Photobleaching Dynamics Research Articles

Structure‐Dependent Photophysics Studied in Single Molecules of Polythiophene Derivatives

We report on the photophysical characterization at the single-molecule level of a graft copolymer consisting of a polythiophene backbone and long polystyrene branches. The presence of the branches prevents the polymer chain from forming a collapsed conformational state. The photophysical properties of the resulting solution-like conformation are studied by measuring single-molecule photobleaching dynamics, emission polarization anisotropy and emission spectra. The results are compared with those obtained on the same polythiophene derivative without the branches. It is found that the presence of the branches is a decisive factor in determining the photophysical properties of the polymers on the single-molecule level.

Anisotropic decay dynamics of photoexcited aligned carbon nanotube bundles

We have performed polarization-dependent ultrafast pump-probe spectroscopy of a film of aligned single-walled carbon nanotube bundles. By taking into account imperfect nanotube alignment, as well as anisotropic absorption cross sections, we quantitatively determined distinctly different photobleaching dynamics for polarizations parallel and perpendicular to the tube axis. For perpendicular polarization, we observe a slow $(1.0--1.5\phantom{\rule{0.3em}{0ex}}\mathrm{ps})$ relaxation process, previously unobserved in randomly oriented nanotube bundles. We attribute this slower dynamics to the excitation and relaxation of surface plasmons in the radial direction of the nanotube bundles.

The Observation of Ultrafast Excited-state Dynamical Evolution In B800- Partially or Completely Released LH2 of Rhodobacter sphaeroides 601 at Room Temperature

Photodynamics of two kinds of peripheral antenna complexes (LU2 of Rhodobacter sphaeroides, native LH2 (RS601) and B800-released LH2 where B800-BChls were partially or completely removed with different pH treatments), were studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results for these samples with different B800/B850 ratios demonstrated that under the excitation around B800 nm, the photoabsorption and photobleaching dynamics were caused by the direct excitation of upper excitonic levels of B850 and excited state of B800 pigments, respectively. Furthermore, the removal of B800 pigments had little effect on the energy transfer processes of B850 interband/intraband transfer.

Observation of ultrafast excited-state dynamical evolution in B800 absorption band of light harvesting 2 (LH2) extracted from Rb. Sphaeroides 601

Photodynamics of two kinds of peripheral antenna complexes light harvesting 2 (LH2) of Rb. Sphaeroides 601, namely the native LH2 and B800-released LH2, where B800-BChls were partially or completely removed with different pH treatments, were studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results for these samples demonstrate that under the excitation around B800 absorption band, the photoabsorption and photobleaching dynamics are caused by the direct excitation of upper excitonic levels of B850 and excited state of B800 pigments, respectively.

Photobleaching Kinetics of Optically Trapped Multilamellar Vesicles Containing Verteporfin Using Two-photon Excitation§

Two-photon excitation photodynamic therapy (TPE-PDT) is being developed as an improved treatment for retinal diseases. TPE-PDT has advantages over one-photon PDT, including lower collateral damage to healthy tissue and more precise delivery of PDT. As with one-photon PDT, there can be local photochemical depletion of oxygen during TPE-PDT. Here, we investigate model systems and live cells to measure local photosensitizer photobleaching and through it, infer local oxygen consumption in therapeutic volumes of the order 1 microm3. Multilamellar vesicles (MLV) and African green monkey kidney (CV-1) cells were used to study the TPE photobleaching dynamics of the photosensitizer, Verteporfin. It was found that in an oxygen-rich environment, photobleaching kinetics could not be modeled using a mono-exponential function, whereas in hypoxic conditions a mono-exponential decay was adequate to represent photobleaching. A biexponential was found to adequately model the oxygen-rich conditions and it is hypothesized that the fast part of the decay is oxygen-dependent, whereas the slower rate constant is largely oxygen-independent. Photobleaching recovery studies in the CV-1 cells support this hypothesis.

Molecular contrast optical coherence tomography: a pump-probe scheme using indocyanine green as a contrast agent

The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (approximately 190 kJ/cm2) is then used to permanently photobleach the ICG molecules--resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, phi(B,cum), defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that phi(B,cum) decreases with fluence as well as with increasing dye concentration. The quantity phi(B,cum) is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.

The influence of intracellular mTHPC concentration upon photobleaching dynamics

In this study, we report the effect of the local photosensitizer concentration upon the dynamics of the singlet oxygen-mediated photobleaching, within formalin-fixed keratinocytes. Although the cells were incubated at a single mTHPC dose, cell-to-cell variations in concentration were defined within the perinuclear region by differences in the initial amplitude of the laser-induced fluorescence emission, located around 652nm. At a fixed laser fluence-rate, it was found that the photobleaching, when plotted as a function of delivered light dose, proceeded more rapidly at higher drug concentration. The mTHPC spectral emission profile is shown to be approximately Lorentzian and remains unchanged as the photobleaching proceeds. This indicates that there is no perturbation of the detected signal due to the inner-filter effect.

Non-exponential bleaching of single bioconjugated Cy5 molecules

Photobleaching behavior of Cy5-conjugated bovine serum albumin (BSA) was investigated on the ensemble and single molecule level. For ensemble studies, a model was developed which accounts for different excitation rates of randomly oriented dye molecules. Both ensemble and single molecule observations yield non-exponential photobleaching dynamics of BSA-Cy5, indicating the existence of multiple bleaching pathways.

Photobleaching of photosynthetic pigments in spinach thylakoid membranes. Effect of temperature, oxygen and DCMU

The time dependence of photobleaching of photosynthetic pigments under high light illumination of isolated spinach thylakoid membranes at 22 and 4 °C was investigated. At 22 °C, the bleaching at 678, 472 and 436 nm was prominent but lowering the temperature up to 4 °C during illumination prevented the pigments from bleaching almost completely. The accelerating effect on pigment photobleaching by the presence of 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea)—(DCMU), a well-known inhibitor of the electron transport and known to prevent photosystem I (PSI) and photosystem II (PSII) against photoinhibitory damage, was also suppressed at low temperature. At 22 °C in the presence and absence of DCMU, the decrease of the absorption at 678 and 472 nm was accompanied by a shift to the shorter wavelengths. To check the involvement of reactive oxygen species in the process, pigment photobleaching was followed in anaerobiosis. The effects of the three different environmental factors—light, temperature and DCMU—on the dynamics of photobleaching are discussed in terms of different susceptibility of the main pigment–protein complexes to photoinhibition.

Ultrafast dynamics of fluorescence-activated CdS nanoparticles in aqueous solutions by femtosecond transient bleaching spectroscopy

The ultrafast dynamics of transient photobleaching and recovery of fluorescence-activated cadmium sulphide (CdS) nanoparticles in aqueous solutions upon treatment with excess cadmium ions have been studied upon excitation at 400 nm with a femtosecond laser system. Three different sized nanoparticles were prepared with a diameter of 5.7, 4.4 and 3.7 nm. The colloids showed strong fluorescence bands at 504, 477 and 458 nm, respectively, which were close to the exciton absorption. Transient photobleaching of less than 1 ps with a peak quite similar to the wavelength of the exciton absorption band was observed for the two larger samples. It was found that the bleaching component with a lifetime of several picoseconds became pronounced upon treatment with excess Cd 2+. A much faster component with a subpicosecond lifetime was also observed. These results strongly suggest that the direct recombination of charge carriers dominates in fluorescence-activated CdS nanoparticles.

Ultrafast dynamics of transient bleaching of surface modified cadmium sulphide nano-particles in Nafion films

Ultrafast dynamics of transient photobleaching and recovery of cadmium sulphide (CdS) nanoparticles in Nafion films before and after treatment with excess cadmium ions have been studied upon excitation at 400 nm with a femtosecond laser system. Both samples gave transient bleaching in less than 1 ps and a multi-exponential recovery in the wavelength region between ca. 410 and 500 nm. Cd 2+ treatment of the nanoparticles resulted in several dramatic changes to the fluorescence and transient bleaching spectra from the films: (1) A new fluorescence band at higher energy appeared in addition to an increase in the ‘defect’ fluorescence band at longer wavelengths. (2) A remarkable increase in the extent of transient bleaching was observed below about 440 nm, which recovered faster than that at longer wavelengths. (3) The recovery rate of the transient bleaching became faster, relative to the untreated nanoparticles, over the whole wavelength region of the bleaching. These results have been interpreted in terms of the recombination of photogenerated electrons and holes from traps or defects in/on the nanoparticles.

The Dynamics of Laser‐Induced Changes in Human Skin Autofluorescence—Experimental Measurements and Theoretical Modeling

To study the temporal dynamics of human skin autofluorescence photobleaching, we measured the autofluorescence spectral changes of skin in vivo during continuous exposure to 442 nm (He-Cd) laser light. Integral intensities were calculated for various spectral wavelength bands and plotted as a function of time. Mathematical analysis of the time function revealed a double-exponential photobleaching process: I(t) = a exp (-t/tau 1) + b exp(-t/tau 2) + c, in which tau 1 and tau 2 differed by an order of magnitude. A hypothesis for the mechanism of the double-exponential photobleaching dynamics was proposed and evaluated using Monte Carlo modeling of light propagation in the skin and autofluorescence escape from skin. By combining the fluorophore microdistributions, Monte Carlo simulation results and the variation in fluorescence decrease parameters (a, b, c, tau 1, tau 2) with increasing exposure intensities a biophysical explanation for the double-exponential photobleaching function was elucidated. The fast decrease term corresponds to laser-induced photobleaching in the stratum corneum, while the slow decrease term represents fluorophore changes in the dermis. The measured autofluorescence photobleaching dynamics can be used to determine the fractional contributions of different skin layers to the total autofluorescence signal measured in vivo.

Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HR1 hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced damage were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photobleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photobleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50-150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced damage. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photobleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Fluorescence and photobleaching dynamics of single light-harvesting complexes.

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 +/- 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only approximately 3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of approximately 0.1 s. About 490 molecules were examined.

Temperature dependence of antenna excitation transport in native photosystem I particles

The temperature dependence of polarized photobleaching dynamics was investigated through 680-nm pump-probe experiments in the Chl a antenna of native photosystem 1 particles (Chl/P700 {approximately} 200) from spinach. The anisotropic decay time is lengthened by an order of magnitude (from {approximately}7 to {approximately}62 ps) when the temperature is reduced from 290 to 38 K; most of this increase occurs between 65 and 38 K. The occurrence of this transition temperature in the tens of kelvin reflects the participation of protein phonons in antenna EET. The isotopic decay kinetics are considerably less temperature sensitive, indicating that the anisotropic and isotropic decays stem from different energy-transfer processes with contrasting temperature dependence. The 38 K photobleaching spectrum at 5 ps exhibits considerably more weighting in the lower energy Chl a spectral forms than the room-temperature spectrum, suggesting that rapid spectral equilibration occurs in the photosystem 1 antenna. In light of the phonon frequency and electron-phonon coupling parameters determined in independent PSI-200 spectral hole-burning experiments, the quantitative temperature dependence int he anisotropic decay times is consistent with a theory for phonon-assisted EET in which the pertinent phonons are independent modes localized about the donor and acceptor chromophores.