Deuterium Isotope Effect in Single Molecule Photophysics and Photochemistry of Hypericin.

The peripherical protons of the dye molecule hypericin can undergo structural interconversion (tautomerization) between different isomers through separated by a low energy barrier with rates that depends sensitively on the interaction with local chemical environment defined by the nature of host material. We investigate the deuterium (D) isotope effect of hypericin tautomerism at the single-molecule level to avoid ensemble averaging in different polymer matrices by a combined spectroscopic and computational approach. In the 'innocent' PMMA matrix only intramolecular isotope effects on the internal conversion channel and tautomerization are observed; while PVA specifically interacts with the probe via H- and D-bonding. This establishes a single molecular picture on intra- and intermolecular nano-environment effects to control chromophore photophysics and -chemistry.

Accumulation and penetration behavior of hypericin in glioma tumor spheroids studied by fluorescence microscopy and confocal fluorescence lifetime imaging microscopy.

Glioblastoma WHO IV belongs to a group of brain tumors that are still incurable. A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell-cell interactions and the extracellular matrix is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O2 activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time > 30 min is required to achieve most suitable conditions for an effective PDT.

Monitoring tautomerization of single hypericin molecules in a tunable optical λ/2 microcavity.

Hypericin tautomerization that involves the migration of the labile protons is believed to be the primary photophysical process relevant to its light-activated antiviral activity. Despite the difficulty in isolating individual tautomers, it can be directly observed in single-molecule experiments. We show that the tautomerization of single hypericin molecules in free space is observed as an abrupt flipping of the image pattern accompanied with fluorescence intensity fluctuations, which are not correlated with lifetime changes. Moreover, the study can be extended to a λ/2 Fabry-Pérot microcavity. The modification of the local photonic environment by a microcavity is well simulated with a theoretical model that shows good agreement with the experimental data. Inside a microcavity, the excited state lifetime and fluorescence intensity of single hypericin molecules are correlated, and a distinct jump of the lifetime and fluorescence intensity reveals the temporal behavior of the tautomerization with high sensitivity and high temporal resolution. The observed changes are also consistent with time-dependent density functional theory calculations. Our approach paves the way to monitor and even control reactions for a wider range of molecules at the single molecule level.

Prediction of Honeydew Contaminations on Cotton Samples by In-Line UV Hyperspectral Imaging.

UV hyperspectral imaging (225 nm-410 nm) was used to identify and quantify the honeydew content of real cotton samples. Honeydew contamination causes losses of millions of dollars annually. This study presents the implementation and application of UV hyperspectral imaging as a non-destructive, high-resolution, and fast imaging modality. For this novel approach, a reference sample set, which consists of sugar and protein solutions that were adapted to honeydew, was set-up. In total, 21 samples with different amounts of added sugars/proteins were measured to calculate multivariate models at each pixel of a hyperspectral image to predict and classify the amount of sugar and honeydew. The principal component analysis models (PCA) enabled a general differentiation between different concentrations of sugar and honeydew. A partial least squares regression (PLS-R) model was built based on the cotton samples soaked in different sugar and protein concentrations. The result showed a reliable performance with R2cv = 0.80 and low RMSECV = 0.01 g for the validation. The PLS-R reference model was able to predict the honeydew content laterally resolved in grams on real cotton samples for each pixel with light, strong, and very strong honeydew contaminations. Therefore, inline UV hyperspectral imaging combined with chemometric models can be an effective tool in the future for the quality control of industrial processing of cotton fibers.

Gradient SERS Substrates with Multiple Resonances for Analyte Screening: Fabrication and SERS Applications.

Surface-enhanced Raman spectroscopy (SERS) provides a strong enhancement to an inherently weak Raman signal, which strongly depends on the material, design, and fabrication of the substrate. Here, we present a facile method of fabricating a non-uniform SERS substrate based on an annealed thin gold (Au) film that offers multiple resonances and gap sizes within the same sample. It is not only chemically stable, but also shows reproducible trends in terms of geometry and plasmonic response. Scanning electron microscopy (SEM) reveals particle-like and island-like morphology with different gap sizes at different lateral positions of the substrate. Extinction spectra show that the plasmonic resonance of the nanoparticles/metal islands can be continuously tuned across the substrate. We observed that for the analytes 1,2-bis(4-pyridyl) ethylene (BPE) and methylene blue (MB), the maximum SERS enhancement is achieved at different lateral positions, and the shape of the extinction spectra allows for the correlation of SERS enhancement with surface morphology. Such non-uniform SERS substrates with multiple nanoparticle sizes, shapes, and interparticle distances can be used for fast screening of analytes due to the lateral variation of the resonances within the same sample.

Anisotropic and Amphiphilic Mesoporous Core-Shell Silica Microparticles Provide Chemically Selective Environments for Simultaneous Delivery of Curcumin and Quercetin.

Porous silica materials are often used for drug delivery. However, systems for simultaneous delivery of multiple drugs are scarce. Here we show that anisotropic and amphiphilic dumbbell core-shell silica microparticles with chemically selective environments can entrap and release two drugs simultaneously. The dumbbells consist of a large dense lobe and a smaller hollow hemisphere. Electron microscopy images show that the shells of both parts have mesoporous channels. In a simple etching process, the properly adjusted stirring speed and the application of ammonium fluoride as etching agent determine the shape and the surface anisotropy of the particles. The surface of the dense lobe and the small hemisphere differ in their zeta potentials consistent with differences in dye and drug entrapment. Confocal Raman microscopy and spectroscopy show that the two polyphenols curcumin (Cur) and quercetin (QT) accumulate in different compartments of the particles. The overall drug entrapment efficiency of Cur plus QT is high for the amphiphilic particles but differs widely between Cur and QT compared to controls of core-shell silica microspheres and uniformly charged dumbbell microparticles. Furthermore, Cur and QT loaded microparticles show different cancer cell inhibitory activities. The highest activity is detected for the dual drug loaded amphiphilic microparticles in comparison to the controls. In the long term, amphiphilic particles may open up new strategies for drug delivery.

Comparison of Whiskbroom and Pushbroom darkfield elastic light scattering spectroscopic imaging for head and neck cancer identification in a mouse model.

The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model's capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine.

Characterization of Pharmaceutical Tablets Using UV Hyperspectral Imaging as a Rapid In-Line Analysis Tool.

A laboratory prototype for hyperspectral imaging in ultra-violet (UV) region from 225 to 400 nm was developed and used to rapidly characterize active pharmaceutical ingredients (API) in tablets. The APIs are ibuprofen (IBU), acetylsalicylic acid (ASA) and paracetamol (PAR). Two sample sets were used for a comparison purpose. Sample set one comprises tablets of 100% API and sample set two consists of commercially available painkiller tablets. Reference measurements were performed on the pure APIs in liquid solutions (transmission) and in solid phase (reflection) using a commercial UV spectrometer. The spectroscopic part of the prototype is based on a pushbroom imager that contains a spectrograph and charge-coupled device (CCD) camera. The tablets were scanned on a conveyor belt that is positioned inside a tunnel made of polytetrafluoroethylene (PTFE) in order to increase the homogeneity of illumination at the sample position. Principal component analysis (PCA) was used to differentiate the hyperspectral data of the drug samples. The first two PCs are sufficient to completely separate all samples. The rugged design of the prototype opens new possibilities for further development of this technique towards real large-scale application.

Simultaneous Determination of Droplet Size, pH Value and Concentration to Evaluate the Aging Behavior of Metalworking Fluids.

Metalworking fluids (MWFs) are widely used to cool and lubricate metal workpieces during processing to reduce heat and friction. Extending a MWF's service life is of importance from both economical and ecological points of view. Knowledge about the effects of processing conditions on the aging behavior and reliable analytical procedures are required to properly characterize the aging phenomena. While so far no quantitative estimations of ageing effects on MWFs have been described in the literature other than univariate ones based on single parameter measurements, in the present study we present a simple spectroscopy-based set-up for the simultaneous monitoring of three quality parameters of MWF and a mathematical model relating them to the most influential process factors relevant during use. For this purpose, the effects of MWF concentration, pH and nitrite concentration on the droplet size during aging were investigated by means of a response surface modelling approach. Systematically varied model MWF fluids were characterized using simultaneous measurements of absorption coefficients µa and effective scattering coefficients µ's. Droplet size was determined via dynamic light scattering (DLS) measurements. Droplet size showed non-linear dependence on MWF concentration and pH, but the nitrite concentration had no significant effect. pH and MWF concentration showed a strong synergistic effect, which indicates that MWF aging is a rather complex process. The observed effects were similar for the DLS and the µ's values, which shows the comparability of the methodologies. The correlations of the methods were R2c = 0.928 and R2P = 0.927, as calculated by a partial least squares regression (PLS-R) model. Furthermore, using µa, it was possible to generate a predictive PLS-R model for MWF concentration (R2c = 0.890, R2P = 0.924). Simultaneous determination of the pH based on the µ's is possible with good accuracy (R²c = 0.803, R²P = 0.732). With prior knowledge of the MWF concentration using the µa-PLS-R model, the predictive capability of the µ's-PLS-R model for pH was refined (10 wt%: R²c = 0.998, R²p = 0.997). This highlights the relevance of the combined measurement of µa and µ's. Recognizing the synergistic nature of the effects of MWF concentration and pH on the droplet size is an important prerequisite for extending the service life of an MWF in the metalworking industry. The presented method can be applied as an in-process analytical tool that allows one to compensate for ageing effects during use of the MWF by taking appropriate corrective measures, such as pH correction or adjustment of concentration.

UV Hyperspectral Imaging as Process Analytical Tool for the Characterization of Oxide Layers and Copper States on Direct Bonded Copper.

Hyperspectral imaging and reflectance spectroscopy in the range from 200-380 nm were used to rapidly detect and characterize copper oxidation states and their layer thicknesses on direct bonded copper in a non-destructive way. Single-point UV reflectance spectroscopy, as a well-established method, was utilized to compare the quality of the hyperspectral imaging results. For the laterally resolved measurements of the copper surfaces an UV hyperspectral imaging setup based on a pushbroom imager was used. Six different types of direct bonded copper were studied. Each type had a different oxide layer thickness and was analyzed by depth profiling using X-ray photoelectron spectroscopy. In total, 28 samples were measured to develop multivariate models to characterize and predict the oxide layer thicknesses. The principal component analysis models (PCA) enabled a general differentiation between the sample types on the first two PCs with 100.0% and 96% explained variance for UV spectroscopy and hyperspectral imaging, respectively. Partial least squares regression (PLS-R) models showed reliable performance with R2c = 0.94 and 0.94 and RMSEC = 1.64 nm and 1.76 nm, respectively. The developed in-line prototype system combined with multivariate data modeling shows high potential for further development of this technique towards real large-scale processes.

Tunable strong coupling of two adjacent optical λ/2 Fabry-Pérot microresonators.

Strong optical mode coupling between two adjacent λ/2 Fabry-Pérot microresonators consisting of three parallel silver mirrors is investigated experimentally and theoretically as a function of their detuning and coupling strength. Mode coupling can be precisely controlled by tuning the mirror spacing of one resonator with respect to the other by piezoelectric actuators. Mode splitting, anti-crossing and asymmetric modal damping are observed and theoretically discussed for the symmetric and antisymmetric supermodes of the coupled system. The spectral profile of the supermodes is obtained from the Fourier transform of the numerically calculated time evolution of the individual resonator modes, taking into account their resonance frequencies, damping and coupling constants, and is in excellent agreement with the experiments. Our microresonator design has potential applications for energy transfer between spatially separated quantum systems in micro optoelectronics and for the emerging field of polaritonic chemistry.

Direct optical detection of cell density and viability of mammalian cells by means of UV/VIS spectroscopy.

The critical process parameters cell density and viability during mammalian cell cultivation are assessed by UV/VIS spectroscopy in combination with multivariate data analytical methods. This direct optical detection technique uses a commercial optical probe to acquire spectra in a label-free way without signal enhancement. For the cultivation, an inverse cultivation protocol is applied, which simulates the exponential growth phase by exponentially replacing cells and metabolites of a growing Chinese hamster ovary cell batch with fresh medium. For the simulation of the death phase, a batch of growing cells is progressively replaced by a batch with completely starved cells. Thus, the most important parts of an industrial batch cultivation are easily imitated. The cell viability was determined by the well-established method partial least squares regression (PLS). To further improve process knowledge, the viability has been determined from the spectra based on a multivariate curve resolution (MCR) model. With this approach, the progress of the cultivations can be continuously monitored solely based on an UV/VIS sensor. Thus, the monitoring of critical process parameters is possible inline within a mammalian cell cultivation process, especially the viable cell density. In addition, the beginning of cell death can be detected by this method which allows us to determine the cell viability with acceptable error. The combination of inline UV/VIS spectroscopy with multivariate curve resolution generates additional process knowledge complementary to PLS and is considered a suitable process analytical tool for monitoring industrial cultivation processes.

Hypericin: Single Molecule Spectroscopy of an Active Natural Drug.

Hypericin is one of the most efficient photosensitizers used in photodynamic tumor therapy (PDT). The reported treatments of this drug reach from antidepressive, antineoplastic, antitumor and antiviral activity. We show that hypericin can be optically detected down to a single molecule at ambient conditions. Hypericin can even be observed inside of a cancer cell, which implies that this drug can be directly used for advanced microscopy techniques (PALM, spt-PALM, or FLIM). Its photostability is large enough to obtain single molecule fluorescence, surface enhanced Raman spectra (SERS), fluorescence lifetime, antibunching, and blinking dynamics. Sudden spectral changes can be associated with a reorientation of the molecule on the particle surface. These properties of hypericin are very sensitive to the local environment. Comparison of DFT calculations with SERS spectra show that both the neutral and deprotonated form of hypericin can be observed on the single molecule and ensemble level.

Low-Cost GRIN-Lens-Based Nephelometric Turbidity Sensing in the Range of 0.1-1000 NTU.

Turbidity sensing is very common in the control of drinking water. Furthermore, turbidity measurements are applied in the chemical (e.g., process monitoring), pharmaceutical (e.g., drug discovery), and food industries (e.g., the filtration of wine and beer). The most common measurement technique is nephelometric turbidimetry. A nephelometer is a device for measuring the amount of scattered light of suspended particles in a liquid by using a light source and a light detector orientated in 90° to each other. Commercially available nephelometers cost usually-depending on the measurable range, reliability, and precision-thousands of euros. In contrast, our new developed GRIN-lens-based nephelometer, called GRINephy, combines low costs with excellent reproducibility and precision, even at very low turbidity levels, which is achieved by its ability to rotate the sample. Thereby, many cuvette positions can be measured, which results in a more precise average value for the turbidity calculated by an algorithm, which also eliminates errors caused by scratches and contaminations on the cuvettes. With our compact and cheap Arduino-based sensor, we are able to measure in the range of 0.1-1000 NTU and confirm the ISO 7027-1:2016 for low turbidity values.

Role of missing carotenoid in reducing the fluorescence of single monomeric photosystem II core complexes.

The fluorescence of monomeric photosystem II core complexes (mPSIIcc) of the cyanobacterium Thermosynechococcus elongatus, originating from redissolved crystals, is investigated by using single-molecule spectroscopy (SMS) at 1.6 K. The emission spectra of individual mPSIIcc are dominated by sharp zero-phonon lines, showing the existence of different emitters compatible with the F685, F689, and F695 bands reported formerly. The intensity of F695 is reduced in single mPSIIcc as compared to single PSIIcc-dimers (dPSIIcc). Crystal structures show that one of the ß-carotene (ß-Car) cofactors located at the monomer-monomer interface in dPSIIcc is missing in mPSIIcc. This ß-Car in dPSIIcc is in van der Waals distance to chlorophyll (Chl) 17 in the CP47 subunit. We suggest that this Chl contributes to the F695 emitter. A loss of ß-Car cofactors in mPSIIcc preparations will lead to an increased lifetime of the triplet state of Chl 17, which can explain the reduced singlet emission of F695 as observed in SMS.

Resolution enhancement for low-temperature scanning microscopy by cryo-immersion.

Here we report a simple way to enhance the resolution of a confocal scanning microscope under cryogenic conditions. Using a microscope objective (MO) with high numerical aperture (NA = 1.25) and 1-propanol as an immersion fluid with low freezing temperature we were able to reach an imaging resolution at 160 K comparable to ambient conditions. The MO and the sample were both placed inside the inner chamber of the cryostat to reduce distortions induced by temperature gradients. The image quality of our commercially available MO was further enhanced by scanning the sample (sample scanning) in contrast to beam scanning. The ease of the whole procedure marks an essential step towards the development of cryo high-resolution microscopy and correlative light and electron cryo microscopy (cryoCLEM).

Strong and Coherent Coupling of a Plasmonic Nanoparticle to a Subwavelength Fabry-Pérot Resonator.

A major aim in experimental nano- and quantum optics is observing and controlling the interaction between light and matter on a microscopic scale. Coupling molecules or atoms to optical microresonators is a prominent method to alter their optical properties such as luminescence spectra or lifetimes. Until today strong coupling of optical resonators to such objects has only been observed with atom-like systems in high quality resonators. We demonstrate first experiments revealing strong coupling between individual plasmonic gold nanorods (GNR) and a tunable low quality resonator by observing cavity-length-dependent nonlinear dephasing and spectral shifts indicating spectral anticrossing of the luminescent coupled system. These phenomena and experimental results can be described by a model of two coupled oscillators representing the plasmon resonance of the GNR and the optical fields of the resonator. The presented reproducible and accurately tunable resonator allows us to precisely control the optical properties of individual particles.

Effect of TMAO and betaine on the energy landscape of photosystem I.

The accumulation of organic co-solvents in cells is a basic strategy for organisms from various species to increase stress tolerance in extreme environments. Widespread representatives of this class of co-solvents are trimethylamine-N-oxide (TMAO) and betaine; these small molecules are able to stabilize the native conformation of proteins and prevent their aggregation. Despite their importance, detailed experimental studies on the impact of these co-solvents on the energy landscape of proteins have not yet been carried out. We use single-molecule spectroscopy at cryogenic temperatures to examine the influence of these physiological relevant co-solvents on photosystem I (PSI) from Thermosynechococcus elongatus. In contrast to PSI ensemble spectra, which are almost unaffected by the addition of TMAO and betaine, statistical analysis of the fluorescence emission from individual PSI trimers yields insight into the interaction of the co-solvents with PSI. The results show an increased homogeneity upon addition of TMAO or betaine. The number of detectable zero-phonon lines (ZPLs) is reduced, indicating spectral diffusion processes with faster rates. In the framework of energy landscape model these findings indicate that co-solvents lead to reduced barrier heights between energy valleys, and thus efficient screening of protein conformations can take place.

Spectroscopic properties of photosystem II core complexes from Thermosynechococcus elongatus revealed by single-molecule experiments.

In this study we use a combination of absorption, fluorescence and low temperature single-molecule spectroscopy to elucidate the spectral properties, heterogeneities and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence emission of photosystem II core complexes (PS II cc) from the cyanobacterium Thermosynechococcus elongatus. At the ensemble level, the absorption and fluorescence spectra show a temperature dependence similar to plant PS II. We report emission spectra of single PS II cc for the first time; the spectra are dominated by zero-phonon lines (ZPLs) in the range between 680 and 705nm. The single-molecule experiments show unambiguously that different emitters and not only the lowest energy trap contribute to the low temperature emission spectrum. The average emission spectrum obtained from more than hundred single complexes shows three main contributions that are in good agreement with the reported bands F685, F689 and F695. The intensity of F695 is found to be lower than in conventional ensemble spectroscopy. The reason for the deviation might be due to the accumulation of triplet states on the red-most chlorophylls (e.g. Chl29 in CP47) or on carotenoids close to these long-wavelength traps by the high excitation power used in the single-molecule experiments. The red-most emitter will not contribute to the fluorescence spectrum as long as it is in the triplet state. In addition, quenching of fluorescence by the triplet state may lead to a decrease of long-wavelength emission.

Enhanced single-molecule spectroscopy in highly confined optical fields: from λ/2-Fabry-Pérot resonators to plasmonic nano-antennas.

While single-molecule fluorescence from emitters with high quantum efficiencies such as organic dye molecules can easily be detected by modern apparatus, many less efficient emission processes such as Raman scattering and metal luminescence require dramatic enhancement to exceed the single-particle detection limit. This enhancement can be achieved using resonant optical systems such as plasmonic particles or nanoantennas, the study of which has led to substantial progress in understanding the interaction of quantum emitters with their electromagnetic environment. This review is focused on the advances in measurement techniques and potential applications enabled by a deeper understanding of fundamental optical interaction processes occurring between single quantum systems on the nanoscale. While the affected phenomena are numerous, including molecular fluorescence and also exciton luminescence and Raman scattering, the interaction itself can often be described from a unified point of view. Starting from a single underlying model, this work elucidates the dramatic enhancement potential of plasmonic tips and nanoparticles and also the more deterministic influence of a Fabry-Pérot microresonator. With the extensive knowledge of the radiative behavior of a quantum system, insight can be gained into nonradiative factors as well, such as energy transfer phenomena or spatial and chemical configurations in single molecules.