Diedrich A. Schmidt

Label‐Free Imaging of Metal–Carbonyl Complexes in Live Cells by Raman Microspectroscopy

The search for novel metal complexes with therapeutic activity, in particular against cancer and infectious diseases, is an active and important area of research in medicinal inorganic chemistry. In addition to the well-studied platinumand ruthenium-based coordination complexes, organometallic compounds have gained considerable importance in recent years. Of those, metal–carbonyl compounds are steadily increasing in interest, with some exhibiting remarkable antitumor activity. The most prominent example is probably the use of such compounds as “solid storage forms” for carbon monoxide. These CO-releasing molecules (CORMs) allow the biological action of this important small molecule messenger to be investigated. To elucidate the biological mode of action of any drug candidate it is mandatory to obtain a detailed picture of the intracellular distribution of such substances and how it evolves over time. Until now, the localization of metal complexes inside cells has been studied using X-ray fluorescence (XRF) 14] and atomic absorption spectroscopy (AAS). While AAS offers high sensitivity but almost no spatial resolution, XRF requires intense X-ray sources such as synchrotrons which will cause damage to biological tissue and is also not routinely available as an analytical technique. Most cellular studies therefore use fluorescence microscopy. Furthermore, this technique requires the additional attachment of a fluorescent label, which might be difficult. Optical excitation can also cause additional problems such as the onset of photochemical reactions or photobleaching. Moreover, the label can alter the biodistribution and properties of the molecule of interest, as recently shown for ruthenium–bipyridyl complexes. Efforts have been made to overcome these limitations by identifying biologically active metal complexes which show inherent fluorescence in vivo, but this has only been possible for a small number of metal–ligand combinations. Thus, it is highly desirable to develop innovative and generally applicable imaging techniques for the study of the uptake and distribution of bioactive metal complexes which do not require any labeling or special photophysical properties but instead use the intrinsic spectroscopic signature of the compound of interest. Raman microspectroscopy is emerging as a powerful noninvasive method to assess and image cellular compartments and processes without further sample preparation or labeling. Since Puppels et al. first showed the feasibility of confocal Raman microspectroscopy for imaging cells, its ability to study whole cells and subcellular organelles such as the nucleus and chromatin, mitochondria, and lipid bodies has been demonstrated by various research groups. Apart from imaging subcellular features, Raman imaging has been used to follow the uptake of molecules by cells. So far, however, these investigations have been restricted to the incorporation of deuterated building blocks as sensitive and specific markers into bio(macro)molecules. Herein, we investigate the uptake and cellular distribution of the new manganese-based CORM [Mn(tpm)(CO)3]Cl (tpm = tris(1-pyrazolyl)methane), which has photoinduceable cytotoxic activity against cancer cells. Metal–carbonyl complexes such as [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations between 1800 and 2200 cm , a region where vibrational signals from the constituents of the cell are negligible. We show that the C O vibrations of this compound can be used as an ideal marker for imaging these complexes in living cancer cells. Although the spectroscopic signature of metal–carbonyl compounds has already been used in bioanalytical techniques such as the carbonyl–metal immunoassay (CMIA), their use in cellular imaging is so far unprecedented, except for an investigation of osmium–carbonyl clusters in dried cells by using infrared microscopy. The IR and Raman spectra of solid [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations at about 1944 and 2050 cm , as expected for local C3v symmetry (Figures S1 and S2 A in the Supporting Information). The different relative intensities of the two peaks can be explained by the distinct selection rules for Raman and IR spectroscopy. The O H stretching vibration localized at about 3400 cm 1 dominates the spectrum of a 2 mm aqueous solution of [Mn(tpm)[*] K. Meister, Dr. D. A. Schmidt, Prof. Dr. M. Havenith Lehrstuhl f r Physikalische Chemie II, Ruhr-Universit t Bochum Universit tsstrasse 150, 44801 Bochum (Germany) E-mail: martina.havenith@rub.de Homepage: www.rub.de/pc2

Rattling in the Cage: Ions as Probes of Sub-picosecond Water Network Dynamics

We present terahertz (THz) measurements of salt solutions that shed new light on the controversy over whether salts act as kosmotropes (structure makers) or chaotropes (structure breakers), which enhance or reduce the solvent order, respectively. We have carried out precise measurements of the concentration-dependent THz absorption coefficient of 15 solvated alkali halide salts around 85 cm(-1) (2.5 THz). In addition, we recorded overview spectra between 30 and 300 cm(-1) using a THz Fourier transform spectrometer for six alkali halides. For all solutions we found a linear increase of THz absorption compared to pure water (THz excess) with increasing solute concentration. These results suggest that the ions may be treated as simple defects in an H-bond network. They therefore cannot be characterized as either kosmotropes or chaotropes. Below 200 cm(-1), the observed THz excess of all salts can be described by a linear superposition of the water absorption and an additional absorption that is attributed to a rattling motion of the ions within the water network. By providing a comprehensive set of data for different salt solutions, we find that the solutions can all be very well described by a model that includes damped harmonic oscillations of the anions and cations within the water network. We find this model predicts the main features of THz spectra for a variety of salt solutions. The assumption of the existence of these ion rattling motions on sub-picosecond time scales is supported by THz Fourier transform spectroscopy of six alkali halides. Above 200 cm(-1) the excess is interpreted in terms of a change in the wing of the water network librational mode. Accompanying molecular dynamics simulations using the TIP3P water model support our conclusion and show that the fast sub-picosecond motions of the ions and their surroundings are almost decoupled. These findings provide a complete description of the solute-induced changes in the THz solvation dynamics for the investigated salts. Our results show that THz spectroscopy is a powerful experimental tool to establish a new view on the contributions of anions and cations to the structuring of water.

Exploring hydrophobicity by THz absorption spectroscopy of solvated amino acids

Although hydrophobicity is a commonly used concept, its microscopic nature, particularly in the context of hydration, is not well understood. Here, we present a study of hydrophobic and hydrophilic solutes using terahertz (THz) spectroscopy and molecular dynamics (MD) simulations. We measured the concentration dependent THz absorption (2.1-2.7 THz) of several amino acids and peptides in aqueous solution. Experimentally, we find a correlation between the change in THz absorption of solvating water and specific properties of the solute such as polarity and hydrophobicity. In addition, we studied the effect of hydrophobic and hydrophilic model particles on water dynamics by MD simulations. We are able to link the vibrational density of states (VDOS) in hydration water around the model particles to the experimentally observed change in THz absorption of solvated amino acids. We find a stronger increase in THz absorption and in the oxygen VDOS of solvating water molecules for the hydrophilic versus hydrophobic solutes. The simulations provide us with a microscopic insight into the change of the hydration dynamics as induced by hydrophobic and hydrophilic solutes. For hydrophobic and hydrophilic model particles a retardation of dynamical processes on the picosecond timescale is found, which is more pronounced for hydrophilic compared to hydrophobic solutes.

Water solvation properties: an experimental and theoretical investigation of salt solutions at finite dilution.

Our combined analysis of first-principle simulations and experiments conducted on salt solutions at finite dilution shows that the high frequency range of the infrared spectrum of an aqueous solution of NaCl displays a shift toward higher frequencies of the stretching band with respect to pure water. We ascribe this effect to a lowering of the molecular dipole moments due to a decrease in the dipole moments of molecules belonging to the first and second solvation shells with respect to bulk water. An analysis of the dipole orientation correlations proves that the screening of solutes is dominated by short-range effects. These jointly experimental and theoretical results are corroborated by the good agreement between calculated and measured dielectric constants of our target solution.

Smart polymer surfaces: mapping chemical landscapes on the nanometre scale

We show that Scattering Infrared Near-field Microscopy (SNIM) allows chemical mapping of polymer monolayers that can serve as designed nanostructured surfaces with specific surface chemistry properties on a nm scale. Using s-SNIM a minimum volume of 100 nm × 100 nm × 15 nm is sufficient for a recording of a “chemical” IR signature which corresponds to an enhancement of at least four orders of magnitudes compared to conventional FT-IR microscopy. We could prove that even in cases where it is essentially difficult to distinguish between distinct polymer compositions based solely on topography, nanophase separated polymers can be clearly distinguished according to their characteristic near-field IR response.

Similar appearance, different mechanisms: xerosis in HIV, atopic dermatitis and ageing

Xerosis is one of the most common dermatologic disorders occurring in the elderly and in patients with atopic dermatitis (AD) and human immunodeficiency virus (HIV) infection. Xerosis has been linked to an impaired skin barrier function of the stratum corneum. Using Raman microspectroscopy, we concentrated on deeper skin layers, viable epidermis and dermis of 47 volunteers and associated molecular alterations to the evolution of xerosis and the skin barrier, for example, lipid, water and antioxidant content. A decrease in lipids within the viable epidermis is found for elderly and HIV‐patients. Lipid and water values of AD patients and their healthy reference group are similar. Decreases in lipids and simultaneous increases in water are found in the dermis for HIV and AD patients in comparison to their healthy reference groups. Excessive levels of epidermal carotenoids, mainly lycopene, in HIV‐patients were found potentially leading to adverse effects such as premature skin ageing.

Combined far- and near-field chemical nanoscope at ANKA-IR2: applications and detection schemes

A newly developed microscopy and nanoscopy station that combines far- and near-field microscopy with other microscopy modalities has recently been integrated at the ANKA-IR2 beamline. The various modalities include broadband synchrotron radiation and tunable laser-based near-field microscopy, atomic force microscopy, Raman microspectroscopy, and confocal laser and fluorescence microscopy. This multi-modal nanoscope is designed to combine a broad array of techniques to study the same sample at the same position. We show some examples that demonstrate several of the available modalities. We also discuss various detection schemes to facilitate sensitive absorption and reaction-kinetic experiments.

Non-invasive nano-imaging of ion implanted and activated copper in silicon

Using vibrational imaging techniques including Fourier-transform infrared (FTIR) synchrotron microscopy,Raman microscopy, and scattering scanning near-field infrared microcscopy (s-SNIM), we mapped a sample of phosphor and copper ions implanted in a high-purity silicon wafer. While Raman microscopy monitors the structural disorder within the implantation fields, the aforementionedinfrared techniques provide a detailed picture of the distribution of the free carriers. On a large scale (tens of micrometers), we visualized the channeling effects of phosphordopants in silicon using FTIRmicroscopy. In comparison, using s-SNIM we were able to image, on a nanometer scale, local variations of the dielectric properties of the silicon substrate due to the activation of copperdopants.

Nano‐spectroscopy and chemical nanoscopy of biomaterials

A scanning near‐field infrared microscopy experimental station will be integrated into the ANKA‐IR2 beamline to combine broadband synchrotron radiation with near‐field microscopy. Other microscopy techniques also available in the station will be compared. We have performed nano‐spectroscopy investigating biomaterials like self‐assembled monolayers and nanoscale lipid membranes. Coherent synchrotron radiation (CSR) at ANKA has been measured to determine power and beam profile for coupling terahertz radiation to the nanoscope.

Using rattling ions to probe sub-ps water network dynamics

We present THz measurements on fifteen solvated alkali halide salts using narrow band THz absorption and broad band THz Fourier transform spectroscopies in order to shed new light on the controversy of salts as kosmotropes (structure breakers) or chaotropes (structure makers).