Single-emitter fluorescence detection is used in diverse fields, from biophysics to quantum optics, to precisely observe processes at the single-molecule level. When performed under fluidic conditions, diffusion can restrict the observation time and detected photon counts, hampering the investigation of both slow and fast phenomena occurring in the molecule. To enhance the optical signal from emitters in a liquid and allow longer observation times, researchers at the Max Planck Institute for the Science of Light (MPL) and the University of Düsseldorf developed and characterized an optofluidic antenna (OFA). The optical design of the OFA was adopted from a planar dielectric antenna. The OFA expands the time range for studying biomolecular dynamics beyond the limit imposed by the translational diffusion time in a laser focus. It collects the photons emitted by individual fluorescent molecules with approximately 85% efficiency, enabling a time resolution in the microsecond (μs) range and allowing conformational changes of individual biomolecules to be observed with the highest temporal resolution. The fabrication of the OFA device is inexpensive and straightforward. The antenna consists of a glass substrate and a layer of water that is several hundred nanometers thick and contains the molecules to be examined. The layer of water is created by a micropipette positioned just a few hundred nanometers above the substrate. The axial boundary of the water layer forces the molecules to diffuse through the center of the laser focus, increasing the brightness of the laser light. The water-air interface slows the diffusion of the molecules and the antenna’s geometry increases the probability that a molecule will return to focus. The researchers characterized the OFA using single-molecule, multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS), and Förster resonance energy transfer (FRET). Using the OFA, they examined the change in conformity of a DNA four-way junction with a molecular mass of about 100 kilodalton (kDa), which is comparable to the size of many proteins and biomolecular machinery used for single-molecule studies. They examined both the slow (milliseconds) and fast (50 μs) dynamics of the DNA four-way junction with real-time resolution. The researchers marked two legs of the DNA four-way junction with a FRET pair. The number of photons emitted by each of the two FRET partners changed with the distance between the two legs. The FRET trajectories revealed the absence of an intermediate conformational state and provided an upper limit for its lifespan. The OFA tracked the dynamics of DNA four-way crossing with a temporal resolution of just a few microseconds. Artistic view of a four-way DNA junction labeled with two fluorescent molecules diffusing inside an optofluidic antenna (OFA). The upper portion of the graphic displays a micropipette where the water meniscus is controlled by the pressure inside the pipette. Courtesy of Dante Yovane. The OFA was found to enhance the fluorescence signal detected from molecules by about 5x per passage. It led to about 7x more frequent returns to the observation volume and it significantly lengthened the diffusion time. The OFA’s efficient collection of photons — an increase of about 2.2-fold — provides access to the optimal photon budget. “Our optofluidic antenna works so well due to the improved photon collection efficiency from slower diffusing molecules in the spatially limited channel,” professor Stephan Götzinger said. The OFA operates in a broad spectral domain and is fault-tolerant to antenna dimensions. It can be readily implemented in existing inverted microscopes and is compatible with other microscopy methods, such as dark-field and interferometric scattering for nanoparticle analysis. It can also be combined with platforms such as plasmonic systems, and with methods that slow down the translational diffusion of analytes, such as trapping, immobilization, and tethering mechanisms. The sensitive, contact-free optical measurements achieved with the OFA provide access to both faster and slower dynamics of biological entities than a regular, bulk fluidic environment. Ease of implementation and compatibility with various microscopy modalities make the OFA a convenient platform for achieving more sensitive single molecule-fluorescence measurements for a range of studies. “The antenna is a powerful device for investigations in the life sciences,” professor Vahid Sandoghdar said. “It is not only easy to use, but can also be easily integrated into many existing microscopy setups.”
