Measured Fluorescence Signals Research Articles

Modular Multi‐Parametric and Portable Readout System for Point‐of‐Care Applications

AbstractA portable, battery‐powered, multi‐purpose readout system for combined optical and thermal measurements is presented. The system is modular with four independent input channels for different measurement schemes, allowing simultaneous multi‐parameter measurements to detect analytes of interest. Combined optical and thermal readout for spectroscopy is implemented by installing two different analog front‐end modules in the four input channels equipped with a micro spectrometer and a high‐resolution temperature sensor, respectively. The readout system is utilized to measure fluorescence signals of a common dye functionalized on gold nanoparticle‐DNA conjugates as a function of the temperature. Gold nanoparticles are modified with Guanine‐rich DNA sequences to perform Förster Resonance Energy Transfer (FRET) measurements. Elongation of DNA sequences at higher temperatures resulted in stronger fluorescence signals, accurately recorded by the portable system. The precision electronic components are chosen for a precise, battery‐powered operation. The size of this versatile, compact, and portable instrument is only 15.6 × 10.6 cm2. An interactive graphical‐user‐interface developed for this system supports point‐of‐care usage. The measurements carried out with the portable system showed a very close match with a commercial set‐up, with deviations less than 1 nm in the optical spectra and less than 0.5 °C in temperature.

Accurate evaluation of drug effect on the LDH activity of live cells: dual measurement of live cell number by fluorescent staining of nucleus and LDH activity by formazan.

Effect of drugs on the intracellular activity of lactate dehydrogenase (LDH) has been measured by using water-soluble tetrazolium (WST). Because the assay is usually conducted in the presence of dead cells, net activity of live cells is not evaluated. Here, we reported the assay of the net intracellular LDH activity of live cells by counting the live cells using fluorescent staining of nucleus. By using a deep red fluorescent dye, dual measurements of fluorescence signal of nucleus and absorbance of WST could be conducted with transparent 96-well-plates. We found that conventional assay in the presence of dead cells overestimate the effect of drugs on the LDH activity.

A novel highly sensitive inner filter effect-based fluorescence immunoassay with quantum dots for bioanalysis of zolbetuximab, a monoclonal antibody used for immunotherapy of gastric and gastroesophageal junction adenocarcinoma

Zolbetuximab (ZOL) is a groundbreaking monoclonal antibody targeting CLDN 18.2, a cancer cell surface protein. It is a first-in-class therapy for gastric and gastroesophageal junction adenocarcinoma. However, there is currently any immunoassay available for bioanalysis of ZOL, hindering its pharmacokinetic studies, therapeutic monitoring, and safety profile refinement. To address this gap, this study presents the development and validation of a novel highly sensitive inner filter effect-based fluorescence immunoassay (IFE-FIA) with quantum dots (QDs) as a probe. This assay enables the quantitative determination of ZOL in plasma samples. The assay involved non-competitive capturing of ZOL from the samples using a specific antigen (CLDN 18.2 protein) immobilized on assay plate microwells. A horseradish peroxidase (HRP)-labelled anti-human IgG was used to measure the immune complex. The assay's detection system relies on the formation of a light-absorbing colored product through an HRP-catalyzed oxidative reaction with the substrate 3,3′,5,5′-tetramethylbenzidine. This light absorption efficiently quenched the fluorescence of QDs via the IFE. The measured fluorescence signals corresponded to the concentrations of ZOL in the samples. The conditions of the IFE-FIA and its detection system were refined, and the optimum procedures were established. Following the guidelines of immunoassay validation for bioanalysis, the assay was validated, and all the validation criteria were acceptable. The assay demonstrates high sensitivity, accurately quantifying ZOL at concentrations as low as 10 ng/mL in plasma samples, with acceptable precision. Importantly, it avoids interferences from endogenous substances and plasma matrix. The recoveries in spiked human plasma ranged from 96.8 % to 104.5 %, with relative standard deviations of 4.1 %–6.5 %. The proposed IFE-FIA represents a valuable tool for quantifying ZOL in clinical settings, enabling assessment of its pharmacokinetics, therapeutic drug monitoring, and safety profile refinement.

Real-time identification of life-threatening necrotizing soft-tissue infections using indocyanine green fluorescence imaging.

Necrotizing soft-tissue infections (NSTIs) are life-threatening infections with a cumulative case fatality rate of 21%. The initial presentation of an NSTI is non-specific, frequently leading to misdiagnosis and delays in care. No current strategies yield an accurate, real-time diagnosis of an NSTI. A first-in-kind, observational, clinical pilot study tested the hypothesis that measurable fluorescence signal voids occur in NSTI-affected tissues following intravenous administration and imaging of perfusion-based indocyanine green (ICG) fluorescence. This hypothesis is based on the established knowledge that NSTI is associated with local microvascular thrombosis. Adult patients presenting to the Emergency Department of a tertiary care medical center at high risk for NSTI were prospectively enrolled and imaged with a commercial fluorescence imager. Single-frame fluorescence snapshot and first-pass perfusion kinetic parameters-ingress slope (IS), time-to-peak (TTP) intensity, and maximum fluorescence intensity (IMAX)-were quantified using a dynamic contrast-enhanced fluorescence imaging technique. Clinical variables (comorbidities, blood laboratory values), fluorescence parameters, and fluorescence signal-to-background ratios (SBRs) were compared to final infection diagnosis. Fourteen patients were enrolled and imaged (six NSTI, six cellulitis, one diabetes mellitus-associated gangrene, and one osteomyelitis). Clinical variables demonstrated no statistically significant differences between NSTI and non-NSTI patient groups (). All NSTI cases exhibited prominent fluorescence signal voids in affected tissues, including tissue features not visible to the naked eye. All cellulitis cases exhibited a hyperemic response with increased fluorescence and no distinct signal voids. Median lesion-to-background tissue SBRs based on snapshot, IS, TTP, and IMAX parameter maps ranged from 3.2 to 9.1, 2.2 to 33.8, 1.0 to 7.5, and 1.5 to 12.7, respectively, for the NSTI patient group. All fluorescence parameters except TTP demonstrated statistically significant differences between NSTI and cellulitis patient groups (). Real-time, accurate discrimination of NSTIs compared with non-necrotizing infections may be possible with perfusion-based ICG fluorescence imaging.

NV color center fluorescence signal measurement based on FPGA architecture

Nitrogen-vacancy (NV) quantum defects in diamond are sensitive detectors of magnetic fields. Then, the existing sensing technologies based on diamond NV centers rely on bulky and expensive instruments, which must be replaced by a more compact design. Here we combine NV quantum sensing technology with integrated circuit technology to create a compact and easy-to-operate platform. Using the existing integrated circuit technology, we integrate the lock-in amplifier, microwave generator and TTL modulator together. with this platform, we can obtain a magnetic sensitivity of 5.72μT/Hz 1/2.

Strain-Modulated and Nanorod-Waveguided Fluorescence in Single Zinc Oxide Nanorod-Based Immunodetection.

Mechanical strain has been shown to be a versatile and tunable means to control various properties of nanomaterials. In this work, we investigate how strain applied to individual ZnO nanorods (NRs) can affect the fluorescence signals originated from external sources of bioanalytes, which are subsequently coupled and guided onto the NRs. Specifically, we determine how factors such as the NR length and protein concentration can influence the strain-induced changes in the waveguided fluorescence intensity along the NRs. We employ a protein of tumor necrosis factor-α (TNF-α) and a fluorophore-labeled antibody in a model immunoassay reaction, after which Alexa488-TNF-α immunocomplex is formed on ZnO NRs. We elucidate the relationships between the types as well as amounts of strain on the NRs and the fluorescence intensity originated from the Alexa488-TNF-α immunocomplexes. We show that tensile (compressive) strain applied to the NR leads to an increase (decrease) in the waveguided fluorescence signals. By assessing important optical phenomena such as fluorescence intensification on nanorod ends (FINE) and degree of FINE (DoF), we confirm their linear dependence with both the types and amounts of strain. Furthermore, the strain-induced changes in both FINE and DoF are found to be independent of protein concentration. We determine that NR length plays a critical role in obtaining high strain-dependence of the measured fluorescence signals. Particularly, we ascertain that longer NRs yield larger changes in both FINE and DoF in response to the applied strain, relative to shorter ones. In addition, longer NRs permit higher linear correlation between the protein concentration and the waveguided fluorescence intensity. These outcomes provide valuable insight into exploiting strain to enhance the detection of optical signals from bioanalytes, thus enabling their quantifications even at ultra-trace levels. Coupled with the use of individual ZnO NRs demonstrated in our measurements, this work may contribute to the development of a miniaturized, highly sensitive biosensor whose signal transduction is best optimized by the application of strain.

Development of a self-contained microfluidic chip and an internet-of-things-based point-of-care device for automated identification of respiratory viruses.

The COVID-19 pandemic greatly impacted the in vitro diagnostic market, leading to the development of new technologies such as point-of-care testing (POCT), multiplex testing, and digital health platforms. In this study, we present a self-contained microfluidic chip integrated with an internet-of-things (IoT)-based point-of-care (POC) device for rapid and sensitive diagnosis of respiratory viruses. Our platform enables sample-to-answer diagnostics within 70 min by automating RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescence detection. The microfluidic chip is designed to store all the necessary reagents for the entire diagnostic assay, including a lysis buffer, a washing buffer, an elution buffer, and a lyophilized RT-LAMP cocktail. It can perform nucleic acid extraction, aliquoting, and gene amplification in multiple reaction chambers without cross-contamination. The IoT-based POC device consists of a Raspberry Pi 4 for device control and data processing, a CMOS sensor for measuring fluorescence signals, a resistive heater panel for temperature control, and solenoid valves for controlling the movement of on-chip reagent solutions. The proposed device is portable and features a touchscreen for user control and result display. We evaluated the performance of the platform using 11 clinical respiratory virus samples, including 5 SARS-CoV-2 samples, 2 influenza A samples, and 4 influenza B samples. All tested clinical samples were accurately identified with high specificity and fidelity, demonstrating the ability to simultaneously detect multiple respiratory viruses. The combination of the integrated microfluidic chip with the POC device offers a simple, cost-effective, and scalable solution for rapid molecular diagnosis of respiratory viruses in resource-limited settings.

Multiway Decomposition Followed by Reconvolution of Fluorescence Time Decay Data.

The use of time-resolved emission spectroscopy (TRES) is increasing as these instruments become more available, and the prices are decreasing, especially with the development of cheaper LED light sources. In this article, we propose a new methodology for analyzing TRES data. It combines two existing methods: PARAFAC and reconvolution. PARAFAC is a soft modeling curve resolution technique which has been extensively applied to steady-state fluorescence data, and reconvolution is the most common method for fitting TRES data. The proposed method is compared to two well-established methods of analyzing these data, namely, global reconvolution and tail fitting. In addition, we compare our approach with the SLICING method proposed in 2021 by Devos et al. which is also based on a soft model, but does not include the reconvolution step. All of these methods follow the assumption that the measured fluorescence signal is a linear combination of the underlying fluorophores. The comparison is based on a measured TRES data set with a mixture of three fluorophores and two sets of simulated data sets with up to four fluorophores. The results show that global fitting works well as long as the signal-to-noise ratio (SNR) is high (more than 15 dB), independent of the spacing between the emission peak maxima. SLICING does not give as good estimates of the time decay, mainly due to the challenge of defining the tail. Our proposed method gives robust and accurate results, outperforming the other techniques in cases with broad instrument response functions and high noise levels with SNRs down to 5 dB.

Virtual blebbistatin: A robust and rapid software approach to motion artifact removal in optical mapping of cardiomyocytes

Fluorescent reporters of cardiac electrophysiology provide valuable information on heart cell and tissue function. However, motion artifacts caused by cardiac muscle contraction interfere with accurate measurement of fluorescence signals. Although drugs such as blebbistatin can be applied to stop cardiac tissue from contracting by uncoupling calcium-contraction, their usage prevents the study of excitation-contraction coupling and, as we show, impacts cellular structure. We therefore developed a robust method to remove motion computationally from images of contracting cardiac muscle and to map fluorescent reporters of cardiac electrophysiological activity onto images of undeformed tissue. When validated on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in both monolayers and engineered tissues, the method enabled efficient and robust reduction of motion artifact. As with pharmacologic approaches using blebbistatin for motion removal, our algorithm improved the accuracy of optical mapping, as demonstrated by spatial maps of calcium transient decay. However, unlike pharmacologic motion removal, our computational approach allowed direct analysis of calcium-contraction coupling. Results revealed calcium-contraction coupling to be more uniform across cells within engineered tissues than across cells in monolayer culture. The algorithm shows promise as a robust and accurate tool for optical mapping studies of excitation-contraction coupling in heart tissue.

Automated Analysis of Telomere Intensity from Quantitative Fluorescence In-Situ Hybridization using CellProfiler

Telomeres are noncoding DNA sequences that protect chromosome ends from degradation. Quantitative fluorescence in situ hybridization (Q-FISH) is the favored technique for measuring telomere length. It can be applied to individual chromosomes in metaphase cells. Much of the currently available software to analyze fluorescence signal intensity as a proxy for telomere length is only commercially available. This study sought to develop a pipeline for the automated analysis of telomere fluorescence intensities in metaphase Q-FISH using modules from the open-source, freely available CellProfiler software. Intensities telomere and centromere were stained with fluorophore-conjugated peptide nucleic acid probes then evaluated using our pipeline. Telomere and centromere intensities were measured in a total of 30 images, giving an average relative telomere intensity of 22.17 %. Overall, our study showed that the pipeline can automatically identify and measure fluorescence signals from telomeres and centromeres in approximately 36 sec/image. However, the pipeline requires high-quality metaphase chromosome images with minimal touching and overlapping of chromosomes. Nevertheless, CellProfiler provides a free and flexible platform for the automated analysis of Q-FISH images that can be easily optimized and shared between researchers

High resolution recording of local field currents simultaneously with sound-evoked calcium signals by a photometric patch electrode in the auditory cortex field L of the chick

BackgroundFunctioning of the brain is based on both electrical and metabolic activity of neural ensembles. Accordingly, it would be useful to measure intracellular metabolic signaling simultaneously with electrical activity in the brain in vivo. New methodWe innovated a PhotoMetric-patch-Electrode (PME) recording system that has a high temporal resolution incorporating a photomultiplier tube as a light detector. The PME is fabricated from a quartz glass capillary to transmit light as a light guide, and it can detect electrical signals as a patch electrode simultaneously with a fluorescence signal. ResultsWe measured the sound-evoked Local Field Current (LFC) and fluorescence Ca2+ signal from neurons labeled with Ca2+-sensitive dye Oregon Green BAPTA1 in field L, the avian auditory cortex. Sound stimulation evoked multi-unit spike bursts and Ca2+ signals, and enhanced the fluctuation of LFC. After a brief sound stimulation, the cross-correlation between LFC and Ca2+ signal was prolonged. D-AP5 (antagonist for NMDA receptors) suppressed the sound-evoked Ca2+ signal when applied locally by pressure from the tip of PME. Comparison with existing methodsIn contrast to existing multiphoton imaging or optical fiber recording methods, the PME is a patch electrode pulled simply from a quartz glass capillary and can measure fluorescence signals at the tip simultaneously with electrical signal at any depth of the brain structure. ConclusionThe PME is devised to record electrical and optical signals simultaneously with high temporal resolution. Moreover, it can inject chemical agents dissolved in the tip-filling medium locally by pressure, allowing manipulation of neural activity pharmacologically.

Optimization of resonant dielectric multilayer for enhanced fluorescence imaging

In the aim of improving the sensitivity in objective-based total internal reflection fluorescence microscopy (ob-TIRF-M), we recently investigated the use of resonant dielectric multilayer (DM) coated coverslips while considering the experimental constraints of commercial ob-TIRF-M. The imaginary part of the refractive index k of the DM top layer material was then shown to be a key parameter to control during the DM optimization in order to limit the discrepancies between theory and experiment. We therefore report here on the development, fabrication and implementation of a specific top layer with a controlled k. This latter was first investigated as a single layer to accurately define its optical properties while adjusting the stoichiometric conditions during deposition. The DM optimization method was then fully revised to include its optical parameters. By doing so, the optimization was freed from the strong dependence on k and we observed a good agreement between the expected and measured fluorescence signal enhancement. This was illustrated by numerically calculating the fluorescence signal enhancement as a function of the top layer k and by comparing it to fluorescent beads imaging over three DM of different k.

Femtosecond two-photon LIF imaging of atomic hydrogen in high-pressure methane-air flames

Atomic hydrogen (H) imaging studies in combustion systems have been limited thus far to atmospheric- or low-pressure flames. The commonly used excitation scheme for two-photon laser-induced fluorescence (TPLIF) of H involves 205-nm deep-ultra-violet (DUV) laser pulses generated using nanosecond (ns), picosecond (ps), or femtosecond (fs) laser systems. Ultrashort pulse, fs excitation schemes are more attractive because of the efficient two-photon excitation and interference-free kHz-rate imaging capability. In this work, we implemented a home-built, direct fourth harmonic generation (FHG) laser system to generate high-energy fs pulses for H imaging in CH4/air flames at pressures up to 10 bar. The high-energy FHG output was capable of overcoming transmission losses of DUV laser pulses through thick optical windows of the pressure vessel. Signal interferences such as photoionization, photolytic production, amplified spontaneous emission, and background chemiluminescence were avoided or minimized during data acquisition and subsequent data processing steps. A quadratic dependence of the H TPLIF signal on the laser energy was observed. The increase in the pressure from 1 to 10 bar showed a rapid decay of the fs H TPLIF signal although it should scale linearly with increasing H number density with pressure ideally at equilibrium conditions. Besides a detailed quenching correction, laser absorption and fluorescence signal trapping remain major issues at elevated pressures. The measured fluorescence signals are compared with the equilibrium H number densities and local H concentrations calculated using the Cantera (0D) and UNICORN (2D) flame codes, respectively, for a range of equivalence ratios, and possible reasons for discrepancies are discussed. Experimentally obtained 1D and 2D spatial distributions of H are well predicted by the UNICORN flame model. Steps towards quantitative single-shot H TPLIF imaging measurements in high-pressure flames are also discussed. Overall, the fs TPLIF imaging technique coupled with a high-efficiency home-built FHG system is a promising diagnostic approach for H imaging in high-pressure flames.

Development of a preclinical CCD-based temperature modulated fluorescence tomography platform.

In preclinical research, fluorescence molecular tomography (FMT) is the most sensitive imaging modality to interrogate whole-body and provide 3D distribution of fluorescent contract agents. Despite its superior sensitivity, its mediocre spatial-resolution has been the main barrier to its clinical translation. This limitation is mainly due to the high scattering of optical photons in biological tissue together with the limited boundary measurements that lead to an undetermined and ill-posed inverse problem. To overcome the limitations of FMT, we previously introduced a novel method termed, Temperature Modulated Fluorescence Tomography (TMFT). TMFT utilizes thermos-sensitive fluorescent agents (ThermoDots) as a key component and localizes them with high-intensity focused ultrasound (HIFU). Scanning the focused HIFU beam having a diameter Ø = 1.3 mm across the tissue while monitoring the variation in the measured fluorescence signals reveals the position of the ThermoDots with high spatial accuracy. We have formerly built a prototype TMFT system that uses optical fibers for detection. In this paper, we present an upgraded version using a CCD camera-based detection that enables non-contact imaging. In this version, the animal under investigation is placed on an ultrasound transparent membrane, which eliminates the need for its immersion in optical matching fluids that were required by the fiber-based system. This CCD-based system will pave the way for convenient and wide-spread use of TMFT in preclinical research. Its performance validation on phantom studies demonstrates that high spatial-resolution (∼1.3 mm) and quantitative accuracy in recovered fluorophore concentration (<3% error) can be achieved.

Three-dimensional quantification of protoporphyrin IX in photodynamic therapy using SFDI/DFT: A pilot experimental validation

Photodynamic therapy (PDT) dosimetry, including light dose, photosensitizer dose and tissue oxygen, has been a research focus in PDT. In this work, we present a three-dimensional (3D) quantification of protoporphyrin IX (PpIX) using combined spatial frequency domain imaging (SFDI) and diffuse fluorescence tomography (DFT). The SFDI maps both the distributions of tissue absorption and scattering properties at three wavelengths and accordingly provides the optical background for DFT and extracts the tissue oxygenation for assessing the therapeutic outcomes, while DFT dynamically monitors the 3D distribution of PpIX dose from measured fluorescence signals for the procedure optimization. A pilot in vivo application in tumor nude models showed that the proposed SFDI/DFT is able to dynamically trace changes in the PpIX concentration and tissue oxygen during the treatment, rendering it a potentially powerful tool for PDT to improve clinical efficacy.

Clinical significance and biomarker potential of MGMT protein measurement in colorectal cancer.

136 Background: O6-methylguanine DNA methyltransferase (MGMT) is the principal repair mechanism of DNA damage created by alkylating agents. Approximately 40% of colorectal cancers (CRC) exhibit MGMT promoter hypermethylation, expected to result in gene silencing. The role of MGMT as a biomarker is poorly understood, and quantitative measurement of MGMT protein has not been performed in CRC. We hypothesized that altered tumor MGMT would affect DNA damage response, modulate adaptive anti-tumor immune responses, and influence survival in CRC. Methods: We used multiplexed quantitative immunofluorescence (QIF) to study 4 clinically annotated retrospective patient cohorts treated from 2000 to 2017 at Yale, consisting of 400 formalin-fixed paraffin-embedded CRC cases represented in tissue microarray format. These included paired tumors and adjacent non-tumor colonic mucosa (n = 112 pairs in Cohort 1), paired primary CRC and lymph node metastases (n = 31 pairs in Cohort 2), and 2 cohorts with primary stage I-IV tumors (n = 250 in Cohort 3; n = 150 in Cohort 4). We established a QIF panel for simultaneous, localized measurement of DAPI (all cells), cytokeratin (CK; tumor epithelial cells), MGMT, γH2AX (DNA damage response), and CD8. The measurement of fluorescent signals in CK-positive tumor cells or CK-negative stromal cells was achieved using co-localization strategies and the AQUA QIF platform. Results: We identified lower MGMT protein levels in CRC cells than in non-tumor colonic epithelium (p &lt; 0.0001), and in lymph node metastases compared to paired primary CRC tumors (p = 0.0411). Using the visual detection threshold, tumor selective MGMT protein downregulation was identified in 20% of cases in Cohorts 3 and 4. Microsatellite instability-high (MSI-H) cases showed decreased tumoral MGMT protein compared to microsatellite stable (MSS) cases (p = 0.002). In Cohorts 3 and 4, low tumoral MGMT was consistently associated with increased CD8+ tumor infiltrating lymphocytes (p = 0.0472; p = 0.0002). The association between MGMT and γH2AX was inconsistent. The association between MGMT and CD8 was significant only in MSS cases (p = 0.0001) but not in MSI-H cases (p = 0.0979), supporting that the effect is not driven by MSI-H status. Tumor-selective MGMT downregulation evidenced by a low tumor-to-stroma ratio was associated with improved progression-free survival and overall survival in both Cohorts 3 and 4, but statistical significance was only seen in Cohort 3. Conclusions: MGMT protein downregulation occurs in 20% of CRCs and is associated with increased adaptive anti-tumor immune responses, mismatch repair (MMR) deficiency and better prognosis. MGMT deficiency may alter DNA repair in tumor cells and mediate the accumulation of antigenic mutations or neopeptides, independent from MMR status. Our results support a biomarker role of MGMT protein and suggest a role for immunotherapy combinations in MGMT deficient tumors.

Automated measurement of fluorescence signals reveals a significant increase of the graft-derived neurite extension in neonates compared to aged rats.

Background– Neural tissue grafting is an acceptable form of cell therapy for brain injury and diseases. However, methods that can evaluate the graft integration and measure axonal extensions in a 3D environment are limited in scale, inconvenient, and operator intensive.Method– We stained grafts with a fluorescent antibody and then quantified the amount of fluorescence through the entire brain. To achieve this, we created an automated computer program designed to sort out authentic staining from background noise without any user input, enabling the analysis of thousands of images.Results– Our program could compensate for variations in the background brightness between images in all animals. Using this program, we show that human induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) progenitor cells integrate better into the striatum of neonates than older rats.Conclusion– Our program can quantify quickly and conveniently the integration of neural grafts in a 3D environment without depending on a blinded human operator. We expect this method to be a useful tool to assess the efficiency of graft-enhancing treatments for neurodegenerative diseases or other neural reconstruction attempts.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Retrieving and Validating Leaf and Canopy Chlorophyll Content at Moderate Resolution: A Multiscale Analysis with the Sentinel-3 OLCI Sensor.

ESA’s Eighth Earth Explorer mission “FLuorescence EXplorer” (FLEX) will be dedicated to the global monitoring of the chlorophyll fluorescence emitted by vegetation. In order to properly interpret the measured fluorescence signal, essential vegetation variables need to be retrieved concomitantly. FLEX will fly in tandem formation with Sentinel-3 (S3), which conveys the Ocean and Land Color Instrument (OLCI) that is designed to characterize the atmosphere and the terrestrial vegetation at a spatial resolution of 300 m. In support of FLEX’s preparatory activities, this paper presents a first validation exercise of OLCI vegetation products against in situ data coming from the 2018 FLEXSense campaign. During this campaign, leaf chlorophyll content (LCC) and leaf area index (LAI) measurements were collected over croplands, while HyPlant DUAL images of the area were acquired at a 3 m spatial resolution. A multiscale validation strategy was pursued. First, estimates of these two variables, together with the combined canopy chlorophyll content (CCC = LCC × LAI), were obtained at the HyPlant spatial resolution and were compared against the in situ measurements. Second, the fine-scale retrieval maps from HyPlant were coarsened to the S3 spatial scale as a reference to assess the quality of the OLCI vegetation products. As an intermediary step, vegetation products extracted from Sentinel-2 data were used to compare retrievals at the in-between spatial resolution of 20 m. For all spatial scales, CCC delivered the most accurate estimates with the smallest prediction error obtained at the 300 m resolution (R2 of 0.74 and RMSE = 26.8 μg cm-2). Results of a scaling analysis suggest that CCC performs well at the different tested spatial resolutions since it presents a linear behavior across scales. LCC, on the other hand, was poorly retrieved at the 300 m scale, showing overestimated values over heterogeneous pixels. The introduction of a new LCC model integrating mixed reflectance spectra in its training enabled to improve by 16% the retrieval accuracy for this variable (RMSE = 10 μg cm–2 for the new model versus RMSE = 11.9 μg cm-2 for the former model).

Evaluating spectral cytometry for immune profiling in viral disease.

In conventional fluorescence cytometry, each fluorophore present in a panel is measured in a target detector, through the use of wide band-pass optical filters. In contrast, spectral cytometry uses a large number of detectors with narrow band-pass filters to measure a fluorophore's signal across the spectrum, creating a more detailed fluorescent signature for each fluorophore. The spectral approach shows promise in adding flexibility to panel design and improving the measurement of fluorescent signal. However, few comparisons between conventional and spectral systems have been reported to date. We therefore sought to compare a modern conventional cytometry system with a modern spectral system, and to assess the quality of resulting datasets from the point of view of a flow cytometry user. Signal intensity, spread, and resolution were compared between the systems. Subsequently, the different methods of separating fluorophore signals were compared, where compensation mathematically separates multiple overlapping fluorophores and unmixing relies on creating a detailed fluorescent signature across the spectrum to separate the fluorophores. Within the spectral data set, signal spread and resolution were comparable between compensation and unmixing. However, for some highly overlapping fluorophores, unmixing resolved the two fluorescence signals where compensation did not. Finally, data from mid- to large-size panels were acquired and were found to have comparable resolution for many fluorophores on both instruments, but reduced levels of spreading error on our spectral system improved signal resolution for a number of fluorophores, compared with our conventional system. Furthermore, autofluorescence extraction on the spectral system allowed for greater population resolution in highly autofluorescent samples. Overall, the implementation of a spectral cytometry approach resulted in data that are comparable to that generated on conventional systems, with a number of potential advantages afforded by the larger number of detectors, and the integration of the spectral unmixing approach. © 2020 International Society for Advancement of Cytometry.