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Super-resolution molecular and functional imaging of nanoscale architectures in life and materials science.

Habuchi S - Front Bioeng Biotechnol (2014)

Bottom Line: Super-resolution (SR) fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems.I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging.These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-)nanotechnology.

View Article: PubMed Central - PubMed

Affiliation: Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology , Jeddah , Saudi Arabia.

ABSTRACT
Super-resolution (SR) fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems. In this review, I describe the current state of various SR fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the SR microscopy. I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging. These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-)nanotechnology.

No MeSH data available.


Super-resolution fluorescence molecular imaging. (A) PALM image, sptPALM image, and diffusion map of a membrane protein, VSVG, in COS7 cells. A photoconvertable fluorescent protein tdEosFP is fused to VSVG. The PALM image was obtained by the temporal activation of tdEosFP–VSVG followed by the localization and reconstruction of the image. The sptPALM image was obtained by tracking the activated tdEosFP–VSVG molecules. The diffusion map was obtained by calculating the diffusion coefficient of individual tdEosFP–VSVG molecules using the diffusion trajectories (Manley et al., 2008). (B) (Left) the first (red) and last (green) localized positions of single actin molecules within dendritic spines. The positions were determined by the sptPALM imaging. A photoactivatable fluorescent protein PAGFP is fused to actin; (right) actin dynamics within individual spines. Orientation and length of the arrows represent direction and velocity of actin flow. They were calculated using the first and last positions of the diffusion trajectories within the radius of 1 camera pixel (111 nm). Gray scale represents molecular density. Vector = 100 nm/s (Frost et al., 2010). (C) (Left) sptPALM image of a DNA-binding protein, DNA polymerase I (Pol), in E. coli cells in the presence of methyl methanesulfonate (MMS). A photoactivatable fluorescent protein PAmCherry is fused to Pol. Inset shows examples of tracks of diffusing Pol (blue) and bound Pol (red); (right) distribution of diffusion coefficient for Pol under constant MMS treatment (Uphoff et al., 2013). Reproduced with permission from Manley et al. (2008), copyright 2008, Nature Publishing Group (A), Frost et al. (2010), copyright 2010, Elsevier (B), and Uphoff et al. (2013), copyright 2013, National Academy of Science, USA (C).
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Figure 4: Super-resolution fluorescence molecular imaging. (A) PALM image, sptPALM image, and diffusion map of a membrane protein, VSVG, in COS7 cells. A photoconvertable fluorescent protein tdEosFP is fused to VSVG. The PALM image was obtained by the temporal activation of tdEosFP–VSVG followed by the localization and reconstruction of the image. The sptPALM image was obtained by tracking the activated tdEosFP–VSVG molecules. The diffusion map was obtained by calculating the diffusion coefficient of individual tdEosFP–VSVG molecules using the diffusion trajectories (Manley et al., 2008). (B) (Left) the first (red) and last (green) localized positions of single actin molecules within dendritic spines. The positions were determined by the sptPALM imaging. A photoactivatable fluorescent protein PAGFP is fused to actin; (right) actin dynamics within individual spines. Orientation and length of the arrows represent direction and velocity of actin flow. They were calculated using the first and last positions of the diffusion trajectories within the radius of 1 camera pixel (111 nm). Gray scale represents molecular density. Vector = 100 nm/s (Frost et al., 2010). (C) (Left) sptPALM image of a DNA-binding protein, DNA polymerase I (Pol), in E. coli cells in the presence of methyl methanesulfonate (MMS). A photoactivatable fluorescent protein PAmCherry is fused to Pol. Inset shows examples of tracks of diffusing Pol (blue) and bound Pol (red); (right) distribution of diffusion coefficient for Pol under constant MMS treatment (Uphoff et al., 2013). Reproduced with permission from Manley et al. (2008), copyright 2008, Nature Publishing Group (A), Frost et al. (2010), copyright 2010, Elsevier (B), and Uphoff et al. (2013), copyright 2013, National Academy of Science, USA (C).

Mentions: Super-resolution localization microscopy has been further combined with single-particle tracking (SPT). SPT is a powerful tool for characterizing the spatiotemporal behavior of individual molecules, especially the diffusional motion of molecules in nanoscopic heterogeneous structures such as cell membranes (Douglass and Vale, 2005) and microporous materials (Kirstein et al., 2007). However, the method can be applied only to relatively low density mapping of the diffusional behavior of individual molecules. This makes it difficult to connect directly the spatiotemporal behavior of the molecules to the nanoscopic structure of the sample. The combination of SPT and SR localization microscopy allows high-density spatial mapping of the diffusional behavior of individual molecules through tracking single fluorescent probe molecules activated in a temporally controlled manner (Figure 4A) (Manley et al., 2008; Giannone et al., 2010; Rossier et al., 2012). This method has been applied to the study of actin molecule dynamics within dendritic spines. While actin plays numerous roles in synaptic transmission, its spatiotemporal behavior has not been well-characterized due to the small (submicrometer) size of the spines. Using this method, the highly heterogeneous velocity of individual actin molecules within the spines has been demonstrated (Figure 4B) (Frost et al., 2010). The same approach has been applied to the study of DNA repair in bacterial cells. When DNA is damaged in a cell, a freely diffusing DNA polymerase (Pol) binds to the damaged site. The spatiotemporal dynamics of DNA repair events have been mapped through high-density tracking of the fluorescently labeled DNA Pol (Figure 4C) (Uphoff et al., 2013). As is evident from these studies, biological processes are precisely regulated by the spatial and temporal dynamics of protein molecules. The tools for quantitative molecular imaging such as cluster analysis, single-molecule counting, and SPT SR microscopy will provide new opportunities to investigate complex biological processes at the molecular level.


Super-resolution molecular and functional imaging of nanoscale architectures in life and materials science.

Habuchi S - Front Bioeng Biotechnol (2014)

Super-resolution fluorescence molecular imaging. (A) PALM image, sptPALM image, and diffusion map of a membrane protein, VSVG, in COS7 cells. A photoconvertable fluorescent protein tdEosFP is fused to VSVG. The PALM image was obtained by the temporal activation of tdEosFP–VSVG followed by the localization and reconstruction of the image. The sptPALM image was obtained by tracking the activated tdEosFP–VSVG molecules. The diffusion map was obtained by calculating the diffusion coefficient of individual tdEosFP–VSVG molecules using the diffusion trajectories (Manley et al., 2008). (B) (Left) the first (red) and last (green) localized positions of single actin molecules within dendritic spines. The positions were determined by the sptPALM imaging. A photoactivatable fluorescent protein PAGFP is fused to actin; (right) actin dynamics within individual spines. Orientation and length of the arrows represent direction and velocity of actin flow. They were calculated using the first and last positions of the diffusion trajectories within the radius of 1 camera pixel (111 nm). Gray scale represents molecular density. Vector = 100 nm/s (Frost et al., 2010). (C) (Left) sptPALM image of a DNA-binding protein, DNA polymerase I (Pol), in E. coli cells in the presence of methyl methanesulfonate (MMS). A photoactivatable fluorescent protein PAmCherry is fused to Pol. Inset shows examples of tracks of diffusing Pol (blue) and bound Pol (red); (right) distribution of diffusion coefficient for Pol under constant MMS treatment (Uphoff et al., 2013). Reproduced with permission from Manley et al. (2008), copyright 2008, Nature Publishing Group (A), Frost et al. (2010), copyright 2010, Elsevier (B), and Uphoff et al. (2013), copyright 2013, National Academy of Science, USA (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Super-resolution fluorescence molecular imaging. (A) PALM image, sptPALM image, and diffusion map of a membrane protein, VSVG, in COS7 cells. A photoconvertable fluorescent protein tdEosFP is fused to VSVG. The PALM image was obtained by the temporal activation of tdEosFP–VSVG followed by the localization and reconstruction of the image. The sptPALM image was obtained by tracking the activated tdEosFP–VSVG molecules. The diffusion map was obtained by calculating the diffusion coefficient of individual tdEosFP–VSVG molecules using the diffusion trajectories (Manley et al., 2008). (B) (Left) the first (red) and last (green) localized positions of single actin molecules within dendritic spines. The positions were determined by the sptPALM imaging. A photoactivatable fluorescent protein PAGFP is fused to actin; (right) actin dynamics within individual spines. Orientation and length of the arrows represent direction and velocity of actin flow. They were calculated using the first and last positions of the diffusion trajectories within the radius of 1 camera pixel (111 nm). Gray scale represents molecular density. Vector = 100 nm/s (Frost et al., 2010). (C) (Left) sptPALM image of a DNA-binding protein, DNA polymerase I (Pol), in E. coli cells in the presence of methyl methanesulfonate (MMS). A photoactivatable fluorescent protein PAmCherry is fused to Pol. Inset shows examples of tracks of diffusing Pol (blue) and bound Pol (red); (right) distribution of diffusion coefficient for Pol under constant MMS treatment (Uphoff et al., 2013). Reproduced with permission from Manley et al. (2008), copyright 2008, Nature Publishing Group (A), Frost et al. (2010), copyright 2010, Elsevier (B), and Uphoff et al. (2013), copyright 2013, National Academy of Science, USA (C).
Mentions: Super-resolution localization microscopy has been further combined with single-particle tracking (SPT). SPT is a powerful tool for characterizing the spatiotemporal behavior of individual molecules, especially the diffusional motion of molecules in nanoscopic heterogeneous structures such as cell membranes (Douglass and Vale, 2005) and microporous materials (Kirstein et al., 2007). However, the method can be applied only to relatively low density mapping of the diffusional behavior of individual molecules. This makes it difficult to connect directly the spatiotemporal behavior of the molecules to the nanoscopic structure of the sample. The combination of SPT and SR localization microscopy allows high-density spatial mapping of the diffusional behavior of individual molecules through tracking single fluorescent probe molecules activated in a temporally controlled manner (Figure 4A) (Manley et al., 2008; Giannone et al., 2010; Rossier et al., 2012). This method has been applied to the study of actin molecule dynamics within dendritic spines. While actin plays numerous roles in synaptic transmission, its spatiotemporal behavior has not been well-characterized due to the small (submicrometer) size of the spines. Using this method, the highly heterogeneous velocity of individual actin molecules within the spines has been demonstrated (Figure 4B) (Frost et al., 2010). The same approach has been applied to the study of DNA repair in bacterial cells. When DNA is damaged in a cell, a freely diffusing DNA polymerase (Pol) binds to the damaged site. The spatiotemporal dynamics of DNA repair events have been mapped through high-density tracking of the fluorescently labeled DNA Pol (Figure 4C) (Uphoff et al., 2013). As is evident from these studies, biological processes are precisely regulated by the spatial and temporal dynamics of protein molecules. The tools for quantitative molecular imaging such as cluster analysis, single-molecule counting, and SPT SR microscopy will provide new opportunities to investigate complex biological processes at the molecular level.

Bottom Line: Super-resolution (SR) fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems.I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging.These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-)nanotechnology.

View Article: PubMed Central - PubMed

Affiliation: Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology , Jeddah , Saudi Arabia.

ABSTRACT
Super-resolution (SR) fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems. In this review, I describe the current state of various SR fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the SR microscopy. I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging. These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-)nanotechnology.

No MeSH data available.