Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL-ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

Optically active fullerenes, including C60 and C70 derivatives carrying organic substituents, are used in a range of applications because of their unique spectroscopic, catalytic, and chiral recognition properties. However, their inherent photoexcited chirality is yet to be elucidated because of their very poor fluorescence quantum yield (Φf). We synthesised a new chiral C70 derivative, X70A, with 20% yield, by reacting bis-borylated xanthene with C70 in a one-step double addition reaction, followed by a successful optical resolution. The isolation of two separate X70A enantiomers was confirmed by mirror-image circular dichroism spectroscopy in the range of 300-750 nm. In toluene, the enantiomeric pair of X70A clearly revealed mirror-image circularly polarised luminescence (CPL) spectra with a high |glum| value of 7.0 ×  10-3 at 690 nm. The first fullerene-based deep-red CPL of X70A should provide a new guideline for the design of chiral nanocarbon materials.

Red-NIR luminescent polymers are principally obtained from petroleum-based derivatives in which emitters, usually a critical raw material such as rare-earth or platinum group metal ions, are embedded. Considering the strong ecological impact of their synthesis and the major risk of fossil fuel energy shortage, there is an urgent need to find alternatives. We describe a luminescent nanocomposite based on red-NIR phosphorescent molybdenum nanoclusters, namely Cs2Mo6I8(OCOC2F5)6, embedded in an eco-friendly cellulose biopolymer matrix that is obtained by a simple solvent casting technique. While homogeneity is kept up to 20 wt% of cluster complex doping, annealing hybrids leads to a large increase of their emission efficiency, as demonstrated by quantum yield measurements.

Efforts have been devoted to screening various prevalent diseases, such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). Real-time polymerase chain reaction (RT-PCR), which is currently the most widely used, has high accuracy, but it requires several facilities and takes a relatively long time to check; so, new testing technology is necessary for a higher test efficiency. A chemiluminescence (CL) sensor is a relatively simple device and suitable as an alternative because it can detect very precise specimens. However, in measurements via CL, the quantitative formulation of reagents that cause color development is important. In the case of mixing using micropipettes, precise analysis is possible, but this technique is limited by uncontrollable errors or deviations in detection amounts. In addition, in using a microfluidic chip to increase field applicability, a syringe pump or other quantification injection tools are required, so problems must be overcome for practical use. Therefore, in this study, a microchip was designed and manufactured to supply a sample of a certain volume by simply blowing air and injecting a sample into the chamber. By utilizing the luminescence reaction of luminol, CuSO4 and H2O2 the performance of the prepared chip was confirmed, and the desired amount of the sample could be injected with a simple device with an error rate of 2% or less. For feasible applications, an experiment was performed to quantitatively analyze thrombin, a biomarker of heart disease. Results demonstrated that biomarkers could be more precisely detected using the proposed microchips than using micropipettes.

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

Temperature is a key parameter in many fields and luminescence-based temperature sensing is a solution for those applications in which traditional (mechanical, electrical, or IR-based) thermometers struggle. Amongst the indicator dyes for luminescence thermometry, Ru(II) polyazaheteroaromatic complexes are an appealing option to profit from the widespread commercial technologies for oxygen optosensing based on them. Six ruthenium dyes have been studied, engineering their structure for both photostability and highest temperature sensitivity of their luminescence. The most apt Ru(II) complex turned out to be bis(1,10-phenanthroline)(4-chloro-1,10-phenanthroline)ruthenium(II), due to the combination of two strong-field chelating ligands (phen) and a substituent with electron withdrawing effect on a conjugated position of the third ligand (4-Clphen). In order to produce functional sensors, the dye has been best embedded into poly(ethyl cyanoacrylate), due to its low permeability to O₂, high temperature sensitivity of the indicator dye incorporated into this polymer, ease of fabrication, and excellent optical quality. Thermosensitive elements have been fabricated thereof as optical fiber tips for macroscopic applications (water courses monitoring) and thin spots for microscopic uses (temperature measurements in cell culture-on-a-chip). With such dye/polymer combination, temperature sensing based on luminescence lifetime measurements allows 0.05 °C resolution with linear response in the range of interest (0⁻40 °C).

Phosphate sensors have been actively studied owing to their importance in water environment monitoring because phosphate is one of the nutrients that result in algal blooms. As with other nutrients, seamless monitoring of phosphate is important for understanding and evaluating eutrophication. However, field-deployable phosphate sensors have not been well developed yet due to the chemical characteristics of phosphate. In this paper, we report on a luminescent coordination polymer particle (CPP) that can respond selectively and sensitively to a phosphate ion against other ions in an aquatic ecosystem. The CPPs with an average size of 88.1 ± 12.2 nm are embedded into membranes for reusable purpose. Due to the specific binding of phosphates to europium ions, the luminescence quenching behavior of CPPs embedded into membranes shows a linear relationship with phosphate concentrations (3-500 μM) and detection limit of 1.52 μM. Consistent luminescence signals were also observed during repeated measurements in the pH range of 3-10. Moreover, the practical application was confirmed by sensing phosphate in actual environmental samples such as tap water and lake water.

A series of anionic heavy lanthanide complexes, involving the N-salicylideneglycinato(2-) Schiff base ligand (salgly) and having the general formula K[Ln(salgly)₂(H₂O)₂]∙H₂O (1-6), where Ln stands for Gd, Tb, Dy, Ho, Er and Tm, was prepared using the one-pot template synthesis. The complexes were thoroughly characterized by elemental and Thermogravimetric/Differential Thermal Analyses (TG/DTA), Fourier Transform Infrared Spectroscopy (FT-IR), and photoluminescence spectroscopies, electrospray-ionization mass spectrometry, and their magnetic properties were studied by temperature-dependent dc magnetic measurements using the superconducting quantum interference device (SQUID). The X-ray structure of the terbium(III) complex (2), representing the unique structure between the lanthanide complexes of N-salicylideneamino acids, was determined. The results of spectral and structural studies revealed the isostructural nature of the prepared complexes, in which the lanthanide ion is octacoordinated by two O,N,O-donor salgly ligands and two aqua ligands. The analysis of magnetic data confirmed that the complexes behave as paramagnets obeying the Curie law. The results of photoluminescence spectral studies of the complexes showed the different origin in their luminescent properties between the solid state and solution. An antenna effect of the Schiff base ligand was observed in a powder form of the complex only, while it acts as a fluorophore in a solution.

In the field of Nanomedicine, there is an increasing demand for new inorganic nanophosphors with low cytotoxicity and efficient loading-release ability of drugs for applications in bioimaging and drug delivery. This work assesses the potentiality of matured Eu-doped citrate-coated carbonated apatite nanoparticles to be used as theranostic platforms, for bioimaging, as luminescent nanoprobes, and for drug delivery applications, using Doxorubicin as a model drug. The drug adsorption isotherm fits the Langmuir-Freundlich (LF) model, showing that the Eu:cit-cAp nanoparticles can carry a maximum of 0.29 ± 0.02 mg Doxo mg Eu:cit-cAp-1 (Qmax). The affinity constant KFL for this binding is 44 ± 2 mL mg-1, and the cooperativity coefficient r is 6 ± 1. The nanoparticle suspensions presented charge reversion from negative to positive after loading with Doxo as revealed by the ζ-potential versus pH characterization. The release of drug from the loaded nanoparticles was found to be strongly pH-dependent, being around 5 wt % at physiological pH 7.4 and 20 wt % at pH 5, in experiments lasting 24 h. Luminescence spectroscopic measurements of Doxo-loaded nanoparticles revealed the increase of luminescence with a decrease in the amount of adsorbed Doxo, due to the so-called inner filter effect. The nanoparticles free of Doxo were cytocompatible when interacted with two human cell lines derived respectively from a gastric carcinoma (GTL-16), and a hepatocarcinoma (Huh7), while Doxo-loaded nanoparticles displayed significant toxicity in a dose-dependent relationship. Therefore, the new nanoassemblies might have a dual function, as nanoprobes in bioimaging by detecting the fate of the nanoparticles in biological environments, and for monitoring the delivery of the drug in such environments, by measuring the rise of the luminescence provided by the desorption of Doxo.

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

Here we present a facile one-pot method to prepare high-quality CdTe nanocrystals in aqueous phase. In contrast to the use of oxygen-sensitive NaHTe or H(2)Te as Te source in the current synthetic methods, we employ more stable sodium tellurite as the Te source for preparing highly luminescent CdTe nanocrystals in aqueous solution. By selecting mercaptosuccinic acid (MSA) as capping agent and providing the borate-citrate acid buffering solution, CdTe nanocrystals with high quantum yield (QY >70% at pH range 5.0-8.0) can be conveniently prepared by this method. The influence of parameters such as the pH value of the precursor solution and the molar ratio of Cd(2+) to Na(2)TeO(3) on the QY of CdTe nanocrystals was systematically investigated in our experiments. Under optimal conditions, the QY of CdTe nanocrystals is even high up to 83%. The biological application of luminescent MSA-CdTe to HEK 293 cell imaging was also illustrated.

Herein, we report the design and synthesis of biocompatible Yb3+ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb3+ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb3+ luminescence signals when the biocompatible Yb3+ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

Comparing the performance of molecular and nanoscale luminophores and luminescent micro- and nanoparticles and estimating achievable signal amplitudes and limits of detection requires a standardizable intensity scale. This initiated the development of the relative MESF (number of molecules of equivalent soluble fluorochromes) and ERF (equivalent reference fluorophores) scales for flow cytometry and fluorescence microscopy. Both intensity scales rely on fluorescence intensity values assigned to fluorescent calibration beads by an intensity comparison to spectrally closely matching fluorophore solutions of known concentration using a spectrofluorometer. Alternatively, the luminophore or bead brightness (B) can be determined that equals the product of the absorption cross section (σa) at the excitation wavelength (σa(λex)) and the photoluminescence quantum yield (Φpl). Thereby, an absolute scale based on fundamental and measurable spectroscopic properties can be realized which is independent of particle size, material, and luminophore staining or labeling density and considers the sensitivity of the optical properties of luminophores to their environment. Aiming for establishing such a brightness scale for light-scattering dispersions of luminescent particles with sizes exceeding a few ten nanometers, we demonstrate how the brightness of quasi-monodisperse 25 nm, 100 nm, and 1 µm sized polystyrene particles (PSP), loaded with two different dyes in varying concentrations, can be obtained with a single custom-designed integrating sphere setup that enables the absolute determination of Φpl and transmittance and diffuse reflectance measurements. The resulting Φpl, σa(λex), imaginary parts of the refractive index, and calculated B values of these samples are given in dependence of the number of incorporated dye molecule per particle. Finally, a unitless luminescence efficiency (LE) is defined allowing for the direct comparison of luminescence efficiencies of particles with different sizes.

Semiconducting single-walled carbon nanotubes (SWCNTs) are an interesting material for strong-light matter coupling due to their stable excitons, narrow emission in the near-infrared region, and high charge carrier mobilities. Furthermore, they have emerged as quantum light sources as a result of the controlled introduction of luminescent quantum defects (sp3 defects) with red-shifted transitions that enable single-photon emission. The complex photophysics of SWCNTs and the overall goal of polariton condensation pose the question of how exciton-polaritons are populated and how the process might be optimized. The contributions of possible relaxation processes, i.e., scattering with acoustic phonons, vibrationally assisted scattering, and radiative pumping, are investigated using angle-resolved reflectivity and time-resolved photoluminescence measurements on microcavities with a wide range of detunings. We show that the predominant population mechanism for SWCNT exciton-polaritons in planar microcavities is radiative pumping. Consequently, the limitation of polariton population due to the low photoluminescence quantum yield of nanotubes can be overcome by luminescent sp3 defects. Without changing the polariton branch structure, radiative pumping through these emissive defects leads to an up to 10-fold increase of the polariton population for detunings with a large photon fraction. Thus, the controlled and tunable functionalization of SWCNTs with sp3 defects presents a viable route toward bright and efficient polariton devices.

Recently, several studies have demonstrated that diaminodicyanoquinone derivatives (DADQs) could present interesting fluorescence properties. Furthermore, some DADQs under the solid state are capable of showing quantum yields that can reach values of 90%. Besides, the diaminodiacyanoquinone core represents a versatile building block propense either to modification or integration into different systems to obtain and provide them unique photophysical features. Herein, we carried out a theoretical study on the fluorescence properties of three different diaminodicyanoquinodimethane systems. Therefore, time-dependent density functional theory (TD-DFT) was used to obtain the values associated with the dipole moments, oscillator strengths, and the conformational energies between the ground and the first excited states of each molecule. The results suggest that only two of the three studied systems possess significant luminescent properties. In a further stage, the theoretical insights were confirmed by means of experimental measurements, which not only retrieved the photoluminescence of the DADQs, but also suggest a preliminary and promising antibacterial activity of these systems.

Semiconductor nanoplatelets (NPLs) are promising materials for nonlinear optical microscopy since they feature good two-photon absorption (TPA) properties, narrow photoluminescence spectra and high quantum yields of luminescence. Nevertheless, the use of semiconductor NPLs is inevitably connected with concerns about heavy metal ion toxicity and their intrinsically hydrophobic character.

We present the synthesis and characterization of two series of mononuclear heteroleptic anionic cycloplatinated(II) complexes featuring terminal cyanide ligand Q+[Pt(C^N)(p-MeC6H4)(CN)]- [C^N = benzoquinolate (bzq), Q+ = K+ 1 and NBu4+ 4; 2-phenylpyridinate (ppy), Q+ = K+ 2 and NBu4+ 5 and 2-(2,4- difluorophenyl)pyridinate (dfppy), Q+ = K+ 3 and NBu4+ 6] and a series of symmetrical binuclear complexes (NBu4)[Pt2(C^N)2(p-MeC6H4)2(μ-CN)] (C^N = bzq 7, ppy 8, dfppy 9). Compounds 5, 6, and 7-9 were further determined by single-crystal X-ray diffraction. There are no apparent intermolecular Pt···Pt interactions owing to the presence of bulky NBu4+ counterion. Slow crystallization of K[Pt(ppy)(p-MeC6H4)(CN)] 2 in acetone/hexane evolves with formation of yellow crystals, which were identified by single-crystal X-ray diffraction methods as the salt complex {[Pt(ppy)(p-MeC6H4)(CN)]2K3(OCMe2)4(μ-OCMe2)2}[Pt(ppy)(p-MeC6H4)(μ-CN)Pt(ppy)(p-MeC6H4)]·2acetone (10), featuring the binuclear anionic unit 8- neutralized by an hybrid inorganic-organometallic coordination polymer {[Pt(ppy)(p-MeC6H4)(CN)]2K3(OCMe2)4(μ-OCMe2)2}+. The photophysical properties of all compounds were recorded in powder, polystyrene film, and solution states with a quantum yield up to 21% for 9 in the solid state. All complexes displayed bright emission in rigid media, and for the interpretation of their absorption and emission properties, density functional theory (DFT) and time-dependent DFT calculations were applied.

Luminescent conjugated polyelectrolytes (LCPs) have emerged as novel stains to detect and distinguish between various amyloidogenic species, including prefibrillar aggregates and mature fibril deposits, both in vitro and in histological tissue samples, offering advantages over traditional amyloid stains. We here use linear dichroism (LD) spectroscopy under shear alignment to characterize interactions between the LCP poly(3-thiophene acetic acid) (PTAA) and amyloid fibrils. The positive signature in the LD spectrum of amyloid-bound PTAA suggests that it binds in the grooves between adjacent protein side-chains in the amyloid fibril core, parallel to the fibril axis, similar to thioflavin-T and congo red. Moreover, using LD we record the absorption spectrum of amyloid-bound PTAA in isolation from free dye showing a red-shift by ca 30 nm compared to in solution. This has important implications for the use of PTAA as an amyloid probe in situ and in vitro and we demonstrate how to obtain optimal amyloid-specific fluorescence read-outs using PTAA. We use the shift in maximum absorption to estimate the fraction of bound PTAA at a given concentration. PTAA binding reaches saturation when added in 36 times excess and at this concentration the PTAA density is 4-5 monomer units per insulin monomer in the fibril. Finally, we demonstrate that changes in LD intensity can be related to alterations in persistence length of amyloid fibrils resulting from changes in solution conditions, showing that this technique is useful to assess macroscopic properties of these biopolymers.

In this work we apply a combination of steady state and time resolved luminescence and absorption spectroscopies to investigate the excited-state dynamics of a recently developed molecular photoswitch, belonging to the hydrazone family. The outstanding properties of this molecule, involving fluorescence toggling, bistability, high isomerization quantum yield and non-negligible two-photon absorption cross section, make it very promising for numerous applications. Here we show that the light induced Z/E isomerization occurs on a fast <1 ps timescale in both toluene and acetonitrile, while the excited state lifetime of the Z-form depends on solvent polarity, suggesting a partial charge transfer nature of its low lying excited state. Time-resolved luminescence measurements evidence the presence of a main emission component in the 500-520 nm spectral range, attributed to the Z-isomer, and a very short living blue-shifted emission, attributed to the E-isomer. Finally, transient absorption measurements performed upon far-red excitation are employed as an alternative method to determine the two-photon absorption cross-section of the molecule.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.