Single-molecule fluorescence detection (SMFD) is able to probe, one molecule at a time, dynamical processes that are crucial for understanding functional mechanisms in biosystems. Fluorescence in the Near-infrared (NIR) offers improved Signal to Noise Ratio (SNR) by reducing the scattering, absorption and autofluorescence from biological cellular or tissue samples, therefore, provides high imaging resolution with increased tissue penetration depth that are important for biomedical applications. However, most NIR-emitters suffer from low quantum yield, the weak NIR fluorescence signal makes the detection extremely difficult.
Plasmonic nanostructures are capable of converting localized electromagnetic energy into free radiation and vice versa. This capability makes them efficient nano-antennas for modulating molecular fluorescence. The plasmonic nano-antenna generally enhances the fluorescence of a nearby molecule by enhancing the excitation rate and the quantum yield of the molecule. In order to optimally enhance the fluorescence, the plasmonic mode of the nano-antenna has to 1) couple strongly to the molecule and 2) radiate strongly to free space. Simultaneously satisfying the two requirements poses a challenge that is impossible to overcome in conventional, symmetric plasmonic nanostructures.
In a new paper published in Light Science & Application, scientists from the State Key Laboratory of Surface Physics, Physics Department of Fudan University, China, establishes a novel, universal approach to enhance single-molecule fluorescence in the NIR regime without compromising the molecule's photostability.
They construct asymmetric nano-antennas consisting of two bars with unequal lengths (Fig. 1) that provide multiple plasmonic modes with tunable resonance frequencies matching both excitation and emission frequencies of the fluorophore. The added tuning parameter, i.e., the ratio of the bar lengths, in such asymmetric structures offers new possibilities to modulate the near-field and far-field properties of the plasmonic modes, thereby further improving both excitation and emission processes. As a result, they experimentally acquire a single-molecule fluorescence enhancement factor up to 405 (Fig. 2), and the corresponding theoretical calculations indicate the quantum yield can be as high as 80%. Because the quantum yield plays a major role in this setup, this enhancement is achieved without sacrificing the molecules' survival time under laser irradiation.
In addition, compared with reference groups of molecules located on glass substrate, the authors have observed a significantly increased photobleaching time in molecules located around asymmetric double-bar nano-antennas (Fig. 3), indicating a much higher number of fluorescence photons emitted by those molecules. The nano-antennas are, therefore, able to drastically suppress photobleaching. Because the local field enhancement does not improve photostability, the suppression comes mainly from the increased quantum yield as a result of competition between photobleaching rate and energy transfer rate to antenna.