Jürgen Ehlert

Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation

Fluorescence of synthetic melanin in the solvents H2O, KOH, ethylene glycol monomethyl ether, and dimethyl sulfoxide has been excited by two-photon absorption at 800 nm, using 120-fs pulses with photon flux densities of ≥1027 cm−2.S−1. Compared to the one-photon (400-nm)-induced fluorescence of melanin, the overall spectral shape is red-shifted and shows a strong environment sensitivity. The decay of the two-photon-induced fluorescence (TPF) of melanin is three-exponential, with a shortest main component of about 200 ps. The results of the TPF studies in line with the unique light absorption property of melanin of a monotonously decreasing absorption spectrum between the near UV-region and the near infrared region indicate that the TPF is realized via stepwise absorption of two 800-nm photons. In comparison to the simultaneous absorption of two photons, the stepwise process needs lower photon flux densities to get a sufficient population of the fluorescent level. This stepwise process offers new possibilities of selective excitation of melanin in skin tissue in a spectral region where there is no overlap with any absorption of another fluorescent tissue component. The first results with different samples of excised human skin tissue (healthy, nevus cell nevi, malignant melanoma) suggest that fluorescence excited in this way yields information on malignant transformation.

Photophysical characterization of the B800-depleted light harvesting complex B850 of Rhodobacter sphaeroides: Implications to the ultrafast energy transfer 800→850 nm

Abstract From the isolated LH2 antenna of Rhodobacter sphaeroides , called B800-850, the bacteriochlorophyll a pigment complement BChl-B800, mainly responsible for the 800 nm absorption band, has been extracted. The remaining complex, with the main NIR absorption peaking at 850 nm (termed pure (p) B850), has a small but distinct sideband in the 800 nm region and contributes there about 20% to the total absorption of the integral B800-850 antenna. In the latter the energy transfer 800→850 nm is per se (at least) biphasic with the pB850 intra-aggregate relaxation as the fastest component known so far ( k ≈10 13 s −1 ).

Substructures and different energy relaxation time within the first electronic transition of pinacyanol

Abstract The S0-S1 absorption band substructure of pinacyanol dissolved in ethanol has been investigated with nonlinear polarisation spectroscopy in the frequency domain (NLPF). The evaluation of the experimental results has been done in the frame of global analysis for a set of four NLPF spectra at two test wavelengths. Four bands with maxima located at 520, 556, 565 and 607 nm have been identified. The energy relaxation after excitation in the 565 nm band is slower (T1 = 13 ps) than in the red-most band (T1 = 7 ps). This behaviour, inverse to usual excess-energy relaxation of organic molecules, suggests that these subbands belong to different isomers of pinacyanol.

Subband analysis of molecular electronic transitions by nonlinear polarization spectroscopy in the frequency domain

Nonlinear polarization spectroscopy in the frequency domain (NLPF) allows a detailed analysis of substructure behind a molecular optical absorption band. Using the χ(3)-approach here we deduce theoretically line shapes of NLPF spectra for systems with more than one optical transition, which contribute to linear as well as excited state absorption. It is possible from such line shapes to decide whether these transitions are independent from each other or belong to a common ground state. We demonstrate further how energy transfer and relaxation paths can be revealed and quantified by a global analysis of NLPF spectra. This is useful to get a comprehensive understanding of ultrafast processes in complex molecular and supermolecular systems.

Stepwise Two-photon Excited Fluorescence from Higher Excited States of Chlorophylls in Photosynthetic Antenna Complexes

Stepwise two-photon excited fluorescence (TPEF) spectra of the photosynthetic antenna complexes PCP, CP47, CP29, and light-harvesting complex II (LHC II) were measured. TPEF emitted from higher excited states of chlorophyll (Chl) a and b was elicited via consecutive absorption of two photons in the Chl a/b Qy range induced by tunable 100-fs laser pulses. Global analyses of the TPEF line shapes with a model function for monomeric Chl a in a proteinaceous environment allow distinction between contributions from monomeric Chls a and b, strongly excitonically coupled Chls a, and Chl a/b heterodimers/-oligomers. The analyses indicate that the longest wavelength-absorbing Chl species in the Qy region of LHC II is a Chl a homodimer with additional contributions from adjacent Chl b. Likewise, in CP47 a spectral form at ∼680 nm (that is, however, not the red-most species) is also due to strongly coupled Chls a. In contrast to LHC II, the red-most Chl subband of CP29 is due to a monomeric Chl a. The two Chls b in CP29 exhibit marked differences: a Chl b absorbing at ∼650 nm is not excitonically coupled to other Chls. Based on this finding, the refractive index of its microenvironment can be determined to be 1.48. The second Chl b in CP29 (absorbing at ∼640 nm) is strongly coupled to Chl a. Implications of the findings with respect to excitation energy transfer pathways and rates are discussed. Moreover, the results will be related to most recent structural analyses.

S1 absorption of chlorophyll-a in the red region

Abstract To clear up controversial results of excited-singlet (S 1 ) absorption of monomeric chlorophyll-a solutions in the red spectral range, non-linear absorption measurements were performed at λ = 670 nm and evaluated in analytic and numerical terms. A large S 1 absorption cross section has been confirmed at 670 nm (σ S 1 (670) = 0.71 σ S o (670) and excited-singlet absorption underlined as the cause of the anomalous short-wave emission of the Chl-a laser. The S 1 , absorption cross sections for Chl-a/dioxan in the range 800–870 nm are given.

Spectral substructure and excitonic interactions in the plant antenna complexes LHC II and CP29 as revealed by non-linear laser spectroscopy

Manifestation and extent of excitonic interactions in the plant light-harvesting complexes LHC II and CP29 have been investigated by nonlinear laser spectroscopy. Nonlinear absorption of 120 fs pulses indicates an increase in absorption cross section in the red wing of the Qy-band of LHC II as compared to monomeric chlorophyll (Chl) a in organic solution. Nonlinear polarization spectroscopy in the frequency domain (NLPF) exploiting also the intensity dependence of the NLPF signal reveals that in LHC II a species emitting at 682 nm is characterized by a 2.2(± 0.8)-fold larger dipole strength than that of monomeric Chl a. NLPF experiments indicate strong excitonic coupling between Chls a and b in CP29. Moreover, the lowest Qy-transition in CP29 (in contrast to LHC II) is assigned to a non-excitonically coupled Chl a. Stepwise two-photon excitation of Chls a and b in LHC II and CP29 with 100 fs pulses in the (red) Qy-region results in a weak blue fluorescence. The dependence of the spectral shape of the blue fluorescence on excitation wavelength together with a comparison to properties of Chls in solution are consistent with the existence of strongly excitonically coupled Chl a/b-heterodimers in LHC II. Implications of the results for the refinement of structural models and intra- as well as inter-antenna energy-transfer mechanisms are discussed.

Instantaneous Fluorescence Quantum Yield—A New Quantity with Characteristic “Fingerprints” for Excited-State Processes

The instantaneous fluorescence quantum yield φins—a new quantity for fluorescence studies defined as the ratio of the fluorescence intensity to the optical density, both measured at the moment of the maximum of the exciting pulse—proves to be a very sensitive function for excited-state processes. Dependent on the excitation intensity φins exhibits characteristic features (maxima/minima) indicating, for example, excited-state absorptions and annihilation processes. φins is therefore more informative as the intensity dependence of the usually utilized fluorescence yield, the information content of which is restricted because this function is hardly structured. In the paper the influences of specific molecular parameters (excited-state absorption cross section, annihilation constant) on φins are given, problems of the experimental accessibility of φins are discussed, and an experimental setup for determination of this new quantity is presented. The application of the method is demonstrated for identification of excited-state absorptions of organic molecules in solution.