The bioenergetic complexes of energy-transducing membranes generate a proton current that powers the synthesis of adenosine triphosphate. Yet, since the early days of the chemiosmotic theory, it has remained elusive and much debated whether the proton motive force (PMF) delocalizes into the bulk solvent surrounding the energy-transducing membrane or if the thermodynamic force is exerted as a localized proton current along the membrane surface. To elucidate the molecular principles underlying protonation dynamics at biological membranes, we combine here proteoliposome experiments with fluorescence correlation spectroscopy and multiscale molecular simulations. We show that ubiquinone (Q10), which is an essential electron carrier of inner mitochondrial membranes, interacts with protons at the membrane, and alters the rate of the protonation reactions along the surface. We find that physiological Q10 concentrations increase the integrity of the liposome membranes to sustain a PMF and enhance the rate of surface protonation reactions of lipid-conjugated pH-sensitive fluorophores, occurring on a microsecond timescale. Our multiscale simulations reveal that the quinone headgroup localizes at the membrane surface and stabilizes protonated water species by cation-π and hydrogen-bonded interactions amplifying the proton exchange on the surface relative to the bulk solvent. We suggest that in addition to the well-established role of quinones as redox mediators in energy-transducing membranes, Q10 also promotes the proton-collecting antenna effect, mediating proton exchange along the membrane and supporting a local proton circuit model. Our combined findings provide molecular insight into propagation of proton currents along biological membranes and reveal key principles underlying the energy conversion mechanisms in biology.

Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Utilizing lanthanide upconversion nanoparticles to convert near-infrared to visible light presents a potential way for fabricating the next generation of low-cost near-infrared imaging sensors. Integrating microlens arrays with upconversion nanoparticles has been shown to be a promising approach for improving the efficiency of upconversion nanoparticles. However, approaches suitable for prototyping and producing microlens arrays to explore optimal device designs are lacking. In this work, we report an approach to fabricating flexible near-infrared-to-visible upconversion devices incorporating upconversion nanoparticles and microlens arrays, which enables easy adjustment of device structures and lens geometry. This is achieved by fabricating flexible films containing upconversion nanoparticles using molding in combination with femtosecond laser 3D printing of lenses, facilitating rapid prototyping for different application scenarios. By adding the microlens array, the intensities of the green (525 and 540 nm) and red (654 nm) upconversion emission bands were enhanced by a factor of 3 and 10, respectively, potentially leading to much reduced detectable near-infrared light intensity.

To overcome challenges in fluorescence labelling of RNA inside living cells we have recently introduced a direct approach using the fluorescent nucleobase analogue 2CNqA. Here we demonstrate its potential for use in fluorescence lifetime imaging (FLIM) to investigate nucleoside metabolism and for metabolic RNA labelling.

Fluorescence-based single-molecule and fluctuation spectroscopy in the near-IR can open avenues for biomolecular dynamic studies in biological media with suppressed autofluorescence and scattering background. However, further implementation is limited by the lower brightness of NIR fluorophores and available single-photon detector technologies that are still to be explored and adapted. Superconducting nanowire single-photon detectors (snSPDs) have found increasing use in quantum optics and optical communication applications thanks to high sensitivity in the near-infraed (NIR), low dark-counts, no after-pulsing, and high time resolution. Here, we present characterization of fluorescence intensity fluctuations from single vesicles and NIR fluorophores based on fluorescence correlation spectroscopy (FCS), specifically taking advantage of these snSPD properties. We present a concept allowing multiplexed readouts based on only one snSPD, in which the emitted photons are separated by their emission wavelength into different optical paths, thereby translating the emission wavelengths into different arrival times onto the snSPD. This concept allows one-laser-one-detector, dual-color fluorescence cross-correlation spectroscopy (FCCS) measurements, with fluorescence intensity fluctuations of two fluorophore species separately analyzed and cross-correlated. It is shown how two fluorophore species in a sample can be distinguished by their different blinking kinetics, fluorescence lifetimes, and/or diffusion properties. Apart from differences in emission spectra, the presented concept for multiplexing using a single detector can also be applied to distinguish emitters by properties such as polarization, coherence lengths, and fluorescence bunching and antibunching signatures. It can also be generalized to other modalities than FCS, including single-molecule detection, confocal microscopy, and imaging.

MINimal photon FLUXes (MINFLUX) offers nanometer localization precision, with lower fluorophore requirements than for other super-resolution microscopy (SRM) techniques. Nonetheless, low localization probabilities hamper its application, and use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. Here, we devised strategies overcoming these limitations. We systematically studied the blinking properties of far-red and NIR cyanine fluorophores, followed by simulations of MINFLUX localizations, over typical time scales (microsecond to 10 milliseconds), sample and excitation conditions for MINFLUX imaging. We identified fluorophore blinking via photoisomerization and photoreduction as the main cause of localization errors, and that use of balanced redox buffers and repetitive excitation beam scans can suppress such errors. Implementing these strategies, we could demonstrate NIR-MINFLUX imaging with nanometer localization precision, thereby also presenting an overall strategy to design optimal sample and excitation conditions, for MINFLUX imaging and for SRM in general.

This study introduces the concept of photophysical structured illumination velocimetry (PP-SIV), verified through comprehensive numerical simulations. PP-SIV can capture two-dimensional (2D) flow velocity fields from a single snapshot image of the emission pattern from luminescent probes, leveraging the suitable photodynamics of the probes and using the applied excitation field pattern as reference. By eliminating the need for any beam or sample scan, PP-SIV has the potential to significantly accelerate the data acquisition process required for velocity field imaging. Furthermore, with excitation patterns applied at different depths, three-dimensional (3D) flow imaging can be potentially achieved. We propose lanthanide-based upconversion nanoparticles (UCNPs), particularly those capable of both absorbing and emitting within the highly biocompatible and transparent NIR-II window (1000-1700 nm), as promising probe candidates for implementing PP-SIV. This concept holds significant potential to pave the way for rapid, three-dimensional (3D) blood flow imaging at sufficient speeds for real-time monitoring of hemodynamic events in the brain.

Exploring lanthanide light upconversion (UC) has emerged as a promising strategy to enhance the near-infrared (NIR) responsive region of silicon solar cells (SSCs). However, its practical application under normal sunlight conditions has been hindered by the narrow NIR excitation bandwidth and the low UC efficiency of conventional materials. Here, we report the design of an efficient multiband UC system based on Ln3+/Yb3+-doped core-shell upconversion nanoparticles (Ln/Yb-UCNPs, Ln3+= Ho3+, Er3+, Tm3+). In our design, Ln3+ ions are incorporated into distinct layers of Ln/Yb-UCNPs to function as near-infrared (NIR) absorbers across different spectral ranges. This design achieves broad multiband absorption withtin the 1100 to 2200 nm range, with an aggregated bandwidth of ~500 nm. We have identified a synthetic electron pumping (SEP) effect involving Yb3+ ions, facilitated by the synergistic interplay of energy transfer and cross-relaxation between Yb3+ and other ions Ln3+ (Ho3+, Er3+, Tm3+). This SEP effect enhances the UC efficiency of the nanomaterials by effectively transferring electrons from the low-excited states of Ln3+ to the excited state of Yb3+, resulting in intense Yb3+ luminescence at ~980 nm within the optimal response region for SSCs, thus markedly improving their overall performance. The SSCs integrated with Ln/Yb-UCNPs with multiband excitation demonstrate the largest reported NIR response range up to 2200 nm, while enabling the highest improvement in absolute photovoltaic efficiency reported, with an increase of 0.87% (resulting in a total efficiency of 19.37%) under standard AM 1.5 G irradiation. Our work tackles the bottlenecks in UCNP-coupled SSCs and introduces a viable approach to extend the NIR response of SSCs.