Ground-State Depletion Microscopy: Detection Sensitivity of Single-Molecule Optical Absorption at Room Temperature

Optical studies of single molecules in ambient environments, which have led to broad applications, are primarily based on fluorescence detection. Direct detection of optical absorption with single-molecule sensitivity at room temperature is difficult because absorption is not a background-freemeasurement and is often complicated by sample scattering. Here we report ground-state depletionmicroscopy for ultrasensitive detection of absorption contrast.We image 20 nm gold nanoparticles as an initial demonstration of this microscopy. We then demonstrate the detection of an absorption signal from a single chromophore molecule at room temperature. This is accomplished by using two tightly focused collinear continuous-wave laser beams at different wavelengths, both within a molecular absorption band, one of which is intensity modulated at a high frequency (>MHz). The transmission of the other beam is found to be modulated at the same frequency due to ground state depletion. The signal of single chromophore molecules scanned across the common laser foci can be detected with shot-noise limited sensitivity. This measurement represents the ultimate detection sensitivity of nonlinear optical spectroscopy at room temperature. SECTION Kinetics, Spectroscopy S ingle-molecule optical detection, imaging, and spectroscopy have had an impact on many disciplines. In particular, room temperature optical detection of single molecules has been used extensively in biological research. Single-molecule optical detection at room temperature dates back to 1976whenHirschfeld reported the use ofanopticalmicroscope to reduceprobevolume, andhence the background signal. He was able to detect individual immobilized protein molecules labeled with tens of fluorophores, demonstrating a single-molecule line-scan image. Similar use of optical microscopes finally allowed single fluorophore sensitivity at room temperature in 1990. It has remained themethod of choice for detecting and imaging singlemolecules in ambient environments to the present. Among the methods that subsequently emerged, single chromophore detection was first achieved by optical absorption measurement at 1.6 K with a sophisticated frequency modulation scheme in 1989. This measurement relied on the large absorption cross-section of the zero-phonon line. It was soon followed by fluorescence detection via excitation at the zero-phonon line, which offered much higher sensitivity by virtue of background free emission detection. These methods, however, were limited only to cryogenic temperatures at which zero-phonon lines exist for a handful of molecules. Imaging of single fluorophores at room temperature was first accomplished with near-field microscopy in 1993. Although a seminal contribution, near-field single molecule imaging had limited applications because of the complexity and perturbation of the near-field probes, and was soon surpassed by much easier far-field single molecule imaging with total internal reflection and confocal microscopy. Surface enhancedRamanscattering is capableofdetecting single molecules, but it requires close contact of molecules with a metal nanostructure, which is difficult to control. High-sensitivity measurements toward single-molecule spectroscopy have been attempted with other contrasts besides fluorescence and Raman scattering, including interferometry, stimulated emission, photothermal and direct absorption measurements. The photothermal method has recently reached single-molecule sensitivity; however, glycerol, an uncommon solvent, was used in order to reduce heat conductivity and increase the refractive index change induced by single-molecule light absorption. The direct absorption method has recently demonstrated single molecule sensitivity. By careful selection of the sample substrate, the authors avoided the major complication of sample scattering for direct absorption measurements. We seek a different approach for detection of single-molecule optical absorption at room temperature. Received Date: October 18, 2010 Accepted Date: October 22, 2010