Finite control set model predictive current control for three phase grid connected inverter with common mode voltage suppression.

This research introduces an advanced finite control set model predictive current control (FCS-MPCC) specifically tailored for three-phase grid-connected inverters, with a primary focus on the suppression of common mode voltage (CMV). CMV is known for causing a range of issues, including leakage currents, electromagnetic interference (EMI), and accelerated system degradation. The proposed control strategy employs a system model that predicts the inverter's future states, enabling the selection of optimal switching states from a finite set to achieve dual objectives: precise current control and effective CMV reduction, a meticulously designed cost function evaluates the potential switching states, balancing the accuracy of current tracking against the necessity to minimize CMV. The approach is grounded in a comprehensive mathematical model that captures the dynamics of CMV within the system, and it utilizes an optimization process that functions in real-time to determine the most suitable control action at each interval, Experimental validations of the proposed FCS-MPCC scheme have demonstrated its effectiveness in significantly improving the performance and durability of three-phase grid-connected inverters, Experimental validations of the proposed (MPC with CMV) scheme have demonstrated its effectiveness in significantly improving the performance and durability of three-phase grid-connected inverters. The proposed method achieved substantial reductions in CMV, notable improvements in current tracking accuracy, and extended system lifespan compared to conventional control methods.

Correlative Effects on Nanoplastic Aggregation in Model Extracellular Biofilm Substances Investigated with Fluorescence Correlation Spectroscopy.

Recent studies show that biofilm substances in contact with nanoplastics play an important role in the aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in the transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics-biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as, e.g., the acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with an ionic strength being realistic for fresh-water conditions. Biomolecule concentrations ranging from 0.5 g/L up to at maximum 21 g/L are considered. We use non-functionalized PS NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA-mediated bridging. In BSA-alginate mixtures, the alginate hampers this interaction, most likely due to alginate-BSA complex formation. In most BSA-alginate mixtures as in alginate alone, NP aggregation is predominantly driven by weaker, pH-independent depletion forces. The stabilizing effect of alginate is only weakened at high BSA contents, when the electrostatic BSA-BSA attraction is not sufficiently screened by the alginate. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single-component biofilm model systems based on comparing the total organic carbon (TOC) content of the extracellular biofilm substances, as usually considered, would have led to a misjudgment of the stability towards aggregation.

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope.

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Two successful pregnancies -in patients taking Volanesorsen for familial chylomicronemia syndrome.

Familial chylomicronemia syndrome (FCS) is a rare inherited disorder characterized by severe hypertriglyceridemia, posing a heightened risk of acute pancreatitis. Recently, Volanesorsen, an APOC3 antisense oligonucleotide, gained approval for FCS treatment in the UK. Caution is advised during pregnancy due to limited safety data, although animal studies show no toxicity/teratogenicity. Two case scenarios are presented: In the first case, a patient with FCS continued Volanesorsen injections without having thrombocytopenia during an unplanned pregnancy until third trimester, maintaining triglyceride control. Upon discovering the pregnancy at 38 weeks, Volanesorsen was ceased, and a low-fat diet reinstated. Despite a heightened risk of pancreatitis, no episodes of pancreatitis occurred during the pregnancy. In the second case, stopping Volanesorsen before conception led to elevated triglycerides, and an episode of acute pancreatitis at 22 weeks, despite strict very low-fat diet and fibrate therapy from 14 weeks. At 23 weeks, Volanesorsen was reintroduced concurrently with regular therapeutic plasma exchange. No further episodes of pancreatitis occurred. In both case, fetal health was maintained throughout pregnancy, fetal scans revealed no anomalies, and planned C-sections delivered healthy babies without congenital issues. Both babies are well and developing normally at 24 and 19 months.

Unveiling the Influence of Brightness Heterogeneity in Fluorescence Correlation Spectroscopy of Perovskite Nanocrystals.

Nanoparticles (NPs), including perovskite nanocrystals (PNCs) with single photon purity, present challenges in fluorescence correlation spectroscopy (FCS) studies due to their distinct photoluminescence (PL) behaviors. In particular, the zero-time correlation amplitude [g2(0)] and the associated diffusion timescale (τD) of their FCS curves show substantial dependency on pump intensity (IP). Optical saturation inadequately explains the origin of this FCS phenomenon in NPs, thus setting them apart from conventional dye molecules, which do not manifest such behavior. This observation is apparently attributed to either photo-brightening or optical trapping, both lead to increased NP occupancy (N) in the excitation volume, consequently reducing the g2(0) amplitude [since g2(0) α 1/N] at high IP. However, an advanced FCS study utilizing alternating laser excitation at two different intensities dismisses such possibilities. Further investigation into single-particle blinking behaviors as a function of IP reveals that the intensity dependence of g2(0) primarily arises from the brightness heterogeneity prevalent in almost all types of NPs. This report delves into the complexities of the photophysical properties of NPs and their adverse impacts on FCS studies.

Intracellular Macromolecular Crowding within Individual Stress Fibers Analyzed by Fluorescence Correlation Spectroscopy.

Introduction: The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in cells is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood. Methods: Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the open cytoplasm, the stress fibers in the open cytoplasm, and those below the nucleus using three kinds of biologically inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients. Results: We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggest that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers. Conclusion: The data presented here would provide a basis for better understanding of the complex protein diffusion in the nonuniform cytoplasm, shedding light on cellular responses to mechanical stress, their local mechanical properties, and reduced turnover in senescent cells.

Combination of autologous FCS and MPG versus each technique alone in the treatment of stable vitiligo.

Follicular cell suspension (FCS) transplantation is a novel surgical method for treating resistant stable vitiligo, whereas mini punch grafting is an established effective method for treating stable vitiligo. The combination of FCS and mini punch grafting is a better strategy for the treatment of resistant stable vitiligo. The aim of the study was to evaluate the efficacy of follicular cell suspension, mini punch grafting, and a combination of both techniques in the treatment of stable vitiligo. This prospective comparative study was conducted on 48 patients with stable vitiligo. They were divided into three equal groups, including group A (treated with follicular cell suspension), group B (treated with mini punch grafting), and group C (treated with the combination of both techniques). All patients were followed-up for six months for the assessment of their therapeutic response regarding clinical outcomes. By comparing the data of the three studied groups, we found that the difference in the degree of re-pigmentation after one and three months of treatment was not significant. However, the progress of re-pigmentation was significantly different after six months of treatment among the three studied groups (P = 0.027). Specifically, re-pigmentation was significantly better in group C than in groups A and B (P = 0.037 and 0.017, respectively), but it was not significantly different between groups A and B.

Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Characterization of peptide-fused protein assemblies in living cells.

Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.

Methods for making and observing model lipid droplets.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 µm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.

Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS.

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and ß1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/µm2.

Transfer of polarity information via diffusion of Wnt ligands in C. elegans embryos.

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.

Food Compass and the challenge of sustainability on the route towards healthful diets.

In order to tackle the global increase in overweight and obesity prevalence, several nutrient profiling systems have been developed; among others, Food Compass Score (FCS) has been designed to encompass multiple domains of food healthfulness. However, environmental sustainability of healthy diets is another crucial dimension which should not be overlooked in the context of human health. The aim of the present study is to assess the association between healthiness and environmental sustainability of food items, using the FCS and Agribalyse databases, respectively. A total of 806 matching food items were identified, grouped in 12 food categories; within each category, differences in median Z-scores between FCS and Single Environmental Footprint (EF) Score were assessed. While Fruits, Legumes and Nuts, Mixed foods, Meat Poultry and Eggs (MPE), Savory and Sweets, and Vegetables showed statistically significant differences (all p < 0.001), Beverages (p = 0.361), Dairy (p = 0.092), Fats and Oils (p = 0.594), Grains (p = 0.436), Sauce and Condiments (p = 0.093), and Seafood (p = 0.241) had similar Food Compass and Single EF Z-scores distributions. These findings underscore a relevant lack of difference between healthfulness and environmental impact of some prominent food categories, such as Grains and Seafood. Therefore, we suggest matching nutrient profiling systems with adequate environmental sustainability indices.

Understanding and manipulating extracellular behaviors of Wnt ligands.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.

Cross-Sectional Quantitative Evaluation of a Novel Patient-Reported Outcome Measure in Familial Chylomicronemia Syndrome.

Background: Familial chylomicronemia syndrome (FCS) is a rare metabolic disorder that impacts physical, emotional, social, and cognitive functioning. The FCS-Symptom and Impact Scale (FCS-SIS) patient-reported outcome (PRO) measure assesses common symptoms and impacts of FCS. This study was conducted to evaluate cross-sectional psychometric properties of the FCS-SIS and its scoring method. Methods: This multisite, cross-sectional, observational study of individuals with FCS was conducted in the United States and Canada. Participants completed a survey composed of 7 PRO measures, including the FCS-SIS, and questions about clinical characteristics and demographics. The structure of the FCS-SIS was evaluated using inter-item and item-scale correlations and internal consistency reliability. Construct, known-groups, and criterion validity were evaluated by examining associations between FCS-SIS item and composite scores and other measures included within the survey. Results: Most of the 33 participants were female (63.6%) and White (78.1%). On average, participants reported first noticing FCS symptoms at ~16 years, with abdominal pain the most frequently reported initial symptom (n=20). Participants reported 2.5 acute pancreatitis attacks on average over the past year. Average FCS-SIS symptom item scores ranged from 1.8 to 3.9 (on a 0-to-10 scale [none-to-worst-possible]) within the 24-hour recall period, with an average Symptom composite score of 2.7. The average impact item scores on the FCS-SIS ranged from 1.6 to 3.0 (on a 0-to-4 scale), with an average Impact composite score of 2.1. Inter-item correlations between the FCS-SIS Symptom items ranged from 0.32 to 0.78. Corrected item-total correlations were highly satisfactory for Impact items, ranging from 0.62 to 0.85. All a priori validity hypotheses were supported by observed correlations and score differences between known groups. Conclusion: The results of this study support the structure, reliability, and validity of the FCS-SIS, laying the psychometric groundwork for longitudinal evaluation of its utility in assessing treatment benefit in FCS clinical studies.

Mechanism of Viral DNA Packaging in Phage T4 Using Single-Molecule Fluorescence Approaches.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Distance-specific functional connectivity strength alterations in human immunodeficiency virus asymptomatic neurocognitive impairment patients: a cross-sectional study.

Background: Asymptomatic neurocognitive impairment (ANI) is the mildest form of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HANDs), and functional connectivity strength (FCS) alternations have been observed in the ANI stage. However, it is not clear whether the FCS alterations are influenced by the anatomical distance. This study sought to investigate distance-specific FCS changes in HIV ANI patients. Methods: In total, 29 patients with HAND and 32 healthy controls (HCs) were enrolled in the study. Between-group differences were detected for short, middle and long range anatomical distance FCS. A correlation analysis was performed to examine the relationship between distance-specific FCS and immunological parameters and neuropsychological tests. A receiver operating characteristic (ROC) analysis was conducted to examine the discriminative performance for HIV ANI patients. Results: In comparison to the HCs, the HAND patients showed increased short-range FCS in the left inferior parietal lobule (IPL), middle-range FCS in the superior temporal gyrus (STG), long-range FCS in the left precuneus (PCC), and decreased FCS in the right postcentral gyrus (PCG) (cluster P<0.05, voxel significance P<0.001). Further, the long-range FCS in the right PCG was negatively correlated with the CD4/CD8 ratio (r=-0.479, 95% confidence interval (CI): -0.735 to -0.104, P=0.015), and the distance-specific FCS also showed good classification performance between the HAND patients and HCs. The left IPL, left STG, right PCG, and left PCC had areas under the curve (AUCs) of 0.875 [95% confidence interval (CI): 0.758-0.949, P<0.0001], 0.806 (95% CI: 0.677-0.900, P<0.0001), 0.855 (95% CI: 0.734-0.935, P<0.0001), and 0.852 (95% CI: 0.754-0.950, P<0.0001), respectively. There was no significant relationship between the distance-specific FCS and the neuropsychological tests. Conclusions: Distance-specific FCS could be used to examine subtle alternations in HIV-infected patients in the ANI stage and help to explain the possible neurophysiological mechanism of HAND.

Food Insecurity Classification in Rural Areas of Mozambique: An Integrated Analysis of Survey-Based Indicators.

National food insecurity early warning systems and food policy interventions need reliable information concerning the classification of food insecurity. The aim of this paper was to produce an acute food insecurity classification in Mozambique, by: i) analyzing food insecurity indicators individually; ii) comparing it with a new integrated analysis of survey-based indicators called the "Matrix Analysis." The Matrix results show more severe classifications than the single indicators for the analyzed districts. The matrix novelty consists on a cross-tabulation of all indicators, allowing a less subjective analysis. Further research is needed on how the Matrix approach could complement national classification systems.

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

RNA Double-Helix Hybridization Measured by Fluorescence Correlation Spectroscopy.

RNA double-strand hybridization is a key player in gene expression regulation. Single-stranded RNA of up to 300 nucleotides forms Watson-Crick base pairs with complementary messenger RNA. Fluorescence-based single-molecule methods allow to study RNA-RNA interaction under physiological conditions. Here is described, how the dissociation constant of RNA double strands can be determined by applying fluorescence correlation spectroscopy.