Characterisation and correction of polarisation effects in fluorescently labelled fibres.

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation-resolved microscopy and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

Plasmon-Enhanced Expansion Microscopy.

Expansion microscopy (ExM) is a rapidly emerging super-resolution microscopy technique that involves isotropic expansion of biological samples to improve spatial resolution. However, fluorescence signal dilution due to volumetric expansion is a hindrance to the widespread application of ExM. Here, we introduce plasmon-enhanced expansion microscopy (p-ExM) by harnessing an ultrabright fluorescent nanoconstruct, called plasmonic-fluor (PF), as a nanolabel. The unique structure of PFs renders nearly 15000-fold brighter fluorescence signal intensity and higher fluorescence retention following the ExM protocol (nearly 76%) compared to their conventional counterparts (<16% for IR-650). Individual PFs can be easily imaged using conventional fluorescence microscopes, making them excellent "digital" labels for ExM. We demonstrate that p-ExM enables improved tracing and decrypting of neural networks labeled with PFs, as evidenced by improved quantification of morphological markers (nearly a 2.5-fold increase in number of neurite terminal points). Overall, p-ExM complements the existing ExM techniques for probing structure-function relationships of various biological systems.

A Plasmonic Fluor-Lightened Microneedle Array Enables Ultrasensitive Multitarget Whole Blood Diagnosis of Anemia in A Paper Origami-Based Device.

This work reports a portable, origami-type paper device with a plasmonic fluor-labeled microneedle sensing module for the multiplexed quantification of anemia biomarkers in whole blood. Sequential steps, including serum separation, target enrichment, and multiplexed readout by a gel imager, are rapidly accomplished with the flexible and highly integrated device. The microneedle array enabled efficient sampling of trace targets from ng mL-1 to pg mL-1 level. Combined with the plasmonic fluor label, the signal is improved by ≈7.6 folds compared with the flat substrate-based assay. The device is applied to simultaneously quantify hemoglobin (Hb), ferritin, folic acid (FA), and vitamin B12 (VB12 ), which are four anemia biomarkers distributed in different environments with different concentration ranges. Featured by the small sample volume (150 µL), short assay time (20 min), low cost (2 $), robust stability, and user-friendliness, the device is promising for the rapid and accurate diagnosis of anemia in real practice.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats.

Estimating Fluor Emission Spectra Using Digital Image Analysis Compared to Spectrophotometer Measurements.

This paper describes a practical method for obtaining the spectra of lights emitted by a fluor in a liquid scintillator (LS) using a digital camera. The emission wavelength results obtained using a digital image were compared with those obtained using a fluorescence spectrophotometer. For general users, conventional spectrophotometers are expensive and difficult to access. Moreover, their experimental measurement setup and processes are highly complicated, and they require considerable care in handling. To overcome these limitations, a feasibility study was performed to obtain the emission spectrum through image analysis. Specifically, the emission spectrum of a fluor dissolved in a liquid scintillator was obtained using digital image analysis. An image processing method was employed to convert the light irradiated during camera exposure into wavelengths. Hue (H) and wavelength (W) are closely related. Thus, we obtained an H-W response curve in the 400~450 nm wavelength region, using a light-emitting diode. Another relevant advantage of the method described in this study is its non-invasiveness in sealed LS samples. Our results showed that this method has the potential to accurately investigate the emission wavelengths of fluor within acceptable uncertainties. We envision the use of this method to perform experiments in chemistry and physics laboratories in the future.

Feasibility Study on the Discrimination of Fluor Concentration in the Liquid Scintillator Using PMT Waveform and Short-Pass Filter.

Neutrinos are difficult to detect because they weakly interact with matter, making their properties least known. The response of the neutrino detector depends on the optical properties of the liquid scintillator (LS). Monitoring any characteristic changes in the LS helps to understand the temporal variation of detector response. In this study, a detector filled with LS was used to study the characteristics of the neutrinos detector. We investigated a method to distinguish the concentrations of PPO and bis-MSB, which are fluors added to LS, through a photomultiplier tube (PMT) acting as an optical sensor. Conventionally, it is very challenging to discriminate the flour concentration dissolved in LS. We employed the information of pulse shape and PMT coupled with the short-pass filter. To date, no literature report on a measurement using such an experimental setup has been published. As the concentration of PPO was increased, changes in the pulse shape were observed. In addition, as the concentration of bis-MSB was increased, a decrease in the light yield was observed in the PMT equipped with the short-pass filter. This result suggests the feasibility of real-time monitoring of LS properties, which are correlated with the fluor concentration, using a PMT without extracting the LS samples from the detector during the data acquisition process.

Analysis of lung stromal expression of the atypical chemokine receptor ACKR2 reveals unanticipated expression in murine blood endothelial cells.

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa-Fluor-labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa-Fluor-labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment.

A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire's disease.

Effects of Viscosity and Refractive Index on the Emission and Diffusion Properties of Alexa Fluor 405 Using Fluorescence Correlation and Lifetime Spectroscopies.

Fluorescence Correlation Spectroscopy (FCS) studies of the interaction of polymers or proteins in solution are strongly affected by the viscosity and refractive index of the medium, and the effects are likely to be more significant with the use of short wavelength excitation (e.g., 405 nm diode lasers). Failing to account for these issues can lead to incorrect measurement of average size, conformational changes, and dynamic behaviour of polymers and proteins. Steady-state, time-resolved, and FCS measurements of Alexa 405 in glycerol:water mixtures were performed to determine its suitability for FCS measurements with 405 nm excitation. The effects of the refractive index and viscosity on the diffusion coefficient and photophysical parameters (lifetime and relative quantum yield) of the fluorophore were determined. Alexa 405 lifetime decreased from 3.55 ns in water to 3.25 ns in a 50:50 glycerol:water mixture, while its diffusion coefficient dropped from 333 ± 16 to 44 ± 1 µm2s- 1. Lifetime data collected from micromolar solutions of Alexa 405 did however also suggest that as solvent polarity decreased, aggregates (excimers) were formed as evidenced by the appearance of a rising edge in the decay plots. The interdependence between lifetime, refractive index, and diffusion coefficient could be accurately fitted by a simple polynomial function indicating that the probe is well behaved and predictable in the glycerol:water model system. Overall, Alexa 405 is a most promising and reliable probe for FCS measurement using violet laser diode excitation sources.

[Dental management of head and neck irradiated patients]. / Prise en charge dentaire des patients irradiés en région cervico-faciale.

The oncological management of head and neck tumours is well known and standardized. Radiotherapy is one of the effective tools. However, it induces major changes in healthy tissues: teeth, gums, mucous membranes, salivary glands and bones. Some, like mucositis, are immediate and often reversible; others, like hyposialia or fibrosis, are late effects and often irremediable. These changes greatly affect oral health and make its management more complex. Dental management also becomes a capital element of the care path but, unfortunately, often remains neglected by the patient but also by some practitioners. It concerns all the stages of the clinical course: initial assessment, cancer treatment itself and long-term follow-up. If neglected, the patient's quality of life will be affected and complications, sometimes serious, such as osteoradionecrosis, may occur. Specific care recommendations for maintaining oral health are mentioned, especially for those patients requiring oral cavity irradiation.

La prise en charge carcinologique des tumeurs cervico-faciales est bien connue et codifiée. La radiothérapie fait partie des outils efficaces proposés. Elle entraîne cependant de profondes modifications tissulaires : dents, gencives, muqueuses, glandes salivaires, os. Certaines, comme la mucite, sont immédiates, et souvent réversibles; d'autres, comme l'hyposialie ou la fibrose, s'installent tardivement et souvent définitivement. Ces remaniements altèrent fortement la santé bucco-dentaire et rendent la prise en charge plus complexe. L'approche dentaire devient ainsi un élément capital du trajet de soins. Elle reste, malheureusement, souvent délaissée par le patient lui-même, mais aussi parfois par le praticien. Cette prise en charge concerne toutes les étapes du parcours : bilan initial, traitement carcinologique en soi et suivi à long terme. Si négligée, la qualité de vie du patient sera affectée et des complications, parfois graves, telle l'ostéoradionécrose, peuvent survenir. Sont évoquées ici des recommandations spécifiques de prise en charge bucco-dentaire dans le décours d'une irradiation portant sur la cavité buccale.

Preliminary study on the application of en bloc resection combined with near-infrared molecular imaging technique in the diagnosis and treatment of bladder cancer.

BACKGROUND: To evaluate the surgical safety of en bloc resection of bladder tumor (ERBT) and the effectiveness of ERBT combined with near-infrared (NIR) imaging technique in the diagnosis and treatment of non-muscle invasive bladder cancer (NMIBC). METHODS: From October 2017 to June 2018, 26 patients newly diagnosed with single NMIBC were included in this retrospectively trial. All patients received ERBT with monopolar current. After surgery, the fresh specimen was incubated with anti-CD47-Alexa Fluor 790, and then imaged under NIR imaging technique. Operative details, intraoperative and postoperative complications of ERBT regarded as safety outcomes, the mean fluorescence intensity (MFI) of tumor tissue and adjacent normal background tissue, and 12 months follow-up data were analyzed. RESULTS: Of 26 collected patients, obturator nerve reflex was occurred in six patients during tumor resection, and only one patient was observed with bladder perforation. In NIR gray image, the gray scale of MFI of tumor tissue were 132.31 ± 6.67 and the adjacent normal background tissue were 52.27 ± 12.09. The result showed a significantly higher MFI signals in tumor tissue compared to adjacent normal background tissue (P < 0.001). The recurrence-free survival rate at 12 month was 96.15%. CONCLUSIONS: ERBT with monopolar current is a safe and feasible technique to treat patients with NMIBC. A integrated bladder tumor tissue-bound anti-CD47-Alexa Fluor 790 was detected under NIR light, and the NIR image indicates that higher MFI signals in surgical margin is a predictive factor for residual tumor in patients with NMIBC after ERBT.

MCR Scaffolds Get Hotter with 18F-Labeling.

Imaging techniques, such as positron emission tomography (PET), represent great progress in the clinical development of drugs and diagnostics. However, the efficient and timely synthesis of appropriately labeled compounds is a largely unsolved problem. Numerous small drug-like molecules with high structural diversity can be synthesized via convergent multicomponent reactions (MCRs). The combination of PET labeling with MCR synthesis of biologically active compounds can greatly simplify radioanalytical and imaging-based analysis. In a proof-of-concept study, we optimized robust on-site radiolabeling conditions that were subsequently applied to several structurally different drug-like MCR scaffolds (e.g., arenes, ß-lactam, tetrazole, and oxazole). These labeled scaffolds were synthesized via pinacol-derived aryl boronic esters (arylBPin) by copper-mediated oxidative 18F-fluorination with radiochemical conversions (RCCs) from 15% to 76%.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4-10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.

2'-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP).

Lipopolysaccaride binding protein (LBP), a glycosylated acute phase protein, plays an important role in the pathophysiology of sepsis. LBP binds with high affinity to the lipid part of bacterial lipopolysaccaride (LPS). Inhibition of the LPS-LBP interaction or blockage of LBP-mediated transfer of LPS monomers to CD14 may be therapeutical strategies to prevent septic shock. LBP is also of interest as a biomarker to identify septic patients at high risk for death, as LBP levels are elevated during early stages of severe sepsis. As a first step toward such potential applications, we isolated aptamers specific for murine LBP (mLBP) by in vitro selection from a library containing a 60-nucleotide randomized region. Modified RNA pools were transcribed in the presence of 2'-fluoro-modified pyrimidine nucleotides to stabilize transcripts against nuclease degradation. As verified for one aptamer experimentally, the selected aptamers adopt a "three-helix junction" architecture, presenting single-stranded 7-nt (5'-YGCTTCY) or 6-nt (5'-RTTTCY) consensus sequences in their core. The best binder (aptamer A011; Kd of 270 nM for binding to mLBP), characterized in more detail by structure probing and boundary analysis, was demonstrated to bind with high specificity to murine LBP.

High affinity binding of amyloid ß-peptide to calmodulin: Structural and functional implications.

Amyloid ß-peptides (Aß) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid ß precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid ß(1-42) HiLyte™-Fluor555, we show in this work that Aß binds with high affinity to CaM through the neurotoxic Aß25-35 domain. In addition, the affinity of Aß for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of Kd of 0.98 ± 0.11 nM obtained for Aß1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aß peptides. A major functional consequence of Aß-CaM interaction is that it slowdowns Aß fibrillation. The novel and high affinity interaction between calmodulin and Aß shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aß in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD.

Development of new hCaM-Alexa Fluor® biosensors for a wide range of ligands.

Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor® 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET). The variation in Tm (range 78.07-81.47 °C; 79.05 to WT) is no larger than two degrees in all cases in regards to CaM WT. The Kds calculated with all biosensors for CPZ and BIMI (a new inhibitor of CaM) are in the range of 0.45-1.86 and 0.69-1.54 µm respectively. All biosensors retain their ability to activate Calcineurin about 70%. Structural models built "in silico" show their possible conformation taking the fluorophores in protein thus we can predict system stability. Finally, these new biosensors represent a biotechnological development applied to an analytical problem, which aims to determine accurately the affinity of inhibitors of CaM without possible interference, to be put forward as possible drugs related to CaM.

Tracking Antibody Distribution with Near-Infrared Fluorescent Dyes: Impact of Dye Structure and Degree of Labeling on Plasma Clearance.

Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab-800CW and -AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

BACKGROUND: The development of reagents to identify and characterize antigen-specific B cells has been challenging. METHODS: We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. RESULTS: In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. CONCLUSIONS: AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination.