Single-Cell Single-Molecule RNA-FISH Combined with Immunofluorescence and High-Speed and High-Resolution Scanning Analysis to Visualize the Reactivation of Latent HIV-1.

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.

Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections.

BACKGROUND: Porcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera. RESULTS: ICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen's κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃. CONCLUSIONS: In summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Quantitative image analysis pipeline for detecting circulating hybrid cells in immunofluorescence images with human-level accuracy.

Circulating hybrid cells (CHCs) are a newly discovered, tumor-derived cell population found in the peripheral blood of cancer patients and are thought to contribute to tumor metastasis. However, identifying CHCs by immunofluorescence (IF) imaging of patient peripheral blood mononuclear cells (PBMCs) is a time-consuming and subjective process that currently relies on manual annotation by laboratory technicians. Additionally, while IF is relatively easy to apply to tissue sections, its application to PBMC smears presents challenges due to the presence of biological and technical artifacts. To address these challenges, we present a robust image analysis pipeline to automate the detection and analysis of CHCs in IF images. The pipeline incorporates quality control to optimize specimen preparation protocols and remove unwanted artifacts, leverages a ß-variational autoencoder (VAE) to learn meaningful latent representations of single-cell images, and employs a support vector machine (SVM) classifier to achieve human-level CHC detection. We created a rigorously labeled IF CHC data set including nine patients and two disease sites with the assistance of 10 annotators to evaluate the pipeline. We examined annotator variation and bias in CHC detection and provided guidelines to optimize the accuracy of CHC annotation. We found that all annotators agreed on CHC identification for only 65% of the cells in the data set and had a tendency to underestimate CHC counts for regions of interest (ROIs) containing relatively large amounts of cells (>50,000) when using the conventional enumeration method. On the other hand, our proposed approach is unbiased to ROI size. The SVM classifier trained on the ß-VAE embeddings achieved an F1 score of 0.80, matching the average performance of human annotators. Our pipeline enables researchers to explore the role of CHCs in cancer progression and assess their potential as a clinical biomarker for metastasis. Further, we demonstrate that the pipeline can identify discrete cellular phenotypes among PBMCs, highlighting its utility beyond CHCs.

Detection of effusion tumor cells under different storage and processing conditions.

BACKGROUND: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. METHODS: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. RESULTS: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. CONCLUSIONS: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Methods to study primary cilia and autophagy in the brain.

Autophagy is an intracellular catabolic pathway that allows proteins, organelles, and pathogens to be recycled. Thus, it is crucial to maintain cell homeostasis, especially important in post-mitotic cells as neurons that cannot dilute cellular damage through mitosis. In the last decade, autophagy has been connected to the primary cilium (PC), a small organelle that acts as a sensory hub and is present in most cell types, including astrocytes and neurons. In this chapter, we briefly describe the state-of-the-art of the interplay between autophagy, PC, and its implications for the brain, in healthy and pathophysiological conditions. Deregulations in autophagy can be monitored by numerous assays, both in vivo and in vitro, and so do changes in PC length/number. Here, we relate a practical and user-friendly description of immunofluorescence methods to study autophagy and PC changes in brain slices, including the tissue preparation, confocal microscopy, image analysis, and deconvolution process.

Statistical Analysis of Multiplex Immunofluorescence and Immunohistochemistry Imaging Data.

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue.

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

Monitoring the interactions between alpha-synuclein and Tau in vitro and in vivo using bimolecular fluorescence complementation.

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-ß (Aß) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

Immunofluorescence and High-Resolution Microscopy Reveal New Insights in Human Globozoospermia.

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques.

Pillar[5]arene-Based Fluorescent Sensor Array for Biosensing of Intracellular Multi-neurotransmitters through Host-Guest Recognitions.

Neurotransmitters are very important for neuron events and brain diseases. However, effective probes for analyzing specific neurotransmitters are currently lacking. Herein, we design and create a supramolecular fluorescent probe (CN-DFP5) by synthesizing a dual-functionalized fluorescent pillar[5]arene derivative with borate naphthalene and aldehyde coumarin recognition groups to identify large-scale neurotransmitters. The developed probe can detect seven model neurotransmitters by generating different fluorescence patterns through three types of host-guest interactions. The obtained signals are statistically processed by principal component analysis, thus the high-throughput analysis of neurotransmitters is realized under dual-channel fluorescence responses. The present probe combines the advantages of small-molecule-based probes to easily enter into living neurons and cross-reactive sensor arrays. Thus, the selective binding enables this probe to identify specific neurotransmitters in biofluids, living neurons, and tissues. High selectivity and sensitivity further demonstrate that the molecular device could extend to more applications to detect and image neurotransmitters.

Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.

Spectrum of renal diseases in the elderly, above age 50.

BACKGROUND: The spectrum of kidney diseases varies in the elderly population with frequent inconsistencies between clinical presentation and histopathological diagnosis. The immunofluorescence (IF) may provide additional information in such situations. AIMS: The purpose was to study the spectrum of kidney diseases in patients above 50 years undergoing renal biopsy and utility of light chain (LC) IF in the diagnosis. SETTINGS AND DESIGN: This was a retrospective, crosssectional, singlecenter-based study. MATERIAL AND METHODS: The clinical details, histopathological findings, and LC IF pattern in native renal biopsy of patients above 50 years were noted. STATISTICAL ANALYSIS: Continuous variables were presented as mean ± standard deviation (SD). Categorical variables were expressed as frequencies and percentages. RESULTS: A total of 205 patients were included in the study. The most common clinical presentation was acute kidney injury/rapidly progressive glomerulonephritis (AKI/RPGN) (49%). Glomerular diseases (72%) were more common. Crescentic glomerulonephritis (21%) and membranous nephropathy (MN) (19%) were the most common glomerulopathy. LC restriction was observed in LC cast nephropathy (LCCN), primary amyloidosis, and LC also helped in classifying the cases of MPGN-type morphology. CONCLUSIONS: AKI/RPGN was the most common indication for renal biopsy in patients above 50 years. Crescentic GN and MN were the frequent glomerular pathology. LC IF is a useful adjunctive tool to classify various renal diseases.

In vitro and in vivo evaluation of AFB1 and OTA-toxicity through immunofluorescence and flow cytometry techniques: A systematic review.

Due to the globalization, mycotoxins have been considered a major risk to human health being the main contaminants of foodstuffs. Among them, AFB1 and OTA are the most toxic and studied. Therefore, the goal of this review is to deepen the knowledge about the toxicological effects that AFB1 and OTA can induce on human health by using flow cytometry and immunofluorescence techniques in vitro and in vivo models. The examination of the selected reports shows that the majority of them are focused on immunotoxicity while the rest are concerned about nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, embryotoxicity, reproductive system, breast, esophageal and lung toxicity. In relation to immunofluorescence analysis, biological processes related to AFB1- and OTA-toxicity were evaluated such as inflammation, neuronal differentiation, DNA damage, oxidative stress and cell death. In flow cytometry analysis, a wide range of assays have been performed across the reviewed studies being apoptosis assay, cell cycle analysis and intracellular ROS measurement the most employed. Although, the toxic effects of AFB1 and OTA have been reported, further research is needed to clarify AFB1 and OTA-mechanism of action on human health.

Circulating platelet-neutrophil aggregates characterize the development of type 1 diabetes in humans and NOD mice.

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.

Design considerations for analyzing protein translation regulation by condensates.

One proposed role for biomolecular condensates that contain RNA is translation regulation. In several specific contexts, translation has been shown to be modulated by the presence of a phase-separating protein and under conditions which promote phase separation, and likely many more await discovery. A powerful tool for determining the rules for condensate-dependent translation is the use of engineered RNA sequences, which can serve as reporters for translation efficiency. This Perspective will discuss design features to consider in engineering RNA reporters to determine the role of phase separation in translational regulation. Specifically, we will cover (i) how to engineer RNA sequence to recapitulate native protein/RNA interactions, (ii) the advantages and disadvantages for commonly used reporter RNA sequences, and (iii) important control experiments to distinguish between binding- and condensation-dependent translational repression. The goal of this review is to promote the design and application of faithful translation reporters to demonstrate a physiological role of biomolecular condensates in translation.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.