Stable jet electrospinning (SJES) enables the incorporation of fluorescent colloidal 2D nanoplatelets (NPLs) into polymer fibers, potentially for optical applications and manipulation of the orientation of the nanoplatelets. Three polymers with excellent optoelectronic properties are used as matrices, namely, poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), and copolycarbonate. The electrospinning parameters are optimized for the production of highly aligned microfibers by SJES. Polarization-resolved fluorescence microscopy is used to determine the orientation of the NPLs within the SJES fibers. For the PMMA fibers spun from a 35 wt % solution, a preferential vertical alignment of the NPL is found. We systematically investigate the origin of the NPL orientation using a series of polymer solutions with well-controlled rheology. During the SJES process, a die swell effect is observed when a highly concentrated viscoelastic polymer solution with a strong elastic component leaves the cannula. The resulting extensional flow causes the anisotropic NPLs to rotate and realign themselves through hydrodynamic forces. Conversely, for concentrations below the overlap concentration, the viscosity drops sharply, and shear thinning disappears, indicating a negligible elastic contribution. The COC and copolycarbonate systems-limited to suboverlap concentrations-exhibited only random NPL orientations. The vertical alignment of the NPLs in the fiber is therefore based on the viscoelastic properties of the non-Newtonian fluid and can be achieved only under strict and specific conditions.

Flat colloidal PbSe QDs (fQDs) represent an innovative class of 2D near-infrared (NIR) photoluminescent QDs, which combine extreme thickness with additional lateral confinement. PbSe fQDs exhibit efficient NIR photoluminescence (860-1550 nm) that is adjustable to the low-loss transmission windows of (optical) fibers and makes them highly promising nanoemitters for fiber-based applications. Here, we demonstrate the incorporation of PbSe fQDs into easy-to-handle functional and stable jet electrospun poly(methyl methacrylate) (PMMA) fibers. Within these electrospun nanocomposites, we find perpendicularly alignedstacks of PbSe fQDs, which give rise to a narrowed and bathochromically shifted photoluminescence (e.g., at 1073 nm, with a quantum yield of 5%) that is caused by an energy transfer into the smallest band gap tail of the PbSe fQD thickness distribution. Embedding PbSe fQDs into solid-state nanocomposite fibers represents an important step forward for implementing near-infrared (NIR)-emitting 2D PbX nanocrystals in fiber optics.

Research on high-efficiency photovoltaic (PV) technologies has consistently improved efficiencies. Yet, laboratory-developed PVs are often far from practical applications due to high material costs. Luminescent solar concentrators (LSCs) can solve this as they use luminophores to direct light from larger areas to little cell materials. However, simple LSCs have very high intrinsic reabsorption, escape cone, and other losses making their combination with high-efficiency PVs unviable. Therefore, systems composed of randomly oriented light-harvesting donor pools, transferring all excitons to a few light-redirecting acceptors aligned parallel to the PV with drastically reduced losses, have been developed (FunDiLight-LSCs). However, these proof-of-principle systems consisted of rather unstable organic molecules. Herein, a novel photostable FunDiLight-LSC based on nanodots as light-harvesting donors and on nanorods as light-redirecting acceptors is introduced. The energy transfer and funneling efficiency in these dots/rods LSCs exceed 90% with escape cone losses potentially below 8%. As the nanoparticles used for the novel LSC are much more stable, combinations of these nanostructured light-harvesting systems with high-efficiency PV will make applications of such photovoltaics in everyday applications significantly more feasible. In this study, a highly efficient luminescent solar concentrator that utilizes photostable nanoparticles is introduced. Pools of nanodots harvest (diffusive) sunlight and funnel it to aligned nanorods that emit the light in distinct directions with high efficiency. This innovation significantly reduces reabsorption and escape cone losses of previous systems, thereby substantially improving the potential of solar concentrators for photovoltaic applications.image (c) 2024 WILEY-VCH GmbH

Using colloidal particles as models to understand processes on a smaller scale is a precious approach. Compared to molecules, particles are less defined, but their architecture can be more complex and so is their long-range interaction. One can observe phenomena that are unknown or much more difficult to realize on the molecular level. The current paper focuses on particle-based surfactants and reports on numerous unexpected properties. The main goal is creating an amphiphilic system with responsiveness in surface activity and associated self-organization phenomena depending on applying an external trigger, preferably a physical field. A key step is the creation of a Janus-type particle characterized by two types of dipoles (electric and magnetic) which geometrically stand orthogonal to each other. In a field, one can control which contribution and direction dominate the interparticle interactions. As a result, one can drastically change the system's properties. The features of ferrite-core organosilica-shell particles with grain-like morphology modified by click chemistry are studied in response to spatially isotropic and anisotropic triggers. A highly unusual aggregation-dissolution-reaggregation sequence w as discovered. Using a magnetic field, one can even switch off the amphiphilic properties and use this for the field-triggered breaking of multiphase systems such as emulsions.

The use of colloidal particles as models to understand processes on a smaller size-scale is a highly valuable approach. Compared to molecules, particles are less defined but their architecture can be more complex, so is their long-range interaction. Sometimes one can observe phenomena which are unknown or much more difficult to realize on the molecular level. The current paper focuses on particle-based surfactants and reports about numerous unexpected properties. The main goal is the creation of an amphiphilic system with responsiveness in surface activity and associated self-organization phenomena depending on the application of an external trigger, preferably a physical field. A key step is the creation of a Janus-type particle characterized by two types of dipoles (electric and magnetic) which geometrically stand orthogonal to each other. In a field, one can control which contribution and which direction dominates the inter-particle interactions and as a result one can change the properties of the system drastically. The features of ferrite-core organosilica-shell particles with grain-like morphology modified by click chemistry is studied in response to spatially isotropic and anisotropic triggers. A highly unusual aggregation-dissolution-reaggregation sequence was discovered. Using a magnetic field, one can even switch off the amphiphilic properties and use this for the field-triggered breaking of multiphase systems such as emulsions.

Stable jet electrospinning (SJES) is a special form of optical fiber generation that prevents chaotic fiber whipping typical for conventional electrospinning procedures. Incorporation of highly emissive semiconductor nanoplatelets (NPLs) in such fibers has very high potential in optical data transmission, optological circuits, fiber lasers, solar light concentrators and many other fields because NPLs exhibit strongly directed emission from their surface plane due to various in-plane transition dipole moments. However, potential orientation control of 2D-NPLs in SJES is entirely unknown as electric fields and various mechanical forces contribute in a complex manner simultaneously. Here, the observation of counter-intuitive yet very beneficial orientation of rectangular CdSe/CdS 2D-NLP in SJES perpendicular to the fiber drawing axis is reported. Scanning electron microscopy, 3D-single particle excitation polarization microscopy, 3D-photogoniometry, polarized emission spectroscopy and small angle X-ray scattering (SAXS) demonstrate aggregation free perpendicular alignment of the NPLs in poly(methyl methacrylate) (PMMA) fibers, resulting in dominant emission in directions parallel to the fiber. It is suggested that the observed vertical alignment is due to normal forces resulting from viscoelastic expansion when the polymer solution leaves the cannula (Barus effect) and that using such perpendicular nano-emitter alignment forces allows for the generation of novel materials also beyond fibers.

Watching events of membrane fusion in real time and distinguishing between intermediate steps of these events is useful for mechanistic insights but at the same time a challenging task. In this chapter, we describe how to use fluorescence cross-correlation spectroscopy and Förster-resonance energy transfer to resolve the tethering and fusion of membranes by SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) as an example. The given protocols can easily be adapted to other membrane proteins to investigate their ability to tether or even fuse vesicular membrane.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions such as protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures such as lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guide of how to set up such a tool.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Transitions into the first excited state of carotenoids, Car S1, are optically forbidden in conventional one-photon excitation (OPE) but are possible via two-photon excitation (TPE). This can be used to quantify the amount of Car S1 to Chlorophyll (Chl) energy transfer in pigment-protein complexes and plants by observing the chlorophyll fluorescence intensity after TPE in comparison to the intensity observed after direct chlorophyll OPE. A parameter, ΦCoupling Car S1-Chl, can be derived that directly reflects relative differences or changes in the Car S1 → Chl energy transfer of different pigment-protein complexes and even living plants. However, very careful calibrations are necessary to ensure similar OPE and TPE excitation probabilities and transition energies. In plants, the exact same sample spot must be observed at the same time. All this is experimentally quite demanding. ΦCoupling Car S1-Chl also corrects intrinsically for direct chlorophyll TPE caused by larger chlorophyll excesses in the complexes, but recently it turned out that in certain TPE wavelengths ranges, its contribution can be quite large. Fortunately, this finding opens also the possibility of determining ΦCoupling Car S1-Chl in a much easier way by directly comparing values in TPE spectra observed at wavelengths that are either more dominated by Cars or Chls. This avoids tedious comparisons of OPE and TPE experiments and potentially allows measurement at even only two TPE wavelengths. Here, we explored this new approach to determine ΦCoupling Car S1-Chl directly from single TPE spectra and present first examples using known experimental spectra from Cars, Chl a, Chl b, LHC II, and PS 1.

Light-harvesting concentrators have a high potential to make highly efficient but precious energy converters, such as multijunction photovoltaics, more affordable for everyday applications. They collect sunlight, including diffusively scattered light, on large areas and redirect it to much smaller areas of the highly efficiency solar cells. Among the best current concepts are pools of randomly oriented light-collecting donor molecules that transfer all excitons to few aligned acceptors reemitting the light in the direction of the photovoltaics. So far, this system has only been realized for the 350–550 nm wavelength range, suitable for AlGaInP photovoltaics. This was achieved by using acceptor molecules that aligned during mechanical stretching of polymers together with donors, that stay random in that very same material and procedure. However, until recently, very little was known about the factors that are responsible for the alignability of molecules in stretched polymers and therefore it was difficult to find suitable donors and acceptors, as well as for other spectral ranges. Recently, a structural parameter was introduced with a high predictivity for the alignability of molecules that contain rigid band-like structures or linear aromatic π-systems. However, for light concentrators in more red spectral ranges, molecular systems often contain larger and extended, planar-like π-systems for which the previously reported parameter is not directly applicable. Here, we present a refined prediction parameter also suitable for larger plane-like structures. The new parameter depends on the number of in-plane atoms divided by out-of-plane atoms as determined by computational geometry optimization and additionally the planar aspect ratio for molecules that contain only in-plane atoms. With the help of this parameter, we found a new system that can efficiently collect and redirect light for the second 500–700 nm AlGaAs layer of current world-record multijunction photovoltaics. Similarly, as the previously reported system for the blue-green layer, it has also overall absorption and re-directioning quantum efficiencies close to 80–100%. Both layers, together, already cover about 75% of the energy in the solar spectrum.

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. SplitGFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than ∼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

SignificanceUsing molecular systems that harvest diffusive sunlight on large areas and funnel it onto much smaller areas of precious high-performance solar cells could pave the way to affordable high-efficiency photovoltaics. Here, we discovered important structural principles of molecules suitable to align diffusive light, the underlying ultrafast depolarization/repolarization dynamics, and a material with significantly higher light harvesting in the peak of the solar spectrum.

Many researchers are recently working on artificial light-harvesters that funnel energy onto high-performance photovoltaics. However, similar to the theoretical photovoltaic Shockley-Queisser limit, there are also physical limits in the maximum efficiency of funneling light energy. Unfortunately, they are very complex and depend on many opposing molecular as well as macroscopic structure factors. For example, higher pigment concentrations absorb more sun-light but lead also to higher, intrinsic reabsorption losses. Molecular orientations increasing incoming light absorption can also increase re-emission losses back into the same direction. Larger spectral absorption ranges collect more sunlight but simultaneously increase losses due to lower emission photon energies. Larger macroscopic areas of light-harvesting material decrease the need for expensive photovoltaic materials but also increase molecular re-absorption losses. Larger pools of energy funneling molecules increase light concentration but also increase energy transfer losses. For finding the optimal overall molecular and macroscopic structure these opposing effects need to be varied within physical feasible boundaries. Here, we present a theoretical assessment that include all necessary molecular and macroscopic parameters and that provided optimized light-harvesting structures representing a new efficiency bench mark. For highest efficiencies, our results indicate that combined organic molecule/quantum particle systems share many of the optimum spectroscopic characteristics.

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of non-modulated back-ground intensity.

Using rotation of linearly polarized illumination from different directions enables reconstructing the full 3D orientation of single molecules. Theoretically, already two projections suffice for a full 3D orientation determination if the intensities are properly calibrated and in the absence of noise. The question arises if a limited set of projections that cannot excite all molecules equally contains already the maximum information on the 3D orientation when also considering photon and experimental noise, or if detecting more projections or even the full angular projection space provides more information. To answer this question, we constructed a device exploring the full angular excitation space available with a distinct microscope objective and tested several different polarization modulation schemes using simulated as well as experimental single molecule data. For noisy experimental data, we found three projections ideal and no significant improvement in angular precision for larger projection numbers or when detecting the full projection space.

We developed three bathochromic, green-light activatable, photolabile protecting groups based on a nitrodibenzofuran (NDBF) core with D-π-A push-pull structures. Variation of donor substituents (D) at the favored ring position enabled us to observe their impact on the photolysis quantum yields. Comparing our new azetidinyl-NDBF (Az-NDBF) photolabile protecting group with our earlier published DMA-NDBF, we obtained insight into its excitation-specific photochemistry. While the "two-photon-only" cage DMA-NDBF was inert against one-photon excitation (1PE) in the visible spectral range, we were able to efficiently release glutamic acid from azetidinyl-NDBF with irradiation at 420 and 530 nm. Thus, a minimal change (a cyclization adding only one carbon atom) resulted in a drastically changed photochemical behavior, which enables photolysis in the green part of the spectrum.

In the past, different methods have been presented to determine the 3D orientation of single molecules in a microscopic set-up by excitation polarization modulation. Using linearly polarized illumination from different directions and thereby measuring different 2D projections enables reconstructing the full 3D orientation. Theoretically, two projections suffice for a full 3D orientation determination if the intensities are properly calibrated. If they are not, a third projection will enable unambiguous orientation measurements. The question arises if three projections already contain the maximum information on the 3D orientation when also considering the limited number of available photons and shot noise in an experiment, or if detecting more projections or even continuously changing the projection direction during a measurement provides more information with an identical number of available photons. To answer this principle question, we constructed a simple device allowing for exploring any projection direction available with a particular microscope objective and tested several different excitation modulation schemes using simulated as well as experimental single molecule data. We found that three different projections in fact already do provide the maximum information also for noisy data. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement. In fluorescence microscopy polarized illumination from different directions enables the determination of the 3D orientation of single molecules by combining the 2D information of different projection directions. Ambiguities that emerge when using only two projections can be eliminated using a third projection. In a systematic study we show that – also considering the limited number of available photons and shot noise in an experiment – three projection directions already contain the maximum information on the 3D orientation. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement.

Collin Steen opened discussion of Tomas Polivka's paper: It would be very interesting to study the excited state dynamics of this E-FCP protein in the context of the whole organism. Dynamics and behavior are frequently altered in an in vivo sample compared to an isolated one. As an example, I'd