ARTICLE | doi:10.20944/preprints201908.0319.v1
Subject: Biology, Other Keywords: Phototoxicity, Photodamage, Super-Resolution Microscopy, Fluorescence
Online: 30 August 2019 (08:22:58 CEST)
Super-Resolution Microscopy enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations.
ARTICLE | doi:10.20944/preprints202002.0343.v1
Subject: Physical Sciences, Optics Keywords: multi-photon processes; quantum optics; photon statistics; fluorescence
Online: 24 February 2020 (02:57:17 CET)
The interaction between single or a fixed number of photons with a single absorber is of fundamental interest in quantum technology. The harnessing of light matter interactions at the single particle limit has several potential applications ranging from quantum communication and quantum metrology to quantum imaging. In this letter, a setup for heralded two-photon absorption at the single absorber level is proposed. The setup is based on a heralded two-photon source utilizing spontaneous parametric down-conversion, entanglement swapping and sum frequency generation for joint detection. The feasibility of the scheme is discussed by reviewing recent achievements in utilizing entangled and correlated photons for two-photon absorption as well as single photon absorption experiments at the limit of single absorbers in the context of applications in imaging (here mainly microscopy) and spectroscopy.
ARTICLE | doi:10.20944/preprints202301.0238.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: induced pluripotent stem cells; embryoid bodies; light-sheet microscopy; multicolor fluorescence in situ hybridization; chromosomes
Online: 13 January 2023 (07:10:46 CET)
Embryoid bodies (EBs) are multicellular three-dimensional (3D) aggregates generated from induced pluripotent stem cells (iPSCs) in suspension and serve as useful biological sources for many downstream applications. Imaging of live EBs has been hampered mainly due to the inherent limitations of the imaging techniques applied to date. This study aimed to image human iPSC (hiPSC) derived EBs to obtain their 3D volume, determining size, morphology, and cell viability from day 7 to 14 using Light Sheet Fluorescence Microscopy (LSFM). Furthermore, chromosomal stability was assessed using Multicolor fluorescence in situ hybridization (M-FISH) from day 8 to 14. EB volume increased from day 7 to 13 which, decreased at day 14. From day 7 to 11, the EBs mainly appeared spherical and morphed into an ellipsoidal shape by day 13. All EBs showed varied external morphologies and larger cavities at day 14. The EB karyotype was diploid 46XY at day 8 and exhibited a low level of aneuploidy from day 10 to 14. This study shows that an increase in cell death affects the morphology and chromosomal stability in EBs derived from hiPSC. We demonstrate that the combination of LSFM and M-FISH helps characterize EBs that will assist future stem cell therapies.
TECHNICAL NOTE | doi:10.20944/preprints202111.0256.v1
Subject: Biology, Other Keywords: planarian; maceration; high-content fluorescence microscopy; formaldehyde fixation; RNA FISH; immunocytochemistry; BrdU; phospho-histone H3; tyramide signal amplification
Online: 15 November 2021 (11:44:53 CET)
High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single cell suspension before fixation and staining. By sharing many reagents with established planarian whole mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment. Recommendations for successful experimental workflows and common sources of errors are discussed.
ARTICLE | doi:10.20944/preprints202211.0199.v1
Subject: Biology, Plant Sciences Keywords: cyanobacteria; photosystem; fast fluorescence kinetics; optical microcavity; fluorescence microscopy
Online: 10 November 2022 (11:05:24 CET)
Photosynthesis is one the most important biological processes on earth, producing life-giving oxygen and is the basis for a large variety of plant products. Measurable properties of photosynthesis provide information about its biophysical state and, in turn, the physiological conditions of a photoautotrophic organism. For instance, chlorophyll fluorescence of an intact photosystem is not linear as in the case of a single fluorescent dye in solution, but shows temporal changes related to the quantum yield of the photosystem. Commercial photosystem analyzers already use the fluorescence kinetics characteristics of photosystems to infer the viability of organisms under investigation. Here, we provide a novel approach based on an optical Fabry-Pérot microcavity or that enables the readout of photosynthetic properties and activity for an individual cyanobacterium. This approach offers a completely new dimension of information, which would normally be lost due to averaging in ensemble measurements obtained from a large population of bacteria.
ARTICLE | doi:10.20944/preprints202104.0573.v1
Subject: Engineering, Automotive Engineering Keywords: fluorescence microscopy; fluorescence emission, malignant tumor, diagnosis, animal experiment
Online: 21 April 2021 (11:47:14 CEST)
A surgical microscope is large in size, which makes it impossible to be portable. The distance between the surgical microscope and the observation tissue is 15–30 cm, and the adjustment range of the right and left of the camera is a maximum of 30°. Therefore, the surgical microscope is generated attenuation (above 58%) of irradiation optical source owing to the long working distance. Moreover, the observation of tissue is affected because of dazzling by ambient light as the optical source power is strong (55 to 160 mW/cm2). Further, observation blind spot phenomena will occur due to the limitations in adjusting the right and left of the camera. Therefore, it is difficult to clearly observe the tumor. In this study, a compact pen-type probe with a portable surgical microscope is presented. The proposed surgical microscope comprises a small and portable pen-type probe that can adjust the working distance between the probe and the observed tissue. In addition, it allows the adjustment of the viewing angle and fluorescence brightness. The proposed probe has no blind spots or optical density loss.
ARTICLE | doi:10.20944/preprints202104.0557.v1
Subject: Engineering, Automotive Engineering Keywords: fluorescence microscopy; fluorescence emission; malignant tumor; diagnosis; animal experiment
Online: 21 April 2021 (08:30:11 CEST)
A surgical microscope is large in size, making portability impossible. The distance between the surgical microscope and the observation tissue is 15 to 30 cm, while the maximum adjustment range of the camera to the right and left is 30°. Therefore, surgical microscopes cause attenuation (above 58%) of the irradiation optical source owing to the long working distance. Moreover, the observation of tissue was dazzled with ambient light because the optical power source was strong (50 to 160 mW/cm2). Owing to the limited ability to adjust the camera to the right and left, a blind spot occurs with a surgical microscope. Therefore, it is difficult to clearly observe a tumor. In this study, a compact pen-type probe with a portable surgical microscope is proposed. The pen-type probe is small with a portable shape, and is capable of adjusting the working distance between itself and the observed tissue. It is also possible to adjust the viewing angle and fluorescence brightness. The proposed pen-type probe has no blind spots or optical density loss.
ARTICLE | doi:10.20944/preprints202109.0229.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: intraoperative photodiagnosis; malignant glioma; fluorescence-guided surgery; intraoperative cytology; fluorescence microscope
Online: 14 September 2021 (10:02:54 CEST)
Objective: Surgical eradication of malignant glioma cells is theoretically impossible. Therefore, reducing the number of remaining tumor cells around the brain-tumor interface (BTI) is crucial for achieving satisfactory clinical results. The usefulness of fluorescence-guided resection for the treatment of malignant glioma was recently reported, but the detection of infiltrating tumor cells in the BTI using a surgical microscope is not realistic. Therefore, we developed an intraoperative rapid fluorescence cytology system, and evaluated its clinical feasibility for the management of malignant glioma. Materials and methods: Twenty-five selected patients with malignant glioma (newly diagnosed: 17; recurrent: 8) underwent surgical resection under photodiagnosis using photosensitizer Talaporfin sodium and a semiconductor laser. Intraoperatively, a crush smear preparation was made from a tiny amount of tumor tissue, and the fluorescence emitted upon 620/660 nm excitation was evaluated rapidly using a compact fluorescence microscope in the operating theater. Results: Fluorescence intensities of tumor tissues measured using a surgical microscope correlated with the tumor cell densities of tissues evaluated by measuring the red fluorescence emitted from the cytoplasm of tumor cells using a fluorescence microscope. A “weak fluorescence” indicated a reduction in the tumor cell density, whereas “no fluorescence” did not indicate the complete eradication of the tumor cells, but indicated that few tumor cells were emitting fluorescence.Conclusion: The rapid intraoperative detection of fluorescence from glioma cells using a compact fluorescence microscope was a useful to evaluate the presence of tumor cells in the resection cavity walls, and provides surgical implications for the more complete resection of malignant gliomas.
ARTICLE | doi:10.20944/preprints202105.0189.v1
Online: 10 May 2021 (13:41:06 CEST)
Fluorescein anisotropy, which is a widely used technique to study the folding state of proteins or affinity of ligands, is used in the present work to study the temperature sensing of fluid in a microchannel, by adding fluorophore in the fluid. Fluorescein was used as a temperature probe, while glycerol-aq. ammonia was used as a working fluid. Fluorescence anisotropy of fluorescein were measured by varying various parameters. Apart from this, a comparison of fluorescence anisotropy and fluorescence intensity is also performed.
REVIEW | doi:10.20944/preprints201709.0160.v1
Subject: Materials Science, Polymers & Plastics Keywords: Nanoparticles; fluorescence; optical imaging
Online: 29 September 2017 (18:36:01 CEST)
Fluorophore molecules can be monitored by fluorescence spectroscopy and microscopy which are highly useful and widely used techniques in cell biology, biochemistry and medicine (e.g., biomarker analysis, immunoassays, cancer diagnosis). Several fluorescent micro- and nanoparticle systems based on block copolymer micelles and cross-linked polymer networks, quantum dots, -conjugated polymers, and dendrimers have been evaluated as optical imaging systems. In this review, we highlight recent advances in the construction of fluorescent single-chain nanoparticles (SCNPs) which are valuable artificial soft nano-objects with tunable, small size (as small as 3 nm). In particular, the main methods currently available to endow SCNPs with fluorescent properties are discussed in detail, showing illustrative examples.
Subject: Physical Sciences, Optics Keywords: fluorescence spectroscopy; fluorescence sensor; olive oil; machine learning; artificial neural networks; quality control
Online: 7 April 2021 (15:18:52 CEST)
Extra virgin olive oil (EVOO) is the highest quality of olive oil and is characterized by highly beneficial nutritional properties. The large increase in both consumption and fraud, for example through adulteration, creates new challenges and an increasing demand for developing new quality assessment methodologies that are easier and cheaper to perform. As of today, the determination of olive oil quality is performed by producers through chemical analysis and organoleptic evaluation. The chemical analysis requires the advanced equipment and chemical knowledge of certified laboratories, and has therefore a limited accessibility. In this work a minimalist, portable and low-cost sensor is presented, which can perform olive oil quality assessment using fluorescence spectroscopy. The potential of the proposed technology is explored by analyzing several olive oils of different quality levels, EVOO, virgin olive oil (VOO), and lampante olive oil (LOO). The spectral data were analyzed using a large number of machine learning methods, including artificial neural networks. The analysis performed in this work demonstrates the possibility of performing classification of olive oil in the three mentioned classes with an accuracy of 100%. These results confirm that this minimalist low-cost sensor has the potential of substituting expensive and complex chemical analysis.
ARTICLE | doi:10.20944/preprints201810.0698.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: optic biosensor; fluorescence; bacterial detection
Online: 30 October 2018 (03:44:11 CET)
The importance of detecting bacteria in various food products is ever-increasing, due to recent food trends that lend themselves to food contamination. Additionally, the detection of probiotics in food products is of increasing importance to consumers, who realize the benefits of probiotics on one’s diet. Existing technologies for detection of bacteria in food are accurate, but most are slow, increasingly costly and unsuitable for applications outside of research laboratories. Optic approaches have recently emerged as an alternative, allowing rapid detection of bacterial presence. This study employs a portable kinetics fluorometer, fabricated in-house, in conjunction with NADH sensitive fluorescence reporter for analysis of various food products. The presence of bacteria is detected in 5 minutes. Both pathogenic and probiotic bacteria were detected in food products, such as raw chicken and beef, spoiled lettuce and contaminated water, yogurt, and kombucha tea. The cellular activity of two probiotic pills was also verified. All samples displayed varying levels of bacterial activity. The study indicates the viability of biosensors being used as an alternate method to detect bacteria in food products – and the viability of a fluorescence-based biosensor to detect viable bacteria. The approach is suitable for both laboratory and field determinations.
ARTICLE | doi:10.20944/preprints202205.0006.v1
Subject: Life Sciences, Biophysics Keywords: structured illumination; fluorescence; brain; multi-camera
Online: 4 May 2022 (12:24:22 CEST)
Fluorescence microscopy provides an unparalleled tool for imaging biological samples. However, producing high-quality volumetric images quickly and without excessive complexity remains a challenge. Here, we demonstrate a simple multi-camera structured illumination microscope (SIM) capable of simultaneously imaging multiple focal planes, allowing for the capture of 3D fluorescent images without any axial movement of the sample. This simple setup allows for the acquisition of many different 3D imaging modes, including 3D time lapses, high-axial-resolution 3D images, and large 3D mosaics.
ARTICLE | doi:10.20944/preprints201810.0190.v1
Subject: Chemistry, Physical Chemistry Keywords: Biomarkers, Dye , Fluorescence, acetate detection, FLIM
Online: 9 October 2018 (15:39:16 CEST)
Development of biomarkers of analytes with interest in clinic is an important field of study. In this work, we synthesized and analyzed the new fluorescent acetate-biomarker, Iso-PG. The mechanism of detection is the acetate buffer mediated proton transfer reaction. The rate constants involved were obtained, and we measured the change in the fluorescence lifetime produced as a consequence of the presence of acetate in the medium. Finally, we checked its potential use as acetate biomarker in synthetic serum
ARTICLE | doi:10.20944/preprints201805.0340.v1
Subject: Biology, Other Keywords: photodynamic therapy; photobleaching; photosensitizers; fluorescence imaging
Online: 24 May 2018 (08:32:27 CEST)
Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light, and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, e.g. PS-CD conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm2, fluence rate: 75 mW/cm2) and (ii) low light fluence and fluence rate (128 J/cm2 and 14 mW/cm2 and 128 J/cm2 and 7 mW/cm2) in photobleaching of the individual chromophores and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.
ARTICLE | doi:10.20944/preprints202104.0239.v1
Subject: Chemistry, Analytical Chemistry Keywords: CDOM; calcium; magnesium; fluorescence quenching; lifetime distribution
Online: 8 April 2021 (12:22:14 CEST)
Revealing the binding properties of calcium ion (Ca2+) and magnesium ion (Mg2+) to chromophoric dissolved organic matter (CDOM) facilities understanding the effect of natural water composition on the photophysics of dissolved organic matter. Steady-state and time-resolved fluorescence spectrometry, and dynamic light scattering were applied to investigate the fluorescence quenching process of CODM by Ca2+ and Mg2+. The binding of Ca2+ and Mg2+ preferred terrestrial CDOM to aquatic CDOM. The fluorescence quenching of CDOM by cations mainly occurred in a static process, which was based on the fact that the decrease of steady-state fluorescence intensity was greater than fluorescence lifetime. The fluorescence quenching was profound under longer excitation and emission wavelength. The binding constant (K, L/mol) for Ca2+ to CDOM from terrestrial source ranged from 4.29 to 5.09 (lgK), which was approximately one order of magnitude higher than that of Mg2+ to CDOM (3.86 to 4.56). Fluorescence decay became faster in the presence of Ca2+ and Mg2+. Lifetime distribution of CDOM excited states shifted to small value side in the presence of metal ions, particularly for Ca2+, indicating fluorescence quenching of CDOM mainly through the interaction of Ca2+/Mg2+ with relatively long-lived fluorophores.
Subject: Life Sciences, Biochemistry Keywords: single particle tracking; single molecule biophysics; fluorescence
Online: 25 January 2021 (12:26:31 CET)
Single particle tracking (SPT) is a powerful class of methods for studying the dynamics of biomolecules inside living cells. The techniques reveal the trajectories of individual particles, with a resolution well below the diffraction limit of light, and from them the parameters defining the motion model, such as diffusion coefficients and confinement lengths. Most existing algorithms assume these parameters are constant throughout an experiment. However, it has been demonstrated that they often vary with time as the tracked particles move through different regions in the cell or as conditions inside the cell change in response to stimuli. In this work, we propose an estimation algorithm to determine time-varying parameters of systems that discretely switch between different linear models of motion with Gaussian noise statistics, covering dynamics such as diffusion, directed motion, and Ornstein-Uhlenbeck dynamics. Our algorithm consists of three stages. In the first stage, we use a sliding window approach, combined with Expectation Maximization (EM) to determine maximum likelihood estimates of the parameters as a function of time. These results are only used to roughly estimate the number of model switches that occur in the data to guide the selection of algorithm parameters in the second stage. In the second stage, we use change detection (CD) techniques to identify where the models switch, taking advantage of the off-line nature of the analysis of SPT data to create non-causal algorithms with better precision than a purely causal approach. Finally, we apply EM to each set of data between the change points to determine final parameter estimates. We demonstrate our approach using experimental data generated in the lab under controlled conditions.
ARTICLE | doi:10.20944/preprints201706.0006.v1
Subject: Physical Sciences, Optics Keywords: fluorescence recovery after photobleaching; fluorescence correlation spectroscopy; single-particle tracking; supported lipid bilayers; membrane curvature engineering; diffusion; molecular shape
Online: 1 June 2017 (08:03:33 CEST)
The biophysical consequences of nanoscale curvature have been challenging to resolve due to size-dependent membrane behavior and the experimental resolution limits imposed by optical diffraction. Recent advances in nanoengineering and super-resolution techniques have enabled new capabilities for creating and observing curvature. In particular, draping supported lipid bilayers over lithographically patterned substrates provides a model system for endocytic pits. The experiments and simulations presented below describe the possible detection of membrane curvature through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), and polarized localization microscopy (PLM). FRAP and FCS depend on diffraction-limited illumination and detection. In particular, a simulation of FRAP shows no effects on lipids diffusion due to a 50 nm diameter membrane bud at any stage in the budding process. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was amplified with curvature-dependent lipid mobility changes. However, PLM and SPT achieve sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of curvature on lipid behavior have been resolved.
REVIEW | doi:10.20944/preprints202112.0331.v1
Subject: Life Sciences, Biochemistry Keywords: Genetic code expansion; unnatural amino acid; fluorescence imaging
Online: 21 December 2021 (12:42:35 CET)
Genetic code expansion has emerged as an enabling tool to provide insight into functions of understudied proteinogenic species such as small proteins and peptides, and to probe protein biophysics in the cellular context. Here we discuss recent technical advances and applications of genetic code expansion in cellular imaging of complex mammalian protein species, along with considerations and challenges upon using the method.
ARTICLE | doi:10.20944/preprints202008.0327.v1
Subject: Earth Sciences, Environmental Sciences Keywords: chlorophyll fluorescence; remote sensing; ecosystems; spring-summer; forest
Online: 14 August 2020 (12:11:37 CEST)
The European heatwave of 2018 led to record-breaking temperatures and extremely dry conditions in many parts of the continent resulting in widespread decrease in agricultural yield, early tree-leaf senescence, and increase in forest fires in Northern Europe. Our study aims to capture the impact of the 2018 European heatwave on terrestrial ecosystem through the lens of a high-resolution solar-induced fluorescence (SIF) data acquired from the Orbiting Carbon Observatory (OCO-2) satellite. SIF is proposed to be a direct proxy for gross primary productivity (GPP) and thus can be used to draw inferences about changes in photosynthetic activity in vegetation due to extreme events. We explore spatial and temporal SIF variation and anomaly during spring and summer months across different vegetation types (agriculture, broadleaved forest, coniferous forest, and mixed forest) during the European heatwave of 2018 and compare it to non-drought conditions (most of Southern Europe). About one-third of Europe’s land area experienced a consecutive spring and summer drought in 2018. Comparing 2018 to mean (2015-2017) conditions, we found a change in intra-spring season SIF dynamics for all vegetation types, with lower SIF during the start of spring followed by an increase in fluorescence from mid-April. Summer, however, showed a significant decrease in SIF. Our results show that particularly agricultural areas were severely affected by the hotter drought of 2018. Furthermore, the intense heat wave in Central Europe showed about 31% decrease in SIF values during July and August as compared to the mean over three previous years. Furthermore, our MODIS and OCO-2 comparative results indicate that especially for forests, OCO-2 SIF has a quicker response and possible higher sensitivity to drought in comparison to MODIS’s fPAR and NDVI when considering shorter reference periods, which highlights the added value of remotely sensed solar-induced fluorescence for studying the impact of drought on vegetation.
ARTICLE | doi:10.20944/preprints201912.0273.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: Capsicum annuum; flavonoids; fluorescence monitoring; bio-waste utilization
Online: 20 December 2019 (11:05:22 CET)
The green biomass of horticultural plants contains valuable secondary metabolites (SM) which can potentially be extracted and sold. When exposed to stress, plants accumulate higher amounts of these SMs, making the extraction and commercialization even more attractive. We evaluated the potential for accumulating of the flavones cynaroside and graveobioside A in leaves of two bell pepper cultivars (Mavras and Stayer) when exposed to salt stress (100 mM NaCl), UVA/B excitation (UVA 4-5 W/m²; UVB 10-14 W/m² for 3 hours per day) or a combination of both stressors. HPLC analyses proved the enhanced accumulation of both metabolites under stress conditions. Cynaroside accumulation is effectively triggered by high-UV stress, whereas graveobioside A contents increase under salt stress. Highest contents were observed in plants exposed to combined stress. Effects of stress on overall plant performance differed significantly between treatments, with least negative impact on aboveground biomass found for high-UV stressed plants. The usage of two non-destructive instruments (Dualex and Multiplex) allowed us to gain insights in ontogenetical effects at the leaf level and temporal development of SM contents over time. Indices provided by those devices correlate fairly with amounts detected via HPLC (Cynaroside: R2 = 0.46 – 0.66; Graveobioside A: R2 = 0.51 – 0.71). The concentrations of both metabolites tend to decrease at leaf level during the ontogenetical development even under stress conditions. High-UV stress is a promising tool for enriching plant leaves with valuable SM without major effects on plant biomass. All data is available online .
ARTICLE | doi:10.20944/preprints201901.0027.v1
Subject: Earth Sciences, Other Keywords: x-ray fluorescence, paleo-depositional environments; porosity correlation
Online: 3 January 2019 (13:54:22 CET)
The Upper Jurassic (Oxfordian Age) Smackover Formation is a significant source for hydrocarbon production in southwest Alabama. Brooklyn Field is in southeast Conecuh County, Alabama and has been a major producer of oil and natural gas for the state. The Smackover is a carbonate formation that is divided into seven distinct lithofacies. In southwest Alabama, the Smackover Formation is heavily influenced by paleotopography from the underlying Paleozoic rocks of the Appalachian system. The goal of this study is to determine elemental ratios in rock core within the Smackover Formation using a X-ray fluorescence (XRF) handheld scanner, to correlate between lithofacies in the Smackover Formation and elementally characterize the upper oolitic grainstone reservoir and the lower thrombolite boundstone. Eight wells were used for the study within Brooklyn Field and Little Cedar Creek fields. Cores from the eight wells were scanned on six-inch intervals. Chemical logs were produced to show elemental weights in relation to depth and lithofacies. Well data collected for chemical signatures within producing zones were correlated to reservoir lithofacies and porosity. Aluminum, silicon, calcium, titanium, and iron were the most significant (>95% confidence level) predictors of porosity and is related to the depositional environment and subsequent diageneses of the strata. XRF data suggests relative enrichments in iron, titanium, and potassium may be related to deposition in relatively restricted marine waters.
COMMUNICATION | doi:10.20944/preprints201809.0613.v1
Subject: Materials Science, Surfaces, Coatings & Films Keywords: fullerene aggregation; organic solar cells; fluorescence; organic photovoltaics
Online: 30 September 2018 (16:35:40 CEST)
We report on the effects of the film morphology on the fluorescence spectra for a thin film including a quinoxaline-based co-polymer (TQ1) and a fullerene derivative (PC70BM). The ratio between the polymer and the fullerene derivative, as well as the processing solvent were varied. Beside the main emission peak at 700 nm in the fluorescence spectra of thin films of this phase-separated blend, a broad emission band is observed with a maximum at 520 - 550 nm. The intensity of this emission band decreases with an increasing degree of mixing in the film and becomes most prominent in thicker films, films with high PC70BM content, and films that were spin-coated from solvents with lower PC70BM solubility. We assign this emission band to aggregated PC70BM.
ARTICLE | doi:10.20944/preprints201805.0332.v1
Subject: Materials Science, Other Keywords: ultra-dilution; droplets; water; evaporation; X-ray fluorescence
Online: 24 May 2018 (05:43:52 CEST)
The analysis of particulate matter (PM) in dilute solutions is an important target for environmental, geochemical and biochemical researches. Here we show how the microdrop technology may allow to control, through the evaporation of small droplets, the deposition of insoluble materials dispersed in a solution on a well-defined area with a specific spatial pattern. Using this technology the superficial density of the deposited solute can be accurately controlled. In particular, it becomes possible to deposit an extremely reduced amount of insoluble material -in the order of few μg- on a confined area, thus allowing a relatively high superficial density to be reached within a limited time. In this work we quantitatively compare the microdrop technique for the preparation of particulate matter samples with the classical filtering technique. After having been optimized, the microdrop technique allows to obtain a more homogeneous deposition and limit sample consumption of a factor ~25. This method is potentially suitable for many novel applications in different scientific fields.
ARTICLE | doi:10.20944/preprints202209.0421.v1
Subject: Materials Science, Nanotechnology Keywords: electrospinning; microfibers; piezoelectric crystals; nitroanilines; fluorescence; functional organic materials
Online: 27 September 2022 (10:22:10 CEST)
N,N-dimethyl-4-nitroaniline (NNDM4NA, C8H10O2N2), is a superelastic and superplastic charge-transfer molecular crystal with a high molecular dipole moment, µ=7.95 D, which crystal-lizes in the acentric polar point group 2. Highly aligned poly-l-lactic acid (PLLA) polymer micro-fibers with embedded NNDM4NA nanocrystals were fabricated using the electrospinning tech-nique. The composite fibers display an extraordinarily high piezoelectric output response, where for a small stress of 5.0x103 Nm-2, an effective piezoelectric voltage coefficient of geff=3.6 VmN-1 was obtained. The fibers were found to display solid state blue fluorescence with a long (147 ns) life-time decay. Furthermore, the composite fibers exhibit an average increase of 67% on the Young modulus reaching 55 MPa, while the tensile strength reaches 2.8 MPa when compared with solely PLLA fibers. The results show that nanocrystals, from small organic molecules, with elastic and piezoelectric properties form hybrid functional 2-dimensional luminescent array which are me-chanical strong and generate high output voltages making them promising for applications in energy harvesting and as solid-state blue emitters.
Subject: Materials Science, Biomaterials Keywords: fluorescence; carbon-dots; red-emitting; bioimaging; light emitting diodes.
Online: 6 July 2021 (10:20:17 CEST)
Carbon dots (C-dots) represent an emerging class of non-toxic nano-emitters that show excitation wavelength dependent photoluminescence (PL) with high quantum yield (QY) and minimal photobleaching. The vast majority of studies focus on C-dots that exhibit the strongest PL emission in the blue/green region of the spectrum, while longer wavelength emissions are ideal for applications such as bioimaging, photothermal and photodynamic therapy and light emitting diodes. Effective strategies to modulate the PL emission of C-dot based systems towards the red end of the spectrum rely on extensive conjugation of sp2 domains, heteroatom doping, solvatochromism, surface functionalization and passivation. Those approaches are systematically presented in this review, while emphasis is given on important applications of red-emissive suspensions, nanopowders and polymer nanocomposites.
ARTICLE | doi:10.20944/preprints202102.0379.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: chlorophyll fluorescence; fog system; greenhouse; energy flux; OJIP; photosynthesis
Online: 17 February 2021 (10:48:01 CET)
The low relative humidity (RH) levels in a greenhouse during the daytime in a strawberry (Fragaria × ananassa Duch) cultivation period negatively affect the growth of strawberry related to photo-physiology. Therefore, this study was conducted to confirm an efficient RH management method by analyzing the phenotypic characteristics related to photo-physiology by controlling the RH in a greenhouse during the daytime with a fog system. Strawberry plants were grown respectively in a greenhouse affected by natural RH changes (control) and in a greenhouse with 40% ~ 50% RH adjusted during the daytime using a fog system. In the greenhouse, with controlled RH, the temperature decreased, and the RH was higher in the initial growth stage of strawberry planting than the control. It was observed a significant increase in the survival rate of the strawberry plant, as well as the incidence of powdery mildew, was lowered. In addition, the photosynthetic rate and OJIP chlorophyll a fluorescence transients related to photosystem II efficiency of strawberry leaves were significantly higher in the fog treatment than in the control. In winter, during the day, the number of days on which the temperature dropped below 20℃ has increased, the greenhouse temperature with controlled RH was lower due to the fog system. When the yield per strawberry plant in January and February was investigated, the control was higher than the RH treatment. Therefore, RH management using a fog system must be controlled at a level where a temperature range is adequate for plant growth, in which the efficient control of these parameters increases strawberry productivity.
Subject: Biology, Anatomy & Morphology Keywords: Leaf monoterpene emission; heath stress; chlorophyll fluorescence; photosynthesis; grapevine
Online: 30 December 2020 (16:51:05 CET)
Rising temperature is among the most remarkably stressful phenomena induced by global climate changes with negative impacts on crop productivity and quality. It has been previously shown that volatiles belonging to the isoprenoid family can confer protection against abiotic stresses. In this work, two Vitis vinifera cv. ‘Chardonnay’ clones (SMA130 and INRA809) differing for a mutation of the DXS gene encoding for 1-deoxy-D-xylulose-5-phosphate (the first dedicated enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway) and involved in the regulation of isoprenoids biosynthesis were investigated in field trials and laboratory experiments. Leaf monoterpene emission, chlorophyll fluorescence and gas-exchange measurements were assessed over three seasons at different phenological stages and either carried out in vivo or controlled conditions under contrasting temperatures. A significant (p<0.001) increase in leaf monoterpene emission was observed in INRA809 when plants were experiencing high temperatures and over two experiments while no differences were recorded for SMA130. Significant variation was observed for the rate of leaf CO2 assimilation under heat stress, with INRA809 maintaining higher photosynthetic rates and stomatal conductance values than SMA130 (p=0.003) when leaf temperature increased above 30°C. At the same time, maximum photochemical quantum yield of PSII (Fv/Fm) was affected by heat stress in the non-emitting clone (SMA130), while the INRA809 showed a significant resilience of PSII under elevated temperature conditions. Consistent data were recorded between field seasons and temperature treatments in controlled environment conditions suggesting a strong influence of monoterpene emission on heat tolerance under elevated temperatures. This work provides further insights on the photoprotective role of isoprenoids under high temperatures in Vitis vinifera and additional studies should focus at unravelling the mechanisms underlying heat tolerance on the monoterpene-emitter grapevine clone.
ARTICLE | doi:10.20944/preprints202007.0056.v1
Subject: Materials Science, General Materials Science Keywords: Aggregation-induced emission; quinones; fluorescence; TD-DFT; quantum yield
Online: 5 July 2020 (08:38:20 CEST)
Since the discovery of the aggregation-induced emission effect in 2001, diaminodicyanoquinone derivatives (DADQs) have presented interesting fluorescence properties, allowing them to be considered fluorescent dyes capable of showing quantum yields above 90%. Besides, the diaminodiacyanoquinone core represents a versatile building block propense either to modification or integration into different systems to obtain and provide them unique photophysical features. Herein, we carried out a theoretical study on the fluorescence properties of three different diaminodicyanoquinodimethane systems. Therefore, time-dependent density functional theory (TD-DFT) was used to obtain the values associated with the dipole moments, oscillator strengths, and the conformational energies between the ground and the first excited states of each molecule. The results suggest that only two of the three studied systems possess significant luminescent properties. In a further stage, the theoretical insights were confirmed by means of experimental measurements, which not only retrieved the luminescence of the DADQs, but also suggest a preliminary and promising antibacterial activity of these systems.
ARTICLE | doi:10.20944/preprints202006.0094.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: CCCP; Mitophagy; Regulatory T cells; Flow cytometry; fluorescence microscopy
Online: 7 June 2020 (15:03:23 CEST)
Objective: To investigate the effects and mechanisms of different concentrations of CCCP on mitophagy in human peripheral blood regulatory T cells. Methods: Tregs were isolated, identified and then grouped, treating with CCCP at a concentration of 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM for 24h in an incubator. Flow cytometry detected the reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial quality, and fluorescence microscopy observed the co-localization of mitochondria and lysosomes in each group. Results: The purity of CD4+CD25+Tregs was (93.36 ± 1.87) %. With the increase of CCCP concentration, the level of ROS gradually increased, while the MMP decreased gradually. About the mitochondria and lysosome fusion, the fluorescence intensity of orange (yellow) was the highest when the concentration of CCCP was in the range of 5-10 μM while decreased with the CCCP concentration continually increasing. The mitochondrial quality decreased with the increase of CCCP concentration. However, there was no significant difference between groups C, D and E. The mitochondrial quality of groups F and G were significantly lower than that of group E. Conclusions: With the concentration of CCCP gradually increased, the level of ROS in Treg cells increased, and MMP decreased, which promoted the mitophagy, mitochondrial quality maintains homeostasis; When ROS accumulated, and MMP decreased significantly, the mitophagy was inhibited, and the mitochondrial quality was significantly decreased.
ARTICLE | doi:10.20944/preprints202005.0496.v1
Subject: Chemistry, Applied Chemistry Keywords: bioorthogonal chemistry; fluorescence; glycosylation; metabolic incorporation; GFP; beta-catenin
Online: 31 May 2020 (19:37:26 CEST)
Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We have developed an original tool enabling labeling and visualization of the cell cycle key-regulator b-catenin in its O-GlcNAcylated form based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of b-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of b-catenin in HeLa cells. Moreover, the changes in O-GlcNAcylation of b-catenin were varied by perturbing global cellular O-GlcNAc levels with inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.
ARTICLE | doi:10.20944/preprints201912.0050.v1
Subject: Chemistry, Inorganic & Nuclear Chemistry Keywords: x-ray crystallography; h-bonding; acetone; fluorescence sensing; dft
Online: 4 December 2019 (12:22:21 CET)
A Zn(II) perchlorate complex has been prepared and characterized by single X-ray crystallography, nuclear magnetic resonance spectroscopy, thermogravimetric analysis, infrared spectroscopy, elemental analysis, and UV-vis spectroscopy. The complex crystallizes in the monoclinic space group P21/c (Z = 4) with a pentacoordinated zinc center. Interestingly, the Zn complex was found be to a potential fluorophore that could sense acetone and other ketones with high selectivity and sensitivity.
COMMUNICATION | doi:10.20944/preprints201809.0589.v1
Subject: Life Sciences, Biochemistry Keywords: squaraine dye; near infrared; fluorescence; chloroquine; malaria; Plasmodium falciparium
Online: 29 September 2018 (10:15:33 CEST)
Chloroquine was among the first of several effective drug treatments against malaria until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine as an antimalarial therapeutic, there is potential in efforts to adapt chloroquine for other clinical applications, such as in combination therapies and in diagnostics. In this context, we designed and synthesized a novel asymmetrical squaraine dye coupled with chloroquine (SQR1-CQ). In this study, SQR1-CQ was used to label live Plasmodium falciparum (P. falciparum) parasite cultures of varying sensitivities towards chloroquine. SQR1-CQ positively stained ring, mature trophozoite and schizont stages of both chloroquine–sensitive and chloroquine–resistant P. falciparum strains. In addition, SQR1-CQ exhibited significantly higher fluorescence, when compared to a chloroquine-BODIPY (borondipyrromethene) conjugate. We also achieved successful SQR1-CQ labelling of P. falciparum directly on thin blood smear preparations. Drug efficacy experiments measuring half-maximal inhibitory concentration (IC50) showed lower concentration of effective inhibition against resistant strain K1 by SQR1-CQ compared to conventional chloroquine. Taken together, the versatile and highly fluorescent labelling capability of SQR1-CQ and promising preliminary IC50 findings potentiates it to be further developed as a promising diagnostic bioimaging tool with drug efficacy against chloroquine-resistant P. falciparum.
Subject: Keywords: Ultrasound imaging; photoacoustic; functional MRI; calcium imaging; rodents; fluorescence imaging
Online: 24 May 2021 (16:12:34 CEST)
In the past decade, the idea that single populations of neurons support cognition and behavior has gradually given way to the realization that connectivity matters, and that complex behavior results from interactions between remote yet anatomically connected areas that form specialized networks. In parallel, innovation in brain imaging techniques has led to the availability of a broad set of imaging tools to characterize the functional organization of complex networks. However, each of these tools poses significant technical challenges and faces limitations, which require careful consideration of their underlying anatomical, physiological and physical specificity. In this review, we focus on emerging methods for measuring spontaneous or evoked activity in the brain. We discuss methods that can measure large-scale brain activity (directly or indirectly) with relatively high temporal resolution, from milliseconds to seconds. We further focus on methods designed for studying the mammalian brain in preclinical models, specifically in mice and rats. This field has seen a great deal of innovation in recent years, facilitated by concomitant innovation in gene editing techniques and the possibility of more invasive recordings. This review aims to give an overview of currently available preclinical imaging methods and an outlook on future developments. This information is suitable for educational purposes and for assisting scientists in choosing the appropriate method for their own research question.
ARTICLE | doi:10.20944/preprints202105.0077.v1
Subject: Materials Science, Biomaterials Keywords: Silk Fibroin; Fluorescence Resonance Energy Transfer (FRET); Confocal Imaging; Electrospinning
Online: 6 May 2021 (12:24:22 CEST)
Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. Confocal imaging is usually per-formed to test the cells behaviour on the construct and in this context the fibroin autofluorescence is regarded as a problem. In addition, the autofluorescence is not intense enough to provide useful morphological images. In fact, to control study the constructs morphology other techniques are used (i.e. SEM, Micro-CT). In this work we propose a method based on the fluorescence energy transfer (FRET) to suppress the fibroin autofluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging.
ARTICLE | doi:10.20944/preprints202104.0174.v1
Subject: Life Sciences, Biochemistry Keywords: Phosphodiesterase (PDE); natural products; inhibitors; Protein Kinase; selectivity; fluorescence polarization
Online: 6 April 2021 (12:07:58 CEST)
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.
ARTICLE | doi:10.20944/preprints202010.0031.v1
Subject: Chemistry, Analytical Chemistry Keywords: primary fatty acid amides; microfluidics; laser induced fluorescence, bioactive lipids
Online: 2 October 2020 (09:16:13 CEST)
The inherent trace quantity of primary fatty acid amides found in biological systems presents challenges for analytical analysis and quantitation, requiring a highly sensitive detection system. The use of microfluidics provides a green sample preparation and analysis technique through small-volume fluidic flow through micron-sized channels embedded in a PDMS device. Microfluidics provides the potential of having a micro total analysis system where chromatographic separation, fluorescent tagging reactions, and detection are accomplished with no added sample handling. This study describes the development and optimization of a microfluidic-laser indued fluorescence (LIF) analysis and detection system that can be used for the detection of ultra-trace levels of fluorescently tagged primary fatty acid amines. A PDMS microfluidic device was designed and fabricated to incorporate droplet-based flow. Droplet microfluidics have enabled on-chip fluorescent tagging reactions to be performed quickly and efficiently, with no additional sample handling. An optimized LIF optical detection system provided fluorescently tagged primary fatty acid amine detection sub-fmol (436 amol) LODs. The use of this LIF detection provides unparalleled sensitivity, with detection limits several orders of magnitude lower than currently employed LC-MS techniques and might be easily adapted for use as a complementary quantification platform for parallel MS-based -omics studies.
ARTICLE | doi:10.20944/preprints201904.0298.v1
Subject: Chemistry, Inorganic & Nuclear Chemistry Keywords: 2,2':6',2''-terpyridine; Salicylidene Schiff bases; Fluorescence sensor; Al3+
Online: 26 April 2019 (10:55:10 CEST)
Two 2,2':6',2''-terpyridine-based Schiff bases (TPySSB and TPySB) have been synthesized. The TPySSB shows remarkable selective ‘off-on’ fluorescence for Al3+ by photoinduced electron transfer (PET) mechanism of sensing. Chemosensor TPySSB binds Al3+ in a 1:2 ratio with an association constant 6.8×105 (R2=0.98) and this 1:2 stoichiometric model is established on Job’s plot and 1H NMR. Compared TPySSB and TPySB, it is of great importance of the existence of salicylidene unit due to its strong binding abilities of both phenol and C=N structure to the Al3+.
ARTICLE | doi:10.20944/preprints201808.0284.v1
Subject: Life Sciences, Biophysics Keywords: adsorbed vaccines, identity, protein conformation, particle sizing, FTIR, SEM, Fluorescence
Online: 16 August 2018 (12:54:59 CEST)
1) Background: Traditionally, complex biological products such as vaccines presented unique challenges to implementation of even rudimentary characterization packages; thus, the product was defined almost exclusively by its manufacturing process. The advances in technology and analytical tools allowed the application of more comprehensive characterization packages for products such as adsorbed combination vaccines, which contain several antigens in a single formulation to protect against more than one disease, and may contain adjuvants and excipients. Aluminum phosphate (AlPO4) is a well-established adjuvant for enhancing the uptake of vaccines and to induce robust immunity against pathogens. During manufacturing, adjuvant is mixed with protein antigens which may in turn impact their higher order structure and stability. 2) Methods: To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM were used. 3) Results: the AlPO4 adjuvant suspension consists of small submicron particles that form a continuous porous surface. Secondary structure alpha-helix and beta-sheet content of DT and TT increased after adsorption to AlPO4 adjuvant, whereas no significant changes were noted for other protein antigens. Interactions were noted between AlPO4 adjuvant and DT, TT, and FHA. 4) Conclusions: here we report for the first time the use of SEM for the visualization of adsorbed multivalent vaccine components. A unique signature profile detected for each multivalent vaccine by FTIR can be used as a lean in-process test to verify vaccine product composition and identity prior to filling.
SHORT NOTE | doi:10.20944/preprints201705.0183.v1
Subject: Chemistry, Organic Chemistry Keywords: benzo[e]indole; benzo[e]imidazo[1,2-a]indole; fluorescence
Online: 25 May 2017 (08:04:28 CEST)
The alkylation of 1,1,2-trimethyl-1H-benzo[e]indole with 2-chloroacetamide, followed by work-up of the reaction mixture with a base and the subsequent treatment of a crude product with acetic acid gives 10a,11,11-trimethyl-10a,11-dihydro-8H-benzo[e]imidazo[1,2-a]indol-9(10H)-one. The structure assignments were based on data from 1H, 13C and 15N NMR spectroscopy. The optical properties of the obtained compound were studied by UV-vis and fluorescence spectroscopy.
ARTICLE | doi:10.20944/preprints201703.0131.v1
Subject: Biology, Plant Sciences Keywords: Rhododendron chrysanthum Pall.; cold stress; chlorophyll fluorescence; photosynthesis; antioxidant enzymes
Online: 17 March 2017 (04:46:21 CET)
Rhododendron chrysanthum Pall., live in Changbai Mountain being exposed to chilling temperature, high light intensities and water scarcity condition. To adapt to the harsh environment, the cold resistance mechanisms of R. chrysanthum have been successfully evolved in the long-term adaptive process. In our present work, the methods of proteomics combined with physiological and biochemical analyses were used to investigate the effects of cold stress on the photosynthesis and antioxidant system of Rhododendron chrysanthum Pall. and the molecular mechanisms involved in cold resistance of plants. A total of 153 photosynthesis related proteins were identified in present work, of which 7 proteins including Rubisco large subunit (rbcL) were up-regulated in experiment group (EG) compared with control group (CG). Simultaneously, four chlorophyll fluorescence parameters were measured in present study. The results showed that the maximum photochemical efficiency of photosystem II (Fv/Fm), actual quantum yield of PSII (Y(II)) and photochemical quenching (qP) were significantly higher in EG, whereas the non-photochemical quenching (NPQ) was notably decreased. Cold stress could lead to a significant reduction in electron transport rate (ETR) accompanied with an increase in excitation pressure (1-qP). The abundance of PetE which involved in the plants photosynthetic electron transfer was also significantly influenced by cold stress. Moreover, the up-regulated expressions and higher levels of enzymatic activities of Glutathione peroxidase (GPX) and Ascorbate peroxidases (APXs) were detected in EG. All these changes which can help plants to survive in low temperature are considered as the crucial parts of cold tolerance mechanisms. These results revealed that photosynthesis and redox adjustment play significant roles in the defense of cold-induced damage.
ARTICLE | doi:10.20944/preprints201608.0137.v1
Subject: Earth Sciences, Environmental Sciences Keywords: heavy metal; contamination assessment; X-ray fluorescence; bus station dusts
Online: 13 August 2016 (09:41:18 CEST)
The objective of this study was to investigate the concentration and spatial distribution patterns of six potentially toxic heavy metal elements (Mn, Zn, Cr, Pb, Cu and Ni) in bus station dusts in the Xifeng district of Gansu province, NW China. The contents were analyzed for Mn, Zn, Cr, Pb, Cu and Ni by using S8 TIGER Brochures wavelength dispersive X-ray fluorescence spectrometry. Geoaccumulation index (Igeo ), enrichment factor (EF), pollution index (PI) and integrated pollution index(IPI) were calculated to evaluate the heavy metal contamination level of bus station dusts. The results indicate that, in comparison with the background values of local soil, bus station dusts in Xifeng have elevated metal concentrations as a whole. The concentrations of heavy metals investigated in this paper are compared with the reported data of other cities. The results show that the arithmetic means of Mn, Zn, Cr, Pb, Cu and Ni are 440.8, 137.9, 60.0, 42.8, 33.5 and 19.8mg kg−1 respectively. The mean values of Igeo reveal the order of Ni<Mn<Cr<Cu<Zn<Pb. The high Igeo and EF for Cu, Zn and Pb in bus station dusts indicate that there is a considerable Cu, Zn and Pb pollution, which mainly originate from traffic and industry activities. The Igeo and EF of Ni, Mn and Cr are low and the assessment results indicate an absence of distinct Ni, Mn and Cr pollution in bus station dusts. The assessment results of PI also support Cu, Zn and Pb in bus station dusts presented middle pollution, and IPI indicates heavy metals of bus station dusts polluted seriously.
ARTICLE | doi:10.20944/preprints202209.0285.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: mesothelioma; chick embryo; CAM; xenograft; bioluminescence; fluorescence; histology; MRI; preclinical; 3Rs
Online: 20 September 2022 (02:48:17 CEST)
Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess therapies using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.
REVIEW | doi:10.20944/preprints202205.0383.v1
Subject: Medicine & Pharmacology, Other Keywords: colorectal; fluorescence; ICG; ICG-NIR; colorectal surgery; intraoperative staining; q-ICG
Online: 27 May 2022 (11:59:25 CEST)
This review looks at the use of indocyanine green (ICG) in colorectal surgery, from a quantitative point of view. The main benefits of the ICG technique in colorectal surgery, can be summarized as follows: a)in the realization of the intraoperative fluorescence angiography as an adjuvant in the process of anastomosis, b)in the fluorescence-guided detection of lymph node metastases in colorectal cancer and, also, the sentinel lymph node technique, which was proven better than formal methods in some studies, c) marking with positive fluorescence a liver nodule as small as "just" 200 tumor cells, d) offering assistance in the diagnosis of a fistula, e)in the possibility to be used for tumor tattooing also, f)providing help in maintaining a clean surgical field and preventing wound infection in abdominoperineal resection. Apart from the qualitative intraoperative use of ICG, the method can be employed in association with quantitative methods, such as maximum intensity, relative maximum intensity, and various parameters of the inflow (time-to-peak, slope, and t1/2max), this latter category being more significantly associated with anastomotic leakage.
ARTICLE | doi:10.20944/preprints202012.0358.v1
Subject: Chemistry, Analytical Chemistry Keywords: Antitumor agents; Fluorescence lifetime imaging; Medicinal chemistry; Metabolic drug; Mitochondrial carrier
Online: 15 December 2020 (08:35:30 CET)
Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of thiophene group as mitochondrial carrier for drugs and fluorescent markers, based on a new concept of nonprotonable, noncharged transporters. We implemented this concept in a medicinal chemistry application by developing an anti-tumoral, metabolic chimeric drug, based on PDHK inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-aimed drugs.
ARTICLE | doi:10.20944/preprints201808.0153.v1
Subject: Chemistry, Analytical Chemistry Keywords: 4-benzoyl pyrazolone; fluorescence sensing; functionalized gold nanoparticles; detection Al3+ ion
Online: 8 August 2018 (03:53:55 CEST)
A new type of gold nanoparticles was synthesized by using benzoyl pyrazolone as a capping agent. The synthesized AuNPs exhibited a spherical shape and a monodisperse and fluorescence emission characteristic peak at 650 nm. The AuNPs –BMPBP was demonstrated as sensitive and selective fluorescent chemosensor for detection of Al 3+ ion. In the presence of Al 3+ ion, the fluorescent emission of BMPBP-AuNPs at 650 nm, increased with an increasing concentration of Al 3+ ion with a low detection limit (2 µM), the proposed method provided to apply determination of Al 3+ ion of tap water with satisfactory results.
ARTICLE | doi:10.20944/preprints202210.0339.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: low-cost fluorescence microscopy; smartphone microscopy; 3D-printed devices; laser-based microscopy
Online: 21 October 2022 (13:48:31 CEST)
Fluorescence microscopy is an important tool for disease diagnosis, often requiring costly optical components, such as fluorescence filter cubes and high-power light sources. Due to its high cost, conventional fluorescence microscopy cannot be fully exploited in low-income settings. Smartphone-based fluorescence microscopy becomes an interesting low-cost alternative, but raises challenges in the optical system. We present the development of a low-cost inverted laser fluorescence microscope, that uses a smartphone to visualize the fluorescence image of biological samples. Our fluorescence microscope uses a laser-based simplified optical filter system, that provides analog optical filtering capabilities of a fluorescence filter cube. Firstly, we validated our inverted optical filtering by visualizing microbeads labeled with three different fluorescent compounds or fluorophores, commonly used for disease diagnosis. Secondly, we validated the disease diagnosis capabilities, by comparing the results of our device with those of a commercial fluorescence microscope. We successfully detected and visualized Trypanosoma cruzi parasites, responsible of the Chagas infectious disease, and the presence of Antineutrophil cytoplasmic antibodies of the ANCA non-communicable autoimmune disease. The samples were labeled with the fluorescein isothiocyanate (FITC) fluorophore, one of the most commonly used for disease diagnosis. Our device provides a 400 X magnification and is at least two orders magnitude cheaper than conventional commercial fluorescence microscopes.
Subject: Life Sciences, Biotechnology Keywords: Drosophila melanogaster; fluorescence resonance energy transfer; Met-lead 1.44 M1; Pb biosensor
Online: 1 October 2021 (11:12:13 CEST)
The heavy metal lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we applied a genetically encoded Förster resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform, and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which represented the presence of Pb in the tested samples in different spatial dimensions. Using iPSC-derived cardiomyocytes, the relationship between beating activity (20–24 beats per minute, bpm) determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 was revealed from simultaneous measurements. Pb (50 μM) affected the beating activity of cardiomyocytes, whereas two drugs that stop the entry of Pb differentially affected this beating activity: verapamil (2 μM) did not reverse the cessation of beating, whereas 2-APB (50 μM) partially restored this activity (16 bpm). The results clearly demonstrate a potential of this biosensor system as an anti-Pb drug screening application. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system (Cha-gal4>UAS-Met-lead 1.44 M1) using fast epifluorescence and high-resolution two-photon 3D FRET ratio image systems. The tissue-specific expression of Pb biosensors provides an excellent opportunity to explore the possible Pb-specific populations within living organisms. We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning and advanced research on Pb neurotoxicology.
REVIEW | doi:10.20944/preprints202102.0163.v1
Subject: Life Sciences, Biochemistry Keywords: ATP; OXPHOS; ATP synthase; mitochondria; biochemical assays; fluorescence microscopy; genetically-encoded biosensors
Online: 5 February 2021 (14:45:04 CET)
ATP is the most universal and essential energy molecule in the eukaryotic cell. This is due to its ability to store energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signalling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolysed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Then, we will explore the palette of genetically-encoded biosensors designed for microscope-based approaches and show how their spatiotemporal resolution opened up the possibility to follow ATP levels and production in living cells. Finally, we will comment on how ATP monitoring is used in preclinical practices, and to what extent genetically-encoded sensors could be used as a promising tool to elucidate pathologies in which ATP is implicated.
ARTICLE | doi:10.20944/preprints202008.0684.v1
Subject: Biology, Ecology Keywords: Chlorophyll Fluorescence; SIF; Drought; Spectral Vegetation Indices; GOME-2; Abiotic Stress; Caatinga
Online: 30 August 2020 (18:24:28 CEST)
Sun-Induced chlorophyll Fluorescence (SIF) relates directly to photosynthesis yield and stress but there are still uncertainties in its interpretation. Most of these uncertainties concern the influences of the emitting vegetation’s structure (e.g., leaf angles, leaf clumping) and biochemistry (e.g., chlorophyll content, other pigments) on the radiative transfer of fluorescent photons. The Caatinga is a large region at northeast Brazil of semiarid climate and heterogeneous vegetation, where such biochemical and structural characteristics can vary greatly even within a single hectare. With this study we aimed to characterize eleven years of SIF seasonal variation from Caatinga vegetation (2007 to 2017) and to study its responses to a major drought in 2012. Orbital SIF data from the instrument GOME-2 was used along with MODIS MAIAC EVI and NDVI. Environmental data included precipitation rate (TRMM), surface temperature (MODIS) and soil moisture (ESA CCI). To support the interpretation of SIF responses we have used red and far-red SIF adjusted by the Sun’s zenith angle (SIF-SZA) and by daily Photosynthetically Active Radiation (dSIF). Furthermore, we have also adjusted SIF through two contrasting formulations using NDVI data as proxy for structure and biochemistry, based on previous leaf-level and landscape level studies: SIF-Yield and SIF-Prod. Data was tested with time-series decomposition, rank correlation, spatial correlation and Linear Mixed Models (LMM). Results show that GOME-2 SIF and adjusted SIF formulations responded consistently to the observed environmental variation and showed a marked decrease in SIF emissions in response to a 2012 drought, that was generally larger than the corresponding NDVI and EVI decreases. Drought sensitivity of SIF, as inferred from LMM slopes, was correlated to land cover at different regions of the Caatinga. This is the first study to show correlation between landscape-level SIF and an emergent property of ecosystems (i.e., resilience), showcasing the value of remotely sensed fluorescence for ecological studies.
COMMUNICATION | doi:10.20944/preprints201810.0136.v2
Subject: Chemistry, Analytical Chemistry Keywords: lead ions; fluorescence detection; ionophore; Benzo-18-Crown-6-ether; drinking water
Online: 30 October 2018 (07:42:15 CET)
Drinking water contamination of lead from various environmental sources, leaching consumer products and intrinsic water-pipe infrastructure is still today a matter of great concern. Therefore, new highly sensitive and convenient Pb2+ measurement schemes are necessary, especially for in-situ measurements at a low-cost. Within this work dye/ionophore/Pb2+ co-extraction and effective water phase de-colorization was utilized for highly sensitive lead measurements and sub-ppb naked-eye detection. A low-cost ionophore Benzo-18-Crown-6-ether was used, and a simple test-tube mix and separate procedure was developed. Instrumental detection limits were in the low ppt region (LOD=3, LOQ=10), and naked-eye detection was 500 ppt. Note, however, that this sensing scheme still has improvement potential as concentrations of fluorophore and ionophore were not optimized. Artificial tap-water samples, leached by a standardized method, demonstrated drinking water application. Implications for this method are convenient in-situ lead ion measurements.
ARTICLE | doi:10.20944/preprints201802.0097.v1
Subject: Earth Sciences, Oceanography Keywords: fluorescence; absorption; chlorophyll-a; remote sensing; hyperspectral; ocean color; IOP; TAPIR; EnMAP
Online: 14 February 2018 (07:11:09 CET)
The Total Algae Peak Integration Retrieval TAPIR relates the chlorophyll-a absorption coefficient at 440 nm (a440) to the reflectance peak near 683 nm induced by chlorophyll-a properties. The two-step retrieval provides both the hyperspectral quantification of the phytoplankton fluorescence and scattering and the estimation of a440 from reflectance signals. Integrating the peak, the Total Algae Peak (TAP) accounts for the variance in the peak's magnitude, shape, and central peak wavelength. TAPIR is a solely optical approach estimating a440 and supports the application of retrieval-independent individual regional bio-optical models afterwards to retrieve the chlorophyll-a concentration. Simulations reveal the major sensitivity on the considered model chlorophyll-a absorption spectrum and its single scattering albedo. Additional water and atmosphere constituents have a low impact. An uncertainty assessment reveals uncertainties of less than 30% for TAPIR a440 greater than 0.8 m-1 and less than 38% for lower a440. In optically complex waters, first validation efforts promise the applicability of TAPIR for high chlorophyll-a concentration estimations in the presence of additional water constituents. The technique is generic and considers external conditions (sun zenith angle, number of measurement bands, surface or satellite measurements, and radiometric quantity). TAPIR applies to all kind of waters including optically complex waters, arctic to tropical regions, and inland, coastal, and open ocean waters. Among other hyperspectral satellite sensors, the Environmental Mapping and Analysis Program (EnMAP) provides sufficient sampling bands for the application of TAPIR.
ARTICLE | doi:10.20944/preprints202007.0039.v1
Subject: Life Sciences, Biotechnology Keywords: Aviation security; biosensor; flow injection assay; monoclonal antibody; fluorescence microscope; lab-on-a-chip; microfluidic systems; antibody labeling; CMOS; diode laser; monolithic column; laser-induced fluorescence detector (LIF)
Online: 3 July 2020 (12:26:26 CEST)
The illegal use of explosives by terrorists and other criminals is an increasing issue in public spaces, such as airports, railway stations, highways, sports arenas, theaters, and other large buildings. Security in these environments can be achieved by a set of different means, including the installation of scanners and other analytical devices to detect ultra-small traces of explosives in a very short time-frame to be able to take action as early as possible to prevent the detonation of such devices. Unfortunately, an ideal explosive detection system still does not exist, which means that a compromise is needed in practice. Most detection devices lack the extreme analytical sensitivity, which is nevertheless necessary due to the low vapor pressure of nearly all explosives. In addition, the rate of false positives needs to be virtually zero, which is also very difficult to achieve. Here we present an immunosensor system based on kinetic competition, which is known to be very fast and may even overcome affinity limitation, which impairs the performance of many traditional competitive assays. This immunosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. In the case of TNT occurring, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled CMOS camera. Liquid handling is performed with high-precision syringe pumps and chip-based mixing-devices and flow-cells. The system achieved limits of detection of 1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of the explosive 2,4,6-trinitrotoluene (TNT). The total assay time is less than 8 min. A cross-reactivity test with 5000 pM solutions showed no signal by PETN, RDX, and HMX. This immunosensor belongs to the most sensitive and fastest detectors for TNT with no significant cross-reactivity by non-related compounds.
ARTICLE | doi:10.20944/preprints202209.0395.v1
Subject: Life Sciences, Biophysics Keywords: chilling requirement; chlorophyll fluorescence; non-photochemical quenching; PAM; photoprotection; stress resilience; winter dormancy
Online: 26 September 2022 (11:01:44 CEST)
Dormancy is a physiological state that confers winter hardiness to and orchestrates phenological phase progression in temperate perennial plants. Weather fluctuations caused by climate change increasingly disturb dormancy onset and release in many plant species including tree crops leading to aberrant growth, flowering, and fruiting. Currently, research in this field is impeded by the lack of affordable non-invasive methods for on-line monitoring of dormancy. We report on an automatic framework for low-cost, long-term, and scalable dormancy studies in deciduous plants. The proposed method is based on continuous near-field sensing of the photosynthetic activity of shoots via pulse-amplitude modulated chlorophyll fluorescence sensors connected remotely to a data processing system. The resulting high-resolution time series of JIP-test parameters indicative of the responsiveness of the photosynthetic apparatus to environmental stimuli are subjected to frequency-domain analysis. The proposed approach allows to overcome the variance coming from diurnal changes of insolation and to derive estimations on the depth of dormancy. Our approach was validated over three seasons in an experimental apple (Malus × domestica Borkh.) orchard by collating the non-invasive estimations with the results of traditional methods (growing of the cuttings obtained from the tress at different phases of dormancy) and the output of commonly used chilling requirement models. We discuss the advantages of the proposed monitoring framework such as prompt detection of freeze damages along with its potential limitations.
ARTICLE | doi:10.20944/preprints202109.0056.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: Aptamer; Fluorescence Resonance Energy Transfer; Heavy Metal Ions; Graphene Oxide; Microfluidic Device; Sensor.
Online: 3 September 2021 (10:29:50 CEST)
Heavy metal contaminants have serious consequences for the environment and human health. Consequently, effective methods for detecting their presence, particularly in water and food, are urgently required. Accordingly, the present study proposes a sensor for the detection of mercury Hg(II) and lead Pb(II) ions using graphene oxide (GO) as a quenching agent and aptamer solu-tion as a reagent. In the proposed device, the aptamer sequences are labeled by FAM and HEX fluorescent dyes, respectively, and are mixed with 500 ppm GO solution in a microfluidic device. The presence of Hg(II) and Pb(II) ions is then detected by measuring the change in the fluores-cence intensity of the GO/aptamer suspension as the aptamer molecules undergo fluorescence resonance energy transfer (FRET). The experimental results show that the aptamer sensors have a linear range of 10~250 nM (i.e., 2.0~50 ppb) for Hg(II) ions and 10~100 nM (i.e., 2.1~20.7 ppb) for Pb(II) ions. Furthermore, the limit of detection is around 2 ppb for both metals, which is signifi-cantly lower than the maximum limits of 6 ppb and 10 ppb prescribed by the World Health Or-ganization (WHO) for Hg(II) and Pb(II) in drinking water, respectively.
CONCEPT PAPER | doi:10.20944/preprints201810.0689.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: 5-aminolevulinic acid, ciprofloxacin, deferiprone, fluorescence, 5-fluorouracil, febuxostat, glioblastoma, photodynamic treatment, temozolomide,
Online: 29 October 2018 (14:12:07 CET)
The CAALA (Complex Augmentation of ALA) regimen was developed with the goal of redressing some of the weaknesses of 5-aminolevulinic acid (5-ALA) use in glioblastoma treatment as it now stands. 5-ALA is approved for use prior to glioblastoma surgery to better demarcate tumor from brain tissue. 5-ALA is also used in intraoperative photodynamic treatment of glioblastoma by virtue of uptake of 5-ALA and its selective conversion to protoporphyrin IX in glioblastoma cells. Protoporphyrin IX becomes cytotoxic after exposure to 410 nm or 635 nm light. CAALA uses four currently marketed drugs - the antibiotic ciprofloxacin, the iron chelator deferiprone, the antimetabolite 5-FU, and the xanthine oxidase inhibitor febuxostat - that all have evidence of ability to both increase 5-ALA mediated intraoperative glioblastoma demarcation and photodynamic cytotoxicity of in situ glioblastoma cells. Data from testing the full CAALA on living minipigs xenotransplanted with human glioblastoma cells will determine safety and potential for benefit in advancing CAALA to a clinical trial.
REVIEW | doi:10.20944/preprints201810.0158.v1
Subject: Life Sciences, Virology Keywords: Hepatitis C virus, Genotyping, Mixed infection, Fluorescence melting curve analysis, Viral Load, Quantification
Online: 8 October 2018 (16:08:48 CEST)
Hepatitis-C is one of the most common viral diseases caused by hepatitis C virus (HCV). It is responsible for millions of deaths each year in the developing world. The common dissemination paths of HCV include the use of contaminated water and transfusion of infected blood. Control of this virus has become a challenge for scientists and health professionals due to its versatility and adaptability in different host environments. Along with other problems, lack of efficient diagnosis, quantification and genotyping of viral strains are the major hindrances in a management of this notorious epidemic. The knowledge of HCV genotype and an amount of virus in patient’s blood are pre-requisites to determine the duration and method of treatment. In this review, we discuss the implications of HCV molecular diagnostic methods and their clinical applications. We conclude that while, several commercial and home-brewed methods are available for this purpose, and there is a visible vacuum for cost effective, robust, sensitive assays that can detect multiple viral genotypes in a single reaction. We are of the view that the level of sensitivity offered by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique is unequivocal as compared to other techniques. Therefore, researchers may explore further possibilities using this technique in the management of HCV.
ARTICLE | doi:10.20944/preprints202010.0261.v1
Subject: Life Sciences, Biochemistry Keywords: Fluorescence immunoassay; sol-gel; 96-well microtiter plate; CdSe/ZnS quantum dots (QDs); progesterone
Online: 13 October 2020 (07:52:14 CEST)
In this study, two heterogeneous fluorescence immunoassays using CdSe/ZnS quantum dot (QD) to label anti-progesterone antibody (P4Ab) for the determination of progesterone (P4) were performed in the wells of a 96-well microtiter plate. First, P4Ab was conjugated to hydrophilic CdSe/ZnS QDs via ethyl-3-(dimethylaminopropyl) carbodiimide(EDC)-N-hydroxysuccinimide) chemistry(NHS) (QDs-P4Ab conjugates). The QDs-P4Ab conjugate was employed as a second antibody in a sandwich assay, where the P4Ab was immobilized onto the 3-aminopropyltrimethoxysilane (APTMS) sol-gel membrane of the wells of a 96-well microtiter plate, and P4 was bound between the immobilized P4Ab and the QDs-P4Ab conjugate. In this assay, the fluorescence intensity of the QDs increased with increasing P4 concentrations. This assay had a detection limit of 553.9 pg/ml and a sensitivity of 18,251.96 pg/ml with a linear range of 2,184.6 – 117,082 pg/ml. In the direct binding assay, P4 was directly bound to the QDs-P4Ab conjugates immobilized onto the APTMS sol-gel membrane of the wells of a 96-well microtiter plate. In this direct binding assay the fluorescence intensity of the QDs decreased with increasing P4 concentrations, and this assay had a linear range of 28.95 – 26,607.7 pg/ml with a detection limit of 3.32 pg/ml and a sensitivity of 987.24 pg/ml. These fluorescence immunoassays have been successfully applied for the determination of P4 in real human serum, and the results were well correlated with those of a certified radioimmunoassay (RIA) method.
ARTICLE | doi:10.20944/preprints201911.0087.v1
Subject: Engineering, Mechanical Engineering Keywords: fluorescence recovery after photo bleaching (FRAP); diffusion coefficient; electric charge; Poly-DADMAC; 3D hydrogel
Online: 8 November 2019 (04:16:49 CET)
The diffusion coefficients of ions are measured in a microchip filled with a cationic charged 3D hydrogel in order to study the effect of cationic charged 3D hydrogel on the diffusivity of ions. In this study, poly-diallyl-dimethyl-ammoniumchloride (poly-DADMAC) is used to produce a 3D hydrogel. Four different fluorophores are used in the 3D hydrogel rhodamine 6G, rhodamine-BSA, fluorescein isothio-cyanate (FITC) and FITC-BSA. The rhodamine 6G and rhodamine-BSA are positively charged (cations), while fluorescein isothio-cyanate (FITC) and FITC-BSA are negatively charged (anions). Two widely used techniques which are short time diffusivity measurement technique and long time diffusivity measurement techniques are used to measure the diffusion coefficients. For the short time measurement, Fluorescence recovery after photo-bleaching (FRAP) is used by a 3D confocal microscope. For the long time measurement, fluorescence images are taken for 11 days to observe a pure diffusivity without any convective movement. As a result, the diffusivity of the cations was found to be lower than that of the anions in the cationic charged hydrogel.
ARTICLE | doi:10.20944/preprints201610.0118.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: tissue-like phantoms; dual-modal imaging; fluorescence lifetime (FLT); FLT imaging (FLIM); diffusion reflection
Online: 27 October 2016 (11:52:21 CEST)
Tissue-like phantoms are widely used as a model for mimicking the optical properties of live tissue. This paper presents the results of a diffusion reflection method as well as fluorescence lifetime imaging microscopy measurements of fluorescein-conjugated gold nanorods in solution as well as inserted in solid tissue-imitating phantoms. A lack of consistency between the fluorescence lifetime results of the solutions and the phantoms raises a question about the ability of tissue-like phantoms to maintain the optical properties of inserted contrast agents.
ARTICLE | doi:10.20944/preprints202112.0228.v1
Subject: Life Sciences, Biophysics Keywords: lipofuscin; retina; retinal pigment epithelium; docosahexaenoate; docosahexaenoic acid; fluorescence; photodegradation; photobleaching; cell viability; endocytic activity
Online: 14 December 2021 (11:41:14 CET)
Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE) where its fluorescence properties are used to assess the retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE, and lipofuscin-laden-cells to visible light, and determine whether an abundant component of lipofuscin, docosahexaenoate (DHA) can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible leads to a decrease of its long-wavelength fluorescence at about 610 nm with concomitant growth of the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure to light of lipofuscin-laden cells leads to loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes of fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra together with quantitation of intensity of long-wavelength fluorescence can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, thereby useful for screening the retina for increased oxidative damage and early AMD-related changes.
Subject: Physical Sciences, Optics Keywords: surface-enhanced fluorescence; quenching; Rhodamine 6G; hot spot; separation layer; high reproducibility; finite difference time domain
Online: 22 December 2019 (13:45:00 CET)
The surface enhanced fluorescence（SEF）detection bases by plasmonic nanopillars array with nanoparticles has opened up a new gate in the application of biological imaging and sensing. The fluorescence enhancement of the probe molecule depends on its position in equilibrium, which is close to the hot spot leading to the electromagnetic field enhancement, but not too close to the metal surface resulting in quenching. Here, a large scale SiO2-Ag-cicada wing SEF substrate was fabricated by magnetron sputtering with correction enhancement factor of 797.6. Thereinto the cicada wing provides the skeleton of the nanopillars array structure, the deposited Ag constructs two kinds of hot spots, and SiO2 forms a separation layer to prevent quenching. Moreover, the substrate exhibited good reproducibility, high sensitivity with low limits of detection (LOD) and high stability for oxidation resistance. We propose that SEF substrate with modification of SiO2 can not only improve the enhancement performance, but also expanding its application in the biological investigations.
ARTICLE | doi:10.20944/preprints201909.0205.v1
Subject: Physical Sciences, Applied Physics Keywords: fluorescent gels; radio-fluorogenic (RFG) gel; tomographic fluorescence imaging; polymer-gel radiation dosimetry; 3D radiation dosimetry
Online: 18 September 2019 (12:54:24 CEST)
Radio-fluorogenic (RFG) gels become permanently fluorescent when exposed to high-energy radiation with the intensity of the emission proportional to the local dose of radiation absorbed. An apparatus is described, FluoroTome 1, that is capable of taking a series of tomographic images (thin slices) of the fluorescence of such an irradiated RFG gel on-site and within minutes of radiation exposure. These images can then be compiled to construct a 3D movie of the dose distribution within the gel. The historical development via a laboratory-bench prototype to a readily transportable, user-friendly apparatus is described. Instrumental details and performance tests are presented.
Subject: Life Sciences, Biophysics Keywords: single-molecule; fluorescence; burst; photon rate; effective detection volume; point-spread function; Brownian; diffusion; simulation; threshold.
Online: 8 July 2019 (14:55:07 CEST)
Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements.
ARTICLE | doi:10.20944/preprints202205.0187.v1
Subject: Arts & Humanities, Art History & Restoration Keywords: historic pigments; colorants; nitrocellulose; X-ray radiography; X-ray fluorescence spectroscopy; infrared Fourier transform spectroscopy; Raman spectroscopy
Online: 13 May 2022 (09:44:35 CEST)
This study applied multiple scientific approaches to establish the significance of an old work of art, Red Guitar, by examining its historical origin and the color materials used in its creation. Furthremore, the study provides thus far unknown pieces of Olga Picasso's family history to be added to her biography. Scientific approaches included digital X-ray radiography, X-ray fluorescence spectroscopy, infrared Fourier transform spectroscopy, Raman spectroscopy, and elemental thermal conductivity analysis. This combination of techniques provided broad confirmation about the time when the painting was created. The work includes colors (white, black, blue, yellow, green, red, and brown/red) and prevalent use of lead-and iron-based historic pigments Chrome Yellow, Yellow Ochre and Red Ochre. It also documents the use of unconventional materials, the colorant Pigment red 4 and nitrocellulose. This investigation led to the conclusion that the art, Red Guitar, is genuine and in accord with Picasso’s work during the first two decades of the 20th century.
REVIEW | doi:10.20944/preprints202101.0471.v1
Subject: Life Sciences, Biochemistry Keywords: aptamer; aptasensor; diagnosis; imaging; sequencing; therapeutics; probes; fluorescence; pathogenic bacteria; cancer cells; monoclonal antibodies; SELEX; nucleic acids
Online: 25 January 2021 (10:18:16 CET)
Issues presented by the application of monoclonal antibodies in diagnostic assays and as curative agents can make the use of such molecules cost-prohibitive and sometimes even unsafe. This has warranted the development of short single-stranded oligonucleotides known as Aptamers. The structural malleability of these short DNA or RNA nucleotide segments allows them to exist in distinct conformations. SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a multi-step process for synthesis of aptamers. Each step of this procedure is governed by a diverse set of factors that influence production efficiency, binding affinity, and specificity of the oligonucleotides. Headway in aptamer research has been made in recent years by the introduction of newer iterations of the SELEX process. A greater number of studies are now being carried out to incorporate aptamers into existing disease detection tools and therapies. An overview has been given first on the key aptamer properties and the process of their production (with its newer iterations), contrasting each of them with that of monoclonal antibodies. Possible manifold applications afforded due to unique aptamer characteristics are also discussed. A keen review is further provided on the design, development and use of fluorescent aptamers in bioimaging, sequencing or profiling, and treatment of pathogenic bacteria and tumor cells.
Subject: Biology, Anatomy & Morphology Keywords: Chlorophyll fluorescence; clary sage; nutrient uptake; oxidative stress; photosynthesis; phytoremediation; phytostabilization; photosynthetic pigments; phenolic content; Zn toxicity
Online: 2 December 2020 (15:36:30 CET)
The responses of the aromatic and medicinal plant Salvia sclarea (clary sage) to 900 µM Zn exposure for 8 days in a hydroponic culture were investigated. The tolerance mechanisms under excess Zn exposure were assessed by changes in nutrient uptake, photosynthetic characteristics and leaf structure. The uptake and the distribution of Zn, as well as some essential nutrient elements such as: Ca, Mg, Fe, Mn and Cu, were examined by inductively coupled plasma mass spectrometry (ICP-MS). The results revealed that Salvia sclarea is a Zn accumulator plant that tolerates significantly high toxic levels of Zn in the leaves by increasing the leaf content of Fe, Ca and Mn ions to protect the photosynthetic function and even stimulate photosystem I (PSI) and photosystem II (PSII) activities. Additionally, the leaf photosynthetic pigments and the total phenolic and anthocyanin content were also studied. Data showed that the exposure to excess Zn significantly increases the synthesis of phenolic compounds in the leaves which plays an important role in the Zn detoxification and protection against oxidative stress. Lipid peroxidation and electrolyte leakage in leaves used as clear indicators for heavy metal damage were slightly increased. All these data highlight that Salvia sclarea is an economically interesting plant for phytoextraction and/or phytostabilization of Zn contaminated soils.
ARTICLE | doi:10.20944/preprints201812.0137.v2
Subject: Life Sciences, Other Keywords: microscopy, fluorescence, machine learning, deep learning, inverse problems, image reconstruction, image restoration, super-resolution, deconvolution, spectral unmixing
Online: 5 February 2019 (10:30:40 CET)
Deep Learning is a recent and important addition to the computational toolbox available for image reconstruction in fluorescence microscopy. We review state-of-the-art applications such as image restoration, super-resolution, and light-field imaging, and discuss how the latest Deep Learning research can be applied to other image reconstruction tasks such as structured illumination, spectral deconvolution, and sample stabilisation. Despite its successes, Deep Learning also poses significant challenges, has often misunderstood capabilities, and overlooked limits. We will address key questions, such as: What are the challenges in obtaining training data? Can we discover structures not present in the training data? And, what is the danger of inferring unsubstantiated image details?
ARTICLE | doi:10.20944/preprints201901.0244.v1
Subject: Materials Science, Surfaces, Coatings & Films Keywords: XRF; X-ray fluorescence; thickness determination; thin film; simulation; XMI-MSIM; electrodeposition; Monte Carlo; galvanic industry; electroplating
Online: 24 January 2019 (08:09:27 CET)
X-ray fluorescence is largely employed in the measurement of the thickness of coatings. Despite of its diffusion, the task is not straightforward because of the complex physics involved that results in high dependence on matrix effects. Thickness quantification is in practice accomplished using the Fundamental Parameters approach, adjusted with empirical measurements of standards with known composition and thickness. This approach has two major drawbacks: i) there are no standards for any possible coating and coating architecture and ii) even relying on standards, the quantification of unknown samples requires the precise knowledge of the matrix nature (e.g., in case of multilayer coatings the thickness and the composition of each underlayer). In this work, we describe a semiquantitative approach to coatings thickness measurement based on the construction of calibration curves through simulated XRF spectra built with Monte Carlo simulations. Simulations have been performed with the freeware software XMI-MSIM. We have assessed the accuracy of the methods by comparing the results with those obtained by i) XRF thickness determination with standards and ii) FIB-SEM cross-sectioning. Then we evaluated which parameters are critical in this kind of indirect thickness measurements.
ARTICLE | doi:10.20944/preprints201810.0456.v1
Subject: Life Sciences, Biochemistry Keywords: chlorophyll fluorescence; J8-1 plum line; mandelonitrile; Prunus domestica; redox signalling; salicylic acid; salt-stress; soluble nutrients
Online: 19 October 2018 (14:57:02 CEST)
Salinity is considered as one of the most important abiotic challenges that affect crop productivity. Plant hormones, including salicylic acid (SA), are key factors in the defence signalling output triggered during plant responses against environmental stresses. We have previously reported in peach a new SA biosynthetic pathway from mandelonitrile (MD), the molecule at the hub of the cyanogenic glucoside turnover in Prunus sp. In this work, we have studied whether this new SA biosynthetic pathway is also present in plum and the possible role this pathway plays in plant plasticity under salinity, focusing on the transgenic plum line J8-1, which displays stress tolerance via an enhanced antioxidant capacity. The SA biosynthesis from MD in non-transgenic and J8-1 micropropagated plum shoots was studied by metabolomics. Then the response of J8-1 to salt stress in presence of MD or Phe (MD precursor) was assayed by measuring: chlorophyll content and fluorescence parameters, stress related hormones, levels of non-enzymatic antioxidants, the expression of two genes coding redox-related proteins, and the content of soluble nutrients. The results from in vitro assays suggest that the SA synthesis from the MD pathway demonstrated in peach is not clearly present in plum, at least under in vitro conditions. Nevertheless, in J8-1 NaCl-stressed seedlings, an increase in SA was recorded as a result of the MD treatment, suggesting that MD could be involved in the SA biosynthesis under NaCl stress conditions in plum plants. We have also shown that the plum line J8-1 was tolerant to NaCl under greenhouse conditions, and this response was somewhat different in MD-treated plants. In that regards, the MD treatment produced an increase in SA, jasmonic acid (JA) and reduced ascorbate (ASC) contents as well as in the coefficient of non-photochemical quenching (qN) and the gene expression of Non-Expressor of Pathogenesis-Related 1 (NPR1) and thioredoxin H (TrxH) under salinity conditions, suggesting a crosstalk between different signalling pathways (NPR1/Trx and SA/JA) leading to salinity tolerance in the transgenic plum line J8-1.
ARTICLE | doi:10.20944/preprints201705.0167.v1
Subject: Chemistry, Organic Chemistry Keywords: N-n-Butyl-4-(N′,N′-dihydroxyethylamino)-1,8-naphthalimide; mono-substituted; di-substituted; synthesis, crystal structure; fluorescence
Online: 23 May 2017 (08:10:02 CEST)
A series of novel N-n-butyl-1,8-naphthalimide derivatives were synthesized via a three-step reaction involving nucleophilic substitution and acylation. All of the compounds were characterized by IR, 1H NMR, 13C NMR, MS, and elemental analysis, and the crystal structure of N-n-butyl-4-[N’,N’-bis(2`,4`-dichlorobenzoyl)ethylamino]-1,8-naphthalimide was determined. The π-π stacking interactions and hydrogen bonds between the two molecular core planes (naphthalimide ring) and the van der Waals forces between the flexible n-butyl groups resulted in a 3D long-chain structure. The UV-vis and fluorescence properties of the title compounds were investigated. The results indicated that the monosubstituted 1,8-naphthalimide derivatives bearing an electron-donating group on the benzene ring or a structure with a larger conjugative effect exhibited enhanced fluorescence properties.
ARTICLE | doi:10.20944/preprints202107.0521.v1
Subject: Chemistry, Analytical Chemistry Keywords: Online detection, security; biosensor; flow injection assay; monoclonal antibody; fluorescence microscope; lab-on-a-chip; microfluidic systems; antibody labeling; CMOS; diode laser; monolithic column; laser-induced fluorescence detector (LIF); low-cost; high-speed; non-competitive immunoassay; immunometric assay
Online: 22 July 2021 (14:13:46 CEST)
The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.
ARTICLE | doi:10.20944/preprints202009.0554.v1
Subject: Earth Sciences, Environmental Sciences Keywords: Remote Sensing; Ocean Colour; Retrievals; Fluorescence; Optical Properties; Satellite; Spectral; Radiative Transfer; optically complex waters; chlorophyll; absorption; scattering
Online: 23 September 2020 (16:39:53 CEST)
The retrieval of sun-induced chlorophyll fluorescence is greatly beneficial to studies of marine phytoplankton biomass, physiology, and composition and is required for user applications and services. Customarily phytoplankton chlorophyll fluorescence is determined from satellite measurements through a fluorescence line-height algorithm using three bands around 680 nm. We propose here a modified retrieval, making use of all available bands in the relevant wavelength range with the goal to improve the effectiveness of the algorithm in optically complex waters. For the Ocean and Land Colour Instrument (OLCI) we quantify a Fluorescence Peak Height from fitting a Gaussian function and related terms into the top-of-atmosphere reflectance bands between 650 and 750 nm. This algorithm retrieves, what we call Fluorescence Peak Height from fitting a Gaussian function upon other terms to top-of-atmosphere reflectance bands between 650 and 750 nm. This approach is applicable to Level-1 and Level-2 data. We find a good correlation of the retrieved fluorescence product to global in-situ chlorophyll measurements, as well as a consistent relation between chlorophyll concentration and fluorescence from radiative transfer modelling and OLCI/in-situ comparison. The algorithm is applicable to complex waters without needing an atmospheric correction and vicarious calibration and features an inherent correction of small spectral shifts, as required for OLCI measurements.
ARTICLE | doi:10.20944/preprints201801.0221.v1
Subject: Life Sciences, Virology Keywords: Zika virus; nucleic acid detection; micro-droplet digital polymerase chain reaction; real-time fluorescence quantitative polymerase chain reaction
Online: 24 January 2018 (04:21:18 CET)
Establishment of diagnostic methods with low detection limits plays a critical role in the maintenance of early diagnosis, prevention of serious neurological complications, and control of the spread of ZIKA. In this study, we established the micro-droplet digital polymerase chain reaction (ddPCR) and real-time fluorescent quantification PCR (qPCR) protocols for the detection of Zika virus based on the NS5 gene. For the Zika standard plasmid, the standard curve of R2 was 0.999, and the amplification efficiency was 92.203%, as determined by qPCR. Both ddPCR and qPCR were positive for cell culture of Zika nucleic acid.The minimum detection limit of ddPCR is 1–2 times lower than qPCR. Moreover, all tests of Dengue virus (1–4 serotypes) were negative in cell culture. Overall, these results suggested than ddPCR may have a lower limit of detection than qPCR.
ARTICLE | doi:10.20944/preprints201704.0007.v1
Subject: Earth Sciences, Environmental Sciences Keywords: High Tatras; trampling; spectroscopy; vegetation indices; fluorescence; Juncus trifidus; Agrostis rupestris; Luzula alpino-pilosa; Oreochloa disticha; Festuca picta
Online: 3 April 2017 (16:44:42 CEST)
This research focuses on the effect of trampling on vegetation in high-mountain ecosystems through the electromagnetic spectrum’s interaction with plant pigments, cell structure, water content and other substances that have a direct impact on leaf properties. The most heavily visited part of the High Tatras in Poland was divided into polygons and, after selecting the dominant species within alpine swards, a detailed analysis of trampled and reference patterns was performed. An ASD FieldSpec 3 was used to acquire high-resolution spectral properties of plants, their fluorescence and the leaf chlorophyll content with the ts-ta temperature index and fraction of accumulated radiation in the range of photosynthesis (fAPAR) used as reference data. The results show that, along tourist trails, vegetation adapts to trampling with the impact depending on the species. A lower chlorophyll value was confirmed by a decrease in fluorescence, and the state of cellular structures was degraded in trampled compared to reference species, with a lower leaf reflectance. Also, at the extreme, trampling can eliminate certain species such as Luzula alpino-pilosa.
ARTICLE | doi:10.20944/preprints202206.0359.v1
Subject: Medicine & Pharmacology, Pathology & Pathobiology Keywords: Kaposi’s sarcoma-associated virus (KSHV); Viral pre-initiation complex (vPIC); Bimolecular fluorescence complementation (BiFC); Artificial intelligence (AI) structure prediction; AlphaFold2
Online: 27 June 2022 (10:15:34 CEST)
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the characterization of the protein-protein interactions of the vPIC factors has not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral factors has not been sufficiently revealed. Here, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Gln36, His41 and Trp170) on ORF18 that were responsible for its interaction with ORF30. The artificial intelligence (AI) system AlphaFold2 predicted that identified four residues are exposed to the surface of ORF18 and located in proximity to each other in the surface of ORF18. Thus, AI-predicted model supports the importance of four residues for binding of ORF18 to ORF30. Our results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.
REVIEW | doi:10.20944/preprints202105.0098.v1
Subject: Life Sciences, Virology Keywords: Campenot, neurons, superior cervical ganglia, dorsal root ganglia, virus, alphaherpesvirus, herpes simplex virus, pseudorabies virus, fluorescence microscopy, cryo electron tomography
Online: 6 May 2021 (15:08:12 CEST)
The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: 1. Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. 2. Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.
ARTICLE | doi:10.20944/preprints201812.0173.v2
Subject: Chemistry, Organic Chemistry Keywords: 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile; X-ray diffraction; DFT; molecular orbital calculations; fluorescence; docking; nNOS; fluorescent ligand
Online: 14 December 2018 (11:41:20 CET)
2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) has been identified as a neurobiological fluorescent ligand that may be used to develop receptor and enzyme binding affinity assays. Compound 1 was synthesised using an optimised microwave irradiation reaction and crystallised from ethanol. Crystallization occurred in the orthorhombic space group P212121 with unit cell parameters: a = 6.4487(12) Å, b = 13.648(3) Å, c = 16.571(3) Å, V = 1458(5) Å3, Z = 4. Density functional theory (DFT) (B3LYP/6-311++G (d,p)) calculations of 1 were carried out. Results showed that the optimised geometry is similar to the crystal structure parameters with a root-mean-squared deviation of 0.143 Å. Frontier molecular orbital energies and net atomic charges are discussed with a focus on potential biological interactions. Docking experiments within the active site of the neuronal nitric oxide synthase (nNOS) protein crystal structure were carried out and analysed. Important binding interactions between the DFT optimised structure and amino acids within the nNOS active site were identified that explain the strong NOS binding affinity reported. Fluorescent properties of 1 were studied using aprotic solvents of different polarities. Compound 1 showed the highest fluorescence intensity in polar solvents with excitation and emission values of 336 nm and 380 nm, respectively.
ARTICLE | doi:10.20944/preprints201808.0298.v1
Subject: Biology, Physiology Keywords: Heterodimeric G protein coupled receptor; saturation transfer difference nuclear magnetic resonance spectroscopy; differential scanning calorimetry; circular dichroism; intrinsic fluorescence spectroscopy
Online: 17 August 2018 (11:15:39 CEST)
Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPRC) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to inadequate methods for producing large homogeneous quantities of the taste-receptor protein and a lack of reliable in vitro assays to precisely measure productive ligand binding modes leading to activity upon their interactions with the receptor protein. We report a multimodal high throughput assays to monitor ligand binding, receptor stability and conformational changes to model the molecular interactions between ligand-receptor. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.
ARTICLE | doi:10.20944/preprints202011.0283.v1
Subject: Biology, Anatomy & Morphology Keywords: Salvia sclarea; chlorophyll fluorescence imaging; non-photochemical quenching; tolerance mechanism; photoprotective mechanism; photosynthesis; reactive oxygen species; toxicity; adaptive response, photochemical efficiency
Online: 9 November 2020 (16:06:01 CET)
Five-day exposure of clary sage (Salvia sclarea) to 100 μM cadmium (Cd) in hydroponics was sufficient to increase Cd concentrations significantly in roots and aboveground parts and affect negatively whole plant levels of calcium (Ca) and magnesium (Mg), since Cd competes for Ca channels, while reduced Mg concentrations are associated with increased Cd tolerance. Total zinc (Zn), copper (Cu), and iron (Fe) uptake increased but their translocation to the aboveground parts decreased possible due to translocation barriers. Despite the substantial levels of Cd in leaves, without any observed defects on chloroplast ultrastructure, an enhanced photosystem II (PSII) efficiency was observed, with a higher fraction of absorbed light energy to be directed to photochemistry (ΦPSΙΙ). The concomitant increase in the photoprotective mechanism of non-photochemical quenching of photosynthesis (NPQ) resulted in an important decrease in the dissipated non-regulated energy (ΦNO), modifying the homeostasis of reactive oxygen species (ROS), through a decreased singlet oxygen (1O2) formation. Thus, when clary sage was exposed to Cd for a short period, tolerance mechanisms were triggered, with PSII photochemistry to be regulated by NPQ in such a way that PSII efficiency to be enhanced. However, exposure to a combination of Cd and high light or for longer duration (8 days) to Cd alone, resulted in an inhibition of PSII functionality pointing out towards Cd toxicity. Thus, the rapid activation of PSII functionality at short time exposures and the inhibition at longer duration suggests a hormetic response and describes these effects in terms of “adaptive response” and “toxicity”, respectively.
ARTICLE | doi:10.20944/preprints202010.0392.v1
Subject: Medicine & Pharmacology, Allergology Keywords: antimicrobial stewardship; detection of bacteria in chronic wounds, chronic wounds, clinical decision support; diagnostic pathway; joint commission; fluorescence imaging; wound clinic
Online: 19 October 2020 (15:35:25 CEST)
Background: In 2014 the World Health Organization (WHO) warned of an emerging world-wide crisis of antibiotic resistant microorganisms. In response, government and professional organizations recommended that health care systems adopt antimicrobial stewardship programs (ASPs). In the United States, the Centers for Medicare Services (CMS) mandated antimicrobial stewardship in the hospital inpatient setting. Effective January 1, 2020, the Joint Commission required ambulatory centers that prescribe antibiotics, such as wound centers, to institute an ASP. Chronic wounds often remain open for months, during which time patients may receive multiple courses of antibiotics and numerous antimicrobial topical treatments. The wound clinician plays an integral role in reducing antimicrobial resistance in the outpatient setting: antibiotics prescribed for skin and soft tissue infections are among the most common in an outpatient setting. One of the most challenging aspects of antimicrobial stewardship in treating chronic wounds is the inaccuracy of bacterial and infection diagnosis. Methods: Joint Commission lists five elements of performance (EP): (1) Identifying an antimicrobial stewardship leader, (2) establishing an annual antimicrobial stewardship goal, (3) implementing evidence-based practice guidelines related to the antimicrobial stewardship goal, (4) providing clinical staff with educational resources related to the antimicrobial stewardship goal, and (5) collecting, analyzing, and reporting data related to the antimicrobial stewardship goal. This article focuses on choosing and implementing an evidence-based ASP goal for 2020. Discussion: Clinical trials have demonstrated the ability of fluorescence imaging (MLiX) to detect clinically significant levels of bacteria in chronic wounds. Combined with clinical examination of signs and symptoms of infection, the MLiX procedure improves the clinician’s ability to diagnose infection and can guide antimicrobial use. In order to satisfy the elements of performance, the MLiX procedure was incorporated into the annual ASP goal for several wound care centers. Clinicians were educated on the fluorescence imaging device and guidelines were instituted. Collection of antimicrobial utilization data is underway.
Subject: Physical Sciences, Applied Physics Keywords: ECR plasmas; warm electrons; self-consistent simulations; experimental benchmarking; volumetric and space-resolved spectra; X-ray fluorescence; bremsstrahlung; ray-tracing Monte Carlo techniques
Online: 5 October 2021 (08:58:06 CEST)
A quantitative analysis of X-ray emission from an electron cyclotron resonance (ECR) plasma was performed to probe the spatial properties of electrons having energy for effective ionisation. A series of measurements were taken by INFN-LNS and ATOMKI, capturing spatially and spectrally resolved X-ray maps as well as volumetric emissions from argon plasma. Comparing the former with model generated maps (involving space-resolved phenomenological electron energy distribution function and geometrical efficiency calculated using ray-tracing MC routine) furnished information on structural aspects of the plasma. Similarly, fitting a model composed of bremsstrahlung and fluorescence to the volumetric X-ray spectrum provided valuable insight into the density and temperature of confined and lost electrons. The latter can be fed back to existing electron kinetics models for simulating more relevant energies, consequently improving theoretical X-ray maps and establishing the method as an excellent indirect diagnostic tool for warm electrons, required for both fundamental and applied research in ECR plasmas.
ARTICLE | doi:10.20944/preprints202201.0380.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: Therapeutic biomarkers; circulating tumor cells; Immunocytochemistry; targeted therapy; Fluorescence in situ hybridization (FISH); Immune Checkpoint Inhibitors; Liquid Biopsy; Hormonal Treatments; Breast Cancer; Lung Cancer
Online: 25 January 2022 (11:31:05 CET)
Biomarker directed selection of targeted anti-neoplastic agents such as immune check-point inhibitors, small molecule inhibitors and monoclonal antibodies form an important aspect of cancer treatment. Immunohistochemistry (IHC) analysis of the tumor tissue is the method of choice to evaluate the presence of these biomarkers. However, a significant barrier to biomarker testing on tissue is the availability of an adequate amount of tissue and need for repetitive sampling due to tumor evolution. Also, tumor tissue testing is not immune to inter- and intra-tumor heterogeneity. We describe the analytical and clinical validation of a Circulating Tumor Cell (CTC) assay to accurately assess the presence of PD-L1 22C3 and PD-L1 28.8, ER, PR and HER2, from patients with solid tumors to guide the choice of suitable targeted therapies. Analytically, the test has high sensitivity, specificity, linearity and precision. Based on a blinded case control study, the clinical sensitivity and specificity for PD-L1 (22C3 and 28.8) was determined to be 90% and 100% respectively. The clinical sensitivity and specificity was 83% and 89% for ER; 80% and 94% for PR; 63% and 89% for HER2 (by ICC); and 100% and 92% for HER2 (by FISH), respectively. The performance characteristics of the test support its suitability and adaptability for routine clinical use.
ARTICLE | doi:10.3390/sci2020046
Subject: Keywords: portable X-ray fluorescence spectrometer (pXRF); Potential Toxic Elements (PTE); lead (Pb), zinc (Zn); copper (Cu); topsoil; sustainable urban drainage systems; SuDS; LID; BMPs; WSUD; GI; SCMs
Online: 13 June 2020 (00:00:00 CEST)
Sustainable urban drainage systems (SuDS) such as swales are designed to collect, store and infiltrate a large amount of surface runoff water during heavy rainfall. Stormwater is known to transport pollutants, such as particle-bound Potential Toxic Elements (PTE), which are known to often accumulate in the topsoil. A portable XRF instrument (pXRF) is used to provide in situ spatial characterization of soil pollutants, specifically lead (Pb), zink (Zn) and copper (Cu). The method uses pXRF measurements of PTE along profiles with set intervals (1 meter) to cover the swale with cross-sections, across the inlet, the deepest point and the outlet. Soil samples are collected, and the In-Situ measurements are verified by the results from laboratory analyses. Stormwater is here shown to be the transporting media for the pollutants, so it is of importance to investigate areas most prone to flooding and infiltration. This quick scan method is time and cost-efficient, easy to execute and the results are comparable to any known (inter)national threshold criteria for polluted soils. The results are of great importance for all stakeholders in cities that are involved in climate adaptation and implementing green infrastructure in urban areas. However, too little is still known about the long-term functioning of the soil-based SuDS facilities.
ARTICLE | doi:10.20944/preprints202105.0480.v1
Subject: Life Sciences, Biochemistry Keywords: camptothecin; drug distribution; drug-membrane interaction; biophysical profiling; biomimetic models; partition coefficient; ADMET/PK prediction; small and wide-angle X-ray diffraction; fluorescence spectroscopy; human serum albumin (HSA)
Online: 20 May 2021 (10:52:33 CEST)
Camptothecin (CPT) is a potent anticancer drug, and its putative oral administration is envisioned although difficult due to physiological barriers that must be overcome. A comprehensive biophysical analysis of CPT interaction with biointerface models can be used to predict some pharmacokinetic issues after oral administration of this or other drugs. To that end, different models were used to mimic the phospholipid composition of normal, cancer, and blood-brain barrier endothelial cell membranes. The logD values obtained indicate that the drug is well distributed across membranes. CPT-membrane interaction studies also confirm the drug’s location at the membrane cooperative and interfacial regions. The drug can also permeate membranes at more ordered phases by altering phospholipid packing. The similar logD values obtained in membrane models mimicking cancer or normal cells imply that CPT has limited selectivity to its target. Furthermore, CPT binds strongly to serum albumin, leaving only 8.05% of free drug available to be distributed to the tissues. The strong interaction with plasma proteins, allied to the large distribution (VDSS=5.75 ± 0.932 L·Kg-1) and tendency to bioaccumulate in off-target tissues, were predicted to be pharmacokinetic issues of CPT, implying the need to develop drug delivery systems to improve its biodistribution.
ARTICLE | doi:10.20944/preprints201812.0185.v1
Subject: Medicine & Pharmacology, Obstetrics & Gynaecology Keywords: Fluorescence in situ hybridization (FISH), Karyotype, array comparative genomic hybridization (aCGH), amniotic fluid (AF), chorionic villus sampling (CVS), aneuploidies, pathogenic copy number variants (pCNV), confined placental mosaicism (CPM), true fetal mosaicism (TFM), pseudo-mosaicism.
Online: 17 December 2018 (09:58:43 CET)
Current prenatal genetic evaluation showed a significantly increase in non-invasive screening and the reduction of invasive diagnostic procedures. To evaluate the diagnostic efficacy on detecting common aneuploidies, structural chromosomal rearrangements and pathogenic copy number variants (pCNV), we performed a retrospective analysis on a case series initially analyzed by aneuvysion fluorescence in situ hybridization (FISH) and karyotyping then followed by array comparative genomic hybridization (aCGH). Of the 386 cases retrieved from the past decade, common aneuploidies were detected in 137 cases (35.5%), other chromosomal structural rearrangements were detected in four cases (1%), and pCNV were detected in five cases (1.3%). The relative frequencies for common aneuploidies suggested a under detection of sex chromosome aneuploidies. Approximately 9.5% of cases with common aneuploidies showed a mosaic pattern. Inconsistent results between FISH and karyotyping were noted in cases with pseudo-mosaicism introduced by culture artifact or variable cellular proliferation from cells with mosaic karyotypic complements under in vitro cell culture. Based on findings from this case series, cell-based FISH and karyotyping should be performed to detect common aneuploidies, structural chromosomal abnormalities, and mosaic pattern. DNA-based aCGH and reflex FISH should be performed to detect and confirm genomic imbalances and pCNV. Practice points to ensure the diagnostic accuracy and efficacy were summarized.
ARTICLE | doi:10.20944/preprints202104.0294.v1
Subject: Chemistry, Analytical Chemistry Keywords: Dimeric copper(I) complexes; PN phosphine ligands; X-ray structures; Combined thermally activated delayed fluorescence (TADF) and phosphorescence; Combined singlet and triplet harvesting; High emission quantum yields; Tunability of photophysical properties; Zero-field splitting (ZFS), Spin-lattice relaxation (SLR); Triplet substate decay components.
Online: 12 April 2021 (12:41:01 CEST)
We present an overview over eight brightly luminescent Cu(I) dimers of the type Cu2X2(PN)3 with X = Cl, Br, I and P^N = 2-diphenylphosphino-pyridine (Ph2Ppy), 2-diphenylphosphino-pyrimidine (Ph2Ppym), 1-diphenylphosphino-isoquinoline (Ph2Piqn) including three new crystal structures (Cu2Br2(Ph2Ppy)3, 1-Br, Cu2I2(Ph2Ppym)3, 2-I, and Cu2I2(Ph2Piqn)3, 3-I). However, we mainly focus on their photo-luminescence properties. All compounds exhibit combined thermally activated delayed fluorescence (TADF) and phosphorescence at ambient temperature. Emission color, decay time, and quantum yield varies over large ranges. For deeper characterization, we select Cu2I2(Ph2Ppy)3, 1-I, showing a quantum yield of 81 %. DFT and SOC-TDDFT calculations provide insight into the electronic structures of the singlet S1 and triplet T1 states. Both stem from metal+iodide-to-ligand charge transfer transitions. Evaluation of the emission decay dynamics, measured from 1.2 ≤ T ≤ 300 K, gives ∆E(S1-T1) = 380 cm-1 (47 meV), a transition rate of k(S1→S0) = 2.25×106 s-1 (445 ns), T1 zero-field splittings, transition rates from the triplet substates, and spin-lattice relaxation times. We also discuss the interplay of S1-TADF and T1-phosphorescence. The combined emission paths shorten the overall decay time. For OLED applications, utilization of both singlet and triplet harvesting can be highly favorable for improvement of the device performance.