An optofluidic antenna for enhancing the sensitivity of single-emitter measurements.

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50 µs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

Insights into protein structure using cryogenic light microscopy.

Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.

Deciphering a hexameric protein complex with Angstrom optical resolution.

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.