Haw Yang

Quantum Dot Nano Thermometers Reveal Heterogeneous Local Thermogenesis in Living Cells

The local temperature response inside single living cells upon external chemical and physical stimuli was characterized using quantum dots as nano thermometers. The photoluminescence spectral shifts from endocytosed quantum dots were used to map intracellular heat generation in NIH/3T3 cells following Ca(2+) stress and cold shock. The direct observation of inhomogeneous intracellular temperature progression raises interesting new possibilities, including further innovations in nanomaterials for sensing local responses, as well as the concept of subcellular temperature gradient for signaling and regulation in cells.

Illuminating the mechanistic roles of enzyme conformational dynamics

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzymes lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AKs domain movements on its catalytic time scale. To quantitatively measure the enzymes entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzymes conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzymes rate-limiting step.

Probing single-molecule dynamics photon by photon

We present the theoretical rationales for data analysis protocols that afford an efficient extraction of conformational dynamics on a broad range of time scales from single-molecule fluorescence lifetime trajectories. Based on correlation analyses, a photon-by-photon approach on one hand provides the highest time resolution, whereas a minimal-binning method on the other hand is most suitable for experiments experiencing external fluorescence intensity variations. Applications of the two methods are illustrated via computer simulations. In cases where fluorescence quenching is either due to Forster fluorescence resonance energy transfer or due to the excited-state electron transfer, the fluorescence lifetime is dependent on donor-acceptor distance, thereby providing a window through which conformational dynamics are revealed. To assist in interpreting experimental data derived from the new protocols, analytical expressions relating fluorescence lifetime fluctuation correlations to a Brownian diffusion model and to an anomalous diffusion model are discussed.

Multiscale complex network of protein conformational fluctuations in single-molecule time series

Conformational dynamics of proteins can be interpreted as itinerant motions as the protein traverses from one state to another on a complex network in conformational space or, more generally, in state space. Here we present a scheme to extract a multiscale state space network (SSN) from a single-molecule time series. Analysis by this method enables us to lift degeneracy—different physical states having the same value for a measured observable—as much as possible. A state or node in the network is defined not by the value of the observable at each time but by a set of subsequences of the observable over time. The length of the subsequence can tell us the extent to which the memory of the system is able to predict the next state. As an illustration, we investigate the conformational fluctutation dynamics probed by single-molecule electron transfer (ET), detected on a photon-by-photon basis. We show that the topographical features of the SSNs depend on the time scale of observation; the longer the time scale, the simpler the underlying SSN becomes, leading to a transition of the dynamics from anomalous diffusion to normal Brownian diffusion.

An accessible approach to preparing water-soluble Mn2+-doped (CdSSe)ZnS (core)shell nanocrystals for ratiometric temperature sensing.

A new synthetic scheme allowing structural modifications to temperature-sensitive and water-soluble D-penicillamine-passivated Mn(2+)-doped (CdSSe)ZnS (core)shell nanocrystals (MnQDs) was reported using air-stable chemicals. The temperature-dependent optical properties of the nanocrystals were tuned by changing their structure and composition--the ZnS shell thickness and the Mn(2+)-dopant concentration. Thick ZnS shells significantly reduce the interference of nonradiative transitions on ratiometric emission intensities. High-dopant concentration affords consistent temperature sensitivity. In addition to the new base structure for quantum dot ratiometric temperature sensing via flexible, glovebox-free routes, the results also underscore the generalizability of the emission intensity ratio scheme for temperature sensing, originally proposed for rare-earth-doped materials.

Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts

We present an apparatus that noninvasively tracks a moving nanoparticle in three dimensions while providing concurrent sequential spectroscopic measurements. The design, based on confocal microscopy, uses a near-infrared laser and a dark-field condenser for illumination of a gold nanoparticle. By monitoring the scattered light from the nanoparticle and using a piezoelectric stage, the system was able to continuously bring the diffusive particle in a glycerol/water solution back to the focal volume with spatial resolution and response time of less than 210nm and a millisecond, respectively.

Real-time chemical imaging of bacterial activity in biofilms using open-channel microfluidics and synchrotron FTIR spectromicroscopy.

Real-time chemical imaging of bacterial activities can facilitate a comprehensive understanding of the dynamics of biofilm structures and functions. Synchrotron-radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy can yield high spatial resolution and label-free vibrational signatures of chemical bonds in biomolecules, but the abundance of water in biofilms has hindered SR-FTIRs sensitivity in investigating bacterial activity. We developed a simple open-channel microfluidic system that can circumvent the water-absorption barrier for chemical imaging of the developmental dynamics of bacterial biofilms with a spatial resolution of several micrometers. This system maintains a 10 microm thick laminar-flow-through biofilm system that minimizes both the imaging volume in liquid and the signal interference from geometry-induced fringing. Here we demonstrate the ability of the open-channel microfluidic platform to maintain the functionality of living cells while enabling high-quality SR-FTIR measurements. We include several applications that show how microbes in biofilms adapt to their immediate environments. The ability to directly monitor and map bacterial changes in biofilms can yield significant insight into a wide range of microbial systems, especially when coupled to more sophisticated microfluidic platforms.

Statistical approaches for probing single-molecule dynamics photon-by-photon

Abstract The recently developed photon-by-photon approach [H. Yang, X.S. Xie, J. Chem. Phys., 2002 (in press)] for single-molecule fluorescence experiments allows measurements of conformational fluctuation with time resolution on a vast range of time scales. In that method, each photon represents a data point, thereby affording better statistics. Here, we utilize the information carried by each detected photon to better differentiate theoretical models for the underlying dynamical processes—including two- and three-state models, and a diffusive model. We introduce a three-time correlation analysis, which is based on time series analyses, and the Kullback–Liebler distance, which is based on information theory principles [Elements of Information Theory, Wiley, New York, 1991]. The feasibility of and general procedures for applying these methods to single-molecule experiments are examined via computer simulations.

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

Guiding a confocal microscope by single fluorescent nanoparticles

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.