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Heterogeneity of the Nuclear Environment Investigated by Superresolution Microscopy and Fluorescence Correlation Spectroscopy
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Sunday, February 12, 2017
multimode fiber and a speckle reducer. This simple illumination achieves a very high uniformity and results in homogeneous measured localization precisions across the field of view, removing statistical bias from quantitative studies. Finally, we present the use of adaptive optics to improve the 3D resolution of supercritical angle localization microscopy (SALM), with the aim to reach isotropic localization precision in the first few hundreds of nanometers above the coverslip. Altogether, these improvements open the way to better sampled and more precise structural studies in superresolution microscopy.
697-Pos Board B462 Versatile Super-Resolution Calibration Standard for Quantifying Protein Copy Number Francesca Cella Zanacchi1, Carlo Manzo2, Angel Sandoval Alvarez3, Nathan D. Derr4, Maria Garcia Parajo2, Melike Lakadamyali1. 1 AFIB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 2 SMB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 3 ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 4Smith College, Northampton, MD, USA. Single molecule based super-resolution microscopy offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. While fluorescent proteins have been extensively characterized for quantitative imaging using calibration standards, similar calibration tools for small organic fluorophores used in conjunction with immunofluorescence are lacking. Within this framework, the development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution techniques such as stochastic optical reconstruction microscopy (STORM). The development of a suitable calibration method represents the best way to address the challenges of molecular counting using superresolution [3,4]. Within this project, we demonstrate that DNA origami in combination with GFP antibodies is a versatile platform for quantifying protein copy number in immunofluorescence based super-resolution microscopy. We show that this calibration method, besides quantifying the average protein copy number in a cell, allows determining the abundance of various oligomeric states. Furthermore, we apply this calibration method to quantify nucleoporins (NUP107) [5] and molecular motors (dynein intermediate chain) [6] in vivo. Overall, we provide a versatile strategy for quantifying a large number of proteins of interest using various labeling approaches. 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62. 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21. 3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91. 4. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016) 5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by SuperResolution Microscopy and Particle Averaging. Science, 2013. 341(6146): p. 655-658. 6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science, 2012. 338(6107): p. 662-5.
698-Pos Board B463 Heterogeneity of the Nuclear Environment Investigated by Superresolution Microscopy and Fluorescence Correlation Spectroscopy Luca Lanzano0 1, Melody Di Bona1,2, Lorenzo Scipioni1,3, Maria J. Sarmento1, Enrico Gratton4, Giuseppe Vicidomini5, Alberto Diaspro1,2. 1 Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2 Department of Physics, University of Genoa, Genoa, Italy, 3Department of Computer Science, Bioengineering, Robotics and Systems Engineering, University of Genoa, Genoa, Italy, 4Biomedical Engineering, University of California, Irvine, CA, USA, 5Molecular Spectroscopy and Microscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy. Genomes are more than one-dimensional entities purely defined by their linear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge
in biology is to decipher the principles of organization of what can be considered, by analogy with human-created libraries, the cell’s primary unit of information storage and retrieval. In this respect, the development of superresolved fluorescence microscopy has provided a new toolbox to peer into the nucleus. For instance, super-resolution microscopy has been recently applied to the investigation of nanoscale chromatin architecture, revealing nucleosome higher-order organization into heterogeneous ‘clutches’ and epigenetically dependent folding motives [Ricci M et al, Cell, (2015); Boettiger A et al, Nature (2016)]. However, dynamic properties of proteins in the nucleus are also critical for their function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy (FCS) has been used to map the dynamics of several proteins in the nucleus with diffraction-limited spatial resolution. Here we combine a novel STEDbased super-resolution method [Lanzano’ L et al, Nat Commun (2015)] with FCS to measure protein dynamics in the nucleus with an improved spatial resolution of about 100 nm. We sample several positions within the nucleus by performing line scanning. The measured spatial and temporal heterogeneity of the dynamics, quantified by a recently introduced algorithm [Scipioni L et al, Biophys J (2016)], is discussed in relation to nuclear organization. 699-Pos Board B464 Analysis of Fibrous Spatial Point Patterns from Single-Molecule SuperResolution Microscopy Data Ruby Peters, Dylan Owen, Juliette Griffie. King’s College London, London, United Kingdom. Unlike conventional microscopy methods which generate pixelated images, SMLM techniques produce images comprising of a list of molecular coordinates - a spatial point pattern (SPP). SMLM methods therefore necessitate a statistical approach to data analysis. SPPs are routinely analyzed using robust cluster analysis algorithms to quantify molecular clustering across diverse biological systems. The analysis of fibrous SPPs however, such as those derived from components of the cytoskeleton, remains relatively understudied. Here, we present statistical methodology based upon a variant of Ripley’s K function to quantitatively assess fibrous patterns generated by SMLM. We demonstrate the technique using various simulated fibrous spatial arrangements and extract spatial descriptors of the pointillist input. We further demonstrate the technique on experimental data acquired using the image reconstruction by integrating exchangeable single-molecule localization (IRIS) approach to SMLM. We quantitatively assess the fibrous distribution of filamentous actin at the T cell immunological synapse, whose structure has been shown to be important for cell morphology, polarization and activation. 700-Pos Board B465 Expanding the Spectral Resolution of Single-Molecule Localization Microscopy with Bodipy-Based Photoswitchable Fluorophores Amy M. Bittel, Ashley Davis, Tao Huang, Xiaolin Nan, Summer L. Gibbs. Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA. Single-molecule localization microscopy (SMLM) has become an important tool for studying molecular biology. Through the use of photoswitchable fluorophores, SMLM can accurately localize individual molecules with 10-20 nm resolution, an order of magnitude better than conventional fluorescence microscopy. While SMLM succeeds in locating individual molecules, it is limited to 4-color emission based imaging due to the standard bandpass filter technology used to generate multicolor images, restricting the number of molecular entities that can be simultaneously localized in a single sample. The development of multi-spectral superresolution microscopy (MSSRM) improves the spectral resolution of SMLM enabling up to 20 color imaging in a single sample. However, MSSRM is currently restricted by the spectrally limited conventional fluorophores with adequate photoswitching for SMLM. Herein, we designed, synthesized and validated a series of novel BODIPY-based fluorophores with appropriate photoswitching for SMLM that span the visible spectrum permitting high-resolution, multi-color images using our MSSRM. The BODIPY-based probes were selected from a 110 member library of compounds synthesized through modification of a core BODIPY FL scaffold with diverse aromatic rings using a solid phase synthetic platform. All BODIPY-based probes were characterized for absorption and emission properties as well as key photoswitching properties to facilitate
Sunday, February 12, 2017
multimode fiber and a speckle reducer. This simple illumination achieves a very high uniformity and results in homogeneous measured localization precisions across the field of view, removing statistical bias from quantitative studies. Finally, we present the use of adaptive optics to improve the 3D resolution of supercritical angle localization microscopy (SALM), with the aim to reach isotropic localization precision in the first few hundreds of nanometers above the coverslip. Altogether, these improvements open the way to better sampled and more precise structural studies in superresolution microscopy.
697-Pos Board B462 Versatile Super-Resolution Calibration Standard for Quantifying Protein Copy Number Francesca Cella Zanacchi1, Carlo Manzo2, Angel Sandoval Alvarez3, Nathan D. Derr4, Maria Garcia Parajo2, Melike Lakadamyali1. 1 AFIB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 2 SMB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 3 ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 4Smith College, Northampton, MD, USA. Single molecule based super-resolution microscopy offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. While fluorescent proteins have been extensively characterized for quantitative imaging using calibration standards, similar calibration tools for small organic fluorophores used in conjunction with immunofluorescence are lacking. Within this framework, the development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution techniques such as stochastic optical reconstruction microscopy (STORM). The development of a suitable calibration method represents the best way to address the challenges of molecular counting using superresolution [3,4]. Within this project, we demonstrate that DNA origami in combination with GFP antibodies is a versatile platform for quantifying protein copy number in immunofluorescence based super-resolution microscopy. We show that this calibration method, besides quantifying the average protein copy number in a cell, allows determining the abundance of various oligomeric states. Furthermore, we apply this calibration method to quantify nucleoporins (NUP107) [5] and molecular motors (dynein intermediate chain) [6] in vivo. Overall, we provide a versatile strategy for quantifying a large number of proteins of interest using various labeling approaches. 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62. 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21. 3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91. 4. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016) 5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by SuperResolution Microscopy and Particle Averaging. Science, 2013. 341(6146): p. 655-658. 6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science, 2012. 338(6107): p. 662-5.
698-Pos Board B463 Heterogeneity of the Nuclear Environment Investigated by Superresolution Microscopy and Fluorescence Correlation Spectroscopy Luca Lanzano0 1, Melody Di Bona1,2, Lorenzo Scipioni1,3, Maria J. Sarmento1, Enrico Gratton4, Giuseppe Vicidomini5, Alberto Diaspro1,2. 1 Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2 Department of Physics, University of Genoa, Genoa, Italy, 3Department of Computer Science, Bioengineering, Robotics and Systems Engineering, University of Genoa, Genoa, Italy, 4Biomedical Engineering, University of California, Irvine, CA, USA, 5Molecular Spectroscopy and Microscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy. Genomes are more than one-dimensional entities purely defined by their linear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge
in biology is to decipher the principles of organization of what can be considered, by analogy with human-created libraries, the cell’s primary unit of information storage and retrieval. In this respect, the development of superresolved fluorescence microscopy has provided a new toolbox to peer into the nucleus. For instance, super-resolution microscopy has been recently applied to the investigation of nanoscale chromatin architecture, revealing nucleosome higher-order organization into heterogeneous ‘clutches’ and epigenetically dependent folding motives [Ricci M et al, Cell, (2015); Boettiger A et al, Nature (2016)]. However, dynamic properties of proteins in the nucleus are also critical for their function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy (FCS) has been used to map the dynamics of several proteins in the nucleus with diffraction-limited spatial resolution. Here we combine a novel STEDbased super-resolution method [Lanzano’ L et al, Nat Commun (2015)] with FCS to measure protein dynamics in the nucleus with an improved spatial resolution of about 100 nm. We sample several positions within the nucleus by performing line scanning. The measured spatial and temporal heterogeneity of the dynamics, quantified by a recently introduced algorithm [Scipioni L et al, Biophys J (2016)], is discussed in relation to nuclear organization. 699-Pos Board B464 Analysis of Fibrous Spatial Point Patterns from Single-Molecule SuperResolution Microscopy Data Ruby Peters, Dylan Owen, Juliette Griffie. King’s College London, London, United Kingdom. Unlike conventional microscopy methods which generate pixelated images, SMLM techniques produce images comprising of a list of molecular coordinates - a spatial point pattern (SPP). SMLM methods therefore necessitate a statistical approach to data analysis. SPPs are routinely analyzed using robust cluster analysis algorithms to quantify molecular clustering across diverse biological systems. The analysis of fibrous SPPs however, such as those derived from components of the cytoskeleton, remains relatively understudied. Here, we present statistical methodology based upon a variant of Ripley’s K function to quantitatively assess fibrous patterns generated by SMLM. We demonstrate the technique using various simulated fibrous spatial arrangements and extract spatial descriptors of the pointillist input. We further demonstrate the technique on experimental data acquired using the image reconstruction by integrating exchangeable single-molecule localization (IRIS) approach to SMLM. We quantitatively assess the fibrous distribution of filamentous actin at the T cell immunological synapse, whose structure has been shown to be important for cell morphology, polarization and activation. 700-Pos Board B465 Expanding the Spectral Resolution of Single-Molecule Localization Microscopy with Bodipy-Based Photoswitchable Fluorophores Amy M. Bittel, Ashley Davis, Tao Huang, Xiaolin Nan, Summer L. Gibbs. Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA. Single-molecule localization microscopy (SMLM) has become an important tool for studying molecular biology. Through the use of photoswitchable fluorophores, SMLM can accurately localize individual molecules with 10-20 nm resolution, an order of magnitude better than conventional fluorescence microscopy. While SMLM succeeds in locating individual molecules, it is limited to 4-color emission based imaging due to the standard bandpass filter technology used to generate multicolor images, restricting the number of molecular entities that can be simultaneously localized in a single sample. The development of multi-spectral superresolution microscopy (MSSRM) improves the spectral resolution of SMLM enabling up to 20 color imaging in a single sample. However, MSSRM is currently restricted by the spectrally limited conventional fluorophores with adequate photoswitching for SMLM. Herein, we designed, synthesized and validated a series of novel BODIPY-based fluorophores with appropriate photoswitching for SMLM that span the visible spectrum permitting high-resolution, multi-color images using our MSSRM. The BODIPY-based probes were selected from a 110 member library of compounds synthesized through modification of a core BODIPY FL scaffold with diverse aromatic rings using a solid phase synthetic platform. All BODIPY-based probes were characterized for absorption and emission properties as well as key photoswitching properties to facilitate