This study analyses the pH-dependent time resolved fluorescence of mCardinal and mNeptune, two red-shifted fluorescent proteins with applications in biomedical imaging. We utilized molecular dynamics (MD) simulations to illuminate the influence of water molecules on the proteins´ photophysical properties.
In mCardinal, the average fluorescence lifetime markedly rises from 0.95 ns at pH 7.0 to 1.25 ns at pH 5.5. Conversely, mNeptune exhibits a constant fluorescence lifetime, showing no pH sensitivity.
Through Decay-Associated Spectra and MD simulations, we correlated mCardinal’s pH-induced lifetime changes with its molecular properties. Despite both proteins being equally stabilized by hydrogen bonds, mCardinal’s chromophore formed more water contacts than mNeptune’s. Additionally, the chromophore’s interactions with specific amino acids varied between the two proteins, suggesting distinct differences in the excited state proton transfer as a crucial mechanism for pH sensitivity.
Photoacoustic (PA) imaging using pump-probe excitation has been shown to detect fluorophores with high specificity. By varying the delay between the two excitation pulses and their wavelengths, the PA signal amplitude can be modulated, yielding three different and fluorophore-specific contrast mechanisms. In this study, PA images were obtained in tissue phantoms containing purified protein solutions using a ring array tomograph and a dual-OPO laser system. The results show that pump-probe excitation can detect multiple fluorescent reporters simultaneously. The method has the potential to recover biophysical parameters, such as pH and ion concentrations.
Polymer-based nanoparticles are promising contrast agents for photoacoustic (PA) imaging as their properties can be tailored to maximize detection sensitivity against the overwhelming endogenous background, while pump-probe excitation and fluence-dependent image acquisition may provide alternative approaches for contrast agent detection instead of multispectral imaging and unmixing. In this study, PA signals were measured in single-chain nanoparticle (SCNP) suspensions at pump and probe wavelengths of 690 nm and 730 nm and showed a strong nonlinear fluence dependence, which provides a unique contrast mechanism for molecular PA imaging.
In concentrated fluorescent solutions, the reabsorption and reemission of fluorescence light affects the temporal shape of the registered emission on the ps to ns time scale. Time-of-flight spectroscopy and intensity based tomography employ light transport models to characterize scattering of biological tissue providing information on the effective path length of the photons and the origin of emission. We propose the evaluation of fluorescence decay curves after reabsorption events to i) determine correction factors for time resolved fluorescence spectroscopy and tomography and ii) to exploit the information in the specific time resolved fluorescence traces to obtain information on the depth of signal generation in tissue and/or determination of reabsorbing structures as for example dye loaded micelles and cell compartments. The expected fluorescence decay curves after reabsorption events were modelled with rate equations for reabsorption and reemission. The fluorescence traces were fitted with the developed model in dependency of fluorophore concentration and path length. The results indicate that reabsorption can be quantitatively determined and depth information can be reconstructed from the time-course of the fluorescence signal. The applicability of the proposed technique to time-resolved fluorescence tomography is discussed.
Photoacoustic pump-probe excitation was used to generate a fluorophore-specific contrast in fluorescent proteins. The measured signals using pump-probe spectroscopy were found to correlate with the absorption and emission spectra of the protein.
Fluorophores, such as exogenous dyes and genetically expressed proteins, exhibit radiative relaxation with long excited state lifetimes. This can be exploited for PA detection based on dual wavelength excitation using pump and probe wavelengths that coincide with the absorption and emission spectra, respectively. While the pump pulse raises the fluorophore to a long-lived excited state, simultaneous illumination with the probe pulse reduces the excited state lifetime due to stimulated emission (SE).This leads to a change in thermalized energy, and hence PA signal amplitude, compared to single wavelength illumination. By introducing a time delay between pump and probe pulses, the change in PA amplitude can be modulated. Since the effect is not observed in endogenous chromophores, it provides a contrast mechanism for the detection of fluorophores via PA difference imaging. In this study, a theoretical model of the PA signal generation in fluorophores was developed and experimentally validated. The model is based on a system of coupled rate equations, which describe the spatial and temporal changes in the population of the molecular energy levels of a fluorophore as a function of pump-probe energy and concentration. This allows the prediction of the thermalized energy distribution, and hence the time-resolved PA signal amplitude. The model was validated by comparing its predictions to PA signals measured in solutions of rhodamine 6G, a well-known laser dye, and Atto680, a NIR fluorophore.
KEYWORDS: Photoacoustic spectroscopy, Absorption, Tissues, Photons, Molecules, Photoacoustic imaging, 3D image processing, Signal generators, Fluorescent markers, In vivo imaging
Photoacoustic imaging has been used to determine the spatial distribution of fluorophores, such as exogenous dyes and genetically expressed proteins, from images acquired in phantoms and in vivo. Most methods involve the acquisition of multiwavelength images and rely on differences in the absorption spectra of the tissue chromophores to estimate the spatial distribution and abundance of the latter using spectral decomposition techniques, such as model based inversion schemes. However, the inversion of 3-D images can be computationally expensive. Experimental approaches to localising contrast agents may therefore be useful, especially if quantification is not essential. This work aims to develop a method for determining the spatial distribution of a near-infrared fluorescent cell marker from images acquired using dual wavelength excitation. The excitation wavelengths coincided with the absorption and emission spectrum of the fluorophore. The contrast mechanism relies on reducing the excited state lifetime of the fluorophore by inducing stimulated emission. This changes the amount of energy thermalized by the fluorophore, and hence the photoacoustic signal amplitude. Since this is not observed in endogenous chromophores, the background may be removed by subtracting two images acquired with and without pulse delay between the pump and probe pulses. To characterise the fluorophore, the signal amplitude is measured in a cuvette as a function of pulse delay, concentration, and fluence. The spatial distribution of the fluorophore is determined from images acquired in realistic tissue phantoms. This method may be suitable for in vivo applications, such as imaging of exogenous or genetically expressed fluorescent cell markers.
Franz-Josef Schmitt, Christoph Theiss, Karin Wache, Justus Fuesers, Stefan Andree, Andrianto Handojo, Anne Karradt, Daniela Kiekebusch, Hans Joachim Eichler, Hann-Jörg Eckert
KEYWORDS: Picosecond phenomena, Luminescence, Antennas, Energy transfer, Molecules, Proteins, Single photon, Visible radiation, Molecular energy transfer, Time metrology
The phototrophic cyanobacterium Acaryochloris marina discovered in 1996 has a unique composition of the light
harvesting system. The chlorophyll (Chl) antenna contains mainly Chl d instead of the usually dominant Chl a and the
Phycobiliprotein (PBP) antenna has a simpler rod shaped structure than in typical cynobacteria [1].
The interaction of the photosynthetic subunits and especially the mechanisms regulating the energy transfer under
different stress conditions are presently interesting and open fields in photosynthesis research.
In this study we use time- and wavelength-resolved single photon counting to investigate the excited states dynamics in
living cells of A.marina. The fluorescence dynamics is synchronistically monitored in the visible and near infrared
spectrum with high signal to noise ratio and short data acquisition times while using low excitation light intensities.
These attributes are necessary to investigate photosynthetic processes in sensitive biological samples, when the light
emission varies due to metabolic changes.
The results suggest a fast excitation energy transfer kinetics of 20-30 ps along the PBP antenna of A.marina followed by
a transfer of about 60 ps to the Chl d antenna.
Cells of A. marina which are stored at 0°C for some time show a decoupling of the PBP antenna, which is partially
reversible when the sample is kept at 25 °C for a short time. Decoupling effects appearing after strong illumination with
white light (1600 W/m2) suggest a mechanism which removes the PBP antenna at different stress conditions to avoid
photo damage of the reaction center of Photosystem II (PS II).
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