Co-reporter:Bing Fu, Benjamin P. Isaacoff, and Julie S. Biteen
ACS Nano September 26, 2017 Volume 11(Issue 9) pp:8978-8978
Publication Date(Web):August 14, 2017
DOI:10.1021/acsnano.7b03420
Plasmonic nanoparticles (NPs) enhance the radiative decay rate of adjacent dyes and can significantly increase fluorescence intensity for improved spectroscopy. However, the NP nanoantenna complicates super-resolution imaging by introducing a mislocalization between the emitter position and its super-resolved emission position. The mislocalization magnitude depends strongly on the dye/NP coupling geometry. It is therefore crucial to quantify mislocalization to recover the actual emitter position in a coupled system. Here, we super-resolve in two and three dimensions the distance-dependent emission mislocalization of single fluorescent molecules coupled to gold NPs with precise distance tuning via double-stranded DNA. We develop an analytical framework to uncover detailed spatial information when direct 3D imaging is not accessible. Overall, we demonstrate that by taking measurements on a single, well-defined, and symmetric dye/NP assembly and by accounting explicitly for artifacts from super-resolution imaging, we can measure the true nanophotonic mislocalization. We measure up to 50 nm mislocalizations and show that smaller separation distances lead to larger mislocalizations, also verified by electromagnetic calculations. Overall, by quantifying the distance-dependent mislocalization shift in this gold NP/dye coupled system, we show that the actual physical position of a coupled single emitter can be recovered.Keywords: dSTORM; emission mislocalization; local surface plasmon resonance; nanoparticle plasmonics; single-molecule imaging; super-resolution microscopy; superlocalization;
Co-reporter:Jessica D. Flynn, Beth L. Haas, and Julie S. Biteen
The Journal of Physical Chemistry C 2016 Volume 120(Issue 37) pp:20512-20517
Publication Date(Web):October 12, 2015
DOI:10.1021/acs.jpcc.5b08049
The sensitivity and resolution of single-molecule fluorescence imaging in biology are mainly limited by two known weaknesses of fluorescent proteins: label brightness and photostability. In this work, we use patterned gold substrates to achieve plasmon-enhanced emission from intrinsically fluorescent proteins in living pathogenic bacteria cells. By coupling membrane-bound single fluorescent protein fusions to the virulence regulator TcpP in living Vibrio cholerae bacteria to extracellular gold nanotriangle arrays, we use plasmonics to improve our measurements of this important question in pathogenesis: how does V. cholerae produce its deadly toxin? Based on a simple experimental geometry, we observe a 1.3× enhancement in the rate of emission and a 1.4× enhancement in the number of photons detected prior to photobleaching. Furthermore, by enhancing both the rate of emission and the total number of photons detected from single-molecule fluorescent probes in live cells, we show that plasmon-enhanced fluorescence is a biocompatible, generalizable path to directly improve the resolution and trajectory lengths of single molecules in live cells.
Co-reporter:Esther A. Wertz, Benjamin P. Isaacoff, and Julie S. Biteen
ACS Photonics 2016 Volume 3(Issue 10) pp:1733
Publication Date(Web):September 20, 2016
DOI:10.1021/acsphotonics.6b00344
The emission properties of fluorescent molecules are strongly affected by proximal plasmonic nanoparticles that act as optical nanoantennas. In particular, fluorescence intensity can be greatly increased by enhancing both the excitation and emission rates of a fluorophore, and the angular and spatial emission pattern from a dye coupled to a plasmonic nanoantenna will be altered. Here, we use single-molecule imaging to measure this shifted emission pattern based on the super-resolution image of cyanine dye molecules coupled to gold nanotriangles. To compare the relative effects of excitation versus emission enhancement on the emission pattern, we vary laser excitation wavelengths, dye emission and absorbance spectra, and local surface plasmon resonance frequency. We demonstrate that the emission pattern is dramatically changed when coupling occurs and that coupling between the dye and gold nanotriangle happens even in the absence of intensity enhancement.Keywords: light−matter interactions; nanoantenna; plasmon-enhanced emission; plasmon-enhanced excitation; single-molecule microscopy; super-resolution imaging
Co-reporter:Stephen A. Lee, Aleks Ponjavic, Chanrith Siv, Steven F. Lee, and Julie S. Biteen
ACS Nano 2016 Volume 10(Issue 9) pp:8143
Publication Date(Web):September 7, 2016
DOI:10.1021/acsnano.6b02863
In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics.Keywords: bacterial biophysics; light-sheet microscopy; nanophotonics; optical sectioning; plasmonic enhancement; single-molecule imaging; super-resolution microscopy
Co-reporter:Dr. Hannah H. Tuson;Alisa Aliaj;Eileen R. Bres;Dr. Lyle A. Simmons;Dr. Julie S. Biteen
ChemPhysChem 2016 Volume 17( Issue 10) pp:1435-1440
Publication Date(Web):
DOI:10.1002/cphc.201600035
Abstract
Single-molecule fluorescence super-resolution imaging and tracking provide nanometer-scale information about subcellular protein positions and dynamics. These single-molecule imaging experiments can be very powerful, but they are best suited to high-copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single-molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single-molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.
Co-reporter:Esther Wertz, Benjamin P. Isaacoff, Jessica D. Flynn, and Julie S. Biteen
Nano Letters 2015 Volume 15(Issue 4) pp:2662-2670
Publication Date(Web):March 23, 2015
DOI:10.1021/acs.nanolett.5b00319
The greatly enhanced fields near metal nanoparticles have demonstrated remarkable optical properties and are promising for applications from solar energy to biosensing. However, direct experimental study of these light-matter interactions at the nanoscale has remained difficult due to the limitations of optical microscopy. Here, we use single-molecule fluorescence imaging to probe how a plasmonic nanoantenna modifies the fluorescence emission from a dipole emitter. We show that the apparent fluorophore emission position is strongly shifted upon coupling to an antenna and that the emission of dyes located up to 90 nm away is affected by this coupling. To predict this long-ranged effect, we present a framework based on a distance-dependent partial coupling of the dye emission to the antenna. Our direct interpretation of these light-matter interactions will enable more predictably optimized, designed, and controlled plasmonic devices and will permit reliable plasmon-enhanced single-molecule nanoscopy.
Co-reporter:Hannah H. Tuson and Julie S. Biteen
Analytical Chemistry 2015 Volume 87(Issue 1) pp:42
Publication Date(Web):November 7, 2014
DOI:10.1021/ac5041346
Co-reporter:Bing Fu
The Journal of Physical Chemistry C 2015 Volume 119(Issue 33) pp:19350-19358
Publication Date(Web):July 23, 2015
DOI:10.1021/acs.jpcc.5b05154
Coupling to metal nanoparticles can increase the fluorescence intensity and photostability of fluorescent probes, and this plasmon-enhanced fluorescence is particularly promising for the dimmer fluorescent proteins common in biological imaging. Here, we measure the intensity distribution of single Cy3.5 dye molecules and mCherry fluorescent proteins one at a time as they adsorb on a conformal surface 4.8–61.0 nm thick over a gold nanorod (NR). The emission intensities for both types of fluorophores depend nonmonotonically on the spacer thickness, and an optimal spacer thickness of ∼10 nm is observed for both fluorophores using two different spacer layer materials. Emission from fluorophores coupled to metal nanoparticles is affected by two competing processes: an enhanced spontaneous decay rate and quenching via nonradiative antenna modes. After averaging over a conformal surface, the product of the simulated enhanced local electric field intensity and the quantum efficiency modification reproduces the experimental 10 nm ideal spacer thickness. Overall, up to a 3.4-fold average enhancement in fluorescence intensity was achieved despite the simple geometry, based on biocompatible, tunable, and economic colloidal gold NRs. This study of the distance dependence of single-molecule plasmon-enhanced fluorescence shows promise for super-resolving cellular membrane proteins naturally positioned above an extracellular substrate.
Co-reporter:Yi Liao;Jeremy W. Schroeder;Burke Gao;Lyle A. Simmons
PNAS 2015 Volume 112 (Issue 50 ) pp:E6898-E6906
Publication Date(Web):2015-12-15
DOI:10.1073/pnas.1507386112
MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies
rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution
microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one
MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome
region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated
with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show
that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition.
Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves
to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS
is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which
is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer
capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize
to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells
as it searches for mismatches.
Co-reporter:Tao Hu, Benjamin P. Isaacoff, Joong Hwan Bahng, Changlong Hao, Yunlong Zhou, Jian Zhu, Xinyu Li, Zhenlong Wang, Shaoqin Liu, Chuanlai Xu, Julie S. Biteen, and Nicholas A. Kotov
Nano Letters 2014 Volume 14(Issue 12) pp:6799-6810
Publication Date(Web):November 17, 2014
DOI:10.1021/nl502237f
Chiral nanostructures exhibit strong coupling to the spin angular momentum of incident photons. The integration of metal nanostructures with semiconductor nanoparticles (NPs) to form hybrid plasmon–exciton nanoscale assemblies can potentially lead to plasmon-induced optical activity and unusual chiroptical properties of plasmon–exciton states. Here we investigate such effects in supraparticles (SPs) spontaneously formed from gold nanorods (NRs) and chiral CdTe NPs. The geometry of this new type of self-limited nanoscale superstructures depends on the molar ratio between NRs and NPs. NR dimers surrounded by CdTe NPs were obtained for the ratio NR/NP = 1:15, whereas increasing the NP content to a ratio of NR/NP = 1:180 leads to single NRs in a shell of NPs. The SPs based on NR dimers exhibit strong optical rotatory activity associated in large part with their twisted scissor-like geometry. The preference for a specific nanoscale enantiomer is attributed to the chiral interactions between CdTe NP in the shell. The SPs based on single NRs also yield surprising chiroptical activity at the frequency of the longitudinal mode of NRs. Numerical simulations reveal that the origin of this chiroptical band is the cross talk between the longitudinal and the transverse plasmon modes, which makes both of them coupled with the NP excitonic state. The chiral SP NR–NP assemblies combine the optical properties of excitons and plasmons that are essential for chiral sensing, chiroptical memory, and chiral catalysis.
Co-reporter:David J. Rowl ; Julie S. Biteen
ChemPhysChem 2014 Volume 15( Issue 4) pp:712-720
Publication Date(Web):
DOI:10.1002/cphc.201300774
Abstract
Single-molecule fluorescence permits super-resolution imaging, but traditional algorithms for localizing these isolated fluorescent emitters assume stationary point light sources. Proposed here are two fitting functions that achieve similar nanometer-scale localization precision as the traditional symmetric Gaussian function, while allowing, and explicitly accounting for, directed motion. The precision of these methods is investigated through Fisher information analysis, simulation and experiments, and the new fitting functions are then used to measure, for the first time, the instantaneous velocity and direction of motion of live bacteria cells. These new methods increase the information content of single-molecule images of fast-moving molecules without sacrificing localization precision, thus permitting slower imaging speeds, and our new fitting functions promise to improve tracking algorithms by calculating velocity and direction during each image acquisition.
Co-reporter:Jessica E. Donehue ; Esther Wertz ; Courtney N. Talicska
The Journal of Physical Chemistry C 2014 Volume 118(Issue 27) pp:15027-15035
Publication Date(Web):June 19, 2014
DOI:10.1021/jp504186n
Single-molecule imaging pushes fluorescence microscopy beyond the diffraction limit of traditional microscopy. Such super-resolution imaging, which relies on the detection of bright, stable fluorescent probes to achieve nanometer-scale resolution, is often hindered in biological systems by dim, blinking fluorescent proteins (FPs). Here, we use gold nanorods and single-molecule fluorescence detection to achieve plasmon-enhanced emission from intrinsically fluorescent proteins. We measure a doubled photon emission rate from the red FP mCherry and detect three times more photons before photobleaching from the photoactivatable FP PAmCherry. We further explore the effect of near-field nanorod interactions on the yellow FP mCitrine, for which the observed emission enhancements cannot overcome measurable quenching. Overall, our work indicates that plasmonic particles improve both the brightness and photostability of FPs and extends the applications of plasmon-enhanced fluorescence to the arena of biological imaging. Furthermore, because gold nanorods are nontoxic, they are promising extracellular imaging substrates for enhancing emission from FP-labeled membrane-bound proteins in live cells.
Co-reporter:Mou-Chi Cheng, Austin T. Leske, Toshiki Matsuoka, Byoung Choul Kim, Jaesung Lee, Mark A. Burns, Shuichi Takayama, and Julie S. Biteen
The Journal of Physical Chemistry B 2013 Volume 117(Issue 16) pp:4406-4411
Publication Date(Web):December 20, 2012
DOI:10.1021/jp307635v
Single-molecule super-resolution microscopy is an emerging technique for nanometer-scale fluorescence imaging, but in vitro single-molecule imaging protocols typically require a constant supply of reagents, and such transport is restricted in constrained geometries. In this article, we develop single-molecule micelle-assisted blink (MAB) microcopy to enable subdiffraction-limit imaging of nanochannels with better than 40 nm accuracy. The method, based on micelles and thiol-related photoswitching, is used to measure nanochannels formed in polydimethylsiloxane through tensile cracking. These conduits are reversibly size-adjustable from a few nanometers up to a micrometer and enable filtering of small particles and linearization of DNA. Unfortunately, conventional techniques cannot be used to measure widths, characterize heterogeneities, or discover porosity in situ. We overcome the access barriers by using sodium dodecyl sulfate (SDS), an ionic surfactant, to facilitate delivery of Cy5 dye and β-mercaptoethanol reducing agent in the confined geometry. These SDS micelles and admicelles have the further benefit of slowing diffusion of Cy5 to improve localization accuracy. We use MAB microscopy to measure nanochannel widths, to reveal heterogeneity along channel lengths and between different channels in the same device, and to probe biologically relevant information about the nanoenvironment, such as solvent accessibility.
Co-reporter:Yi Liao, Seong K. Yang, Kyoungmoo Koh, Adam J. Matzger, and Julie S. Biteen
Nano Letters 2012 Volume 12(Issue 6) pp:3080-3085
Publication Date(Web):May 2, 2012
DOI:10.1021/nl300971t
The diffusion of individual Nile red molecules in three different crystalline microporous coordination polymers (MCPs) is visualized with single-molecule fluorescence microscopy. By localizing molecules with high spatial resolution, the trajectories of the diffusing dyes are reconstructed with nanometer-scale precision. A detailed analysis of these tracks reveals different dynamics and guest–host interactions in each crystal as well as distinct motion types within the same system, suggesting the presence of structural heterogeneities in local environments.
Co-reporter:Justin S. Lenhart, Monica C. Pillon, Alba Guarné, Julie S. Biteen, Lyle A. Simmons
Research in Microbiology (January 2016) Volume 167(Issue 1) pp:4-12
Publication Date(Web):1 January 2016
DOI:10.1016/j.resmic.2015.08.006
DNA mismatch repair (MMR) is responsible for correcting errors formed during DNA replication. DNA polymerase errors include base mismatches and extra helical nucleotides referred to as insertion and deletion loops. In bacteria, MMR increases the fidelity of the chromosomal DNA replication pathway approximately 100-fold. MMR defects in bacteria reduce replication fidelity and have the potential to affect fitness. In mammals, MMR defects are characterized by an increase in mutation rate and by microsatellite instability. In this review, we discuss current advances in understanding how MMR functions in bacteria lacking the MutH and Dam methylase-dependent MMR pathway.
Co-reporter:David J. Rowland, Hannah H. Tuson, Julie S. Biteen
Biophysical Journal (24 May 2016) Volume 110(Issue 10) pp:
Publication Date(Web):24 May 2016
DOI:10.1016/j.bpj.2016.04.023
By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of single-molecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques.
Co-reporter:David J. Rowland, Julie S. Biteen
Chemical Physics Letters (16 April 2017) Volume 674() pp:
Publication Date(Web):16 April 2017
DOI:10.1016/j.cplett.2017.02.052
•A commonly used single particle tracking analysis technique is improved.•We greatly reduce the number of fitting parameters required to estimate diffusion.•The estimation precision and fitting robustness is improved.Single-molecule super-resolution imaging and tracking can measure molecular motions inside living cells on the scale of the molecules themselves. Diffusion in biological systems commonly exhibits multiple modes of motion, which can be effectively quantified by fitting the cumulative probability distribution of the squared step sizes in a two-step fitting process. Here we combine this two-step fit into a single least-squares minimization; this new method vastly reduces the total number of fitting parameters and increases the precision with which diffusion may be measured. We demonstrate this Global Fit approach on a simulated two-component system as well as on a mixture of diffusing 80 nm and 200 nm gold spheres to show improvements in fitting robustness and localization precision compared to the traditional Local Fit algorithm.
Co-reporter:Julie S. Biteen
Biophysical Journal (1 October 2013) Volume 105(Issue 7) pp:
Publication Date(Web):1 October 2013
DOI:10.1016/j.bpj.2013.08.022
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