David P. Fromm

Methods of single-molecule fluorescence spectroscopy and microscopy

Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal reflection, as well as confocal and near-field optical scanning microscopies are described. In order to extract the maximum amount of information from an experiment, a wide array of properties of the emission can be recorded, such as polarization, spectrum, degree of energy transfer, and spatial position. Whatever variable is measured, the time dependence of the parameter can yield information about excited state lifetimes, photochemistry, local environmental fluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to the breadth of applications now appearing, single-molecule spectroscopy and microscopy may be viewed as useful new tools for the study of dynamics in complex systems, especially where ensemble averaging or lack of synchronization may obscure the details of the process under study.

Exploring the chemical enhancement for surface-enhanced Raman scattering with Au bowtie nanoantennas

Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >10(7), much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules.

Spectral analysis of strongly enhanced visible light transmission through single C-shaped nanoapertures

We designed, fabricated, and characterized single C-shaped apertures in an Au film, resonant in the visible regime. Our C-shaped apertures showed transmission enhancement of 13–22 times over a square aperture of the same area and suggest as high as 106 enhancement over square apertures that are designed to produce the same near-field spot size. Spectra from individual apertures demonstrate the ability to tune this resonance over 70nm by scaling the dimensions of the apertures. This shows the C aperture to be a versatile tool for gaining high-resolution, enhanced transmission through single subwavelength apertures at optical wavelengths.

Field enhancement and resonance in optical antennas

Optical antennas are studied as probes for near-field optical microscopy. Finite difference time domain calculations indicate an intensity enhancement of more than 3 orders of magnitude. Optical antennas are fabricated and tested with good agreement between experiment and theory.

Biomolecular applications of single-molecule measurements: kinetics and dynamics of a single-enzyme reaction

In this work we describe preliminary experiments in which we have used ultra-sensitive fluorescence microscopy to observe the dynamics of individual enzyme molecules acting upon a substrate. The enzyme, (beta) -galactosidase from E.coli, is specifically immobilized onto a glass substrate while maintaining its functionality. The immobilized protein degrades a fluorogenic substrate to produce a fluorescent product, whose generation can be observed in real time. Individual copies of (beta) -galactosidase can be observed for many minutes, allowing the measurement of a large number of successive substrate turnover events. A rudimentary analysis of these turnovers using autocorrelation functions is presented, and a strong heterogeneity in reaction rates between different molecules is observed. In addition, the challenges inherent in successful surface immobilization of proteins for single-molecule experiments are discussed.

Spectral study of enhanced transmission through single C-shaped nano-apertures

C-shaped apertures in a metal film, were made to be resonant at visible wavelengths. They showed transmission enhancement of 22 times over a square aperture of the same area. This resonance was tuned over 70 nm