Advanced fluorescence-based fluctuation methods for biosensing

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