The environment-sensitive fluorescence emission of Laurdan (6-lauroyl-2-dimethylaminonaphthalene) underlies its widespread application in studies of biomolecular assemblies. The first part of this invited contribution clarifies the relevant photophysical concepts of Laurdan. Based on an overview of the literature on the characterization of extracellular vesicles (EVs) using Laurdan, the second part advances Laurdan's use by introducing time-resolved emission spectra (TRES) for EV characterization. As diagnostic applications require distinguishing between EV subtypes, and given that tetraspanins are major protein constituents of EVs, we characterized Laurdan fluorescence in EVs isolated from various tetraspanin knockout cell lines. To resolve differences between these EVs, we recorded TRES and analyzed them using the standard time-dependent fluorescence shift approach. In addition, we applied spectral phasor analysis to the TRES data-a combination that, as far as we are aware, has not previously been explored. This combined approach offers clear advantages over previously employed steady-state methods, as it can distinguish EVs with different protein profiles and provides comprehensive information on both compositional heterogeneity and lipid phase state. Inspection of the TRES phasor trajectories further suggests that Laurdan is sensitive not only to the lipid matrix but also to nonlipid constituents of EVs, such as membrane-associated proteins.

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1's activity has been suggested to be connected to two molecular concepts, which are, however, lacking experimental proof: a) enhanced binding affinity of Gal-1 toward membranes containing monosialotetrahexosylganglioside (GM1) over disialoganglioside GD1a and b) cross-linking of GM1's by homodimers of Gal-1. We provide evidence about the specificity and the nature of the interaction of Gal-1 with model membranes containing GM1 or GD1a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 indeed binds specifically to GM1 and not to GD1a when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 20 μM). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM1 cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM1 and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM1/Gal-1 equilibria toward the membrane-bound homodimeric Gal-1.

Optical properties of molecules change drastically as results of interactions with surrounding environments as observed in solutions, clusters and aggrergates. Here we make 7,7,8,8-tetracyanoquinodimethane (TCNQ) highly luminescent by encapsulating it in crystalline melamine. Coloured single crystals are synthesized by slow evaporation of a solution of melamine and TCNQ in water and tetrahydrofuran. Single crystal X-ray diffraction reveals the lattice structure of pure melamine, meaning that the colour is of impurities. Both mass spectrometry and UV-Vis spectroscopy combined with density-functional theory calculations elucidate that the impurity species are neutral TCNQ and its oxidation product, dicyano-$p$-toluoyl cyanide anion (DCTC$^-$), whose concentrations in a melamine crystal can be controlled by adjusting the molar ratio between melamine and TCNQ in the precursor solution. Fluorescence exciation-emission wavelength mappings on the precursor solutions illustrate dominant emissions from DCTC$^-$ while the emission from TCNQ is quenched by the resonance energy transfer to DCTC$^-$. On the contrary, TCNQ in crystalline melamine is a bright fluorophore whose emission wavelength centered at 450 nm and slow fluorescence lifetimes of about 2 ns. Our method of encapsulating molecules into transparent melamine would make many other molecules fluorescencent in solids.

The goal of this contribution is threefold:

Recent advancements in isolation and stacking of layered van der Waals materials have created an unprecedented paradigm for demonstrating varieties of 2D quantum materials. Rationally designed van der Waals heterostructures composed of monolayer transition-metal dichalcogenides (TMDs) and few-layer hBN show several unique optoelectronic features driven by correlations. However, entangled superradiant excitonic species in such systems have not been observed before. In this report, it is demonstrated that strong suppression of phonon population at low temperature results in a formation of a coherent excitonic-dipoles ensemble in the heterostructure, and the collective oscillation of those dipoles stimulates a robust phase synchronized ultra-narrow band superradiant emission even at extremely low pumping intensity. Such emitters are in high demand for a multitude of applications, including fundamental research on many-body correlations and other state-of-the-art technologies. This timely demonstration paves the way for further exploration of ultralow-threshold quantum-emitting devices with unmatched design freedom and spectral tunability.

Diamond optical centers have recently emerged as promising single-photon sources for quantum photonics. Particularly, negatively charged silicon vacancy (SiV-) centers show great promise due to their narrow zero-phonon emission line present also at room temperature. However, due to fabrication tolerances it is challenging to prepare directly photonic structures with optical modes spectrally matching the emission of SiV- centers. To reach the spectral overlap, photonic structures must typically undergo complicated post-processing treatment. In this work, suspended photonic crystal cavities made of polycrystalline diamond are engineered and more than 2.5-fold enhancement of the SiV- center zero-phonon line intensity via coupling to the cavity photonic mode is demonstrated. The intrinsic non-homogeneous thickness of the diamond thin layer within the sample is taken as an advantage that enables reaching the spectral overlap between the emission from SiV- centers and the cavity modes without any post-processing. Even with lower optical quality compared to monocrystalline diamond, the fabricated photonic structures show comparable efficiency for intensity enhancement. Therefore, the results of this work may open up a promising route for the application of polycrystalline diamond in photonics.

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.

Silicon vacancy (SiV) centers are optically active defects in diamond. The SiV centers, in contrast to nitrogen vacancy (NV) centers, possess narrow and efficient luminescence spectrum (centered at ≈738 nm) even at room temperature, which can be utilized for quantum photonics and sensing applications. However, most of light generated in diamond is trapped in the material due to the phenomenon of total internal reflection. In order to overcome this issue, we have prepared two-dimensional photonic crystal slabs from polycrystalline diamond thin layers with high density of SiV centers employing bottom-up growth on quartz templates. We have shown that the spectral overlap between the narrow light emission of the SiV centers and the leaky modes extracting the emission into almost vertical direction (where it can be easily detected) can be obtained by controlling the deposition time. More than 14-fold extraction enhancement of the SiV centers photoluminescence was achieved compared to an uncorrugated sample. Computer simulation confirmed that the extraction enhancement originates from the efficient light-matter interaction between light emitted from the SiV centers and the photonic crystal slab.

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2-methylacrylic acid 2-[(2-(dimethylamino)ethyl)methylamino]ethyl ester44} (POEGMA70-b-P(OEGMA10-co-DAMA44). Block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (R(H) ≈ 65-85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment.

This volume focuses on Time-Correlated Single Photon Counting (TCSPC), a powerful tool allowing luminescence lifetime measurements to be made with high temporal resolution, even on single molecules. C

Detection sensitivity from the ultraviolet to the near infrared spectral region is a key parameter to meet today's demand for handling smallest analyte amounts and short measurement times in the optical evaluation of miscellaneous samples. The introduction of single photon counting based data acquisition has proven to yield a major sensitivity increase and very high dynamic range - it is the ideal method for measuring weak luminescence. We present the hardware and handling optimization of a state of the art spectrometer for steady-state and time-resolved fluorescence measurements. The high sensitivity of the spectrometer was shown by measurements of popular fluorescent dyes as well as the Raman spectrum of water under well defined and reproducible conditions. The achieved sensitivity allows us to quantify singlet oxygen generation and to characterize the singlet oxygen phosphorescence decay, a prerequisite when studying photosensitisers like porphyrins and phthalocyanines used for example in photodynamic therapy (PDT). Moreover, with the help of an integrating sphere fluorescence quantum yields of low fluorescent samples like Ru(bpy)3 in water can be determined very precisely. The fibre connection of the spectrometer to a time-resolved fluorescence microscope (MicroTime100/200) was also realized. The combination of the advantages of both setups makes it e.g. possible to perform 2D-lifetime imaging with a freely tunable detection window for low luminescent samples even far into the near infrared region. The measurements with such a combination give not only the spectral and lifetime information of a luminescent sample but also the spatial information which is especially important for hetergeneous samples.

We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.

The free charge carrier lifetime is a highly sensitive parameter that can be used for analyzing the function of semiconductor devices and monitoring the quality of wafer materials. In this context, time-resolved photoluminescence (TRPL) is presented as a technique for directly determining the free charge carrier lifetime with pulsed diode laser excitation and time-correlated single photon counting, employing highly sensitive single-photon avalanche detectors (SPADs). The full range in time-scales of charge carrier dynamics can be addressed with the capability to resolve luminescence lifetimes from approximately 50 ps up to several hundred microseconds. This is achieved with an instrument response function (IRF) as short as 100 ps and the capability of adjustable repetition rates in the pulsed laser excitation that can be adapted to the luminescence lifetime of the material. The technique is capable of correlating spectral information concerning material specific band gap transitions and transmission edges with the respective luminescence lifetimes in a specific spectral channel. This is particularly valuable for the analysis of multi-component systems. Furthermore, the general instrumentation can be combined with a raster scanning based microscope setup, which can be configured to cover lateral resolutions down to sub-μm scale and scan ranges from 100 microns up to several centimeters [1].

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

Efficient signal generation in DNA-based assays requires understanding of the influence of fluorophores interactions on the spectroscopic properties. The resulting changes in fluorescence intensity, quantum yield, emission anisotropy, and fluorescence lifetime provide straightforward tools for the study of molecular dynamics and interaction between labels and nucleic acids. Searching for bright fluorescent reporters for rolling circle amplification (RCA) as efficient signal enhancement strategy for biological formats, we investigated the spectroscopic properties of seven dyes: cyanines, rhodamines, and BODIPYs. They spectrally resemble Cy3, the most frequently used fluorophore in biodetection formats, and are measured in six samples (free dye, dye-dUTP, internally labeled ssDNA and dsDNAsingle- and triple-labeled) using steady-state and time-resolved fluorometry. Special emphasis was dedicated to characterizing the nature of the interaction of these fluorophores differing in dye class, charge, and rigidity. Our results suggest dye charge and structure as main factors governing the dyes interactions, with DY-555 and Cy3B presenting the best candidates for our envisaged signal amplification strategy. This label comparison underlines the importance of a proper understanding of structure-property relations and dyebiomolecule interactions for reporter choice and presents a road map towards the design and interpretation of experiments using these labels on DNA of known sequence.