Unique Fluorescence Signatures Research Articles

Determination of the Course of Cyan Fluorescence of Pseudomonas aeruginosa with a Handheld Bacterial Imaging Device.

Chronic wound infections are of clinical concern as they often lead to high rates of mortality and morbidity. A point-of-care handheld bacterial fluorescence imaging has been designed to detect the auto-fluorescent characteristics of most clinically relevant species of bacteria. This device causes most species of bacteria to exhibit red fluorescence due to the production of exoproduct porphyrins. One of the most significant contributors to the pathogenicity of chronic wounds is the pathogen Pseudomonas aeruginosa, and interestingly, this organism exhibits an additional unique cyan fluorescence signature. There is an over 90% positive predictive value that, when a chronic wound exhibits cyan fluorescence with the bacterial fluorescence imaging device, the wound will harbor P. aeruginosa. This project seeks to understand what genetic factor(s) contribute to the cyan phenotype observed.

Multi-wavelength Fluorescence Monitoring of Faecal Contamination in Waters: A Laboratory-based Quantification

Near real-time monitoring of faecal indicator bacteria (FIB) in waters is currently not feasible, and current monitoring methods require field sampling and laboratory testing that inhibits decision-making within a relevant timeframe. While recent studies identified the potential of using specific fluorescence regions for FIB monitoring, sufficient accuracy often requires site-specific calibration due to minor variations in fluorescence peak locations. In this study, a series of lab experiments were completed to address some of the selectivity issues. Specifically, the study explored correlations between wavelength-specific fluorescence signals acquired through fluorescence excitation-emission matrices (EEM) and the amount of E. coli K-12 (E. coli) and E. faecalis (enterococci) in exponential and stationary phase broth cultures. Subsequently, the experiments quantified how the addition of known concentrations of L-tryptophan amplifies an indole pulse, specifically its concentration and the corresponding fluorescence properties. Results show unique peak excitation/emission (λex/λem) wavelengths (± 5 nm) in EEMs for E. coli cell pellet and in M9 broth (~ 280/ ~ 327 nm), enterococci cell pellet (~ 276/ ~ 324 nm), L-tryptophan (~ 278/ ~ 343 nm and ~ 298/ ~ 344 nm), and indole (~ 232/ ~ 321 nm). The findings demonstrate that L-tryptophan concentrations in E. coli broth were reduced. At the same time, the indole content increased throughout the initiation phase to the stationary phase of the bacteria growth curve, with the peak indole pulse occurring approximately at the time of transition from the exponential to stationary phase. Such unique fluorescence signatures for not only FIB but also indole (whose pulse can be triggered by L-tryptophan) provide foundations for developing reliable and near real-time in situ FIB sensors.

Instant identification of hydrocarbon spill types using laser-induced fluorescence and associated hyperspectral imaging

Marine hydrocarbon (HC) spills seriously harm the marine ecosystem and necessitate the creation of effective cleanup strategies. For efficient treatment, it is crucial to accurately identify the type of HC implicated in the spill. Diesel oil, gasoline, lubrication oil, and waste oil were chosen as the experimental starting materials since they are prevalent HC kinds. This study reports on the facile differentiation of diesel fuel and gasoline to engine oil via induced fluorescence by a laser. The tested material was stimulated with a 450-nm blue laser source. The fluorescence characteristic of each tested HC spill sample was captured via a hyperspectral imaging camera. Diesel fuel could be identified via their unique fluorescence signatures at 470 nm and 515 nm. Gasoline was found to be a less photoluminescent active material, with characteristic signals at 465 nm and 585 nm. At 505 nm, lubricating oil had the typical fluorescence signal. On the whole study spectrum, there are no re-emitted frequencies for the waste oil. Fluorescence signals secured good discrimination between fuel and engine oil of HC chemical structures. Signal intensity can contribute further discrimination. Fluorescence intensity at selected photoluminescence wavelengths secured further grouping between fuel and oil. Our imaging approach would provide investigators with the ability to define the HC and choose a mitigation strategy for fast HC spill identification. Aerial systems with a customized common camera that only functions at a certain wavelength filter with our methodology based on laser-induced fluorescence may be effectively developed for HC spill detecting applications.

High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding.

The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.

Multiplex Quantitative Polymerase Chain Reaction Test to Identify SARS-CoV-2 Variants.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes which fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here we describe a method using the Inhibitor-Tolerant RT-qPCR kit, developed by Meridian Bioscience kit which allows simultaneous real-time quantitation of the UK, South Africa, and Brazil SARS-CoV-2 variants.

A Microscopic and Metabolomic Description of Stip-affected Tissue in New Mexico Pod-type Pepper

Stip is a physiological disorder that affects certain pepper (Capsicum annuum) cultivars, most notably bell-pod types. It has been attributed in the literature to nutrient imbalances, temperature extremes, and/or other environmental stressors. Symptoms present as brown, black, and yellow ovoid-shaped necrotic lesions ≈0.5 to 1.2 cm long by 0.5 cm wide. Between 2014 and 2015, symptomatic and asymptomatic pods were harvested from 15 commercial farms in southern New Mexico. Fluorescent microscopy comparisons of harvested symptomatic tissue revealed a unique fluorescent signature and the absence of chlorophyll. A new spectral peak centered around 560 nm was observed in symptomatic tissue. High-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS) analyses of these tissues detected significant differences in 13 metabolites, of which several have been associated with fruit maturation and/or senescence. This report represents the first combination of a detailed microscopic description and metabolite profile of field-grown symptomatic plants with this disorder.

Directly Quantifiable Biotinylation Using a Water-Soluble Isatoic Anhydride Platform.

Isatoic anhydride (IA) has been shown to be a useful platform for quantifiable bioconjugation. The elaboration of a water-soluble isatoic anhydride-based platform with biotin offers readily quantifiable biotinylation reagents through nondestructive methods of quantification. The incorporation of functionality is directly quantified using the reagent's unique absorbance or fluorescence signature, located outside the biological window. Several biotinylation reagents are prepared with various linker lengths, and the quantification of biotinylated proteins is demonstrated and compared to results from the traditional HABA assay.

Multiplexed optical barcoding of cells via photochemical programming of bioorthogonal host-guest recognition.

Modern chemical and biological studies are undergoing a paradigm shift, where understanding the fate of individual cells, in an apparently homogeneous population, is becoming increasingly important. This has inculcated a growing demand for developing strategies that label individual cells with unique fluorescent signatures or barcodes so that their spatiotemporal trajectories can be mapped in real time. Among various approaches, light-regulated methods employing photocaged fluorophores have received particular attention, owing to their fine spatiotemporal control over labelling. However, their multiplexed use to barcode large numbers of cells for interrogating cellular libraries or complex tissues remains inherently challenging, due to the lack of multiple spectrally distinct photoactivated states in the currently available photocaged fluorophores. We report here an alternative multiplexable strategy based on optically controlled host–guest recognition in the cucurbit[7]uril (CB[7]) system that provides spatial control over the positioning of fluorophores to generate distinct barcodes in ‘user-defined’ cells. Using a combination of three spectrally distinct CB[7]-conjugated fluorophores and by sequentially performing cycles of photoactivation and fluorophore encoding, we demonstrate 10-color barcoding in microtubule-targeted fixed cells as well as 7-color barcoding in cell surface glycan targeted live MCF7 cells.

DdRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform

Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a “one-color-one-target” approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.

Highly Responsive Fluorescent Assemblies Allow for Unique, Multiparametric Sensing of the Phospholipid Membrane Environment.

Despite decades-long extensive research, probes that provide a comprehensive description of the lipid membrane microenvironment are still lacking. Here, a "smart" pyrene-terpyridine probe for multiparametric sensing of lipid membranes is reported. The complexity of the associated local microenvironment can be described by the distinct features of the probe fluorescence. The self-assembly of the probe molecules in phospholipid bilayers was sensitive to membrane order and phase state. The self-assembled probes showed a unique emission, influenced by dye-dye interactions and excited-state charge transfer. Moreover, this emission was sensitive to interfacial hydration, with very specific changes in emission wavelengths and fluorescence lifetimes upon variation of lipid compositions and properties. In parallel, changes in the lipid order and hydration affected the ground-state interactions in the dye aggregates and, thus, could be measured through ratiometric changes in the excitation and emission readouts. In addition, fluorescence anisotropy measurements provided another way to study the nature of dye aggregates in lipid bilayers. Overall, this report demonstrates how multiple aspects of the membrane microenvironment can be sensed through the unique fluorescence signatures of this "smart" probe in lipid membranes, and it establishes a new paradigm in lipid-membrane sensing.

Unity Makes Strength: How Aggregation‐Induced Emission Luminogens Advance the Biomedical Field

AbstractFluorescent bioprobes play an important role in preclinical and clinical practice. During the past few years, a distinguished type of organic luminogen with aggregation‐induced emission (AIE) characteristics has shown great potential in construction of advanced fluorescent bioprobes for biological process monitoring and disease theranostics. The AIE luminogens (AIEgens) possess high brightness, excellent photostability, low toxicity, and tailorable functionality, catering to a variety of biomedical tasks. Thanks to the unique fluorescent signature, these AIEgens are ideal candidates to design stimuli‐responsive light‐up probes and highly emissive fluorescent nanoparticles. This review especially focuses on the exploration of smart/functional AIEgen‐based bioprobes for biomedical studies to promote the development of this promising research field. The recent advances of such bioprobes in specific biomedical applications in terms of bacteria imaging and suppressing, targeted cellular/intracellular organelle imaging and ablation, as well as in vivo blood vessel imaging and disease theranostics are summarized. It is hoped that the review will not only provide insights into the design and preparation of advanced bioprobes, but also inspire greater enthusiasm from the researchers working in biomedicines and life sciences to pay attention to and use AIEgens.

High-Throughput Signaling Profiling in Blood Platelets by Multiplexed Phosphoflow Cytometry.

Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples. These uniquely labeled samples can be combined for simultaneous antibody staining and acquisition. During software analysis, multiplexed samples can be differentiated by their distinct fluorescence intensities and analyzed as if they had been acquired individually. Multiplexing eliminates intersample variation, increases statistical robustness, and allows 4-96 samples to be processed with no appreciable increase in antibody consumption or runtime. The method can be performed on washed platelets, platelet-rich plasma (PRP), and whole blood. Its inherent versatility can fulfil wide-ranging experimental requirements from simple dose titrations to complex pharmacologic screens.

Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes.

The discovery of cell division tracking properties of 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE) by Lyons and Parish in 1994 led to a broad range of new methods and numerous important biological discoveries. After labeling, CFSE is attached to free amine groups and intracellular proteins in the cytoplasm and nucleus of a cell, and halves in fluorescence intensity with each round of cell division, enabling enumeration of the number of divisions a cell has undergone. A range of popular division tracking dyes were subsequently developed, including CellTrace Violet (CTV), making available the green fluorescent channel previously occupied by CFSE. More recently, CellTrace Yellow (CTY) and CellTrace Far Red (CTFR), each with unique fluorescence properties, were introduced. In a comparison, we found that the fluorescence values of both dyes were well separated from autofluorescence, and enabled a greater number of divisions to be identified than CTV, before this limit was reached. These new dyes provided clear and well‐separated peaks for both murine and human B lymphocytes, and should find wide application. The range of excitation/emission spectra available for division tracking dyes now also facilitates multiplexing, that is, the labeling of cells with different combinations of dyes to give a unique fluorescence signature, allowing single cell in vitro and in vivo tracking. The combinatorial possibilities are significantly increased with these additional dyes.

Chlorophyll Fluorescence Imaging Uncovers Photosynthetic Fingerprint of Citrus Huanglongbing.

Huanglongbing (HLB) is one of the most destructive diseases of citrus, which has posed a serious threat to the global citrus production. This research was aimed to explore the use of chlorophyll fluorescence imaging combined with feature selection to characterize and detect the HLB disease. Chlorophyll fluorescence images of citrus leaf samples were measured by an in-house chlorophyll fluorescence imaging system. The commonly used chlorophyll fluorescence parameters provided the first screening of HLB disease. To further explore the photosynthetic fingerprint of HLB infected leaves, three feature selection methods combined with the supervised classifiers were employed to identify the unique fluorescence signature of HLB and perform the three-class classification (i.e., healthy, HLB infected, and nutrient deficient leaves). Unlike the commonly used fluorescence parameters, this novel data-driven approach by using the combination of the mean fluorescence parameters and image features gave the best classification performance with the accuracy of 97%, and presented a better interpretation for the spatial heterogeneity of photochemical and non-photochemical components in HLB infected citrus leaves. These results imply the potential of the proposed approach for the citrus HLB disease diagnosis, and also provide a valuable insight for the photosynthetic response to the HLB disease.

One-Step Identification of Antibody Degradation Pathways Using Fluorescence Signatures Generated by Cross-Reactive DNA-Based Arrays.

Therapeutic antibodies are prone to degradation via a variety of pathways during each stage of the manufacturing process. Hence, a low-cost, rapid, and broadly applicable tool that is able to identify when and how antibodies degrade would be highly desirable to control the quality of therapeutic antibody products. With this goal in mind, we have developed signature-based sensing system to discriminate differently degraded therapeutic antibodies. The use of arrays consisting of conjugates between nanographene oxide and fluorophore-modified single-stranded DNAs under acidic pH conditions generated unique fluorescence signatures for each state of the antibodies. Multivariate analyses of the thus obtained signatures allowed identifying (i) common features of native, denatured, and visibly aggregated antibodies, (ii) complicated degradation pathways of therapeutic omalizumab upon time-course heat-treatment, and (iii) the individual compositions of differently degraded omalizumab mixtures. As the signature-based sensing has the potential to identify a broad range of degraded antibodies formed by different kinds of realistic stress types, this system may serve as the basis for high-throughput assays for the screening of antibody manufacturing processes.

Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome

Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.

Multiplex Single Nucleotide Polymorphism Analyses.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

Identification of detergents for forensic fiber analysis.

Trace fibers are an important form of trace evidence, and identification of exogenous substances on textile fibers provides valuable information about the origin of the fiber. Laundering textiles can provide a unique fluorescent spectral signature of the whitening agent in the detergent that adsorbs to the fiber. Using fluorescence microscopy, the spectral characteristics of seven detergents adsorbed to single fibers drawn from laundered textiles were investigated, and principal component analysis of clusters was used to characterize the type of detergent on the fiber. On dyed nylon fibers, spectra from eight different detergent pairs could be resolved and washed validation fibers correctly classified. On dyed acrylic fibers, five different detergent pairs could be resolved and identified. Identification of the detergent type may prove useful in matching a trace fiber to its bulk specimen of origin.

Leptosperin is a distinct and detectable fluorophore in Leptospermum honeys

New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270–365nm excitation–emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.