title: Planar dielectric antennas for applications in bio-physics
Creators: name: Morales Inostroza, Luis Esteban Alejandro
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2023
Subjects: subject: Dielectric antenna
subject: Photon collection efficiency
subject: DDC Classification::5 Naturwissenschaften und Mathematik :: 50 Naturwissenschaften :: 500 Naturwissenschaften und Mathematik

description: The detection of single molecules in solution enables the study of heterogeneities naturally present in biomolecules that are otherwise hidden in the ensemble average. In the last decades, single-molecule fluorescence methods have been widely implemented to monitor the dynamic exchange between different conformations of individual biomolecules, such as proteins, DNA junctions, and other complex molecular machines. However, the time resolution obtained with these methods is limited by the total number of photons that can be detected per unit of time. Thus, efficient light detection is critical in extracting information from fast dynamic processes. This thesis demonstrates the use of planar dielectric antennas for studying dynamic processes of individual biomolecules in solution with improved time resolution and prolonged observation times. The so-called optofluidic antenna consists of a thin layer of water arranged over a cover glass and capped with air. When molecules diffuse inside the optofluidic antenna, we observe a 4.5-fold enhancement of the brightness per molecule compared to the case of diffusion in bulk solution. This enhancement in photon collection is attributed to the superior collection efficiency provided by the optofluidic antenna. Furthermore, we found two significant effects on the molecules’ Brownian motion when diffusing inside the optofluidic antenna. First, the geometrical constraints posed by the boundaries of the antenna increase the probability of a specific analyte returning to the observation volume. To understand these observations, we performed Monte-Carlo simulations to reproduce the diffusion of the analytes inside the optofluidic antenna. Furthermore, rigorous finite-difference time-domain (FDTD) simulations were performed to show that the lateral dimensions of the observation volume and maximum intensity produced by a confocal excitation inside the optofluidic antenna are not dramatically modified. The second effect is caused by the interaction of the analytes with the water/air interface, which slows the analytes’ translational diffusion, resulting in up to a 400-fold increase in the characteristic observation time compared to the case of diffusion in bulk solution. Next, we studied the internal dynamics of a Holliday Junction inside the optofluidic antenna using fluorescence resonant energy transfer (FRET) to demonstrate the applicability of our novel method to analyzing a biologically relevant sample. We confirmed the previous observations and obtained a 4-fold enhancement in the donor’s and the acceptor’s brightness when measuring with the optofluidic antenna. This result demonstrates the extreme broadband nature of the antenna design since the donor and acceptor dyes are spectrally separated by more than 150 nm, and the enhancement factor is nevertheless nearly identical. A critical point when studying biologically relevant samples is to ensure that the measurement device does not perturb the internal degrees of freedom of the analyte. Using single-molecule multiparameter detection measurements, we show that the conformational states of the Holliday junction are unaffected when it diffuses inside the optofluidic antenna. Moreover, using species-filtered fluorescence correlation spectroscopy (sFCS), we corroborate that the dynamic exchange rate between the different states of the Holliday junction remains unperturbed inside the optofluidic antenna relative to an open solution. A further advantage of measuring inside the optofluidic antenna is the longer observation time. A problem that arises when studying a slow dynamic process is that when the exchange between conformational states becomes slow compared to the characteristic diffusion time, the information about the dynamics between the states is not accessible. We demonstrate the use of the optofluidic antenna to identify intermediate populations in slow dynamic processes by investigating a Holliday junction in solution at 1 mM of magnesium chloride. In this regime, standard methods are only partially effective in identifying the active exchange between the Holliday Junction’s conformations and can only identify quasi-static states. However, when measuring inside the optofluidic antenna, the Holliday Junction diffuses half as quickly; thus, the transition between the conformations becomes visible. Our novel collection strategy is broadly applicable to various fluorescence-related techniques and other approaches such as dark-field microscopy, phase contrast microscopy, and interferometric scattering microscopy (iSCAT). Moreover, our design of the optofluidic antenna can be readily implemented in commercially available inverted microscopes.