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3064-Pos Board B756 Optically Modulated Fluorescent Proteins Enhance Sensitivity in Live Cell Imaging Amy E. Jablonski 1 , Irina Issaeva 1 , Yen-Cheng Chen 1 , Jung-Cheng Hsiang 1 , Russell B. Vegh 1 , Pritha Bagchi 1 , Bettina Bommarius 2 , Andreas S. Bommarius 2 , Laren M. Tolbert 1 , Christoph J. Fahrni 1 , Robert M. Dickson 1 . 1 Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA, 2 Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA. Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular structure and processes. Although the palette of fluorescent pro- teins has revolutionized detection and dynamics in molecular and cellular biology, the limited brightness, low spectral discrimination, and high cellular autofluorescence continue to limit applications. In contrast to photoswitch- based methods, we have developed a spectroscopic method to recover fluores- cence from photoaccessible dark states via long-wavelength secondary laser co-illumination. We selectively recover the higher energy fluorescence by modulating this secondary laser without modulating background autofluores- cence. Using hypothesized photoreversible isomerizations of the chromophore, we have identified specific fluorescent protein mutants, ranging from blue to red, capable of optical modulation. Employing these methods, we have demon- strated the ability to recover the modulated signal, with >10-fold improvement, from background in live cells. Such modulation schemes enable new imaging modalities for probing intracellular kinetics and equilibria of low-abundance proteins. 3065-Pos Board B757 Mechanisms of Multiphoton Bleaching of Red Fluorescent Proteins Mikhail Drobizhev 1 , Caleb Stoltzfus 1 , Thomas Hughes 2 , Igor Topol 3 , Lauren M. Barnett 2 , Geoffrey R. Wicks 1 , Aleksander Rebane 1 . 1 Physics, Montana State Univ, Bozeman, MT, USA, 2 Cell Biology and Neuroscience, Montana State Univ, Bozeman, MT, USA, 3 SAIC-Frederick, Inc., Frederick, MD, USA. Two-photon laser scanning microscopy (TPLSM) has several advantages over one-photon confocal microscopy, including deeper tissue penetration, higher signal-to-background ratio, and less photodamage in the out-of-focus volume. However, due to very high instantaneous light intensities in the focal volume, the probability of further, stepwise resonant photon(s) absorption increases dramatically, leading to very efficient bleaching of a probe. To deal with this challenge one has to understand the underlying mechanisms. Here we measured the power dependence of multiphoton bleaching rates of several red fluorescent proteins expressed in live E. coli cells under two-photon microscope condi- tions. To clarify the photophysical mechanisms, we also used much lower repe- tition rate (1 kHz) and different pulse durations in experiments in vitro with Ti:Sa amplifier excitation. Our experimental data supported by quantum mechanical calculations of the chromophore in protein environment are consis- tent with the mechanism of ultrafast (<150 fs) singlet-singlet stepwise absorp- tion of one or two additional photons following initial simultaneous two-photon absorption. In the DsRed2 protein, the third photon absorption most probably results in an ultrafast electron transfer (ET) from the anionic chromophore to an excited Rydberg state of a nearby positive amino acid residue (e.g. K163þ). The transient radical state of the chromophore tends to accept an elec- tron from the deprotonated E215- amino acid, thus promoting the first step of the recently established decarboxylation reaction. In mFruits proteins, the third photon promotes an ET from the chromophore to the 4S Rydberg state of nearby K70þ residue with its subsequent photoionization by the fourth photon. 3066-Pos Board B758 Quantitative Imaging of Protein Secretions from Single Cells in Real Time Marc Raphael, Joseph Christodoulides, James Delehanty, James Long, Jeff Byers. Naval Research Laboratory, Washington, DC, USA. Cell-to-cell signaling often involves the secretion of proteins, which creates spatial and temporal concentration profiles in the extra-cellular environment for the receiving cells to detect and interpret. Real-time measurements of secreted protein concentrations at the single cell level are thus fundamental to understanding these communications pathways but have proven difficult to realize in practice. Here we present a label-free technique based upon nanoplas- monic imaging and high-affinity binding which enabled the measurement of individual cell secretions in real time. When applied to the detection of anti- body secretions from individual hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell. The technique is incorporated on to a wide-field microscope which enables real-time transmitted light and fluorescence imaging of the cells as well. We anticipate this technique will be broadly applicable to the real-time characterization of both paracrine and autocrine signaling pathways. 3067-Pos Board B759 A Fluorescence Approach to Discrimination of Aggregated Amyloid Proteins Kevin J. Cao 1 , Kristyna Elbel 1 , Christina Sigurdson 2 , Emmanuel Theodorakis 1 , Jerry Yang 1 . 1 Chemistry & Biochemistry, UC San Diego, La Jolla, CA, USA, 2 Pathology, UC San Diego, La Jolla, CA, USA. We present the design and synthesis of a new class of fluorophores with sensi- tivity to both environment viscosity as well as environment polarity, a property known as solvatochromism. We recently reported the ability of these fluoro- phores to discriminate, based on fluorescence emission, protein amyloid aggre- gate deposition in ex vivo neuronal tissue (Cao et al., J. Am. Chem. Soc., 2012, 134, 17338-17341). Here, we have further modeled the solvatochromic rela- tionship of these probes with environmental permittivity and have used these results to extrapolate the permittivity values of the probe binding pockets within several amyloid proteins. Additionally, we have begun to examine the utility of these fluorophores as potential diagnostic tools for detection of neuro- degenerative pathologies. 3068-Pos Board B760 Live Synaptic Mapping of Vertebrate Whole Brain with Light Sheet Microscopy and Endogenously Labeled Synapsin-2B Protein Andrey Andreev, John M. Choi, Le A. Trinh, Thai V. Truong, Scott E. Fraser. Translational Imaging Center, University of Southern California, Los Angeles, CA, USA. A synaptic map of vertebrate brain is a crucial step for understanding the molecular basis for neuronal networks development and modification. How- ever, conventional two-photon, point-scanning microscopy presents challenges for in vivo, whole-animal studies of brain synaptic structure and dynamics in higher organism due to reduced axial resolution, and inadequate temporal res- olution for developmental or behavioral studies, as well as excessive photo- damage to the sample. Application of two-photon, light sheet microscopy overcomes these barriers, approaching isotropic 3D resolution and faster volume scan speed (>10x), while simultaneously reducing phototoxic effects. Using a protein trap zebrafish line, in which Synapsin-2B is expressed as a fluo- rescent fusion protein from the endogenous locus, combined with two-photon light sheet microscopy enables live, synaptic mapping of the whole vertebrate brain. This work provides the basis for further research in imaging brain dynamics and development, for example fast, high-resolution whole-brain activity imaging. 3069-Pos Board B761 A Multi-Emitter Localization Comparison of 3D Superresolution Imaging Modalities Sheng Liu, Keith A. Lidke. University of New Mexico, Albuquerque, NM, USA. Single-molecule localization-based superresolution imaging is complicated by emission from multiple emitters overlapping at the detector. The potential for overlapping emitters is even greater for 3D imaging than for 2D imaging due to the large effective ‘volume’ of the 3D point spread function (PSF). Overlapping emission can be accounted for in the estimation model, recovering the ability to localize the emitters, but with the caveat that the localization precision has a dependence on the amount of overlap from other emitters. It is interesting to consider if a particular 3D imaging modality has a significant advantage in facilitating the position estimation of overlapping emitters. We compare vari- ants of two commonly used and easily implemented imaging modalities for 3D single-molecule imaging: astigmatic imaging; dual focal plane imaging; and the combination of the two approaches dual focal plane with astigmatism. We quantify the ability for a particular 3D modality to facilitate estimation with overlapping emitters using the Crame ´r-Rao lower bound (CRLB). The CRLB is used to calculate the theoretical best localization precision under a multi-emitter estimation model. We investigate the performance of these 3D modalities under a wide range of conditions including various distributions of collected photons per frame, background counts, pixel sizes, and camera read-out noise values. Tuesday, February 18, 2014 607a

Optically Modulated Fluorescent Proteins Enhance Sensitivity in Live Cell Imaging

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3064-Pos Board B756Optically Modulated Fluorescent Proteins Enhance Sensitivity in Live CellImagingAmy E. Jablonski1, Irina Issaeva1, Yen-Cheng Chen1, Jung-Cheng Hsiang1,Russell B. Vegh1, Pritha Bagchi1, Bettina Bommarius2,Andreas S. Bommarius2, Laren M. Tolbert1, Christoph J. Fahrni1,Robert M. Dickson1.1Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA,USA, 2Chemical & Biomolecular Engineering, Georgia Institute ofTechnology, Atlanta, GA, USA.Fluorescence microscopy is a widely used non-invasive tool for investigatingintracellular structure and processes. Although the palette of fluorescent pro-teins has revolutionized detection and dynamics in molecular and cellularbiology, the limited brightness, low spectral discrimination, and high cellularautofluorescence continue to limit applications. In contrast to photoswitch-based methods, we have developed a spectroscopic method to recover fluores-cence from photoaccessible dark states via long-wavelength secondary laserco-illumination. We selectively recover the higher energy fluorescence bymodulating this secondary laser without modulating background autofluores-cence. Using hypothesized photoreversible isomerizations of the chromophore,we have identified specific fluorescent protein mutants, ranging from blue tored, capable of optical modulation. Employing these methods, we have demon-strated the ability to recover the modulated signal, with>10-fold improvement,from background in live cells. Such modulation schemes enable new imagingmodalities for probing intracellular kinetics and equilibria of low-abundanceproteins.

3065-Pos Board B757Mechanisms of Multiphoton Bleaching of Red Fluorescent ProteinsMikhail Drobizhev1, Caleb Stoltzfus1, Thomas Hughes2, Igor Topol3,Lauren M. Barnett2, Geoffrey R. Wicks1, Aleksander Rebane1.1Physics, Montana State Univ, Bozeman, MT, USA, 2Cell Biology andNeuroscience, Montana State Univ, Bozeman, MT, USA, 3SAIC-Frederick,Inc., Frederick, MD, USA.Two-photon laser scanning microscopy (TPLSM) has several advantages overone-photon confocal microscopy, including deeper tissue penetration, highersignal-to-background ratio, and less photodamage in the out-of-focus volume.However, due to very high instantaneous light intensities in the focal volume,the probability of further, stepwise resonant photon(s) absorption increasesdramatically, leading to very efficient bleaching of a probe. To deal with thischallenge one has to understand the underlying mechanisms. Here we measuredthe power dependence of multiphoton bleaching rates of several red fluorescentproteins expressed in live E. coli cells under two-photon microscope condi-tions. To clarify the photophysical mechanisms, we also used much lower repe-tition rate (1 kHz) and different pulse durations in experiments in vitro withTi:Sa amplifier excitation. Our experimental data supported by quantummechanical calculations of the chromophore in protein environment are consis-tent with the mechanism of ultrafast (<150 fs) singlet-singlet stepwise absorp-tion of one or two additional photons following initial simultaneous two-photonabsorption. In the DsRed2 protein, the third photon absorption most probablyresults in an ultrafast electron transfer (ET) from the anionic chromophore toan excited Rydberg state of a nearby positive amino acid residue (e.g.K163þ). The transient radical state of the chromophore tends to accept an elec-tron from the deprotonated E215- amino acid, thus promoting the first step ofthe recently established decarboxylation reaction. In mFruits proteins, the thirdphoton promotes an ET from the chromophore to the 4S Rydberg state ofnearby K70þ residue with its subsequent photoionization by the fourth photon.

3066-Pos Board B758Quantitative Imaging of Protein Secretions from Single Cells in Real TimeMarc Raphael, Joseph Christodoulides, James Delehanty, James Long,Jeff Byers.Naval Research Laboratory, Washington, DC, USA.Cell-to-cell signaling often involves the secretion of proteins, which createsspatial and temporal concentration profiles in the extra-cellular environmentfor the receiving cells to detect and interpret. Real-time measurements ofsecreted protein concentrations at the single cell level are thus fundamentalto understanding these communications pathways but have proven difficult torealize in practice. Here we present a label-free technique based upon nanoplas-monic imaging and high-affinity binding which enabled the measurement ofindividual cell secretions in real time. When applied to the detection of anti-body secretions from individual hybridoma cells, the enhanced time resolutionrevealed two modes of secretion: one in which the cell secreted continuouslyand another in which antibodies were released in concentrated bursts thatcoincided with minute-long morphological contractions of the cell. From the

continuous secretion measurements we determined the local concentration ofantibodies at the sensing array closest to the cell. The technique is incorporated

on to a wide-field microscope whichenables real-time transmitted light andfluorescence imaging of the cells aswell. We anticipate this technique willbe broadly applicable to the real-timecharacterization of both paracrine andautocrine signaling pathways.

3067-Pos Board B759

A Fluorescence Approach to Discrimination of Aggregated AmyloidProteinsKevin J. Cao1, Kristyna Elbel1, Christina Sigurdson2,Emmanuel Theodorakis1, Jerry Yang1.1Chemistry & Biochemistry, UC San Diego, La Jolla, CA, USA, 2Pathology,UC San Diego, La Jolla, CA, USA.We present the design and synthesis of a new class of fluorophores with sensi-tivity to both environment viscosity as well as environment polarity, a propertyknown as solvatochromism. We recently reported the ability of these fluoro-phores to discriminate, based on fluorescence emission, protein amyloid aggre-gate deposition in ex vivo neuronal tissue (Cao et al., J. Am. Chem. Soc., 2012,134, 17338-17341). Here, we have further modeled the solvatochromic rela-tionship of these probes with environmental permittivity and have used theseresults to extrapolate the permittivity values of the probe binding pocketswithin several amyloid proteins. Additionally, we have begun to examine theutility of these fluorophores as potential diagnostic tools for detection of neuro-degenerative pathologies.

3068-Pos Board B760Live Synaptic Mapping of Vertebrate Whole Brain with Light SheetMicroscopy and Endogenously Labeled Synapsin-2B ProteinAndrey Andreev, John M. Choi, Le A. Trinh, Thai V. Truong,Scott E. Fraser.Translational Imaging Center, University of Southern California, LosAngeles, CA, USA.A synaptic map of vertebrate brain is a crucial step for understanding themolecular basis for neuronal networks development and modification. How-ever, conventional two-photon, point-scanning microscopy presents challengesfor in vivo, whole-animal studies of brain synaptic structure and dynamics inhigher organism due to reduced axial resolution, and inadequate temporal res-olution for developmental or behavioral studies, as well as excessive photo-damage to the sample. Application of two-photon, light sheet microscopyovercomes these barriers, approaching isotropic 3D resolution and fastervolume scan speed (>10x), while simultaneously reducing phototoxic effects.Using a protein trap zebrafish line, in which Synapsin-2B is expressed as a fluo-rescent fusion protein from the endogenous locus, combined with two-photonlight sheet microscopy enables live, synaptic mapping of the whole vertebratebrain. This work provides the basis for further research in imaging braindynamics and development, for example fast, high-resolution whole-brainactivity imaging.

3069-Pos Board B761A Multi-Emitter Localization Comparison of 3D Superresolution ImagingModalitiesSheng Liu, Keith A. Lidke.University of New Mexico, Albuquerque, NM, USA.Single-molecule localization-based superresolution imaging is complicated byemission from multiple emitters overlapping at the detector. The potential foroverlapping emitters is even greater for 3D imaging than for 2D imaging due tothe large effective ‘volume’ of the 3D point spread function (PSF). Overlappingemission can be accounted for in the estimation model, recovering the ability tolocalize the emitters, but with the caveat that the localization precision has adependence on the amount of overlap from other emitters. It is interesting toconsider if a particular 3D imaging modality has a significant advantage infacilitating the position estimation of overlapping emitters. We compare vari-ants of two commonly used and easily implemented imaging modalities for3D single-molecule imaging: astigmatic imaging; dual focal plane imaging;and the combination of the two approaches� dual focal plane with astigmatism.We quantify the ability for a particular 3D modality to facilitate estimationwith overlapping emitters using the Cramer-Rao lower bound (CRLB). TheCRLB is used to calculate the theoretical best localization precision under amulti-emitter estimation model. We investigate the performance of these 3Dmodalities under a wide range of conditions including various distributionsof collected photons per frame, background counts, pixel sizes, and cameraread-out noise values.