Benjamin Dietzek

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

Second-harmonic generation (SHG) microscopy is a fantastic tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. In fact, SHG combines the advantages of a non-linear microscopy approach with a coherent modality able to probe molecular organization. In this manuscript we review the physical concepts describing SHG from collagen, highlighting how this optical process allows to probe structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward - backward SHG detection, we then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods, highlighting advantages and limitations of the methods applied to tissue imaging and to potential clinical applications.

A possible mechanism for the emergence of an additional band gap due to a Ti–O–C bond in the TiO2–graphene hybrid system for enhanced photodegradation of methylene blue under visible light

Here we report the experimental and theoretical study of two TiO2–graphene oxide (TG) and TiO2–reduced graphene oxide (TR) composites synthesized by a facile and ecological route, for enhanced visible light (∼470 nm) photocatalytic degradation of Methylene Blue (MB) (99% efficiency), with high rate constant values (1800% over bare TiO2). TG couples TiO2 nanopowder with Graphene Oxide (GO) while TR couples it with reduced graphene oxide (RGO). The present study, unlike previous reports, discusses never-before-reported double absorption edges obtained for both TG (3.51 eV and 2.51 eV) and TR (3.42 eV and 2.39 eV) composites, which represents the reason behind feasible visible light (2.56 eV) induced photocatalysis. TiO2 domains in the composites dominate the higher band edge, while GO/RGO domains explain the lower band edge. Formation of Ti–O–C bonds in both TG and TR drives the shifting upwards of the valence band edge and reduction in band gap. Further, these bonds provide a conductive pathway for charge carriers from TiO2 nanopowder to the degraded species via the GO/RGO matrix, resulting in decreased charge carrier recombination in TiO2 and enhanced efficiency. To attest that the developed theory is correct, density function theory (DFT) calculations were performed. DFT obtained energetics and electronic structures support experimental findings by demonstrating the role of the Ti–O–C bond, which results in double band edge phenomenon in composites. Finally, the mechanism behind MB degradation is discussed comprehensively and the effect of the weight percent of GO/RGO in the composite on the rate constant and photodegradation efficiency has been studied experimentally and explained by developing analytical equations.

Automated classification of healthy and keloidal collagen patterns based on processing of SHG images of human skin

All-optical microspectroscopic and tomographic tools have a great potential for the clinical investigation of human skin and skin diseases. However, automated optical tomography or even microscopy generate immense data sets. Therefore, in order to implement such diagnostic tools into the medical practice in both hospitals and private practice, there is a need for automated data handling and image analysis ideally implementing automized scores to judge the physiological state of a tissue section. In this contribution, the potential of an image processing algorithm for the automated classification of skin into normal or keloid based on second-harmonic generation (SHG) microscopic images is demonstrated. Such SHG data is routinely recorded within a multimodal imaging approach. The classification of the tissue implemented in the algorithm employs the geometrical features of collagen patterns that differ depending on the constitution, i.e., physiological status of the skin.

Photocatalytic Hydrogen Evolution Driven by [FeFe] Hydrogenase Models Tethered to Fluorene and Silafluorene Sensitizers

It is successfully shown that photocatalytic proton reduction to dihydrogen in the presence of a sacrificial electron donor, such as trimethylamine (TEA) and ascorbate, can be driven by compact sensitizer-catalyst dyads, that is, dithiolate-bridged [FeFe] hydrogenase models tethered to organic sensitizers, such as fluorenes and silafluorenes (1 a-4 a). The sensitizer-catalyst dyads 1 a-4 a show remarkable and promising catalytic activities as well as enhanced stabilities during photocatalysis performed under UV-light irradiation. The photocatalysis was carried out both in non-aqueous and aqueous media. The latter experiments were performed by solubilizing the photocatalysts within micelles formed by either sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB). In this study a turnover number of 539 (7 h) is achieved under optimized conditions, which corresponds to an exceptionally high turnover frequency of 77 h-1 . Theoretical investigations as well as emission decay experiments were performed to understand the observed phenomena together with the mechanisms of photocatalytic H2 generation.

Raman Spectroscopic Insights into the Chemical Gradients within the Wound Plug of the Green Alga Caulerpa taxifolia

The invasive unicellular green macroalga Caulerpa taxifolia has spread dramatically in the Mediterranean Sea over the last decades. Its success is based on rapid plug formation after wounding, to prevent the loss of cell content. This quick and efficient process involves the rapid transformation of the secondary metabolite caulerpenyne to the reactive 1,4‐dialdehyde oxytoxin 2, which acts as a protein crosslinker. The main metabolites of the wound plug were identified as proteins, caulerpenyne derivatives, and sulfated polysaccharides. Because of a methodological deficit, however, the detailed distribution of the compounds within the wound plug of C. taxifolia was unknown. This study demonstrates the suitability of FT‐Raman spectroscopy for the noninvasive in vivo determination of caulerpenyne and its derivatives, as well as β‐carotene, from signals with special spectral features within the wound plug and the adjacent intact alga tissue, with a resolution of 100 μm. FT‐Raman spectra allowed four different zones with distinct chemical compositions around the region of wounds to be characterized. Gradients of the investigated metabolites within the wound plug and the alga could be determined. Moreover, various caulerpenyne derivatives could be identified spectroscopically, and this has led to a mechanistic proposal for the internal and the external wound plug formation.

A program for automatically predicting supramolecular aggregates and its application to urea and porphin

Not only the molecular structure but also the presence or absence of aggregates determines many properties of organic materials. Theoretical investigation of such aggregates requires the prediction of a suitable set of diverse structures. Here, we present the open‐source program EnergyScan for the unbiased prediction of geometrically diverse sets of small aggregates. Its bottom‐up approach is complementary to existing ones by performing a detailed scan of an aggregates potential energy surface, from which diverse local energy minima are selected. We crossvalidate this approach by predicting both literature‐known and heretofore unreported geometries of the urea dimer. We also predict a diverse set of dimers of the less intensely studied case of porphin, which we investigate further using quantum chemistry. For several dimers, we find strong deviations from a reference absorption spectrum, which we explain using computed transition densities. This proof of principle clearly shows that EnergyScan successfully predicts aggregates exhibiting large structural and spectral diversity.

Non-linear imaging and characterization of atherosclerotic arterial tissue using combined SHG and FLIM microscopy

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view, especially for what is concerning collagen content and organization because collagen plays a crucial role in plaque vulnerability. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Non-linear microscopy techniques offer the potential for providing morpho-functional information on the examined tissues in a label-free way. In this study, we employed combined SHG and FLIM microscopy for characterizing collagen organization in both normal arterial wall and within atherosclerotic plaques. Image pattern analysis of SHG images allowed characterizing collagen organization in different tissue regions. In addition, the analysis of collagen fluorescence decay contributed to the characterization of the samples on the basis of collagen fluorescence lifetime. Different values of collagen fiber mean size, collagen distribution, collagen anisotropy and collagen fluorescence lifetime were found in normal arterial wall and within plaque depositions, prospectively allowing for automated classification of atherosclerotic lesions and plaque vulnerability. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

Non-linear imaging and characterization of atherosclerotic arterial tissue using combined two photon fluorescence, second-harmonic generation and CARS microscopy

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires a morpho-functional approach. Multimodal non-linear microscopy has the potential to bridge this gap by providing morpho-functional information on the examined tissues in a label-free way. Here we employed multiple non-linear microscopy techniques, including CARS, TPF, and SHG to provide intrinsic optical contrast from various tissue components in both arterial wall and atherosclerotic plaques. CARS and TPF microscopy were used to respectively image lipid depositions within plaques and elastin in the arterial wall. Cholesterol deposition in the lumen and collagen in the arterial wall were selectively imaged by SHG microscopy and distinguished by forward-backward SHG ratio. Image pattern analysis allowed characterizing collagen organization in different tissue regions. Different values of fiber mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

Impact of drying procedure on the morphology and structure of TiO2 xerogels and the performance of dye sensitized solar cells

Different morphologies of TiO2 photoelectrodes for dye sensitized solar cells were obtained by using TiO2 gels dried in normal conditions (TiO2amb) and in CO2 atmosphere at high pressure (TiO2press). After a subsequent calcination, the powders were processed as pastes and drop-casted on conductive glass in order to prepare photoanodes for dye sensitized solar cells. N719 commercial dye was used as sensitizer in all the experiments. The structure and morphology of the processed TiO2 materials were investigated via X-ray diffraction, scanning electron microscopy, transmission electron microscopy and N2 adsorption/desorption measurements. The dye adsorption capacity of the photoanodes was tested using ultraviolet-visible absorption spectroscopy. The photovoltaic performances of the dye sensitized solar cells were investigated using current/voltage curves (I/V), open circuit photovoltage decay measurements and electrochemical impedance spectroscopy. The conversion efficiency (η) and short circuit density (Jsc) for TiO2amb were 1.90 % and 5 mA/cm2 respectively, while the TiO2press cell exhibited a 40 and 50 % increase in Jsc and η values respectively. This was correlated with increased dye loading capacities due to a broader distribution of pore size towards the mesopore region.Graphical Abstract

Response to the Comments by L. O. Björn on our Paper “Catalytic Efficiency of a Photoenzyme—An Adaptation to Natural Light Conditions”

NADPH:protochlorophyllide oxidoreductase (POR) is one of only two enzymes in nature, in which the enzymatic activity is switched on by the absorption of light. The pre-formation of the enzyme–substrate complex in the dark and the initiation of catalysis by light make the POR enzyme an excellent model for studying the initial, ultrafast steps of enzymatic reactions in real time. Throughout the last decade a series of such studies have been reported. They all include recombinant POR enzymes, which were expressed in Escherichia coli and reconstituted to the ternary enzyme complexes by addition of the substrate and coenzyme, protochlorophyllide (PChlide) and NADPH, respectively. In addition, the thermodynamics of substrate/coenzyme binding as well as the complete POR catalytic cycle have been investigated by spectroscopic techniques in conjunction with low-temperature and stopped flow methods. All these experiments were also performed on recombinant enzymes expressed in Escherichia coli or on a pigment-free, monomeric enzyme isolated from etiolated oat (Avena sativa) seedlings. As in the studies cited above, we have used purified recombinant POR enzymes in our experiments aimed to determine the quantum yield for the POR catalyzed photoreduction. In the article of L. O. Bjçrn the results obtained from those enzymes are subjected to criticisms in so far as they would differ from those published more than 40 years ago. However, most of the differences can be explained by the different types and aggregation states of the enzymes examined. In the work referred by L. O. Bjçrn, POR bound to natural membranes and POR isolated from etiolated plant material was investigated. There is strong evidence that POR associates with membranes under natural conditions and exists in an aggregated state as dimer or larger sized oligomer. In a similar manner, the enzymes obtained from the extraction of etiolated plant material, PChlide holochromes, form high molecular weight complexes (~600 kDa), too. 21, 23] The photochemistry of aggregated PChlide-POR complexes is impaired by pigment–pigment interactions, energy transfer between PChlide and chlorophyllide (the product of the enzymatic reaction) and further affected by protein–lipid interactions. Nowadays, considerable effort has therefore been made to substitute the aggregated POR complexes by recombinant monomeric enzymes, which are catalytically active. The reason is that such minimal photoactive POR complexes consisting of the natural constituents—photoenzyme, substrate, and cofactor—should allow a more precise insight into the molecular mechanism of POR. As outlined in the supplement of our paper, the POR enzymes used in our experiments are monomers and were obtained from heterologous expression of the POR A and POR B gen from barley (Hordeum vulgare) and the POR gen from Synechocystsis. The enzymes from barley do not include the transit peptide, which is only necessary to import the precursor proteins from the cytoplasm into plastids. The sequence of the Synechocystis POR corresponds to that of the strain PCC 6803. The enzymes were purified to homology and reconstituted with the substrate PChlide and the cofactor NADPH. The molecular weights of the enzyme monomers correspond to those calculated from the amino acid sequences (POR A ~37 kDa, POR B and Synechocystis POR ~37.5 kDa). PChlide, necessary for the reconstitution of the photoactive enzymes, was isolated from etiolated oat seedlings and finally purified by reversedphase HPLC to a chemically pure form. The absorbance spectra of the reconstituted POR-PChlideNADPH complexes are strictly reproducible and, as can be seen from Figure S1 of our manuscript L. O. Bjçrn is referring to, not disturbed by scattered light. They exhibit the characteristic features of a porphyrin-like compound with a prominent B or Soret band in the blue spectral region at 438 [Bx,y (0,0)] nm and two weak shoulders at 418 [By (0,1)] and 390 nm [By (0,2)] . The strongest Q-band appears at 630 nm [Qy(0,0)] and is accompanied by two sub-bands at 577 [Qy(0,1)] and 535 nm [Qy(0,2)] . The fluorescence excitation spectra reveal the well-resolved structure of the absorbance bands independent of the fluorescence wavelength. This shows that only one single ground-state species of PChlide exists in the ternary POR complexes and points to the high purity of the reconstituted enzymes under investigation. Moreover, the binding of PChlide to the POR enzymes induces shifts in the 77 K fluorescence [a] Prof. Dr. M. Schmitt, Prof. Dr. B. Dietzek, Prof. Dr. J. Popp Friedrich-Schiller-Universit t, Institut f r Physikalische Chemie Helmholtzweg 4, 07743 Jena (Germany) Fax: (+ 49) (0)3641-948302 E-mail : juergen.popp@uni-jena.de [b] Dr. G. Hermann Friedrich-Schiller-Universit t Jena, Institut f r Biochemie und Biophysik Philosophenweg 12, 07743 Jena (Germany) [c] Prof. Dr. B. Dietzek, Prof. Dr. J. Popp Institut f r Photonische Techologien e. V. Albert-Einstein-Str. 9, 07745 Jena (Germany)