Various approaches are used in fluorescence-based biological research to enhance the signal-to-background ratio (SBR). One general approach is to move to NIR wavelengths for excitation, where the background from cellular autofluorescence (AF) is minimal. The use of NIR fluorophores offers several additional advantages, including higher penetration depth and lower phototoxicity from lower photon energy. However, the low sensitivity of standard detectors in the NIR range is a major factor hindering the widespread use of NIR fluorophores. The low quantum yield and shorter lifetime of NIR fluorophores further exacerbate this challenge. Some detectors, like superconducting nanowire single-photon detectors (SNSPDs), are designed for improved operation in the NIR range. However, they are associated with significant costs. In Paper II, a dual-channel detection possibility with a single SNSPD was demonstrated to further add to their potential. Lock-in amplification is another commonly used method for enhancing the SBR. However, lock-in detection also amplifies any unintended laser light from scattering or reflection since it is modulated at the same reference frequency. Contributions from scattered laser light can introduce a significant background in tissue imaging where emission filters often cannot easily suppress this background. In Paper III, the nonlinear properties of lanthanide-doped upconversion nanoparticles (UCNPs) were exploited as frequency mixers to generate new frequency components, which could then be filtered out by a Fast Fourier Transform of the emission signal. Moreover, additional low-frequency beating signals could be generated by modulated excitation with more than one base modulation frequency. These results open for background-free imaging based on UCNPs and using low-speed detectors, including cameras. Although the two aforementioned studies focused on the advantages of excitation and detection within the NIR spectral range, thereby aiming to remove AF, the benefits of label-free studies based on AF itself should not be overlooked. The amino acid tyrosine is one of the abundant but dim sources of AF in the human body. Its emission can also contain valuable information about the local environment, particularly via its blinking properties reflecting its photophysical state transitions. This information has remained inaccessible by use of established methods such as fluorescence correlation spectroscopy (FCS). FCS analyses fluorescence intensity fluctuations from brightly fluorescent molecules in low concentrations and requires single-molecule detection (SMD) conditions, making FCS studies of dim tyrosine-containing molecules extremely difficult. Instead, the transient state (TRAST) monitoring technique can be used, allowing the exploration of their photophysics and extraction of related environmental information. TRAST, not requiring SMD conditions, provides the flexibility to be applied to dim samples where low fluorescence intensity signals can be compensated by increasing their concentration. In Paper I, TRAST measurements on tyrosine were demonstrated and applied to follow the conformational state of the tyrosine-containing protein Calmodulin. Exploiting AF is particularly valuable because it offers a label-free option, avoiding perturbations that may follow from fluorophore labeling. Yet, labeling with external fluorophores can also add further information to fluctuation-based methods, such as TRAST and FCS. In Paper IV, studying the co-enzyme Q₁₀ on fluorescein-labeled unilamellar vesicles allowed direct observation of proton exchange kinetics and of proton collecting antenna (PCA) effects of Q₁₀ in the vesicle membrane. In this case, although Q₁₀ is itself dimly AF, PCA effects were better observed through changes in protonation relaxation of the pH-sensitive fluorescein fluorophore label upon variation of the Q₁₀ concentrations in the vesicle membranes.

Inom fluorescensbaserad biologisk forskning används olika metoder för att förbättra signal/bakgrundsförhållandet (SBR). Ett allmänt tillvägagångssätt är att övergå till NIR-våglängder för excitation, där bakgrunden från cellulär autofluorescens (AF) är minimal. Användningen av NIR-fluoroforer ger flera ytterligare fördelar, bland annat högre penetrationsdjup och lägre fototoxicitet på grund av lägre fotonenergier. Den låga känsligheten hos standarddetektorer i NIR-området är dock en viktig faktor som hindrar en utbredd användning av NIR-fluoroforer. Det låga kvantutbytet och den kortare livslängden hos NIR-fluoroforer innebär ytterligare utmaningar. Vissa detektorer, t.ex. supraledande, med nanotrådsbaserade enfotondetektorer (SNSPD), är konstruerade för att fungera bättre i NIR-området. De är dock förknippade med betydande kostnader. I Paper II demonstrerades en möjlighet till tvåkanalsdetektering med en enda SNSPD för att ytterligare öka deras potential. Lock-in-förstärkning är en annan vanligt förekommande metod för att förbättra SBR. Lock-in-detektering förstärker dock även oavsiktligt laserljus från spridning eller reflektion eftersom det moduleras med samma referensfrekvens. Bidrag från spritt laserljus kan ge en betydande bakgrund vid vävnadsavbildning där emissionsfilter ofta inte enkelt kan undertrycka denna bakgrund. I Paper III utnyttjades de olinjära egenskaperna hos lantanid-dopade uppkonverteringsnanopartiklar (UCNPs) som frekvensblandare för att generera nya frekvenskomponenter, som sedan kunde filtreras fram genom en snabb Fourier-transformation av emissionssignalen. Dessutom kunde ytterligare lågfrekventa pulssignaler genereras genom modulerad excitation med mer än en basmoduleringsfrekvens. Dessa resultat öppnar för bakgrundsfri avbildning baserad på UCNPs och med hjälp av låghastighetsdetektorer, inkluderande även kameror. Även om de två ovannämnda studierna fokuserade på fördelarna med att excitera och detektera i NIR-spektralområdet och därmed ta bort AF, bör fördelarna med inmärkningsfria studier baserade på AF i sig inte förbises. Aminosyran tyrosin är en av de rikliga men svaga källorna till AF i människokroppen. Dess emissioner kan också innehålla värdefull information om den lokala miljön, särskilt via dess blinkande egenskaper som återspeglar dess fotofysiska tillståndsövergångar. Denna information har förblivit otillgänglig för etablerade metoder som fluorescens korrelationsspektroskopi (FCS). FCS analyserar fluktuationer i fluorescensintensitet från starkt fluorescerande molekyler i låga koncentrationer och kräver SMD-förhållanden (single-molecule detection), vilket gör FCS-studier av svaga tyrosininnehållande molekyler extremt svåra. Istället kan en teknik för att följa transienta tillstånd (TRAST) användas, vilken gör det möjligt att utforska dessa moleculers fotofysik och utvinna relaterad omgivningsinformation. TRAST, som inte kräver SMD-förhållanden, ger flexibiliteten att tillämpas på svagt emitterande prover där signaler med låg fluorescensintensitet kan kompenseras genom att öka deras koncentration. I Paper I demonstrerades TRAST-mätningar på tyrosin, vilka sedan tillämpades för att följa konformationstillståndet hos det tyrosininnehållande proteinet Calmodulin. Att utnyttja AF är särskilt värdefullt eftersom det erbjuder ett märkningsfritt alternativ, där man undviker störningar som kan uppstå till följd av fluoroformärkning. Märkning med externa fluoroforer kan dock också ge ytterligare information till fluktuationsbaserade metoder, som TRAST och FCS. I Paper IV möjliggjorde studier av co-enzymet Q₁₀ på fluoresceinmärkta unilamellära vesiklar direkt observation av protonutbyteskinetik och av PCA-effekter (proton collecting antenna) av Q₁₀ i vesikelmembranet. I det här fallet, även om Q₁₀ i sig är svagt AF, observerades PCA-effekter bättre genom förändringar i protoneringsrelaxationen hos den pH-känsliga fluorescein-fluoroforen vid variation av Q₁₀-koncentrationerna i vesikelmembranen.

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.

The amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies, based on the intensity, lifetime, wavelength and/or polarization of their emitted fluorescence. Similar to most fluorescent organic molecules, these amino acids can undergo transitions into dark meta-stable states, such as triplet and photo-radical states. On the one hand, these transitions limit the fluorescence signal, but they are also highly environment-sensitive and can offer an additional set of parameters, reflecting interactions, folding states, and immediate environments around the proteins. In this work, by analyzing the average intensity of tyrosine emission under different excitation modulations with the transient state monitoring (TRAST) technique, we explored the photo physics of tyrosine as a basis for such environment-sensitive readouts. From how the dark state transitions of tyrosine varied with excitation intensity and solvent conditions we first established a photophysical model for tyrosine. Next, we studied Calmodulin (containing two tyrosines), and how its conformation is changed upon calcium binding. From these TRAST experiments, performed with 280 nm time-modulated excitation, we show that tyrosine dark state transitions clearly change with the calmodulin conformation, and may thus represent a useful source of information for (label-free) analyses of protein conformations and interactions.

We have developed an Ir(PPy)₃ photoredox-catalyzed cross-coupling reaction that allows installation of quinoxalinones at the C7 position of thiazolino ring-fused 2-pyridones (TRPs) under mild conditions. The methodology tolerates various substituted quinoxalinones and biologically relevant substituents on the C8 position of the TRP. The TRP scaffold has large potential in the development of lead compounds, and while the coupled products are interesting from a drug-development perspective, the methodology will be useful for developing more potent and drug-like TRP-based candidates.

In lanthanide-doped upconversion nanoparticles (UCNPs), the concentration of emitter ions, also known as activator ions, is usually limited to 1 - 5 mol% due to concentration quenching effects. This circumstance limits the luminescent efficiency of UCNPs' and their use in a variety of application areas. Earlier studies have attributed the activator concentration quenching to migration of energy to the nanoparticle surface, while indicating that cross-relaxation between activator ions had a minor role therein. In this work, we carried out comparative studies on Er3+-doped and Yb3+-Er3+ codoped UCNPs and could, in contrast to this notion, prove a general adverse effect of cross-relaxation between activator ions, here Er3+ ions, on up -conversion luminescence (UCL). The direct result of the cross-relaxation is that the energy of the excitation light is accumulated into a low-lying excited state of Er3+ in the infrared region, so forming a "low-lying excited state energy trap ". As a result, the excitation energy is used for generating down-conversion lu-minescence or for indirectly facilitating UCL channels that are directly related to the low-lying excited state energy trap. The identified effect can be used to regulate UCL channels to achieve a concentrated UCL band that is more favorable for certain applications, e.g., biological imaging.

Lanthanide upconversion nanoparticles (UCNPs) have beenextensivelyexplored as biomarkers, energy transducers, and information carriersin wide-ranging applications in areas from healthcare and energy toinformation technology. In promoting the brightness and enrichingthe functionalities of UCNPs, core-shell structural engineeringhas been well-established as an important approach. Despite its importance,a strong limiting issue has been identified, namely, cation intermixingin the interfacial region of the synthesized core-shell nanoparticles.Currently, there still exists confusion regarding this destructivephenomenon and there is a lack of facile means to reach a delicatecontrol of it. By means of a new set of experiments, we identify andprovide in this work a comprehensive picture for the major physicalmechanism of cation intermixing occurring in synthesis of core-shellUCNPs, i.e., partial or substantial core nanoparticle dissolutionfollowed by epitaxial growth of the outer layer and ripening of theentire particle. Based on this picture, we provide an easy but effectiveapproach to tackle this issue that enables us to produce UCNPs withhighly boosted optical properties.

Nd3+-sensitized upconversion nanoparticles (UCNPs) can be excited by both 980 and 808 nm light, which is regarded as a particularly advantageous property of these particles. In this work, we demonstrate that the nanoparticles can exhibit significantly different response when excited at these two excitation wavelengths, showing dependence on the intensity of the excitation light and the way it is distributed in time. Specifically, with 808 nm excitation saturation in the emitted luminescence is more readily reached with increasing excitation intensities than upon 980 nm excitation. This is accompanied by delayed upconversion luminescence (UCL) kinetics and weaker UCL intensities. The different luminescence response at 808 and 980 nm excitation reported in this work is relevant in a manifold of applications using UCNPs as labels and sensors. This could also open new possibilities for multi-wavelength excitable UCNPs for upconversion color display and in laser-scanning microscopy providing selective readouts and sub-sectioning of samples.