Real-time observation of single molecules by confocal fluorescence microscopy

Abstract

The ability to detect, identify, and manipulate individual molecules offer exciting possibilities in many fields, including chemical analysis, materials research, and the biological sciences. A particularly powerful approach is to combine the exquisite sensitivity of laser-induced fluorescence and the spatial localization and imaging capabilities of diffraction-limited or near-field optical microscopes. Unlike scanning tunneling microscopy (STM) and atomic force microscopy (AFM), which lack molecular specificity, optical spectroscopy and microscopy techniques can be used for real-time monitoring and molecular identification at nanometer dimensions or in ultrasmall volumes.We report the use of confocal fluorescence microscopy coupled with a diffraction-limit laser beam and a high-efficiency photodiode for real-time detection of single fluorescent molecules in solution at room temperature. Rigler and Eigen have also demonstrated single-molecule detection with a confocal microscope and fluorescence correlation spectroscopy. The probe (or sampling) volume is effectively an elongated cylinder, with its radius being determined by optical diffraction and length by spherical aberration.