Single Line Imaging Method (SLIM) for Biochemical Applications
Loading...
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
Reuse License
Abstract
Single-particle dark-field microscopy enables the study of molecular interactions, determining binding constants and measuring dynamic processes. However, the number of nanoparticles that can be detected simultaneously combined with high time resolution is still a requirement that limits the method's applicability in the presence of spatiotemporal heterogeneity of the sample under investigation. Therefore, I have developed a new approach to detect the change of the plasmon signal. Instead of employing the spectral information, I switched to use intensity changes at a fixed wavelength. The detection of intensities simplifies the setup, allows for time- and spatial resolution, and extends the range of sensors for dark-field microscopy since the method is independent of resonance phenomena. I have emphasized these advantages by obtaining previously inaccessible information on long time scales, visualizing dynamic processes of molecular interactions with plasmonic nanosensors, and made out-of-resonance dielectric nanoparticles accessible for dark-field microscopy.
I have shown that intensity changes are suited for measuring the adsorption of macromolecules on nanoparticles with the advantage of reducing the overall measurement time up to a factor of two. I also derived new quantities to describe the plasmon sensitivity in such an intensity scheme, which distinguishes between the various contributions: Rayleigh scattering, dielectric contrast, plasmon shift, and frequency-dependent plasmon bulk damping. The sensitivity parameters, intensity bulk sensitivity SI and intensity surface sensitivity ŞI, allow characterizing and optimizing the sensor's performance. Furthermore, I found that the basic description of the figure of merit (FOM) underestimates the nanoparticles' performance below the interband transition.
To highlight that plasmonic nanoparticles serve as label-free sensors for visualizing dynamic processes, I investigated the Min system, and I observed that on long time scales (24 hours) the wave pattern, the oscillation period and the synchronization of the wave highly depend on the membrane composition. Such data was previously not accessible because commonly used fluorescence labels are not photostable, thus, limiting the observation time.
In addition, the intensity-based readout allows utilizing out-of-resonance nanoparticles as sensing elements that have not been addressed so far. I showed in a systematic study that out-of-resonance nanoparticles are similar in their performance in comparison to the commonly used gold nanorods. This new class of sensors for dark-field microscopy has the advantage that the nanosensors can be made out of any transparent material, e.g. many oxides, lipid vesicles, or polymer beads, making studies of biological macromolecules, such as the formation of a protein corona, easier to access.