Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation

Tuesday, February 10, 2015 1802-Symp Mechanisms of Pressure Effects in Biology: From Proteins to Live Bacteria Catherine Ann Royer. Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA. Nearly 80% of the terrestrial biosphere is found in the ocean at depths over 1000 meters. Most of this space is dark and cold, such that the organisms that live there have had to adapt to high pressure, low temperature and lack of light. These parameters are known to have an enormous effect on bio-molecular struc- ture and dynamics, and presumably cellular physiology. In particular, pressures of up to 1000 bar are reached in the deepest parts of the ocean. Understanding how organisms adapt to such extreme environments requires and understanding of how pressure effects bio-molecules and the bio-molecular basis of physiolog- ical responses to pressure. We have pursued these questions using model sys- tems and state of the art biophysical approaches. In this talk I will present our recent results on defining the molecular mechanisms of pressure-induced un- folding of proteins and the in vivo response of E. coli to pressure shock. 1803-Symp What Limits Microbial Growth at High Pressure? Doug Bartlett. Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA. Elevated hydrostatic pressure is an important but relatively unstudied thermody- namic parameter that has influenced the evolution and distribution of life on and in Earth. Piezophiles are deep-sea and deep-subsurface microorganisms whose pressure optima for growth exceeds atmospheric pressure. In this presentation multiple lines of inquiry will be described which provide new details on piezo- philes and piezophily. This will include the isolation and characterization of pie- zophilic and piezotolerant microbes from the Challenger Deep within the Mariana Trench and from the Mid-Cayman Rise hydrothermal vent system. Mi- crobes recently isolated which are capable of growth above the known upper pres- sure limit for life (130 megapascals) will be described. Comparative analyses of genomes obtained from single cells extracted from trenches, from pure cultures of piezophiles, and from one deep trench metagenome indicate that life at high pres- sure includes an expansion of regulatory features and of pathways of carbon and energy acquisition. Curiously deep ocean microbes also contain a high proportion of genes encoding aquaporins, channels used for water and solute transport in and out of the cells. The possible significance of this discovery to the interplay be- tween osmotic pressure and hydrostatic pressure will be discussed. Finally, laboratory-based directed evolution experiments will be described in which cells of the mesophile Escherichia coli which are capable of enhanced growth at high pressure and decreased growth at atmospheric pressure have been isolated. Ana- lyses of a mutant derived from this selection indicates that changes in membrane physical state brought about by the introduction of increased proportions of un- saturated fatty acids are necessary but not sufficient for the resulting evolutionary changes driving the cells in the direction of piezophily. The time is now ripe to utilize the tools of biophysics to examine the basis and limits of life at high pressure. Platform: Optical Microscopy and Super-Resolution Imaging II 1804-Plat Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation Luca Lanzano 1 , Ivan Coto Hernandez 1 , Marco Castello 1 , Enrico Gratton 2 , Alberto Diaspro 1 , Giuseppe Vicidomini 1 . Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA. The visualization at the nanoscale level inside cells is a fundamental need in mo- lecular biology. The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribu- tion of the molecules inside the detection volume of the microscope can be en- coded within the fluorescence dynamics and can be decoded by resolving the signal into its dynamics components [1]. We present here a robust and general method, based on the phasor analysis [2], to spatially sort the fluorescent photons on the basis of the associated molecular dynamics and without making use of any fitting procedure. In a specific implementation of this method, we generate spatially controlled gradients in the fluorescence lifetime by stimulated emission [3]. The separation of the time-resolved fluorescence components sorts photons according to their spatial positions. Spatial resolution can be increased indefi- nitely by increasing the number of resolved components up to a maximum, pre- 359a dictable number, determined by the amount of noise. The method also isolates any uncorrelated background signal. We demonstrate that this spectroscopy- based method provides background-free nanoscale imaging of subcellular struc- tures, opening new routes in super-resolution microscopy based on the encoding of spatial information through manipulation of molecular dynamics. We discuss advantages and limitations considering application of the method to the imaging of sparse cytoskeletal structures and large scale organization of chromatin. References [1] Enderlein J. Breaking the diffraction limit with dynamic saturation optical microscopy. Applied Physics Letters 87,094105(2005). [2] Digman MA, Caiolfa VR, Zamai M & Gratton E. The phasor approach to fluorescence lifetime imaging analysis. Biophysical journal 94,L14(2008). [3] Vicidomini G et al. Sharper low-power STED nanoscopy by time gating. Nature methods 8,571(2011). 1805-Plat Investigating Cellular Focal Adhesions on Nano-Patterned Substrates with Dual Color Photo-Activated Localization Microscopy Hendrik G. Deschout 1 , Michelle A. Baird 2 , Michael W. Davidson 2 , Joachim P. Spatz 3 , Aleksandra Radenovic 1 . Institute of Bioengineering, EPFL, Lausanne, Switzerland, 2 Florida State University, Tallahassee, FL, USA, 3 Max Planck Institute for Intelligent Systems, Stuttgart, Germany. It is essential for cells to be able to adhere to, move within, and sense the extra- cellular matrix. Focal adhesions are one of the most important means through which cells achieve this goal. These are sites where trans-membrane proteins called integrins bind to specific peptides in the extracellular matrix. Inside the cells, these integrins are linked to the actin cytoskeleton through other proteins that belong to the focal adhesions. In total, there are at least 150 proteins involved in the assembly and functioning of focal adhesions [1]. This complexity, together with a size in the order of one micron or lower, strongly limits the capability of conventional imaging techniques to resolve the inner structure of focal adhesions. The recently developed single-molecule localization microscopy techniques, such as photo-activated localization microscopy, are more suitable for this pur- pose [2]. Another development that is currently contributing to a more detailed investigation of focal adhesions is the use of nano-engineered substrates as an extracellular matrix [3]. By precisely tuning the chemical and physical properties of such substrates, subtle changes in the behavior of focal adhesions can be pro- voked. Combining both approaches, we use dual color photo-activated localiza- tion microscopy [4] to investigate the co-localization between integrins and other focal adhesion proteins in cells that adhere to substrates with different nano- structured patterns of cyclic arginine-glycine-aspartic peptides [5]. References [1] Zaidel-bar et al. Nature Cell Biology 2007, 9: 858-867. [2] Tabarin et al. ChemPhysChem 2014, 15: 606-618. [3] Geiger et al. Nature Reviews Molecular Cell Biology 2009, 10: 21-33. [4] Annibale et al. Optical Nanoscopy 2012, 1:9. [5] Huang et al. Nano Letters 2009, 9: 1111-1116. 1806-Plat Quantitative Analysis of Nanoscale Lipid Bilayer Modifications via Second Harmonic Generating Probes Erick K. Moen 1 , Hope Beier 2 , Andrea Armani 3 , Bennett Ibey 4 . EE-Electrophysics, University of Southern California, Los Angeles, CA, USA, 2 Radio Frequency Bioeffects Branch, Human Effectiveness Directorate, Air Force Research Laboratory - Fort Sam Houston, San Antonio, TX, USA, 3 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA, 4 Radio Frequency Bioeffects Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Fort Sam Houston, San Antonio, TX, USA. Second Harmonic Generation (SHG) microscopy is an effective tool for study- ing the order of symmetry-breaking interfaces, such as lipid bilayers. Recently, we have shown that disruptions in the interfacial nature of the membrane can be studied with the addition of lipophilic SHG probes, specifically Di-4- ANEPPDHQ (Di-4). In addition to the conformational information provided by the SHG signal, this particular probe can be used to determine lipid phase and transmembrane voltage through the dye’s fluorescence behavior. We now strengthen our technique by changing the polarity of the incident laser beam from linear to circular polarization. The modification provides SHG signal around the circumference of the cell in every optical slice, while main- taining a sufficient signal-to-noise ratio. Utilizing nanosecond pulsed electric fields (nsPEFs) of varying pulse widths and intensities as a tool for provoking spatially and temporally minute perturbations in the cell membrane, we observe membrane poration on the nano-scale. To verify our results, we adapted a