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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.
Film formation of self synthesized Polymer EPM–g–VTMDS (ethylene–propylene rubber, EPM, grafted with vinyltetramethyldisiloxane, VTMDS) was studied regarding bonding to adhesion promoter vinyltrimethoxysilane (VTMS) on oxidized 18/10 chromium/nickel–steel (V2A) stainless steel surfaces. Polymer films of different mixed solutions including commercial siloxane and silicone, dimethyl, vinyl group terminated crosslinker (HANSA SFA 42100, CAS# 68083-19-2, 0.35 mmol Vinyl/g) and platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complex Karstedt's catalyst (ALPA–KAT 1, CAS# 68478-92-2) were spin coated on V2A stainless steel surfaces with adsorbed VTMS thin layers in order to analyze film formation of EPM–g–VTMDS at early stages. Surface topography and chemical bonding of the high performance polymers on different oxidized V2A surfaces were investigated with X–ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and surface enhanced Raman spectroscopy (SERS). AFM and SEM as well as XPS results indicated that the formation of the polymer film proceeds via growth of polymer islands. Chemical signatures of the essential polymer contributions, linker and polymer backbones, could be identified using XPS core level peak shape analysis and also SERS. The appearance of signals which are related to Si–O–Si can be seen as a clear indication of lateral crosslinking and silica network formation in the films on the V2A surface.
We study three-color Förster resonance energy transfer (triple FRET) between three spectrally distinct fluorescent dyes, a donor and two acceptors, which are embedded in a single polystyrene nanosphere. The presence of triple FRET energy transfer is confirmed by selective acceptor photobleaching. We show that the fluorescence lifetimes of the three dyes are selectively controlled using the Purcell effect by modulating the radiative rates and relative fluorescence intensities when the nanospheres are embedded in an optical Fabry–Pérot microcavity. The strongest fluorescence intensity enhancement for the second acceptor can be observed as a signature of the FRET process by tuning the microcavity mode to suppress the intermediate dye emission and transfer more energy from donor to the second acceptor. Additionally, we show that the triple FRET process can be modeled by coupled rate equations, which allow to estimate the energy transfer rates between donor and acceptors. This fundamental study has the potential to extend the classical FRET approach for investigating complex systems, e.g., optical energy switching, photovoltaic devices, light-harvesting systems, or in general interactions between more than two constituents.
Direct observation of structural heterogeneity and tautomerization of single hypericin molecules
(2021)
Tautomerization is a fundamental chemical reaction which involves the relocation of a proton in the reactants. Studying the optical properties of tautomeric species is challenging because of ensemble averaging. Many molecules, such as porphines, porphycenes, or phenanthroperylene quinones, exhibit a reorientation of the transition dipole moment (TDM) during tautomerization, which can be directly observed in single-molecule experiments. Here, we study single hypericin molecules, which is a prominent phenanthroperylene quinone showing antiviral, antidepressive, and photodynamical properties. Observing abrupt flipping of the image pattern combined with time-dependent density functional theory calculations allows drawing conclusions about the coexistence of four tautomers and their conversion path. This approach allows the unambiguous assignment of a TDM orientation to a specific tautomer and enables the determination of the chemical structure in situ. Our approach can be applied to other molecules showing TDM reorientation during tautomerization, helping to gain a deeper understanding of this important process.
Scanning Near-Field Optical Microscopy (SNOM) has developed during recent decades into a valuable tool to optically image the surface topology of materials with super-resolution. With aperture-based SNOM systems, the resolution scales with the size of the aperture, but also limits the sensitivity of the detection and thus the application for spectroscopic techniques like Raman SNOM. In this paper we report the extension of solid immersion lens (SIL) technology to Raman SNOM. The hemispherical SIL with a tip on the bottom acts as an apertureless dielectric nanoprobe for simultaneously acquiring topographic and spectroscopic information. The SIL is placed between the sample and the microscope objective of a confocal Raman microscope. The lateral resolution in the Raman mode is validated with a cross section of a semiconductor layer system and, at approximately 180 nm, is beyond the classical diffraction limit of Abbe.
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.
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.
Monitoring tautomerization of single hypericin molecules in a tunable optical λ/2 microcavity
(2022)
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.
One-pot synthesis of micron partly hollow anisotropic dumbbell shaped silica core-shell particles
(2016)
A facile method is described to prepare micron partly hollow dumbbell silica particles in a single step. The obtained particles consist of a large dense part and a small hollow lobe. The spherical dense core as well as the hollow lobe are covered by mesoporous channels. In the case of a smaller lobe these channels are responsible for the permeability of the shell which was demonstrated by confocal imaging and spectroscopy.
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).