PostprintThis is the accepted version of a paper published in Analytical Chemistry. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Chmyrov, V., Spielmann, T., Hevekerl, H., Widengren, J. (2015) Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells. Corresponding AuthorJerker Widengren: jerker@biomolphysics.kth.se.ABSTRACT Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins, and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540) was first analyzed by Fluorescence Correlation Spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics could then also be monitored of MC540 in lipid vesicles, and the influence of lipid polarity, membrane curvature and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photo-induced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. Based on these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics, and that the TRAST imaging technique can provide spatial maps of photo-induced isomerization as well as both photo-induced and thermal back-isomerization, resolving differences in local membrane micro-viscosity in live cells.

Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.

Inter-or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleedthrough, thereby tolerating a high excess (up to 100 fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50 % in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in-vitro and in-vivo FRET-based spectroscopy and imaging.

ABSTRACT. Luminescent conjugated polyelectrolytes (LCPs) can upon interaction with biological macromolecules change their luminescent properties, and thereby serve as conformation-and interaction-sensitive biomolecular probes. However, to exploit this in a more quantitative manner, there is a need to better understand the photophysical processes involved. We report studies of the conjugated pentameric oligothiophene derivative p-FTAA, which changes optical properties with different p-FTAA concentrations in aqueous buffers, and in a pH and oxygen saturation dependent manner. Using dynamic light scattering, luminescence spectroscopy and fluorescence correlation spectroscopy, we find evidence for a monomer-dimer equilibrium, for the formation of large clusters of p-FTAA in aqueous environment, and can couple aggregation to changed emission properties of oligothiophenes.In addition, we observe the presence of at least two dark transient states, one presumably being a triplet state. Oxygen was found to statically quench the p-FTAA fluorescence, but also to promote molecular fluorescence by quenching dark transient states of the p-FTAA molecules. Taken together, this study provides knowledge of fluorescence and photophysical features essential for applying p-FTAA and other oligothiophene derivatives for diagnostic purposes, including detection and staining of amyloid aggregates.

Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study.The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.