Julie S. Biteen

Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function

We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 μm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

Spectral tuning of plasmon-enhanced silicon quantum dot luminescence

In the presence of nanoscale silver island arrays, silicon quantum dots exhibit up to sevenfold luminescence enhancements at emission frequencies that correspond to the collective dipole plasmon resonance frequency of the Ag island array. Using electron-beam lithography to alter the pitch and particle diameter, this wavelength-selective enhancement can be varied as the metal array resonance wavelength is tuned from 600 to 900 nm. The luminescence intensity enhancement upon coupling is attributed to an increase in the radiative decay rate of the silicon quantum dots.

Three‐Dimensional Super‐Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism

Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. We quantify these dynamics and determine the FtsZ depolymerization time to be <100 ms. We image the Z-ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre-divisional stage.

Cy3-Cy5 Covalent Heterodimers for Single-Molecule Photoswitching

Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.

Tools for the Microbiome: Nano and Beyond

The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.

Single-Molecule Super-Resolution Microscopy Reveals How Light Couples to a Plasmonic Nanoantenna on the Nanometer Scale

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.

Self-Organization of Plasmonic and Excitonic Nanoparticles into Resonant Chiral Supraparticle Assemblies

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.

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.

Single-molecule motions and interactions in live cells reveal target search dynamics in mismatch repair

Significance We integrated single-molecule superresolution imaging with biochemical and genomic approaches to understand how the mismatch repair protein MutS efficiently identifies DNA mismatches during real time in living cells. We show that MutS molecules move fast, exploring the entire nucleoid, but can transition to a slow-moving population that is localized at the replisome even before a mismatch is produced. We show that bacterial MutS must initiate mismatch binding in very close proximity to the replisome. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Our results provide fundamental insight into the searching behavior of single MutS molecules during DNA replication in live cells. 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.