New Aspects Regarding the Fluorescence Spectra of Melanin and Neuromelanin in Pigmented Human Tissue Concerning Hypoxia.

Melanin is a crucial pigment in melanomagenesis. Its fluorescence in human tissue is exceedingly weak but can be detected through advanced laser spectroscopy techniques. The spectral profile of melanin fluorescence distinctively varies among melanocytes, nevomelanocytes, and melanoma cells, with melanoma cells exhibiting a notably "red" fluorescence spectrum. This characteristic enables the diagnosis of melanoma both in vivo and in histological samples. Neuromelanin, a brain pigment akin to melanin, shares similar fluorescence properties. Its fluorescence can also be quantified with high spectral resolution using the same laser spectroscopic methods. Documented fluorescence spectra of neuromelanin in histological samples from the substantia nigra substantiate these findings. Our research reveals that the spectral behavior of neuromelanin fluorescence mirrors that of melanin in melanomas. This indicates that the typical red fluorescence is likely influenced by the microenvironment around (neuro)melanin, rather than by direct pigment interactions. Our ongoing studies aim to further explore this distinctive "red" fluorescence. We have observed this red fluorescence spectrum in post-mortem measurements of melanin in benign nevus. The characteristic red spectrum is also evident here (unlike the benign nevus in vivo), suggesting that hypoxia may contribute to this phenomenon. Given the central role of hypoxia in both melanoma development and treatment, as well as in fundamental Parkinson's disease mechanisms, this study discusses strategies aimed at reinforcing the hypothesis that red fluorescence from (neuro)melanin serves as an indicator of hypoxia.

From Melanocytes to Melanoma Cells: Characterization of the Malignant Transformation by Four Distinctly Different Melanin Fluorescence Spectra (Review).

The melanin fluorescence emitted by pigment cells of the human skin has been a central research topic for decades, because melanin, on the one hand, protects against (solar) radiation in the near-UV range, whereas on the other hand, melanocytes are the starting point for the malignant transformation into melanoma. Until recently, however, melanin fluorescence was not accessible in the context of conventional spectroscopy, because it is ultraweak and is overshadowed by the more intense so-called autofluorescence of endogenous fluorophores. The advent of a new method of laser spectroscopy has made this melanin fluorescence measurable in vivo. A stepwise two-photon absorption with 800 nm photons is used, which more selectively excites melanin (dermatofluoroscopy). Our review summarizes the experimental results on melanin fluorescence of the four types of cutaneous pigment cells from healthy and malignant tissues. Outstanding is the finding that different types of melanocytes (i.e., melanocytes of common nevi, versus dysplastic nevi or versus melanoma cells) show characteristically different fluorescence spectra. The possibilities of using this melanin fluorescence for melanoma diagnosis are shown. Moreover, the uniform fluorescence spectra emitted by different melanoma subtypes are essential. Conclusions are drawn about the molecular processes in the melanosomes that determine fluorescence. Finally, experimental suggestions for further investigations are given.

Dermatofluoroscopy Is Also for Redheads a Sensitive Method of Early Melanoma Detection.

BACKGROUND: Caucasians with red hair and fair skin have a remarkably increased risk of malignant melanoma compared to non-redhead Caucasians. OBJECTIVES: With the aim of a reliable melanoma diagnosis in redheads, the application of dermatofluoroscopy was analyzed in 16 patients with red hair. Most of them had been included in a clinical dermatofluoroscopy study for patients with the suspicion of melanoma. We examined whether the 25 lesions of the redheads showed the same characteristic melanin fluorescence spectra for dysplastic nevi and melanomas as those of non-redhead Caucasians or whether there was a different fluorescence pattern. This is important in view of the known significantly altered ratio of eumelanin to pheomelanin in the skin of redheads. METHODS: More than 8,000 spatially resolved fluorescence spectra of 25 pigmented lesions were measured and analysed. The spectra were excited by the stepwise absorption of two 800-nm photons (principle of dermatofluoroscopy). Furthermore, the fluorescence spectra of eumelanin and pheomelanin in hair samples were determined in the same way. RESULTS: The evaluation revealed that the melanin fluorescence spectra of dysplastic nevi and melanomas of redheads have the same spectral characteristics as those of non-redhead Caucasians. An accompanying result is that dermatofluoroscopy shows identical fluorescence spectra for eumelanin and pheomelanin. CONCLUSIONS: Dermatofluoroscopy proves to be a reliable diagnostic method also for redheads. Our results also explain our recent finding that there is a uniform fluorescence spectroscopic fingerprint for melanomas of all subtypes, which is of particular interest for hypomelanotic and apparently amelanotic melanomas containing pheomelanin.

[Fluorescence detection to diagnose melanoma : An alternative objective method for dermatohistology]. / Fluoreszenzbasierte Melanomdiagnostik : Eine flankierende objektive Methode zur Dermatohistologie.

BACKGROUND: Dermatohistological assessment is the gold standard in the diagnosis of melanoma. As a subjective method, this depends, among other things, on the expertise of the examiner. AIM: A new objective method of investigation-dermatohistofluoroscopy-aims to improve the diagnostic reliability of melanoma diagnosis. MATERIALS AND METHODS: The ultra-weak spectrally resolved fluorescence of melanin of pigment-bearing skin cells is a reliable indicator of the malignancy of the tissue to be diagnosed. Using a special laser spectroscopic method, this fluorescence can be measured on histological specimen (and also on tissue in vivo and on excidate). RESULTS AND DISCUSSION: Melanocytes from normally pigmented skin, nevomelanocytes from benign and dysplastic nevi, and melanoma cells each show characteristic, different fluorescence spectra. If these cell types are shown spatially resolved in different colors on the preparation to be diagnosed, they give the histologist an objective basis for the diagnosis, even in difficult cases, e.g., the so-called stumbling blocks of melanoma diagnosis such as Spitzoid tumors. Currently, the method can only be used for Fitzpatrick skin types 1 and 2. In addition, a separate second measurement signal at 400 nm can be used to identify tumor breakthrough through the basement membrane. This signal is collagen bound, so it also appears in amelanotic melanomas, to which the method is otherwise inherently inapplicable.

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.

Melanin and Neuromelanin Fluorescence Studies Focusing on Parkinson's Disease and Its Inherent Risk for Melanoma.

Parkinson's disease is associated with an increased risk of melanoma (and vice versa). Several hypotheses underline this link, such as pathways affecting both melanin and neuromelanin. For the first time, the fluorescence of melanin and neuromelanin is selectively accessible using a new method of nonlinear spectroscopy, based on a stepwise two-photon excitation. Cutaneous pigmentation and postmortem neuromelanin of Parkinson patients were characterized by fluorescence spectra and compared with controls. Spectral differences could not be documented, implying that there is neither a Parkinson fingerprint in cutaneous melanin spectra nor a melanin-associated fingerprint indicating an increased melanoma risk. Our measurements suggest that Parkinson's disease occurs without a configuration change of neuromelanin. However, Parkinson patients displayed the same dermatofluorescence spectroscopic fingerprint of a local malignant transformation as controls. This is the first comparative retrospective fluorescence analysis of cutaneous melanin and postmortem neuromelanin based on nonlinear spectroscopy in patients with Parkinson's disease and controls, and this method is a very suitable diagnostic tool for melanoma screening and early detection in Parkinson patients. Our results suggest a non-pigmentary pathway as the main link between Parkinson's disease and melanoma, and they do not rule out the melanocortin-1-receptor gene as an additional bridge between both diseases.

Femtosecond spectroscopy of native and carotenoidless purple-bacterial LH2 clarifies functions of carotenoids.

EET between the two circular bacteriochlorophyll compartments B800 and B850 in native (containing the carotenoid rhodopin) and carotenoidless LH2 isolated from the photosynthetic purple sulfur bacterium Allochromatium minutissimum was investigated by femtosecond time-resolved transient absorption spectroscopy. Both samples were excited with 120-fs laser pulses at 800 nm, and spectral evolution was followed in the 720-955 nm range at different delay times. No dependence of transient absorption in the B800 band on the presence of the carotenoid rhodopin was found. Together with the likewise virtually unchanged absorption spectra in the bacteriochlorophyll Q(y) region, these observations suggest that absence of rhodopin does not significantly alter the structure of the pigment-protein complex including interactions between bacteriochlorophylls. Apparently, rhodopin does also not accelerate B800 to B850 EET in LH2, contrary to what has been suggested previously. Moreover, "carotenoid-catalyzed internal conversion" can also be excluded for the bacteriochlorophylls in LH2 of A. minutissimum. Together with previous results obtained with two-photon fluorescence excitation spectroscopy, it can also be concluded that there is neither EET from rhodopin to B800 nor (back-)EET from B800 to rhodopin.

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae.

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a "building block" approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving "optically dark" states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).

Study protocol for a prospective, non-controlled, multicentre clinical study to evaluate the diagnostic accuracy of a stepwise two-photon excited melanin fluorescence in pigmented lesions suspicious for melanoma (FLIMMA study).

INTRODUCTION: Non-invasive, nanosecond, stepwise two-photon laser excitation of skin tissue was shown to induce melanin fluorescence spectra that allow for the differentiation of melanocytic nevi from cutaneous melanoma. METHODS AND ANALYSIS: This prospective, non-controlled, multicentre clinical study is performed to evaluate the diagnostic performance of the stepwise two-photon excited melanin fluorescence in the detection of cutaneous melanoma. The comparator will be the histopathological diagnosis. A total of 620 pigmented skin lesions suspicious for melanoma and intended for excision will be enrolled. ETHICS AND DISSEMINATION: Ethics approval was provided by the local ethics committees of the medical faculties of the University of Tuebingen, Heidelberg and Berlin. STUDY REGISTRATION: The FLIMMA study NCT02425475.

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.

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 approximately 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 approximately 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 approximately 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.

Pigment-pigment interactions in PCP of Amphidinium carterae investigated by nonlinear polarization spectroscopy in the frequency domain.

Peridinin-chlorophyll a-protein (PCP) is a unique antenna complex in dinoflagellates that employs peridinin (a carotenoid) as its main light-harvesting pigment. Strong excitonic interactions between peridinins, as well as between peridinins and chlorophylls (Chls) a, can be expected from the short intermolecular distances revealed by the crystal structure. Different experimental approaches of nonlinear polarization spectroscopy in the frequency domain (NLPF) were used to investigate the various interactions between pigments in PCP of Amphidinium carterae at room temperature. Lineshapes of NLPF spectra indicate strong excitonic interactions between the peridinin's optically allowed S(2) (1Bu(+)) states. A comprehensive subband analysis of the distinct NLPF spectral substructure in the peridinin region allows us to assign peridinin subbands to the two Chls a in PCP having different S(1)-state lifetimes. Peridinin subbands at 487, 501, and 535 nm were assigned to the longer-lived Chl, whereas a peridinin subband peaking at 515 nm was detected in both clusters. Certain peridinin(s), obviously corresponding to the subband centered at 487 nm, show(s) specific (possibly Coulombic?) interaction between the optically dark S(1)(2A(g)(-)) and/or intramolecular charge-transfer (ICT) state and S(1) of Chl a. The NLPF spectrum, hence, indicates that this peridinin state is approximately isoenergetic or slightly above S(1) of Chl a. A global subband analysis of absorption and NLPF spectra reveals that the Chl a Q(y)-band consists of two subbands (peaking at 669 and 675 nm and having different lifetimes), confirmed by NLPF spectra recorded at high pump intensities. At the highest applied pump intensities an additional band centered at S(1)/ICT transition of peridinin.

Two-photon excited fluorescence from higher electronic states of chlorophylls in photosynthetic antenna complexes: a new approach to detect strong excitonic chlorophyll a/b coupling.

Stepwise two-photon excitation of chlorophyll a and b in the higher plant main light-harvesting complex (LHC II) and the minor complex CP29 (as well as in organic solution) with 100-fs pulses in the Q(y) region results in a weak blue fluorescence. The dependence of the spectral shape of the blue fluorescence on excitation wavelength offers a new approach to elucidate the long-standing problem of the origin of spectral "chlorophyll forms" in pigment-protein complexes, in particular the characterization of chlorophyll a/b-heterodimers. As a first result we present evidence for the existence of strong chlorophyll a/b-interactions (excitonically coupled transitions at 650 and 680 nm) in LHC II at ambient temperature. In comparison with LHC II, the experiments with CP29 provide further evidence that the lowest energy chlorophyll a transition (at approximately 680 nm) is not excitonically coupled to chlorophyll b.

Excitonic coupling of chlorophylls in the plant light-harvesting complex LHC-II.

Manifestation and extent of excitonic interactions in the red Chl-absorption region (Q(y) band) of trimeric LHC-II were investigated using two complementary nonlinear laser-spectroscopic techniques. Nonlinear absorption of 120-fs pulses indicates an increased absorption cross section in the red wing of the Q(y) band as compared to monomeric Chl a in organic solution. Additionally, the dependence of a nonlinear polarization response on the pump-field intensity was investigated. This approach reveals that one emitting spectral form, characterized by a 2.3(+/-0.8)-fold larger dipole strength than monomeric Chl a, dominates the fluorescence spectrum of LHC-II. Considering available structural and spectroscopic data, these results can be consistently explained assuming the existence of an excitonically coupled dimer located at Chl-bindings sites a2 and b2 (referring to the original notation of W. Nühlbrandt, D.N. Wang, and Y. Fujiyoshi, Nature, 1994, 367:614-621), which must not necessarily correspond to Chls a and b). This fluorescent dimer, terminating the excitation energy-transfer chain of the LHC-II monomeric subunit, is discussed with respect to its relevance for intra- and inter-antenna excitation energy transfer.

Spectral substructure and excitonic interactions in the minor photosystem II antenna complex CP29 as revealed by nonlinear polarization spectroscopy in the frequency domain.

CP29 (the lhcb4 gene product), a minor photosystem II antenna complex, binds six chlorophyll (Chl) a, two Chl b, and two to three xanthophyll molecules. The Chl a/b Q(y) absorption band substructure of CP29 (purified from spinach) was investigated by nonlinear polarization spectroscopy in the frequency domain (NLPF) at room temperature. A set of NLPF spectra was obtained at 11 probe wavelengths. Seven probe wavelengths were located in the Q(y) spectral region (between 630 and 690 nm) and four in the Soret band (between 450 and 485 nm). Evaluation of the experimental data within the framework of global analysis leads to the following conclusions: (i) The dominant Chl a absorption (with a maximum at 674 nm) splits into (at least) three subbands (centered at 660, 670, and 681.5 nm). (ii) In the Chl b region two subbands can be identified with maxima located at 640 and 646 nm. (iii) The lowest energy Q(y) transition (peaking at 681.5 nm) is assigned to a Chl a which only weakly interacts with other Chl aor b molecules by incoherent Förster-type excitation energy transfer. (iv) Pronounced excitonic interaction exists between certain Chl a and Chl b molecules, which most likely form a Chl a/b heterodimer. The subbands centered at 640 and 670 nm constitute a strongly coupled Chl a/b pair. The findings of the study indicate that the currently favored view of spectral heterogeneity in CP29 being due essentially to pigment-protein interactions has to be revised.