Gustav Persson

Transient state imaging for microenvironmental monitoring by laser scanning microscopy.

Photoinduced transient dark states are exhibited by practically all common fluorophores. These relatively long-lived states are very sensitive to the local environment and thus highly attractive for microenvironmental imaging purposes. However, because of methodological constraints, their sensitivity has to date been very sparsely exploited. Here, a concept based on spatio-temporal modulation of the excitation intensity is presented that can image these states via their photodynamic fingerprints. With the use of a standard laser scanning microscope, it unites the outstanding environmental sensitivity of the transient state parameters with the high sensitivity of the fluorescence readout and is easily implemented. For demonstration, triplet state images of liposomes with different internal environments were generated. These images provide an example of how local environmental differences can be resolved, which are not clearly distinguishable via other fluorescence parameters. Having minor instrumental and sample constraints the concept can be foreseen to provide several new, useful, and independent fluorescence-based parameters in biomolecular imaging.

Interactions between a luminescent conjugated oligoelectrolyte and insulin during early phases of amyloid formation.

Aggregates of misfolded proteins play an important role in diseases such as Alzheimers. Here it is demonstrated how the anionic oligothiophene p-FTAA interacts with and influences pre-fibrillar protein assemblies during the earlier stages of in vitro fibrillation. Conjugated polythiophenes have previously been demonstrated to detect and discriminate between different types of protein aggregates and also introduce luminescent or conductive properties to these nanoscale fiber structures. Fluorescence spectroscopy, DLS, TEM and FCS are employed to follow the interplay between p-FTAA and insulin during in vitro fibrillation.

Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies

Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form. Two lasers are used alternatingly to excite predominantly the protonated and the deprotonated form of the dye. The alternating excitation modulation scheme is combined with detection gating, and is based on a recently developed concept that allows extraction of correlation data for all correlation times regardless of the chosen modulation period. The scheme can thus be applied without concern for the time-scales of the molecular dynamic processes to be studied. By this combined discrimination based on both excitation and emission, spectral cross-talk is dramatically reduced and a very distinct and unambiguous anticorrelation can be recorded in the correlation curves as a consequence of the proton exchange. The strong discrimination power makes the approach applicable also to ratiometric dyes with less pronounced spectral shifts. It should also be useful in combination with ratiometric dyes sensitive to other ambient conditions and ions, such as the biologically very important calcium ion.

Modulation Filtering Enables Removal of Spikes in Fluorescence Correlation Spectroscopy Measurements without Affecting the Temporal Information

The appearance of intensity spikes in measurements is a common problem in fluorescence correlation spectroscopy (FCS) studies of biological samples. In this work, we present a new method for generating artifact-free correlation curves from fluorescence traces that have undergone spike removal. This method preserves the temporal information throughout the measurement and properly represents the correlation between events separated by removed spikes. The method was validated using experimental data. The proposed algorithm is demonstrated herein to be generally applicable, but it is particularly powerful for cases where spikes occur frequently.

Dark States in Ionic Oligothiophene Bioprobes—Evidence from Fluorescence Correlation Spectroscopy and Dynamic Light Scattering

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.

Modulated or alternating excitation in fluorescence correlation spectroscopy

We have previously shown that formation of triplet states and other photo-induced states can be controlled by modulating the excitation with pulse widths and periods in the range of the transition times of the involved states. However, modulating the excitation in fluorescence correlation spectroscopy (FCS) measurements normally destroys correlation information and induces ringing in the correlation curve. We have introduced and experimentally verified a method to retrieve the full correlation curves from FCS measurements with modulated excitation and arbitrarily low fraction of active excitation. Modulated excitation applied to FCS experiments was shown to suppress the triplet build-up more efficiently than reducing excitation power with continuous wave excitation. The usefulness of the method was demonstrated by measurements done on fluorescein at different pH, where suppression of the triplet significantly facilitates the analysis of the protonation kinetics. Using a fluorophore where the protonation-coupled fluorescence intensity fluctuations are due to spectral shifts, introduction of two-color alternating excitation and spectral crosscorrelation can turn the protonation component of the correlation curve into an anti-correlation and further facilitate the distinction of this component from those of other processes.

Transient state microscopy: a new tool for biomolecular imaging

Photoinduced transient dark states are exhibited by practically all common fluorophores. However, their information content has to date only been sparsely exploited due to methodological constraints. Here, a new concept is presented and verified that can monitor and image these states via their photodynamic fingerprints. It unites the environmental sensitivity of these states with the sensitivity of fluorescence-based detection. For demonstration, triplet state images of liposomes in different environments were generated, showing how local environmental differences can be resolved, not clearly distinguishable via other fluorescence parameters. The concept can provide several new, useful and independent fluorescence-based parameters in biomolecular imaging.