Fluorescence Imaging Measurements Research Articles

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.

Tin-based perovskite films fabricated by chemical vapor deposition for photodetector application

Tin-based perovskite materials have shown great potential applications in environment-friendly optoelectronic devices. However, it is difficult to control the film growth process by solution method due to the rapid formation speed of the film. In this work, tin-based perovskite (FASnI3) films were prepared by chemical vapor deposition method. The film is mainly composed of thin nanoplates when the deposition temperature is 600 °C. The film shows good antioxidant properties by comparing the ratio between Sn4+ and Sn2+. Time-resolved fluorescence imaging measurements clearly reveal the fluorescence lifetime inhomogeneity in the film. The photodetectors based on tin-based perovskite films show good photo response properties, with a switching ratio of 808. This work provides new opportunities for non-lead perovskite thin film preparation and optoelectronic application.

Improved intraoperative identification of close margins in oral squamous cell carcinoma resections using a dual aperture fluorescence ratio approach: first in-human results.

Surgical excision is the main treatment for solid tumors in oral squamous cell carcinomas, where wide local excision (achieving a healthy tissue margin of around the excised tumor) is the goal as it results in reduced local recurrence rates and improved overall survival. No clinical methods are available to assess the complete surgical margin intraoperatively while the patient is still on the operating table; and while recent intraoperative back-bench fluorescence-guided surgery approaches have shown promise for detecting "positive" inadequate margins (), they have had limited success in the detection of "close" inadequate margins (1 to 5mm). Here, a dual aperture fluorescence ratio (dAFR) approach was evaluated as a means of improving detection of close margins. The approach was evaluated on surgical specimens from patients who were administered a tumor-specific fluorescent imaging agent (cetuximab-800CW) prior to surgery. The dAFR approach was compared directly against standard wide-field fluorescence imaging and pathology measurements of margin thickness in specimens from three patients and a total of 12 margin locations (1 positive, 5 close, and 6 clear margins). The area under the receiver operating characteristic curve, representing the ability to detect close compared to clear margins () was found to be 1.0 and 0.57 for dAFR and sAF, respectively. Improvements in dAFR were found to be statistically significant (). These results provide evidence that the dAFR approach potentially improves detection of close surgical margins.

Study of the photobleaching phenomenon to optimize acquisition of 3D and 4D fluorescence images. A special scenario for trilinear and quadrilinear models

Emission (3D) and excitation-emission (4D) fluorescence images allow covering wide excitation and emission spectral ranges and, hence, provide very complete information for a good characterization and location of fluorophores in samples. However, when the acquisition time of the image is too long, degradation of the fluorescence signal of compounds and sample photodamage can occur due to photobleaching. This phenomenon is due to the long exposure time of the sample to the light source and can hinder the detection and the proper characterization of the fluorophores in samples.The main purpose of this research is providing a methodology to obtain and interpret the information of fluorescence images for the characterization of samples without suffering the consequences of photobleaching. Such a goal implies a first thorough knowledge of the photobleaching phenomenon to adapt the fluorescence imaging measurement for an optimal characterization of the fluorophores present in samples.The proposed approach relies first on a study of time-series of 3D or 4D fluorescence images to characterize spatially and spectroscopically the fluorophores present in the samples and their photobleaching behaviour. Since photobleaching is fluorophore-dependent, the unmixing algorithm Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) is applied to the set of fluorescence images acquired as a function of time to understand the specific behaviour of every fluorophore. The characteristics of the photobleaching phenomenon and the nature of the fluorescence measurement offer a challenging scenario to look for adapted implementations of trilinear and quadrilinear models within the MCR framework. From the results obtained, appropriate instrumental settings are adopted for an image acquisition that allows the correct spatial and spectroscopic characterization of fluorophores in samples.To test the potential of this methodology, the characterization of thin cross-sections of the Oryza sativa (commonly called rice) root have been studied due to the co-occurrence of several natural fluorophores in vegetal tissues.

A SERS/fluorescence dual-mode immuno-nanoprobe for investigating two anti-diabetic drugs on EGFR expressions.

A surface-enhanced Raman scattering (SERS)/fluorescence dual-mode nanoprobe was proposed to assess anti-diabetic drug actions from the expression level of the epidermal growth factor receptor (EGFR), which is a significant biomarker of breast cancers. The nanoprobe has a raspberry shape, prepared by coating a dye-doped silica nanosphere with a mass of SERS tags, which gives high gains in fluorescence imaging and SERS measurement. The in situ detection of EGFR on the cell membrane surfaces after drug actions was achieved by using this nanoprobe, and the detection results agree with the enzyme-linked immunosorbent assay (ELISA) kit. Our study suggests that rosiglitazone hydrochloride (RH) may be a potential drug for diabetic patients with breast cancer, while the anti-cancer effect of metformin hydrochloride (MH) is debatable since MH slightly promotes the EGFR expression of MCF-7 cells in this study. This sensing platform endows more feasibility for highly sensitive and accurate feedback of pesticide effects at the membrane protein level.

Feedback-based wavefront shaping for weak light with lock-in beat frequency detection.

Feedback-based wavefront shaping is a promising and versatile technique for enhancing the contrast of a target signal through highly scattering media. However, this technique can fail for low optical signals such as fluorescence and Raman signals or in a reflection setup because the trend in weak feedback signals can be easily overwhelmed by noise. To address this challenge, we develop a technique based on a single acousto-optic deflector (AOD) to create a signal with a selected beat frequency from optical signals that can serve as feedback, in which the phase distribution of various radio frequency components of the driving signal for the AOD is optimized for wavefront shaping. By shifting incident light frequency with the AOD, the feedback signal at a selected beat frequency can be measured with a high signal-to-noise ratio (SNR) by a lock-in amplifier, thus enabling the enhancement of weak target signals through highly scattering media. It is found that the method of lock-in beat frequency detection can significantly improve fluorescence imaging and Raman spectral measurements in a reflection setup, and thus could be potentially used for in vivo measurements.

Pluronic stabilized conjugated polymer nanoparticles for NIR fluorescence imaging and dual phototherapy applications

• Pluronic stabilized conjugated polymer nanoparticles (CP-NP-Plu) are prepared. • CP-NP-Plu present photothermal conversion efficiency of 61% under NIR irradiation. • 2-fold increase of singlet oxygen generation quantum yield due to encapsulation with Plu • NIR fluorescence imaging ability validated at single nanoparticle level. Besides their intensive application as solar photovoltaic materials, low bandgap conjugated polymers have been recently exploited in cell imaging and phototherapeutic applications due to their photoactivity in the near-infrared (NIR) spectral domain, also known as the “biologically transparent window”. Herein, Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b′]dithiophene)-alt-4,7(2,1,3-benzothiadiazole) (PCPDTBT) conjugated polymer was used to prepare novel NIR activatable theranostic nanoparticles, via nanoprecipitation method, for fluorescence imaging and dual photothermal-photodynamic therapy (PTT-PDT) applications. To overcome the drawback of hydrophobicity of PCPDTBT, the approach of encapsulating the obtained nanoparticles with a layer of Pluronic F127 amphiphilic block co-polymer was herein applied. The Pluronic layer not only provides excellent colloidal stability, but also ensures enhanced fluorescence emission, improved photothermal heating and singlet oxygen generation ability of the nanoparticles in aqueous dispersion. Specifically, upon irradiation with a NIR laser at 785 nm, the obtained nanoparticles exhibit good thermal response and excellent photostability leading to a photothermal conversion efficiency of 61%. Moreover, a near two-fold increase in the singlet oxygen generation quantum yield due to encapsulation with Pluronic is also detected. Furthermore, fluorescence imaging measurements on single nanoparticle immobilized on glass substrate reveal their potential as fluorescence contrast agents. The enhanced fluorescence and improved therapeutic properties of our nanoparticles suggest the possibility to be employed as NIR activatable theranostic agents via dual phototherapy combined with high-resolution fluorescence tracking.

Internal and external hydrodynamics of Taylor cone under constant and alternating voltage actuation

Electrohydrodynamic atomization (EHDA) is carried out in the Taylor cone mode for generating unimodal particle distribution, which can be achieved by either constant voltage actuation (CVA) or alternating voltage actuation (AVA). The present study reports an experimental investigation of the flow field both inside and outside the Taylor cone using light sheet fluorescence imaging and time-resolved particle image velocimetry measurements. Liquid ethanol is used as the working fluid and the amplitude of both constant and alternating electric potential difference is set at the same value, i.e., VC=VA=5.2 kV with an actuation frequency of 200 Hz in the case of alternating EHDA. The hydrodynamic behavior both inside and outside the Taylor cone is presented for the first time. The flow field measurements demonstrate meridional circulation from the nozzle exit toward the apex of the Taylor cone along the generatrix followed by flow from the apex of the Taylor cone along the central axis. A symmetric toroidal vortex is observed inside the Taylor cone in the case of CVA and an asymmetric toroidal vortex is observed for AVA. The flow field shows streamline-like flow in the ambient medium from the nozzle toward the ground electrode along the interface of the Taylor cone jet for CVA. In contrast, two vortical structures are observed around the apex of the Taylor cone for AVA. The velocity profile near the liquid–air interface of the Taylor cone indicates no direct correlation between the flow field inside the Taylor cone with the flow of the ambient medium. This difference may be attributed to the corona wind generated due to asymmetric electrode configuration. The unsteady flow field generated by alternating EHDA has great potential for enhanced heat transfer using spray cooling.

Enhanced Displacement of Phase Separating Liquid Mixtures in 2D Confined Spaces

Displacing liquids in a confined space is important for technological processes ranging from porous membrane separation to CO2 sequestration. The liquid to be displaced usually consists of multiple components with different solubilities in the displacing liquid. Phase separation and chemical composition gradients in the liquids can influence the displacement rate. In this work, we investigate the effects of liquid composition on the displacement process of ternary liquid mixtures in a quasi-2D microchannel where liquid–liquid phase separation occurs concurrently. We focused on model ternary mixtures containing 1-octanol (a model oil), ethanol (a good solvent), and water (a poor solvent). These mixtures are displaced with water or with an ethanol aqueous solution. As a comparison, for some experiments, water was displaced by ternary mixtures. The bright-field and fluorescence imaging measurements reveal distinct phase separation behaviors. The spatial distribution of subphases arising from phase separation and the displacement rates of the solution are impacted by the initial ternary solution composition. The boundary between the solution and displacing liquid changes from a defined interface to a diffusive interface as the initial 1-octanol composition in the solution is reduced. The displacement rate also varies non-linearly with the initial 1-octanol composition. The slowest displacement rate arises at intermediate 1-octanol concentration, where a stable three-zone configuration forms at the boundary. At very low 1-octanol concentration, the displacement rate is fast, associated with droplet formation and motion driven by the chemical concentration gradients formed during phase separation. The excess energy provided from phase separation may contribute to the enhanced displacement at intermediate to high 1-octanol concentrations but not at low 1-octanol concentration with enhancement from induced flow in confinement. The knowledge gained from this study highlights the importance of manipulating phase separation to enhance mass transport in confinement for a wide range of separation processes.

One-step synthesis of red emission multifunctional carbon dots for label-free detection of berberine and curcumin and cell imaging

In this work, the red emission multifunctional carbon dots (R-CDs) were prepared via one-pot hydrothermal strategy of neutral red (NR) and ethylenediamine (EDA) for the label-free detection of berberine and curcumin, cell imaging, and fluorescent flexible film. The as-fabricated R-CDs not only possess good water dispersibility and excellent fluorescence stability, but also were successfully employed as a photoluminescent nanoprobe for label-free monitoring of berberine (BRH) and curcumin (Cur) based on dynamic quenching and internal filter effect (IFE), respectively. More importantly, as-proposed R-CDs displayed outstanding cellular permeability and lower cytotoxicity for cellular applications, which was consistent with the results of confocal fluorescence imaging and cell viability measurement of SMMC7721 cells. Thus, the multifunctional R-CDs may provide a rich tool library for biosensing and cellular imaging reagent applications. Interestingly, R-CDs were also used to manufacture R-CDs/PVA composites as fluorescent flexible films. To the best of our knowledge, this is the first demonstration of a label-free multifunctional fluorescent nanoprobe for berberine and curcumin based on red emission CDs.

Fuel placement and atomization inside a gas-turbine fuel injector at realistic operating conditions

The atomization process and fuel placement inside a gas turbine fuel injector are investigated at realistic operating conditions to understand the role of individual flow processes and hardware components. A high pressure, high temperature spray test facility is developed that can simulate a wide range of aero-engine operating conditions. The fuel injector is a swirl cup - dual orifice nozzle combination which is used in the rich dome combustors of various aero-engines. The internal flow field is captured using time-resolved laser induced fluorescence imaging and phase Doppler interferometry measurements. A stage-wise characterization is adopted to track the spray formation as the flow evolves through the swirl cup. The fuel placement at the cup exit is mainly decided by pilot nozzle spray characteristics and the relative momentum exchange between the gas and liquid phases. At the swirl cup exit, two class of droplets are formed: (1) droplets originated directly from the pilot nozzle, which are convected downstream by the primary air swirl, (2) droplets originated from the rim, which are atomized by the shear layer. The relative percentage of these two droplet classes depends on the pilot nozzle operation at low and high power conditions. The effect of primary spray and wall-filming are minimal towards improving the atomization. In both low and high power operating cases, the counter-rotating shear layer interactions are identified as the major mechanism that is improving the atomization process in swirl cup.

Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers.

Annexins are soluble membrane-binding proteins that associate in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms crystalline networks that seal or stabilize small membrane tears. Herein, we model this biochemical mechanism to simulate membrane healing at microcavity array supported, transversally asymmetric, lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). Varying annexin V concentration, lipid composition, and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochemical impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was observed over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochemical impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.

Smartphone based on-chip fluorescence imaging and capillary flow velocity measurement for detecting ROR1+ cancer cells from buffy coat blood samples on dual-layer paper microfluidic chip

Diagnosis of hematological cancer requires complete white blood cell count, followed by flow cytometry with multiple markers, and cytology. It requires substantial time and specialized training. A dual-layer paper microfluidic chip was developed as a quicker, low-cost, and field-deployable alternative to detect ROR1+ (receptor tyrosine-like orphan receptor one) cancer cells from the undiluted and untreated buffy coat blood samples. The first capture layer consisted of a GF/D glass fiber substrate, preloaded with cancer specific anti-ROR1 conjugated fluorescent particles to its center for cancer cell capture and direct smartphone fluorescence imaging. The second flow layer was comprised of a grade 1 cellulose chromatography paper with wax-printed four channels for wicking and capillary flow-based detection. The flow velocity was used as measure of antigen concentration in the buffy coat sample. In this manner, intact cells and their antigens were separated and independently analyzed by both imaging and flow velocity analyses. A custom-made smartphone-based fluorescence microscope and automated image processing and particle counter software were developed to enumerate particles on paper, with the limit of detection of 1 cell/μL. Flow velocity analysis showed even greater sensitivity, with the limit of detection of 0.1 cells/μL in the first 6 s of assay. Comparison with capillary flow model revealed great alignment with experimental data and greater correlation to viscosity than interfacial tension. Our proposed device is able to capture and on-chip image ROR1+ cancer cells within a complex sample matrix (buffy coat) while simultaneously quantifying cell concentration in a point-of-care manner.

Remarkable Boron Delivery Of iRGD-Modified Polymeric Nanoparticles For Boron Neutron Capture Therapy.

PurposeBoron neutron capture therapy (BNCT) is an emerging binary radiotherapy, which is limited for application due to the challenge of targeted delivery into tumor nowadays. Here, we propose the use of iRGD-modified polymeric nanoparticles for active targeted delivery of boron and doxorubicin (DOX) in BNCT.Methods10B-enriched BSH was covalently grafted to PEG-PCCL to prepare 10B-polymer, then surface-modified with iRGD. And, DOX was physically incorporated into polymers afterwards. Characterization of prepared polymers and in vitro release profile of DOX from polymers were determined by several methods. Cellular uptake of DOX was observed by confocal microscope. Accumulation of boron in cells and tissues was analyzed by ICP-MS. Biodistribution of DOX was studied by ex vivo fluorescence imaging and quantitative measurement. Tumor vascular normalization of Endostar for promoting delivery efficiency of boron on refractory B16F10 tumor was also studied.ResultsThe polymers were monodisperse and spheroidal in water with an average diameter of 24.97 nm, which were relatively stable at physiological pH and showed a sustained release of DOX, especially at endolysosomal pH. Enhanced cellular delivery of DOX was found in iRGD-modified polymer group. Cellular boron uptake of iRGD-modified polymers in A549 cells was remarkably raised fivefold (209.83 ng 10B/106 cells) compared with BSH. The polymers represented prolonged blood circulation, enhanced tumor accumulation of 10B against BSH, and favorable tumor:normal tissue boron concentration ratios (tumor:blood = 14.11, tumor:muscle = 19.49) in A549 tumor-bearing mice 24 hrs after injection. Both fluorescence imaging and quantitative measurement showed the highest tumor accumulation of DOX at 24 hrs after injecting of iRGD-modified polymers. Improvement of vascular integrity and reduction of vascular mimicries were found after Endostar injection, and raised tumor accumulation of boron as well.ConclusionThe developed nanoparticle is an inspiring candidate for the safe clinical application for BNCT.

Black Lipid Membranes: Challenges in Simultaneous Quantitative Characterization by Electrophysiology and Fluorescence Microscopy

Horizontal black lipid membranes (BLMs) enable optical microscopy to be combined with the electrophysiological measurements for studying ion channels, peptide pores, and ionophores. However, a careful literature review reveals that simultaneous fluorescence and electrical recordings in horizontal BLMs have been rarely reported for an unclear reason, whereas many works employ bright-field microscopy instead of fluorescence microscopy or perform fluorescence imaging and electrical measurements one after another separately without truly exploiting the advantage of the combined setup. In this work, the major causes related to the simultaneous electrical and fluorescence recordings in horizontal BLMs are identified, and several solutions to counteract the issue are also proposed.

Simultaneous high-speed imaging of temperature, heat-release rate, and multi-species concentrations in turbulent jet flames.

We report the high-speed imaging of multi-species and multi-parameter combustion diagnostics for turbulent non-premixed jet flames using a three-legged burst-mode laser system. Simultaneous OH/CH2O planar laser-induced fluorescence and Rayleigh-scattering imaging measurements at a 10-kHz rate are obtained. OH and CH2O concentrations, flame temperatures, and heat-release rates are simultaneously acquired in two-dimensions at 10 kHz.

Two-dimensional μXAFS analysis for accumulated uranium in kidneys of rats exposed to uranyl acetate

Kidneys are critical targets of uranium exposure, meaning that effective analytical methods for characterizing uranium accumulated in kidneys are required in toxicological studies of uranium. In this work, synchrotron radiation micro-X-ray fluorescence (SR-μXRF) imaging and two-dimensional μXAFS measurements were combined to analyze renal section specimens. By histochemical staining of the serial sections, the distribution of uranium chemical condition within the uranium-concentrated areas of the proximal tubules, which are toxic target sites of uranium, were obtained with 1 µm × 1 µm spatial resolution.

Jiedu Sangen Decoction Reverses Epithelial-to-mesenchymal Transition and Inhibits Invasion and Metastasis of Colon Cancer via AKT/GSK-3β Signaling Pathway.

Ethnopharmacology relevance: Jiedu Sangen Decoction (JSD), an empirical prescription of Traditional Chinese Medicine (TCM), has been reported to inhibit invasion and metastasis of colon cancer in our previous study. The aim of this study was to investigate the mechanism of JSD-triggered inhibition of invasion and metastasis in colon cancer.Methods: In vitro, AKT1 knockdown (si-AKT1) or overexpression (oe-AKT1) cells were successfully constructed both in SW480 and SW620 cell lines. Si-AKT1 and oe-AKT1 cells were then treated with or without JSD. Cell invasion, metastasis potential and expression of epithelial-mesenchymal transformation (EMT)-related and AKT1/GSK-3β proteins were then observed by wound healing, transwell, and western blot assays. In vivo, liver metastasis model mice were developed by inoculating SW480 cells. After JSD diet intervention, living fluorescence imaging and weight measurements were carried out to investigate JSD induced inhibition effects on liver metastasis of colon cancer. Immunohistochemistry and western blot assays were performed to observe tissue features and detect protein expression.Results: Invasion and metastasis potential, as well as EMT of colon cancer, can be markedly inhibited by JSD treatment or AKT1 knockdown, while enhanced by AKT1 overexpression. JSD-induced inhibition effects were significantly weakened when AKT1 was knocked down, while clearly enhanced when AKT1 was overexpressed. Additionally, JSD could lead to an increase in expression of E-cadherin, and a decrease in expression of N-cadherin, Vimentin, p-AKT1, AKT1, p- GSK-3β, Snail, Slug, and Twist in colon cancer cells.Conclusion: JSD reverses EMT and inhibits invasion and metastasis of colon cancer through the AKT/GSK-3β signaling pathway.

Climate specific accelerated ageing tests and evaluation of ageing induced electrical, physical, and chemical changes

AbstractAs the PV market shows enormous potential with huge growth rates especially in climatic‐sensible regions, specific artificial ageing test procedures are a key point for an efficient and fast product development of new PV modules/materials optimized for the use in specific climatic regions. Based on the definition of four climate profiles (dry and hot—arid, moderate, humid, and hot—tropical and high irradiation—alpine), a program was worked out with 14 climate specific test conditions for accelerated ageing tests. The big challenge in this respect was the adaption/advancement of existing standard procedures for PV modules/components testing in a way that reliable testing for certain climatic conditions optimized PV modules is possible. The time‐dependent repeated application of combined climatic and environmental stresses (temperature, temperature cycles, humidity, irradiation, mechanical load, salt mist) was used to induce performance losses, material degradation, and failures in test modules which resemble those effects occurring in real‐life PV installations under comparable climatic and environmental conditions. For this demanding task, a large number of identical test modules with respect to composition and module design was manufactured. A detailed nondestructive analysis/characterization of all modules was performed: (1) before; (2) during (six intermediate stages); and (3) after the accelerated ageing test. The nondestructive characterization methods used to follow the module's ageing processes throughout the whole accelerated ageing procedure were current‐voltage characteristics measurements and electroluminiscence images for the electrical performance evaluation and ultraviolet fluorescence (spectroscopic and imaging) measurements, Fourier transform infrared spectroscopy as well as colour measurements of the backsheets outer layer for recording of chemical changes of the encapsulant and backsheet. The electrical and material characterization data were incorporated in an optimized database. As stated above, a set of three identical modules was exposed together in the respective climate specific ageing tests and subsequently characterized in order to increase statistical reliability of the measuring results. The analysis of the data and first approaches of advanced data treatment have already clearly shown that the electrical and material degradation of the test modules is dependent on (1) the type and combination, (2) duration, and (3) mode (sequential versus constant) of the stresses applied. On the one hand, the simulation of environmental stresses like heavy snow and wind load and enhanced frequency of temperature cycling resulting in cell cracks and cell connector breakage could be demonstrated. Additional treatment in salty atmosphere, on the other hand, did not show an accelerating effect on degradation on the electrical or material side. The accelerating effect of enhanced temperature, humidity, or additional irradiation on the degradation of power and materials could be shown very well. Direct evidence for the formation of acetic acid and lead‐acetate in the encapsulant ethylene vinyl acetate after prolonged damp‐heat exposure could be found and directly related to cell corrosion effects paralleled by power losses.

Mode-Selective Raman Imaging of Dopamine-Human Dopamine Transporter Interaction in Live Cells.

Dopamine (DA) is the catecholamine neurotransmitter which interacts with dopamine receptors (DARs) to generate dopaminergic signals in the nervous system. Dopamine transporter (DAT) interacts with DA to maintain DA's homeostasis in synaptic and perisynaptic space. DAT and DARs have great importance in the central nervous system (CNS) because they are associated with the targeted binding of drugs. Interactions of DA, its analogue with DARs, or DAT have been studied extensively to understand the mechanism of the dopaminergic signaling process and several neurodegenerative diseases, including schizophrenia, Parkinson's diseases, addiction, attention deficit hyperactivity disorder, and bipolar disorder. However, there is still a lack of a risk-free, label-free, and minimally invasive imaging approach to probe the interaction between DA and DAT or DARs. Here, we probed the DA, human dopamine transporter (hDAT), and DA-hDAT interactions in live cells using combined approach of two-photon excited (2PE) fluorescence imaging and mode-selective Raman measurement. We utilized the signature Raman peak at 1287 cm-1 to probe the location of DA and 807 and 1076 cm-1 to probe the DA-hDAT interaction in live cells. We found that the combined approach of mode-selective Raman imaging, 2PE fluorescence imaging, and computational methods is successful to probe and confirm the DA-hDAT interactions in living cells. The probing of the interactions of DARs or DAT with DA or other targeting drugs is crucial for the diagnosis and cure of several neurodegenerative diseases. Also, this analytical approach could be extended to probe other types of protein-ligand interactions.