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Super Resolution Microscopy with Induced Optical Fluctuation
Monday, February 29, 2016 874-Symp Towards the Reconstitution of Minimal Cell Division Petra Schwille. Max-Planck-Institut fur Biochemie, Martinsried, Germany. What would be the minimal molecular machinery required to divide a cell? This obviously depends on many factors, such as the size and shape of the cell and the composition of its contents and boundaries. It also depends on the ability of this machinery to self-organize and self-assemble, and to overcome the thermodynamic and kinetic barriers for such a significant morphological transition. Inspired by the divisome of the E.coli bacterial cell, we presently aim to reconstitute the first steps towards the positioning and contraction of a ring-like filamentous structure in the middle of a model cell compartment. In our minimal system, the MinCDE proteins oscillate between the two compartment poles and thereby faithfully position cytoskeletal FtsZ filaments to the center zone of a rod-like membrane compartment. I will discuss how the spatial regulation of the different elements depend on molecular properties of the underlying proteins and the spatial features of the membrane compartment. Building on the insights gained from this model, perspectives to engineer a minimal modular system for cell geometry recognition and regulation will be given.
Platform: Optical Microscropy and SuperResolution Imaging II 875-Plat Towards Single Molecule Imaging of Fluorescence Anisotropy Viviane Devauges, Simon P. Poland, James Monypenny, Anthony H. Keeble, Andrew J. Beavil, Simon M. Ameer-Beg. King’s College London, London, United Kingdom. Given the complexity of biological systems, information on a single molecule basis is crucial to accurately probe molecular properties. In fact, single molecule imaging provides real time conformational dynamics, which can underline heterogeneity in molecular distribution in terms of dipole orientations, spectra or intramolecular distances, in both stable and unstable systems. In addition to these structural data, functional information, in real time, can also be accessed and reaction kinetics determined. Fluorescence anisotropy is a method of contrast, which by quantifying the fluorescence depolarization, can access the fluorophore’s rotational correlation times or quantify energy transfer (e.g. FRET). This is another technique to probe the fluorophore’s environment in terms of viscosity, interactions between molecules and ligand-substrate binding. Here, we present an adapted and optimized Total Internal Reflection Fluorescence (TIRF) microscope combined with steady state fluorescence anisotropy detection for single molecule imaging. Our set-up is used for FRET imaging of GFP/mRFP FRET biosensors by measuring the depolarisation of the acceptor upon donor excitation. Preliminary data aiming towards single molecule imaging with a fluorescence anisotropy read-out, made by measurements on purified Immunoglobilin E (IgE) FRET biosensors, are presented and future prospects discussed. 876-Plat A Method for Estimating Unknown Parameters from Particle Tracking Experiments Trevor T. Ashley, Sean B. Andersson. Mechanical Engineering, Boston University, Boston, MA, USA. Optimal estimation of motion parameters (e.g. diffusion coefficients) from single and multiple particle tracking data has developed substantially over the past several years. The standard approach for estimating these parameters typically involves first localizing the particle(s) within each frame, assembling a timeseries of estimated positions, calculating the mean squared displacement (MSD), and finally fitting a known function to the resulting MSD. It has been shown, however, that this approach may lead to erroneous results due the potential subjectivity of the fit [1]. Recently, a powerful method based on the Maximum Likelihood framework was described in [2]. This method, however, cannot estimate parameters other than diffusion coefficients, examples of which include confinement lengths and tether stiffnesses. In addition, the aforementioned method dissociates the localization procedure from the estimation procedure which are inherently coupled processes. In this work, we present a technique which leverages recent work from [3] in which the Expectation Maximization (EM) algorithm is used in conjunction with Sequential Monte Carlo (SMC) methods to simultaneously estimate unknown fixed parameters in addition to time-varying states. Here, we demonstrate the applicability of this technique to the problem of tracking a moving particle undergoing complex modes of motion, including confined diffusion and elastic tethering. [1] M. J. Saxton. ‘‘Single-Particle Tracking: The Distribution of Diffusion Coefficients.’’ Biophysical Journal, vol. 72, no. 4, pp. 1744-1753, 1997.
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[2] X. Michalet and A. J. Berglund. ‘‘Optimal Estimation of Diffusion Coefficients.’’ Physical Review E, vol. 85, 2012. [3] T. B. Scho¨n, A. Wills, B. Ninness. ‘‘System Identification of Nonlinear State--Space Models.’’ Automatica, vol. 47, no. 1, pp. 39-49, 2011. 877-Plat Super Resolution Microscopy with Induced Optical Fluctuation MinKwan Kim, Chung-Hyun Park, Yong-Hoon Cho, YongKeun Park. Korea Advanced Institute of Science and Technology, Daejeon, Korea, Republic of. In conventinal microscopy, resolution is fundamentally limited by diffraction barrier. However, as phenomena within size of diffraction limit become important in many field, diffraction barrier becomes a big issue in conventional microscopy. To overcome this, many researchers have developed super-resolution methods using physical principles. Among them, lately, Super Resolution Optical fluctuation Imaging (SOFI) method developed by T. Dertinger et al, provides super-resolution image using statistical analysis of temporal optical fluctuation that comes from the blinking property of fluorophores. Because SOFI only requires opical fluctuation of fluorophores without any special equipment, it can be directly applied to conventional microscopy for superresolution imaging. However, because intrinsic blinking property of fluorophores is uncontrollable, SOFI has practical challenges and limitations in some applications, including the limitation of available fluorophores and camera speed. To overcome these limitations, we proposed a new concept of SOFI using optical fluctuation induced by the illumination light with random patterns. Because the optical flcutuation does not come from intrinsic blinking property of fluorophore, but comes from the illumination light in this method, the blinking becomes a controllable property using external method. We employ speckle patterns as random patterns because speckle pattern has interesting statistical properties such as correlation. To verify the super-resolution capability of our proposed method, simple model simulation with only two flurophores was analytically solved using equations of SOFI and speckle pattern statistics. Also, to experimentally demonstrate it, diffuser and motorized stage were added in conventional microscopy to generate speckle patterns and statistical properties of speckle patterns. Using simulation and experiment, we theoretically and experimentally demonstrate the super-resolution imaging capability of our proposed method. Furthermore, proposed method gives superresolution image of samples labelled with non-blinking fluorophores. 878-Plat Ultra-High Resolution Three Dimensional Imaging using 4Pi-SMSN throughout Whole Cells Fang Huang1,2, George Sirinakis1, Edward S. Allgeyer1, Lena Schroeder1, Whitney C. Duim1, Joerg Bewersdorf1,3. 1 Department of Cell Biology, Yale University, New Haven, CT, USA, 2 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA, 3Department of Biomedical Engineering, Yale University, New Haven, CT, USA. Major advances in biology have been tightly linked with innovations in microscopy. A major hurdle over the last ~100 years is the limited resolution of light microscopy. The advent of single molecule switching nanoscopy (SMSN, also known as PALM/STORM/FPALM) has overcome this fundamental limit by improving the resolution of fluorescence microscopy (250-700 nm) by a factor of ten. This method routinely achieves 20-40 nm lateral resolution and 50-80 nm resolution in the axial direction. While the inferior axial resolution largely restricts the biological discoveries to two-dimensional observations, interferometric SMSN (iPALM or 4Pi-SMSN) achieves unprecedented 10 to 20 nm axial resolution by coherently combining single-molecule emissions in a two opposing-objective setup. It allowed, for the first time, the molecular anatomy of focal adhesions to be mapped with nanometer precision. However, the physical principle of 4Pi geometry limits this approach to isolated structures in thin samples because the single molecule interference pattern repeats every 250 nm in depth. While most of biological processes happen deep in the cellular volume, driving ultra-high resolution imaging deeper into the cell will lead to a new wave of biological discoveries. Here, we present whole-cell 4Pi-SMSN resulting from the confluence of multiple innovations. Our system, for the first time, allowed super-resolution imaging of a ~10 mm thick sample using 4Pi geometry achieving 10-15 nm resolution throughout the depth. This resolution is 20-50 times higher than conventional microscopy with imaging depth improved by 10-40 fold from the state of art technology of interferometric SMSN. It enables ultra-high resolution three-dimensional imaging for vast majority of the subcellular structures. We demonstrate applications in a range of delicate cellular structures including: bacteriophages, ER, mitochondria, nuclear pore complexes, primary cilia, Golgi complex-associated COPI vesicles, and synaptonemal complexes in whole mouse spermatocytes.
Platform: Optical Microscropy and SuperResolution Imaging II 875-Plat Towards Single Molecule Imaging of Fluorescence Anisotropy Viviane Devauges, Simon P. Poland, James Monypenny, Anthony H. Keeble, Andrew J. Beavil, Simon M. Ameer-Beg. King’s College London, London, United Kingdom. Given the complexity of biological systems, information on a single molecule basis is crucial to accurately probe molecular properties. In fact, single molecule imaging provides real time conformational dynamics, which can underline heterogeneity in molecular distribution in terms of dipole orientations, spectra or intramolecular distances, in both stable and unstable systems. In addition to these structural data, functional information, in real time, can also be accessed and reaction kinetics determined. Fluorescence anisotropy is a method of contrast, which by quantifying the fluorescence depolarization, can access the fluorophore’s rotational correlation times or quantify energy transfer (e.g. FRET). This is another technique to probe the fluorophore’s environment in terms of viscosity, interactions between molecules and ligand-substrate binding. Here, we present an adapted and optimized Total Internal Reflection Fluorescence (TIRF) microscope combined with steady state fluorescence anisotropy detection for single molecule imaging. Our set-up is used for FRET imaging of GFP/mRFP FRET biosensors by measuring the depolarisation of the acceptor upon donor excitation. Preliminary data aiming towards single molecule imaging with a fluorescence anisotropy read-out, made by measurements on purified Immunoglobilin E (IgE) FRET biosensors, are presented and future prospects discussed. 876-Plat A Method for Estimating Unknown Parameters from Particle Tracking Experiments Trevor T. Ashley, Sean B. Andersson. Mechanical Engineering, Boston University, Boston, MA, USA. Optimal estimation of motion parameters (e.g. diffusion coefficients) from single and multiple particle tracking data has developed substantially over the past several years. The standard approach for estimating these parameters typically involves first localizing the particle(s) within each frame, assembling a timeseries of estimated positions, calculating the mean squared displacement (MSD), and finally fitting a known function to the resulting MSD. It has been shown, however, that this approach may lead to erroneous results due the potential subjectivity of the fit [1]. Recently, a powerful method based on the Maximum Likelihood framework was described in [2]. This method, however, cannot estimate parameters other than diffusion coefficients, examples of which include confinement lengths and tether stiffnesses. In addition, the aforementioned method dissociates the localization procedure from the estimation procedure which are inherently coupled processes. In this work, we present a technique which leverages recent work from [3] in which the Expectation Maximization (EM) algorithm is used in conjunction with Sequential Monte Carlo (SMC) methods to simultaneously estimate unknown fixed parameters in addition to time-varying states. Here, we demonstrate the applicability of this technique to the problem of tracking a moving particle undergoing complex modes of motion, including confined diffusion and elastic tethering. [1] M. J. Saxton. ‘‘Single-Particle Tracking: The Distribution of Diffusion Coefficients.’’ Biophysical Journal, vol. 72, no. 4, pp. 1744-1753, 1997.
175a
[2] X. Michalet and A. J. Berglund. ‘‘Optimal Estimation of Diffusion Coefficients.’’ Physical Review E, vol. 85, 2012. [3] T. B. Scho¨n, A. Wills, B. Ninness. ‘‘System Identification of Nonlinear State--Space Models.’’ Automatica, vol. 47, no. 1, pp. 39-49, 2011. 877-Plat Super Resolution Microscopy with Induced Optical Fluctuation MinKwan Kim, Chung-Hyun Park, Yong-Hoon Cho, YongKeun Park. Korea Advanced Institute of Science and Technology, Daejeon, Korea, Republic of. In conventinal microscopy, resolution is fundamentally limited by diffraction barrier. However, as phenomena within size of diffraction limit become important in many field, diffraction barrier becomes a big issue in conventional microscopy. To overcome this, many researchers have developed super-resolution methods using physical principles. Among them, lately, Super Resolution Optical fluctuation Imaging (SOFI) method developed by T. Dertinger et al, provides super-resolution image using statistical analysis of temporal optical fluctuation that comes from the blinking property of fluorophores. Because SOFI only requires opical fluctuation of fluorophores without any special equipment, it can be directly applied to conventional microscopy for superresolution imaging. However, because intrinsic blinking property of fluorophores is uncontrollable, SOFI has practical challenges and limitations in some applications, including the limitation of available fluorophores and camera speed. To overcome these limitations, we proposed a new concept of SOFI using optical fluctuation induced by the illumination light with random patterns. Because the optical flcutuation does not come from intrinsic blinking property of fluorophore, but comes from the illumination light in this method, the blinking becomes a controllable property using external method. We employ speckle patterns as random patterns because speckle pattern has interesting statistical properties such as correlation. To verify the super-resolution capability of our proposed method, simple model simulation with only two flurophores was analytically solved using equations of SOFI and speckle pattern statistics. Also, to experimentally demonstrate it, diffuser and motorized stage were added in conventional microscopy to generate speckle patterns and statistical properties of speckle patterns. Using simulation and experiment, we theoretically and experimentally demonstrate the super-resolution imaging capability of our proposed method. Furthermore, proposed method gives superresolution image of samples labelled with non-blinking fluorophores. 878-Plat Ultra-High Resolution Three Dimensional Imaging using 4Pi-SMSN throughout Whole Cells Fang Huang1,2, George Sirinakis1, Edward S. Allgeyer1, Lena Schroeder1, Whitney C. Duim1, Joerg Bewersdorf1,3. 1 Department of Cell Biology, Yale University, New Haven, CT, USA, 2 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA, 3Department of Biomedical Engineering, Yale University, New Haven, CT, USA. Major advances in biology have been tightly linked with innovations in microscopy. A major hurdle over the last ~100 years is the limited resolution of light microscopy. The advent of single molecule switching nanoscopy (SMSN, also known as PALM/STORM/FPALM) has overcome this fundamental limit by improving the resolution of fluorescence microscopy (250-700 nm) by a factor of ten. This method routinely achieves 20-40 nm lateral resolution and 50-80 nm resolution in the axial direction. While the inferior axial resolution largely restricts the biological discoveries to two-dimensional observations, interferometric SMSN (iPALM or 4Pi-SMSN) achieves unprecedented 10 to 20 nm axial resolution by coherently combining single-molecule emissions in a two opposing-objective setup. It allowed, for the first time, the molecular anatomy of focal adhesions to be mapped with nanometer precision. However, the physical principle of 4Pi geometry limits this approach to isolated structures in thin samples because the single molecule interference pattern repeats every 250 nm in depth. While most of biological processes happen deep in the cellular volume, driving ultra-high resolution imaging deeper into the cell will lead to a new wave of biological discoveries. Here, we present whole-cell 4Pi-SMSN resulting from the confluence of multiple innovations. Our system, for the first time, allowed super-resolution imaging of a ~10 mm thick sample using 4Pi geometry achieving 10-15 nm resolution throughout the depth. This resolution is 20-50 times higher than conventional microscopy with imaging depth improved by 10-40 fold from the state of art technology of interferometric SMSN. It enables ultra-high resolution three-dimensional imaging for vast majority of the subcellular structures. We demonstrate applications in a range of delicate cellular structures including: bacteriophages, ER, mitochondria, nuclear pore complexes, primary cilia, Golgi complex-associated COPI vesicles, and synaptonemal complexes in whole mouse spermatocytes.