Analysis of Fluorescence Flicker as a Tool to Monitor Proton Transport and Biomolecular Interactions

The overall focus of this thesis is on fluorescence flicker processes of fluorescent molecules, e.g. protonation-deprotonation or singlet-triplet electronic state transitions, intrinsic or generated by their interaction with their environment, monitored by fluorescence spectroscopy. Understanding proton migration along membranes and membrane proteins in cells is essential for understanding energy metabolism. It has been seen that certain membrane-spanning proton-transporter proteins in the respiratory chain in the mitochondrial inner membrane take up protons faster than the rate limited by diffusion. To explain these observations it has been suggested that there is a proton-collecting antenna, consisting of negatively and protonatable residues on the surface of these proteins, which increases the rate of uptake. Using fluorescence correlation spectroscopy and artificial biological membranes the proton collecting antenna effect is verified, as well as the proton migration properties on these membranes at various surface buffer concentrations. Fluorescence flicker due to singlet-triplet electronic state transitions in a fluorescent molecule is interesting because of the long transition time between the two states. This means that the molecule has a long time to interact with the local environment, and can therefore be used as a microenvironmental sensor. A novel method for monitoring photo-induced, transient, long-lived, non- or weakly fluorescent states, e.g. the triplet state, was developed. With this method, only the time averaged intensity is detected and used for determining the triplet state kinetics. This method has several advantages, in particular it lends itself well for parallelization, over traditional methods including fluorescence correlation spectroscopy.QC 2010112

Monitoring Proton Exchange and Triplet States with Fluorescence

Fluorescent molecules commonly shift to transient dark states, induced bylight or triggered by chemical reactions. The transient dark states can beused as probes of the local environment surrounding the fluorescent molecules,and are therefore attractive for use in biomolecular applications. Thisthesis explores the use and development of novel fluorescence spectroscopictechniques for monitoring transient dark states.This work demonstrates that kinetic information regarding photoinduced transient dark states of fluorescent molecules can be obtained from the time-averaged fluorescence intensity of fluorescent molecules subject totemporally modulated illumination. Methods based on this approach havethe advantage that the light detectors can have a low time resolution, which allows for parallelization and screening of biomolecular interactions withhigh throughput. Transient state images are presented displaying local environmental differences such as those in oxygen concentration and quencher accessibility.Analysis of the fluorescence intensity fluctuations resulting from thetransitions to and from transient dark states can be used to obtain information regarding the transition rates and occupancy of the transient darkstates. Fluorescence fluctuation analysis was used to reveal rates of protonbinding and debinding to single fluorescent molecules located close to biological membranes and protein surfaces. The results from these studies show that the proton exchange rate increases dramatically when the fluorescent molecule is close to the membrane.QC 2010080

Sammanställning av kunskap om effekterna av IKT-medierade tjänster för resenärsinformation för kollektiva transporter och effekter av styva tidtabeller.

Information och kommunikationsteknologi (IKT)-medierade tjänster för resenärsinformation har uttryckts erbjuda möjligheter till att upprätthålla kollektiva transporters attraktivitet vid resursbegränsningar. Att erbjuda avgångar enligt styvatidtabeller har länge ansetts göra kollektiva transporter mer attraktiva för resenärer men är från ett produktionsperspektiv resurskrävande. En litteraturstudie har på uppdrag av Trafikverket utförts i syfte att sammanfatta kunskap kring effekter av IKT-medierade tjänster för resenärsinformation. Fokus har lagts på hur sådana tjänster skulle kunna påverka värdet av styva tidtabeller. För att kunna avgöra vad hos IKT-medierade tjänster för resenärsinformation som skulle påverka värdet avstyva tidtabeller har även kunskap om styva tidtabellers effekter sammanfattats. Resultat från denna litteraturstudie visar bland annat att mängden referentgranskade studier om effekter av styva tidtabeller är begränsad samt att det finns en något störremängd referentgranskade studier om effekter av IKT-medierade tjänster för kollektivtrafikresenärer. De effekter som har beskrivits i de identifierade studierna i har dock inte visat sig kvantifierbart jämförbara. Frågan rekommenderas därför studerasytterligare för att i framtiden kunna stötta ekonomiska analysmetoder och kalkylprinciper.Effekter av ICT-medierad resenäresinformation för kollektiva transporte

Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements

For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.QC 20100617 Uppdaterad från manuskript till artikel (20110110). Tidigare titel: Recovery of Photo-Induced Reversible Dark States Utilized for Molecular Diffusion Measurements</p

Iodide as a Fluorescence Quencher and Promoter-Mechanisms and Possible Implications

In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.QC 20101206</p

Iodide as a Triplet State Promoter and Quencher - Mechanisms and Possible Implications

In this work, Fluorescence Correlation Spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet state decay by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state build-up of one dye and reduce it for another, red-shifted dye. The anticorrelated, spectrally separable responses of two dyes to the presence of one and the same agent are likely to provide a useful readout for biomolecular interaction and micro-environmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an anti-oxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the anti-oxidative properties of KI that play a role

Iodide as a Triplet State Promoter and Quencher –Mechanisms and Possible Implications

In this work, Fluorescence Correlation Spectroscopy(FCS) was used to investigate the effects of potassium iodide(KI) on the electronic state population kinetics of arange of organic dyes in the visible wavelength range. Apartfrom a heavy atom effect promoting intersystem crossing tothe triplet states in all dyes, KI was also found to enhancethe triplet state decay by a charge-coupled deactivation.This deactivation was only found for dyes with excitationmaximum in the blue range, not for those with excitationmaxima at wavelengths in the green range or longer. Consequently,under excitation conditions sufficient for tripletstate formation, KI can promote the triplet state build-up ofone dye and reduce it for another, red-shifted dye. The anticorrelated,spectrally separable responses of two dyes to thepresence of one and the same agent are likely to provide auseful readout for biomolecular interaction and micro-environmentalmonitoring studies. In contrast to the typicalnotion of KI as a fluorescence quencher, the FCS measurementsalso revealed that when added in micromolar concentrationsKI can act as an anti-oxidant, promoting the recoveryof photo-oxidized fluorophores. However, in millimolarconcentrations KI also reduces intact, fluorescently viablefluorophores to a considerable extent. In aqueous solutions,an optimal concentration of KI of approximately 5 mM canbe defined at which the fluorescence signal is maximized.This concentration is not high enough to allow full tripletstate quenching. Therefore, as a fluorescence enhancementagent, it is primarily the anti-oxidative properties of KI thatplay a role.QC2010061

Iodide as a Triplet State Promoter and Quencher –Mechanisms and Possible Implications

In this work, Fluorescence Correlation Spectroscopy(FCS) was used to investigate the effects of potassium iodide(KI) on the electronic state population kinetics of arange of organic dyes in the visible wavelength range. Apartfrom a heavy atom effect promoting intersystem crossing tothe triplet states in all dyes, KI was also found to enhancethe triplet state decay by a charge-coupled deactivation.This deactivation was only found for dyes with excitationmaximum in the blue range, not for those with excitationmaxima at wavelengths in the green range or longer. Consequently,under excitation conditions sufficient for tripletstate formation, KI can promote the triplet state build-up ofone dye and reduce it for another, red-shifted dye. The anticorrelated,spectrally separable responses of two dyes to thepresence of one and the same agent are likely to provide auseful readout for biomolecular interaction and micro-environmentalmonitoring studies. In contrast to the typicalnotion of KI as a fluorescence quencher, the FCS measurementsalso revealed that when added in micromolar concentrationsKI can act as an anti-oxidant, promoting the recoveryof photo-oxidized fluorophores. However, in millimolarconcentrations KI also reduces intact, fluorescently viablefluorophores to a considerable extent. In aqueous solutions,an optimal concentration of KI of approximately 5 mM canbe defined at which the fluorescence signal is maximized.This concentration is not high enough to allow full tripletstate quenching. Therefore, as a fluorescence enhancementagent, it is primarily the anti-oxidative properties of KI thatplay a role.QC2010061