Correspondence Author -Tjaart P. J. Krüger, tjaart.kruger@up.ac.za.Abstract-Plasmonic coupling of metallic nanoparticles and adjacent pigments can dramatically increase the brightness of the pigments due to the enhanced local electric field. Here, we demonstrate that the fluorescence brightness of a single plant light-harvesting complex (LHCII) can be significantly enhanced when coupled to single gold nanorods (AuNRs). The AuNRs utilized in this study were prepared via chemical reactions, and the hybrid system was constructed using a simple and economical spin-assisted layer-by-layer technique.Enhancement of fluorescence brightness of up to 240-fold was observed, accompanied by a 109-fold decrease in the average (amplitude-weighted) fluorescence lifetime from approximately 3.5 ns down to 32 ps, corresponding to an excitation enhancement of 63-fold and emission enhancement of up to 3.8-fold. This large enhancement is due to the strong spectral overlap of the longitudinal localized surface plasmon resonance of the utilized AuNRs and the absorption or emission bands of LHCII. This study provides an inexpensive strategy to explore the fluorescence dynamics of weakly emitting photosynthetic light-harvesting complexes at the single molecule level.

Real‐time feedback‐driven single‐particle tracking (RT‐FD‐SPT) is a class of techniques in the field of single‐particle tracking that uses feedback control to keep a particle of interest in a detection volume. These methods provide high spatiotemporal resolution on particle dynamics and allow for concurrent spectroscopic measurements. This review article begins with a survey of existing techniques and of applications where RT‐FD‐SPT has played an important role. Each of the core components of RT‐FD‐SPT are systematically discussed in order to develop an understanding of the trade‐offs that must be made in algorithm design and to create a clear picture of the important differences, advantages, and drawbacks of existing approaches. These components are feedback tracking and control, ranging from simple proportional‐integral‐derivative control to advanced nonlinear techniques, estimation to determine particle location from the measured data, including both online and offline algorithms, and techniques for calibrating and characterizing different RT‐FD‐SPT methods. Then a collection of metrics for RT‐FD‐SPT is introduced to help guide experimentalists in selecting a method for their particular application and to help reveal where there are gaps in the techniques that represent opportunities for further development. Finally, this review is concluded with a discussion on future perspectives in the field.

Real-time feedback-driven single-particle tracking is a technique that uses feedback control to enable single-molecule spectroscopy of freely diffusing particles in native or near-native environments. A number of different real-time feedback-driven single-particle tracking (RT-FD-SPT) approaches exist, and comparisons between methods based on experimental results are of limited use due to differences in samples and setups. In this study, we used statistical calculations and dynamical simulations to directly compare the performance of different methods. The methods considered were the orbital method, the knight‘s tour (grid scan) method, and MINFLUX, and we considered both fluorescence-based and interferometric scattering (iSCAT) approaches. There is a fundamental trade-off between precision and speed, with the knight’s tour method being able to track the fastest diffusion but with low precision, and MINFLUX being the most precise but only tracking slow diffusion. To compare iSCAT and fluorescence, different biological samples were considered, including labeled and intrinsically fluorescent samples. The success of iSCAT as compared to fluorescence is strongly dependent on the particle size and the density and photophysical properties of the fluorescent particles. Using a wavelength for iSCAT that is negligibly absorbed by the tracked particle allows for an increased illumination intensity, which results in iSCAT providing better tracking for most samples. This work highlights the fundamental aspects of performance in RT-FD-SPT and should assist with the selection of an appropriate method for a particular application. The approach used can easily be extended to other RT-FD-SPT methods.

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Super-resolution microscopy and single molecule fluorescence spectroscopy require complementary and mutually exclusive experimental strategies that optimize either spatial or temporal resolution, making their combined implementation a challenge. With a GPU-driven, camera-based measurement strategy we demonstrate that it is possible to achieve this in 5 minutes (including measurement time) using the epidermal growth factor receptor (EGFR) as our model. EGFR, a transmembrane receptor tyrosine kinase, is a crucial regulator of proliferation and survival, and its mutations and dysregulated signalling have been implicated in many cancers. However, a clear understanding of its oligomerization state, dynamics, and interactions with the actin cytoskeleton and other plasma membrane components has been elusive, and we have investigated these in live CHO-K1 cells using TIRF microscopy. Simultaneous dual-channel spatiotemporal super-resolution measurements were performed using the actin marker Lifeact along with EGFR. By optimizing the extraction of various parameters through spatial or temporal binning, a single data set is used for multi-parametric analysis involving imaging fluorescence correlation spectroscopy (ImFCS) to study EGFR dynamics, number and brightness analysis (N&B) to determine EGFR oligomerization, FCS diffusion law to determine sub-resolution EGFR localization, and super-resolution radial fluctuations (SRRF) to super-resolve actin fibres. Cross-correlation analysis between any two parameters provides new biological knowledge that cannot be obtained from separate sequential measurements. A combination of measurements involving resting cells, ligand addition, drug treatments, and EGFR mutants revealed that the domain localization of EGFR in membranes is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions, but is largely independent of EGFR-actin interactions. Our approach does not require specialized instrumentation, can be implemented with other combinations of fluorescence techniques, and thus is easily applicable and useful to answer a wide variety of biological questions.