration data, a lookup table can be created for the extraction of the actual axial position. In the open-source QuickPALM plugin for ImageJ (Henriques et al. 2010), the standard deviations of the calibration PSF in x and y are determined and the known z-position is plotted against σx − σy. The obtained straight line serves to look up the z-coordinates during the course of a measurement.Fig. 2

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