Peter M. Goodwin

Alterations of Single Molecule Fluorescence Lifetimes in Near-Field Optical Microscopy

Fluorescence lifetimes of single Rhodamine 6G molecules on silica surfaces were measured with pulsed laser excitation, time-correlated single photon counting, and near-field scanning optical microscopy (NSOM). The fluorescence lifetime varies with the position of a molecule relative to a near-field probe. Qualitative features of lifetime decreases are consistent with molecular excited state quenching effects near metal surfaces. The technique of NSOM provides a means of altering the environment of a single fluorescent molecule and its decay kinetics in a repeatable fashion.

Single-Molecule Fluorescence Analysis in Solution

Over the past five years, several groups have developed the capability to detect and identify single fluorescent molecules in solution as the molecules flow through a focused laser beam. The history of the approach to single-molecule detection in fluid solution is shown in Fig. 1. Approximately one dozen molecular species have been detected at this level of sensitivity. Fluorescence-based, single-molecule detection techniques are expected to have a significant impact in fields where fluorescence detection and quantification are broadly applied, e.g., analytical chemistry, biology, and medicine. Single-molecule detection is a new way of doing analytical chemistry, and new applications will arise. In this article, we describe our approach to single-molecule detection and explore assays that can be done at the single-species level that would be difficult or impossible with bulk measurements.

Base-Directed Formation of Fluorescent Silver Clusters

Small silver clusters that form with short oligonucleotides are distinguished by their strong fluorescence. Previous work showed that red and blue/green emitting species form with the cytosine oligonucleotide dC12. To understand how the bases and base sequence influence cluster formation, the blue/green emitting clusters that form with the thymine-containing oligonucleotides dT12, dT4C4T4, and dC4T4C4 are discussed. With dT12 and dT4C4T4, variations in the solution pH establish that the clusters associate with the N3 of thymine. The small clusters are bound to the larger DNA template, as demonstrated by fluorescence anisotropy, circular dichroism, and fluorescence correlation spectroscopy (FCS) studies. For dT4C4T4, FCS studies showed that approximately 50% of the strands are labeled with the fluorescent clusters. Absorption spectra and the gas dependence of the fluorescence show that nonfluorescent clusters also form following the reduction of the silver cation - oligonucleotide conjugates. Fluorescent cluster formation is favored by oxygen, thus indicating that the DNA-bound clusters are partially oxidized. To elaborate the sequence dependence of cluster formation, dC4T4C4 was studied. Cluster formation depends on the oligonucleotide concentration, and higher concentrations favor a red emitting species. A blue/green emissive species dominates at lower concentrations of dC4T4C4, and it has spectroscopic, physical, and chemical properties that are similar to those of the clusters that form with dT12 and dT4C4T4. These results suggest that cytosine- and thymine-containing oligonucleotides stabilize a preferred emissive silver cluster.

Shape Evolution and Single Particle Luminescence of Organometal Halide Perovskite Nanocrystals

Organometallic halide perovskites CH3NH3PbX3 (X = I, Br, Cl) have quickly become one of the most promising semiconductors for solar cells, with photovoltaics made of these materials reaching power conversion efficiencies of near 20%. Improving our ability to harness the full potential of organometal halide perovskites will require more controllable syntheses that permit a detailed understanding of their fundamental chemistry and photophysics. In this manuscript, we systematically synthesize CH3NH3PbX3 (X = I, Br) nanocrystals with different morphologies (dots, rods, plates or sheets) by using different solvents and capping ligands. CH3NH3PbX3 nanowires and nanorods capped with octylammonium halides show relatively higher photoluminescence (PL) quantum yields and long PL lifetimes. CH3NH3PbI3 nanowires monitored at the single particle level show shape-correlated PL emission across whole particles, with little photobleaching observed and very few off periods. This work highlights the potential of low-dimensional organometal halide perovskite semiconductors in constructing new porous and nanostructured solar cell architectures, as well as in applying these materials to other fields such as light-emitting devices and single particle imaging and tracking.

Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence

The detection and measurement of fluorescence lifetimes of single Rhodamine‐110 molecules in a flowing, aqueous sample stream is described. Time‐correlated single‐photon counting, used in combination with mode‐locked picosecond pulsed excitation, allows the detection of single fluorescent molecules in the presence of significant solvent Raman and Rayleigh backgrounds. The fluorescence lifetime of a detected molecule is estimated from the record of arrival times (relative to the excitation pulse) of photons detected during the molecule’s passage through the ∼1 pL excitation volume.

Effect of shell thickness and composition on blinking suppression and the blinking mechanism in ‘giant’ CdSe/CdS nanocrystal quantum dots

We recently developed an inorganic shell approach for suppressing blinking in nanocrystal quantum dots (NQDs) that has the potential to dramatically improve the utility of these fluorophores for single-NQD tracking of individual molecules in cell biology. Here, we consider in detail the effect of shell thickness and composition on blinking suppression, focusing on the CdSe/CdS core/shell system. We also discuss the blinking mechanism as understood through profoundly altered blinking statistics. We clarify the dependence of blinking behavior and photostability on shell thickness, as well as on interrogation times. We show that, while the thickest-shell systems afford the greatest advantages in terms of enhanced optical properties, thinner-shell NQDs may be adequate for certain applications requiring relatively shorter interrogation times. Shell thickness also determines the sensitivity of the NQD optical properties to aqueous-phase transfer, a critical step in rendering NQDs compatible with bioimaging applications. Lastly, we provide a proof-of-concept demonstration of the utility of these unique NQDs for fluorescent particle tracking.

Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries.

Excitation of fluorescence with total internal reflection (TIR) excitation yields very low background scattered light and good signal-to-background contrast. The background and its associated noise can be made low enough to detect single fluorescent molecules under ambient conditions. In this paper, different TIR geometries were compared for excitation and detection of single rhodamine 6G (R6G) molecules at air-silica interfaces and single B-phycoerythrin proteins at water-silica interfaces. Through-objective, objective-coverslip, and prism-based TIR geometries were investigated. The signal-to-background ratio (SBR) and the number of photons detected before photobleaching (Nb) were optimum in different geometries. The greatest image contrast was obtained when using prism-TIR (SBR = 11.5), but the largest number of detected signal photoelectrons was obtained by using through-objective TIR for R6G-air-silica ( = 10(4)). The results were discussed in terms of the TIR field enhancements and the modified dipole emission pattern near a dielectric interface. The SBR and total detected photons are important parameters for designing photon-limited experiments.

Three-dimensional tracking of individual quantum dots

We describe an instrument that extends the state of the art in a single-molecule tracking technology, allowing extended observations of single fluorophores and fluorescently labeled proteins as they undergo directed and diffusive transport in three dimensions. We demonstrate three-dimensional tracking of individual quantum dots undergoing diffusion for durations of over a second at velocities comparable to those of intracellular signaling processes.

Time-resolved three-dimensional molecular tracking in live cells.

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.

A maximum likelihood estimator to distinguish single molecules by their fluorescence decays

Abstract We have developed a maximum likelihood estimator to distinguish between similar molecules at the single molecule level based upon fluorescence decay measurements. Time resolved fluorescence measurements for single Rhodamine 6G and tetramethylrhodamine isothiocyanate molecules in fluid flow are derived from time-correlated single photon counting. A maximum likelihood estimator is developed and applied to data from a mixture of molecules. Single molecules are identified and distinguished by their fluorescence time decays. Comparison is made between identification error rates and theoretical predictions. To our knowledge, this is the first reported example of single molecule identification by fluorescence decay in a mixture.