Dieter Leupold

Two-photon excitation of alkyl-substituted magnesium phthalocyanine: radical formation via higher excited states

Abstract The photophysical properties of tetra-(tert-butyl)-phthalocyanato-magnesium (t 4 -PcMg) in solution and microheterogeneous systems (liposomes and micelles) were investigated. Radical cation formation occurs in chloroform during UV excitation in the presence of an electron acceptor (CBr 4 ). The same result is achieved by two-step absorption in the singlet manifold using pulsed excitation at λ exc =670 nm, which is of interest from the viewpoint of photon delivery through the therapeutic window of tissues. To obtain a deeper insight into the photophysics leading to radical cation formation via the higher excited singlet state, the transient spectra and singlet—singlet absorption cross-sections were determined. In addition to strong excited state absorption within the spectral range of the Q x -band, relatively large absorption cross-sections were also found in regions with low ground state absorption. The importance of these transitions for an effective two-colour excitation regime is discussed with regard to new start mechanisms for photodynamic laser tumour theraphy.

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.

Femtosecond two-photon excited fluorescence of melanin.

Abstract. Fluorescence of synthetic melanin in dimethyl sulfoxide has been excited by two‐photon absorption at 800 run, using 120 fs pulses with photon flux densities 1027 cm 2 s1. The shortest main component of the three‐exponential decay of fluorescence is 200 ± 2 ps. The overall spectral shape is red‐shifted with respect to the 400 nm excited fluorescence. Two‐photon excited melanin fluorescence also has been measured from excised samples of healthy human skin tissue. Because of the selectivity of melanin excitation via resonant two‐photon absorption, it is hypothesized that fluorescence excited in this way may yield information on malignant transformation.

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra‐weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two‐photon excitation, melanin‐dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two‐photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.

Spectroscopic properties of potential sensitizers for new photodynamic therapy start mechanisms via two-step excited electronic states

Three substituted tetraazaporphyrins, octa‐(4‐tert‐butylphenyl)‐tetrapyrazinoporphyrazine, tetra‐(4‐tert‐butyl)phthalocyanine and tetra‐(4‐tert‐butyi)phthalocyanatomagnesium (t4‐PcMg), were spectroscopically checked in solutions and liposomes with respect to suitability as potential sensitizers of a possible new start mechanism for photodynamic therapy (PDT) from a stepwise excited higher singlet state. This PDT start mechanism was recently proposed to overcome the problem of O2 (1δ2)‐caused cutaneous phototoxicity in PDT.

Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence

Because of the increasing incidence of skin cancer, interest in using the autofluorescence of skin tissue as a noninvasive tool for early diagnosis is enforced. Focus is especially on malignant melanotic melanoma. On the basis of a newly developed method to selectively excite melanin fluorescence of skin tissue by stepwise two-photon excitation with nanosecond laser pulses at 810 nm, we have investigated information from this melanin fluorescence with respect to the differentiation of pigmented lesions. A distinct difference in the melanin fluorescence spectrum of malignant melanoma (including melanoma in situ) when compared to that of benign melanocytic lesions (i.e., common nevi) has been found for freshly excised samples as well as for histopathological samples. There is also specific fluorescence from dysplastic nevi. In this way, early detection of malignant melanoma is possible.

Fluorescence and excited state absorption in modified pigments of bacterial photosynthesis a comparative study of metal-substituted bacteriochlorophylls a

Abstract A series of transmetalated bacteriochlorophylls, where the central magnesium has been replaced by Pd, Ni, Zn, Cd, Cu, have been investigated by linear and non-linear laser spectroscopic methods. A strong dependence of the fluorescence on the central metal was obtained. Differences in fluorescence lifetimes and quantum yields are caused mainly by different efficiencies of intersystem crossing. Strong excited state absorptions were found, in particular, the Pd-compound has a strong transient absorption in the 550–670 nm range. The results are of interest with respect to a possible oxygen-free sensitizing action of Pd-BPhe a for photodynamic tumor therapy.

Quantitative comparison of excited state properties and intensity- dependent photosensitization by rose bengal.

A quantitative (multistep) excitation-deactivation model of rose bengal (RB) has been developed which includes energy and electron transfer to oxygen and the possibility of photoproduct formation via higher excited triplet-singlet states. The model is based on previous measurements of non-linear absorption (NLA) and emission of RB with picosecond pulses at 532 nm and on NLA measurements with nanosecond pulses. A coupled rate equation and photon transport equation approach for non-linear light-matter interaction is used. The resulting term scheme with all relevant excited state parameters confirms that (i) in the first excited state of RB, relevant absorption at 532 nm takes place only in the triplet, and (ii) the previously reported intensity dependence of RB-sensitized enzyme inhibition is well modelled by the intensity-dependent RB-T1 population and (as the main process) subsequent energy transfer to form singlet oxygen.

Introduction of a 60 fs deactivation channel in the photosynthetic antenna LH1 by Ni-bacteriopheophytin a

Abstract Forty femtosecond pump and probe investigations in the 870 nm absorption band of the reconstituted core antenna, LH1, from Rhodobacter sphaeroides , in which varying amounts of Ni– bacteriopheophytin replace part of the native bacteriochlorophyll, show a tremendous shortening of the ground state recovery time with increasing amount of exchanged pigments. In the Ni–bacteriopheophytin containing antenna, a 60 fs deactivation channel has been found, which originates from a one-exciton state delocalized over the whole LH1 of about 20 pigment molecules. The 60 fs channel is interpreted as internal conversion in Ni–bacteriopheophytin.

Intensity-dependent enzyme photosensitization using 532 nm nanosecond laser pulses.

Abstract— The intensity dependence of the rose bengal (RB)‐photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 μM RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than ∼ 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet‐triplet absorption coefficient was determined to be 1.9 × 104M−1 cm−1 at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico‐mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.