In addition to use traditional fluorescence parameters for biological studies,the main focus of this thesis is on the monitoring of photoinduced fluorophoretransient states. These dark states, such as the triplet state, redox states andphotoisomers that are formed upon excitation, are long-lived in comparisonto the fluorescent singlet state and therefor more sensitive to the surroundingenvironment and their sensitivity are to biological relevant conditions. Themain method used to study these states is called transient state monitoring(TRAST) and based on laser-modulation over the sample. Different modalities using this approach, are explored to characterize blinking kinetics offluorophores in varying contexts. A multiparameter confocal laser-scanningsetup with TCSPC and correlation capabilities is used as a complement to thementioned method and used solely in one of the works. It is motivated howthe transient states adds dimensions of information. They can with advantagebe used as an additional fluorescence read-out in imaging and spectroscopicstudies of fluorophore tagged biological samples.

In paper I, the autofluorescent co-enzyme NAD(P)H was studied byTRAST, showing a redox sensitivity. A two-photon excitation (2PE) scanning TRAST approach was established. With this label-free approach, cellular images reflecting local redox environments could be obtained and visualizechanges in the environment after disturbing the cellular metabolism.

In paper II, single-molecule confocal TCSPC with pulsed interleavedexcitation and burst-analysis was used to study loading yields of exosomemimetic nanovesicles.

In paper III, the photodynamics of two dyes used in photodynamic therapy (PDT) were characterized. It was motivated how TRAST can be used asa method for live-monitoring in PDT and be used as feed-back to optimizetreatment. A fiber-based TRAST approach was demonstrated and used tomeasure the fluorophores in tissue.

Paper IV and paper V focuses on the photodynamics of the cyaninedyes Cy7 and Cy5. A second emissive photoisomerized state in addition tothe ground state (trans) is found and confirmed by various methods, amongthem an approach where TRAST-curves can be selectively be produced fromdifferent bands of the full emission spectrum. This method was establishedduring this work. Differences between fluorescence correlation spectroscopy(FCS) and TRAST are discussed and included in the analysis of data. Varioussensing parameters of Cy7 are explored.

In paper VI it is shown how the blinking dynamics from the photoinduced transient states can be used for fluorescence barcoding and muliplexing.FCS, laser-modulated TRAST and flow-based TRAST, where the sample isflowing through the excitation light, are used to demonstrate this encodingdimension of free dyes and tagged vesicles. Labelled cells are imaged, wheremultiple colors can be produced by the fluorophore specific blinking characteristics.

In recent decades, photoluminescence properties of single molecules and ions have opened new possibilities for studies on smaller size scales, below the light-diffraction limit. In this thesis, the advantages of such single photon emitters were harnessed and studied mainly in the field of biophysics, but also, in investigations within solid state photonics. The first aspect encompassed studies on extracellular vesicles (EVs) and nanovesicles derived from red blood cell (RBC) membranes which were bioengineered for drug delivery applications. RBC-derived vesicles demonstrated high biocompatibility and low immunogenicity, offering superior production scalability over physiological EVs. However, thorough physical characterizations of such nanovesicles are yet to be developed. Such investigations are essential for their clinical deployment, for therapeutic and diagnostic purposes. Initially, the morphology and size-stability of vesicles were investigated by applying atomic force microscopy and dynamic light scattering. These studies demonstrated the size heterogeneity and agglomeration tendencies of the vesicles. Comparative studies on physiological EVs revealed a higher size stability, while RBC-produced vesicles showed about 50 % reversible agglomerations. Secondly, a dual-colour coincident fluorescent burst (DC-CFB) experimental analysis technique was developed. DC-CFB was then used to characterize and profile the cargo-loading yields of bioengineered nanovesicles, overcoming challenges related to their small size (below the diffraction limit), their inherent heterogeneity, and the presence of free, non-encapsulated cargoes. The developed methodology was then applied to explore the loading with relatively small single nucleotides (dUTP) as well as larger antibody (Ab) molecules, motivated by the prospective role of such EVs and EV-mimetic bioengineered vesicles as nanocarriers of therapeutic drugs. The studies demonstrated consistent average loading yields of around 14-20 % for both cargo types (dUTP and Ab) into both vesicle categories, i.e., EVs and RBC-derived vesicles. Additionally, the analysis capability of the DC-CFB technique at single-vesicle and single-molecule levels, afforded analyses of the number of loaded molecules inside each vesicle, and how this number varied with the vesicle size. On average, this number was found to be greater than two, for both cargo types. Overall, the developed techniques based on fluorescence single photon counting provided a comprehensive assessment of the drug loading properties of nanovesicles. Such bioengineered nanocarriers have a disruptive potential for pharmaceutical applications. The last part of the thesis investigates the realm of solid-state on-chip photon emitters. Specifically, it considers the integration of erbium ions, exhibiting photoluminescent emission in the telecommunication C-band, into thin film lithium niobate (TFLN) waveguides. An Er-ion implantation process compatible with integrated optical circuits in x-cut TFLN was developed and the erbium photoluminescence properties were investigated versus temperature. These preliminary studies provide a foundation for future integration of Er single photon emitters into TFLN-based photonic components.

Under de senaste årtiondena har fotoluminescens-egenskaper hos enskilda molekyler och joner öppnat nya möjligheter till studier på mindre storleksskalor, under ljusdiffraktionsgränsen. I den här avhandlingen utnyttjades och studerades fördelarna med sådana enskilda foton-källor främst inom biofysik, men också för undersökningar inom fasta tillståndets fotonik. Det första aspekten omfattade studier av extracellulära vesiklar (EVs) och nanovesiklar, genererade från röda blodkroppars (RBC) membran, och utformade för applikationer rörande leverans av läkemedel i kroppen. De RBC-genererade vesiklarna uppvisade hög biokompatibilitet och låg immunogenicitet och erbjöd en överlägsen uppskalbarhet för produktion jämfört med fysiologiska EVs. Emellertid måste noggranna fysiska karakteriseringar av sådana nanovesiklar ännu utvecklas. Sådana undersökningar är nödvändiga för att i förlängningen kunna använda denna typ av vesiklar klinikst, för terapeutiska och diagnostiska ändamål. Inledningsvis undersöktes morfologin och storleksstabiliteten hos vesiklarna genom atomkraftsmikroskopi och dynamisk ljusspridning. Dessa studier visade på storleksheterogenitet och agglomereringstendenser hos vesiklarna. Jämförande studier av fysiologiska EVs avslöjade deras förhållandevis högre storleksstabilitet, medan RBC-producerade vesiklar uppvisade reversibla agglomerationer i omkring 50 % av fallen. För det andra utvecklades en experimentell analysmetod med tvåfärgs-avläsning av sammanfallande fluorescens-signaler (DC-CFB). Metoden användes sedan för att karakterisera och profilera molekylupptaget hos de bioteknologiskt framtagna nanovesiklarna. Därmed var det möjligt att hantera vesiklarnas ringa storlek (under diffraktionsgränsen), deras inneboende heterogenitet och närvaron av fria, icke-inkapslade molekyler. Den utvecklade metoden tillämpades därefter för att utforska upptaget i vesiklarna av relativt små nukleotider (dUTP) såväl som av större antikroppsmolekyl (Ab), motiverat utifrån en möjlig framtida användning av sådana EVs och EV-mimetiska vesiklar som nanobärare av terapeutiska läkemedel. Studierna visade genomgående genomsnittliga upptag runt 14-20 % för båda molekyl-typerna (dUTP och Ab), i både EVs och i de RBC-tillverkade vesiklarna. Dessutom möjliggjorde DC-CFB-tekniken analyser på enstaka-vesikel och enstaka-molekylnivå, av antalet upptagna molekyler inuti varje vesikel i förhållande till dess storlek. I genomsnitt var detta antal större än två för de båda upptagna molekyl-typerna. Sammantaget gav de utvecklade teknikerna, baserade på detektion av enstaka fluorescens--fotoner, en omfattande bedömning av kapaciteten för läkemedelsupptag hos nanovesiklar. Bioteknologiska nanotransportörer av detta slag har en betydande potential för farmaceutiska tillämpningar. Den sista delen av avhandlingen undersöker området för fasta tillståndets on-chip-fotonemitterare. Mer specifikt undersöks integrationen av erbiumjoner, som uppvisar fotoluminescerande emission i telekommunikations-C-bandet, i tunnfilmer av litiumniobat (TFLN) vågledare. En Er-jonimplanteringsprocess som är kompatibel med integrerade optiska kretsar I x-delat TFLN utvecklades och erbiums fotoluminescensegenskaper undersöktes i förhållande till temperaturen. Dessa inledande studier utgör en grund för framtida integration av enskilda fotonemitterare av Er i TFLN-baserade fotoniska komponenter.