Fluorophore Research Articles

Imaging Flow Cytometry in HIV Infection Research: Advantages and Opportunities

The human immunodeficiency virus (HIV) is a type of retrovirus that infects humans and belongs to the Lentivirus group. Despite the availability of effective treatments, HIV infections are still increasing in some parts of the world, according to the World Health Organization (WHO). Another major challenge is the growing problem of HIV becoming resistant to drugs. This highlights the importance of ongoing research to better understand HIV and find new ways to stop the virus from spreading in the body. Scientists use a variety of methods to study HIV, including techniques from molecular and cellular biology. Many of these methods rely on fluorescent dyes to help visualize specific parts of the virus or infected cells. This article focuses on a technique called imaging flow cytometry, which is particularly useful for studying HIV. Imaging flow cytometry is unique because it not only measures fluorescence (light emitted by the dyes) but also captures images of each cell being analyzed. This allows researchers to see where the fluorescence is located within the cell and to study the cell’s shape and structure in detail. Additionally, this method can be combined with machine learning to analyze large amounts of data more efficiently.

PÓSTER] New fluorescent Schiff base modified nanocellulose-based chemosensors for the selective detection of Fe3+, Zn2+ and Cu2+ in semi-aqueous media and application in seawater sample

Cellulose is the most abundant linear carbohydrate bio-polymer in nature. It’s well-known as the most renewable, biocompatible, biodegradable, cost-effective and eco-friendly. In recent years, the shift from MCC to nanocellulose NC has rapidly attracted a great attention and has gained much interest as a new class of nanoscale biopolymers and as the new renewable source from both academia and industry researchers due to its promising potential in applications. NC is specifically characterized by a great dispersibility, high aspect ratio, large surface area, high mechanical strength, high crystallinity and thermal expansion coefficient. Thus, Schiff bases with an azomethine or imine (-C=N-) group have been extensively designed and used in field of cation species sensing, anions, proteins and organic substances, because of their simple preparation, high yield and easy structural modifications that can coordinate different analytes via the pair of electrons of azomethine nitrogen (-C=N) that make various complexes. Fluorometric chemosensors/probes are very effective for the detection and quantification of heavy metal ions, being the recognition simple process, that leads to high sensitivity, distinguished selectivity and excellent efficiency at low-cost. However, these small molecules are sometimes unstable in the surrounding changes in solutions. So, a new alternative has recently proposed using Schiff base-modified cellulose. For example, WANG et al. (2023) [1] developed a fluorescent coumarin derivative grafted dialdehyde cellulose-based probe for the detection of Fe3+. In this study, four new fluorescent dyes, namely, S1: (PDA-DANC), S2: (SAL-PDA-DANC), S3: (BrSAL-PDA-DANC) and S4: (ClSAL-PDA-DANC) have been successfully synthesized from (DANC) for the detection of heavy metal. The microstructural and photophysical properties of (NC), (DANC) and the synthesized dyes were investigated by FT-IR, SEM-EDX, XRD, TGA, DLS and photoluminescence.

Higher Rates of Visualization for Axillary Reverse Mapping Using Indocyanine Green Fluorescence Compared With Blue Dye.

Successful axillary reverse mapping (ARM) during lymph node surgery for breast cancer has the potential to reduce risk of lymphedema. Standard of care uses blue dye for ARM; however, recent imaging advances with near-infrared indocyanine green (ICG) fluorescence has demonstrated potential to improve intraoperative ARM imaging. The objective was to determine the feasibility of using ICG fluorescence through the OnLume Avata System for ARM. Breast cancer patients undergoing axillary lymph node dissection and were to undergo ARM were enrolled. Lymphatic structures were visualized using ICG fluorescence and blue dye. Real-time fluorescence images were acquired with the OnLume Avata System preincision, intraoperatively, and post dissection during the ARM. Preincision images were quantitatively analyzed for lymphatic fluorescence signal in terms of contrast-to-noise ratio. Imaging data were evaluated in terms of binary visualization rates and signal-to-background ratio. Lymph nodes, lymphatic vessels, and lymph pooling were observed with fluorescence more frequently than blue dye. For seven out of eight cases, at least one vessel was visualized near the axilla preincision. In all eight cases, ICG fluorescence was noted during the procedure with five cases visualizing intact lymphatics at the end of the procedure. The ambient-light compatibility of the imager allowed the surgeon to operate with image guidance throughout the ARM procedure. The Avata demonstrated superior identification and visualization with ICG when compared to blue dye for visualizing lymphatic structures in real time with minimal disruption to the clinical workflow.

Nanofibers loaded with trichromatic fluorescent dyes for enhancing the electro-optical properties of PDLC for application in colored smart windows

Nanofibers loaded with trichromatic fluorescent dyes for enhancing the electro-optical properties of PDLC for application in colored smart windows

Characterization of Extracellular Vesicles from Streptococcus thermophilus 065 and Their Potential to Modulate the Immune Response.

Bacteria can release membrane-derived nanoparticles made of lipid bilayers, so-called extracellular vesicles (EVs), which can carry diverse cargo and are important for microbe-microbe and microbe-host interactions. Here, we studied the production of EVs by Streptococcus thermophilus 065, the protein composition of the EVs, and how the produced EVs impact the immune response in vitro. Cultures of S. thermophilus grown for 6h at 40°C in M17 broth with 2% lactose reached high biomass yields and a high level of EVs quantified by lipophilic fluorescent dye staining. Proteome analysis of the isolated EVs revealed a high abundance of membrane-associated binding proteins of ABC transporters, ribosomal proteins, and glycolytic enzymes. In addition, phage proteins were found to be present in the EVs, which suggests a low-level expression of prophage genes during growth most likely supporting the release of EVs without causing cell lysis. The role of prophage activation was confirmed in an experiment with the addition of mitomycin C resulting in the expression of phage proteins including holin and endolysin causing a drop in culture OD and concomitant EV release. Subsequent in vitro immune assays using non-activated and activated human peripheral blood mononuclear cells (PBMCs) showed immune regulation in both cases upon exposure to S. thermophilus EVs and producer cells. This study shows the capacity of S. thermophilus EVs to act as immune modulators and opens the possibility for their use as postbiotics.

Microplastics and Nanoplastics Detection Using Flow Cytometry:Challenges and Methodological Advances with Fluorescent Dye Application

Microplastics and Nanoplastics Detection Using Flow Cytometry:Challenges and Methodological Advances with Fluorescent Dye Application

BODIPY fluorescent dyes for selective staining of intracellular organelles

BODIPY fluorescent dyes for selective staining of intracellular organelles

Fluorescence Behavior of Cyanine Fluorophore Cy3 Confined in Anodic Porous Alumina: Advanced Surface Analysis

In this study, an advanced analysis of the three-dimensional (3-D) surface microtexture of anodic porous alumina (APA) arrays was conducted to investigate their efficacy in confining cyanine fluorophore Cy3. The microstructural characterization of APA plays a pivotal role in enhancing the understanding of the material's surface properties, especially when applied to biological arrays and fluorescent dye confinement. To achieve this, Atomic Force Microscopy (AFM) was employed to obtain precise 3-D surface microtexture data. These measurements were complemented by stereometric analyses, adhering to ISO 25178-2: 2012 standards, ensuring a robust quantitative and qualitative evaluation of the surface micromorphology. The analysis revealed a clear correlation between surface roughness parameters and micromorphological changes as a function of varied processing conditions. Notably, the increase in certain fabrication parameters led to significant alterations in the superficial texture, with observable changes in the fractal dimension of the surface structure. The APA arrays, fabricated using a specialized anodization technique followed by a tailored annealing procedure, exhibited extraordinarily large pore sizes, ranging from 1 to 2 μm, which are well-suited for accommodating the Cy3 dye molecules. These pore dimensions offer an enhanced capacity for dye confinement, providing a stable environment for the cyanine fluorophore while promoting optimal fluorescence behavior.

Synthesis of fluorescent dyes for polyolefin fibers and their fluorescence emission properties

Synthesis of fluorescent dyes for polyolefin fibers and their fluorescence emission properties

Investigating the binding of fluorescent probes to a trypanosomal-tRNA synthetase: A fluorescence spectroscopic and molecular dynamics study.

Given the high prevalence of Chagas disease in the Americas, we targeted the unique arginyl-tRNA synthetase of its causative agent Trypanosoma cruzi. Among their many possible uses, naphthalene-derived fluorescent ligands, such as ANS and bis-ANS, may be employed in pharmacokinetic research. Although ANS and bis-ANS have become prominent fluorescent probes for protein characterization, the structural and spectroscopic characteristics of protein-ANS/bis-ANS complexes remain largely unknown. Both fluorescent dyes bind to either the folded or partially folded hydrophobic regions of proteins. Additionally, they serve to identify molten globule-like intermediates. These probes have been used to study the folding problems of protein structures and the mechanisms of protein-protein interactions. ANS and bis-ANS exhibited significant enhancement and blue shift in their emission spectra upon binding to TcArgRS, the primary enzyme responsible for attaching l-arginine to its corresponding tRNA. Through fluorescence spectroscopy and computational studies, we concluded that bis-ANS binds more tightly to TcArgRS and that ATP affects bis-ANS fluorescence signal. Thus, these probes are useful resources for studying the intricate intermolecular relationships between proteins in terms of their structure, function, and mechanism. Our study provides a framework for identifying the hydrophobic regions present in TcArgRS. The utilization of hydrophobic patches on proteins for drug targeting is noteworthy because they can assist in identifying regions on the surface of proteins that are likely to interact with ligands. These patches help identify hotspot residues that play a vital role in determining binding affinity. Drugs are mainly small and hydrophobic in nature, and they target protein surfaces which have complementary properties. In this study, we elucidated the potential of TcArgRS as a target for combating trypanosomal diseases and extending life expectancy.

Sensitizing K-Ras G12D mutant colorectal patient-derived organoids via ferroptosis pathway.

211 Background: Colorectal cancer remains the third most fatal form of cancer for males and females combined, with K-Ras mutations present in approximately 40% of all cases. These mutations drive cancer progression and confer resistance to conventional therapies, underscoring the need for novel treatment strategies. Ferroptosis, a regulated non-apoptotic form of cell death characterized as iron-dependent lipid peroxidation, has emerged as a target for cancer therapy. A study published in 2023 found that K-Ras mutant pancreatic ductal adenocarcinoma and non-small cell lung cancer cells lack ferroptosis induced lipid peroxidation and concurrently upregulated ferroptosis suppressing protein (FSP-1). It is unknown if K-Ras mutant colorectal tumors also evade ferroptosis-induced cell death as a method of cancer progression and therapy resistance. This study aims to measure the effects of inhibiting K-Ras G12D, the most common K-Ras mutation found in CRC, on ferroptosis through a patient derived colorectal cancer organoid (PDO) model. We hypothesize that blocking K-Ras G12D using a MRTX-1133, a K-Ras G12D specific inhibitor, can induce ferroptosis and synergistically work with ferroptosis inducers. Methods: As a pilot study, two patient derived organoid lines were established. Surveillance screening and whole exome sequencing was performed to verify K-Ras mutation. CRC9T has wild-type and CRC30T has mutant K-Ras. Additionally, Columbia Pathology Core confirmed tissues used to generate organoids were moderately differentiated colorectal adenocarcinoma. CTG-3D assays were performed to measure IC50 using ferroptosis inducers RSL3, iFSP, and K-Ras inhibitor MRTX-1133. Organoids were cultured in standard gut media for 72 hours, treated, and collected after 72 hours. After treatment, organoids were imaged and stained with BODIPY-C11, a lipid peroxidation fluorescence dye. Flow cytometry was performed, and results were analyzed using FlowJo to show ferroptosis-induced lipid peroxidation in stained organoids. Results: Inhibiting K-Ras G12D and inducing ferroptosis showed a synergistic effect on ferroptosis-induced lipid peroxidation. CRC9T was more sensitive (IC50 RLS3: 2.96 uM, iFSP 3.46 uM) to ferroptosis inducers than CRC30T (IC50: RSL3: 5.68 uM, iFSP: 8.6 uM). Blocking K-Ras in was only efficacious in CRC30T (IC50: MRTX1133: 9.73 uM). Cells from the mutant line showed greatest lipid peroxidation when combining ferroptosis inducers with K-Ras G12D inhibitors in flow cytometry analysis. Conclusions: These findings demonstrate a synergistic effect when blocking K-Ras G12D and inducing ferroptosis and align with previous studies that showed a connection between ferroptosis suppression and oncogenic KRAS expression. We plan to validate these findings in other patient derived organoid model systems and to assess the effects of pan-KRAS inhibition and ferroptosis inducer on non-K-Ras G12D PDOs.

Comparing the Efficacy of XP-endo Finisher with Irrigation Activation Methods on Sealer Penetration: An in Vitro Confocal Laser Scanning Microscopy Study

Aim: The irrigation method is effective on the dentinal tubule penetration (DTP) of endodontic pastes, which is important for treatment outcome. The purpose of this study was to compare the effect of different irrigation techniques on the DTP of bioceramic endodontic paste with CLSM. Materials and Methods: A total of 52 mandibular premolar root canals were shaped with the ProTaper Next files and were divided randomly into four experimental groups ( n = 13) according to the final irrigation method with 17% EDTA and 5.25% NaOCl as follows: Group 1: Conventional needle irrigation (CNI), Group 2: sonic activation (SA), Group 3: passive ultrasonic activation (PUA), Group 4: XP-endo Finisher (XPF). After final irrigation, root canals were obturated with a bioceramic sealer labeled with the fluorescent dye. The roots were sectioned horizontally at three different levels, and 1 ± 0.1 mm thick sections were obtained to examine under CLSM. The penetration area (PA), maximum tubule penetration (MTP), and the percentage of penetration (PP) values were statistically evaluated with the two-way repeated measurements ANOVA test. The Tukey (HSD) test was carried out for intergroup comparisons ( p < .01). The relationship between the MTP, PP, and PA at each root level was evaluated using Pearson’s correlation test. Results: In the middle sections, the penetration area of the sealer was higher in the SA group (7.120 ± 1.470) compared to the other groups ( p < .01). Penetration area was higher in the XPF group (11.830 ± 1.370) in the coronal section compared to CNI (7.034 ± 0.926) ( p < .01). No significant difference was observed between the MTP and PP values of the experimental groups ( p > .01). DTP of sealer was statistically similar for each irrigation method in the apical section. The coronal regions showed higher DTP values compared with the apical regions. There was a linear relationship between the PA and MTP values at all root levels of experimental groups ( p < .05). The linear relationship between the PA and PP was not significant in the XP group ( p > .05). Conclusion: The penetration area of SA was larger than others in the middle section, and XPF was superior to CNI in the coronal section. DTP of the sealer in the apical regions was lower than in the coronal regions.

Embedding Carbon Nanotubes in Artificial Cells Enhances Probe Transfer.

Monitoring intracellular biomarkers is crucial for clinical disease diagnosis. However, the majority of signaling molecules face difficulties in slow transference across the cell membrane to reach intracellular detection sites, limiting their application in clinical settings. This study proposes an artificial cell-based signal probe transfer-enhanced sensing strategy to effectively detect microRNAs (miRNAs) by embedding carbon nanotubes (CNTs) in artificial cell membranes. The liposome-based artificial cell is constructed to protect the signal probes such as nucleic acids, metal ions, and fluorescent dyes. CNTs are embedded in artificial cell membranes and employed as artificial channels to enhance intercellular signal probe transduction and mass transfer. Through CNTs-mediate cell-cell signal probe transmission, the probes pass through the liposome membrane interface and fuse into target cells, selectively hybridizing with the intracellular target miRNAs, triggering a sensing process and resulting in enhanced fluorescence signal. Furthermore, molecular dynamics simulations are carried out to prove the enhancement of CNTs-mediated cell-cell fusion. This strategy demonstrates excellent analytical performance by quantitatively detecting let-7a miRNA and visualizing it in living colon cancer cells. These findings hold great significance in promoting and accelerating cell-cell signal probe transmission and enabling effective sensing of intracellular biomarkers for diagnostic purposes.

Two-photon excited fluorescence with shaped laser pulses for refractive beams

We report two-photon excited fluorescence of dyes for refracted laser beams by utilizing tailored laser pulses. A fluorescence contrast difference due to phase shaping could be achieved between different coumarin dyes. Particularly, an increased contrast difference is obtained for configurations close to the Brewster angle. Furthermore, by using a subsequent deformable phase plate for spatial shaping it was possible to precisely adjust the laser beam for controlled refraction at the liquid dye surface. A polarization-dependent refraction was observed when directing the shaped laser beam on the curved liquid adhesion meniscus close to the cuvette wall. This results in a refraction-dependent contrast difference. The presented method could be utilized for surface-sensitive biophotonic imaging applications.Graphic abstract

Virtual staining from bright-field microscopy for label-free quantitative analysis of plant cell structures

The applicability of a deep learning model for the virtual staining of plant cell structures using bright-field microscopy was investigated. The training dataset consisted of microscopy images of tobacco BY-2 cells with the plasma membrane stained with the fluorescent dye PlasMem Bright Green and the cell nucleus labeled with Histone-red fluorescent protein. The trained models successfully detected the expansion of cell nuclei upon aphidicolin treatment and a decrease in the cell aspect ratio upon propyzamide treatment, demonstrating its utility in cell morphometry. The model also accurately documented the shape of Arabidopsis pavement cells in both wild type and the bpp125 triple mutant, which has an altered pavement cell phenotype. Metrics such as cell area, circularity, and solidity obtained from virtual staining analyses were highly correlated with those obtained by manual measurements of cell features from microscopy images. Furthermore, the versatility of virtual staining was highlighted by its application to track chloroplast movement in Egeria densa. The method was also effective for classifying live and dead BY-2 cells using texture-based machine learning, suggesting that virtual staining can be applied beyond typical segmentation tasks. Although this method still has some limitations, its non-invasive nature and efficiency make it highly suitable for label-free, dynamic, and high-throughput analyses in quantitative plant cell biology.

Evaluating the In Situ Effects of Whole Protein Coronas on the Biosensing of Antibody-Immobilized Nanoparticles Using Two-Color Fluorescence Nanoparticle Tracking Analysis

The formation of protein coronas around engineered nanoparticles (ENPs) in biological environments is critical in nanomedicine, as these coronas significantly influence the biological behavior of ENPs. Despite extensive research on protein coronas, understanding the in situ influence of whole (soft plus hard) protein coronas has remained challenging. In this study, we demonstrate a strategy to assess the in situ effects of whole coronas on the model biosensing of anti-IgG using IgG-conjugated gold nanoparticles (IgG-AuNPs) through fluorescence nanoparticle tracking analysis (F-NTA), which enables the selective tracking of fluorescent particles within complex media. In our approach, anti-IgG and IgG-AuNPs were labeled with distinct fluorescent dyes. The accordance in hydrodynamic diameter distributions observed at two different wavelengths verifies the successful capture of anti-IgG on the IgG-AuNPs. The counting of fluorescent anti-IgG within the size distribution allows for a quantitative assessment of biosensing efficiency. This method was applied to evaluate the effects of four protein coronas—human serum albumin, high-density lipoproteins, immunoglobulin G, and fibrinogen—as well as their mixture across varying incubation times and concentrations. The results suggest that the physical presence of whole protein coronas surrounding the IgG-AuNPs may assist the biosensing interaction in situ rather than screening it.

A beginner’s guide to differential scanning fluorimetry

Differential scanning fluorimetry (DSF) is a technique used to determine a protein’s stability. In a typical DSF experiment, a purified protein is heated in the presence of a fluorescent dye and the resulting temperature vs fluorescence plot is used to calculate its apparent melting temperature. This technique can be used to study a wide range of phenomena, including binding of a protein to ligands or cofactors and the effects of point mutations or buffers on a protein’s relative stability. The advantage of DSF is that it can be used in low volume and high-throughput such that hundreds or thousands of conditions can be rapidly tested. This guide provides an introduction to DSF and guidance on assay development and data interpretation, including how to avoid common artifacts.

Bright and Specific Targeting of Metastatic Lymph Nodes in Orthotopic Mouse Models of Gastric Cancer with a Fluorescent Anti-CEA Antibody.

Gastric cancer poses a major diagnostic and therapeutic challenge. Improved visualization of tumor margins and lymph node metastaseswith tumor-specific fluorescent markers could improve outcomes. To establish orthotopic modelsof gastric cancer, one million cells of the humangastric cancer cell line, MKN45, were suspended in 50 μl of equal parts PBS and Matrigel and injected into the nude mouse stomach with a 29-gauge needle. Tumors were allowed to grow for 8-12 weeks before performing imaging studies. For tumor labeling, M5A (humanized anti-CEA mAb) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Mice were randomized to receive 50 μg of M5A-IR800 (n = 14) or 50 μg of IgG-IR800 (n = 14) intravenously and were imaged 72 hours later. Fluorescence imaging was performed using the LI-COR Pearl Imaging System. Forty-two lymph nodes were collected from 28 mice, of which 59.5% were found to contain metastatic gastric cancer cells on pathologic examination. In mice that received M5A-IR800, there was a statistically significant difference in the mean fluorescence signal for cancer-positive lymphnodes at 0.431 (SE ± 0.224) compared with 0.105 (SE ± 0.009) for cancer-negative nodes (p: 0.002). For IgG-IR800, there was no significant difference in the mean fluorescence signal for cancer-positive nodes (0.057) compared with cancer-negative nodes (0.064), p-value 0.677. Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright labeling of lymph nodes containing metastatic gastric cancer. This tumor-specific fluorescent antibody is a promising clinical tool for identifying lymph nodes containing metastatic gastric cancer.

Near-infrared spectroscopy discriminates mass-reared sterile and wild tsetse flies.

Monitoring the efficacy of the sterile insect technique (SIT) programs, it is desirable to discriminate between wild and sterile tsetse males captured in monitoring traps. Currently, this is primarily achieved by marking sterile males with fluorescent dye powder before release, and identifying them using a fluorescence camera and/or microscope. However, the accuracy of this method is limited due to defective marking and wild flies contaminated with a few dye particles in the monitoring traps. Molecular techniques have been developed to discriminate doubtful flies, but they are expensive for endemic countries. Here, we investigate the ability of a new generation monitoring tool, Near-Infrared Spectroscopy (NIRS), to discriminate between laboratory-reared Glossina palpalis gambiensis males and their field counterparts. NIRS was able to discriminate wild males from laboratory-reared males with 86% accuracy. Notably, the prediction accuracy improved to 88% when the laboratory-reared flies had been irradiated. These findings suggest that NIRS can successfully identify tsetse flies even when UV camera identification is inconclusive. However, further studies are needed to expand the training dataset and include additional environmental variables before validating NIRS as a complementary method for future tsetse eradication programs.

P-1839. Elucidating Key Interactions Between Macrophages and the Fungal Pathogen Coccidioides

Abstract Background In the environment, the fungal pathogen Coccidioides grows as hyphae, yet when its spores (arthroconidia) are inhaled by a host, they develop instead into a parasitic form called the spherule. Spherules can be elicited in vitro in the presence of specific media, elevated temperatures, and high CO2, yet what triggers this transition in vivo and the role of host immune cells in this transition remains largely unknown. Methods We performed live imaging of Coccidioides posadasii Silveira arthroconidia in the presence or absence of murine bone marrow derived macrophages isolated from wildtype (C57BL/6) mice. Images were taken hourly over a 3-day time course. We recorded the time at which hyphae first appeared, the number of spherules at the final time point, and average size of spherules at the final time point. We utilize fluorescent dyes to determine if arthroconidia are phagocytosed and to assess host cell death. RNAseq was performed on RNA isolated from arthroconidia cultured with macrophages over 48hrs. Experiments were conducted at 37C and 5% CO2. Results We observed that the presence of macrophages strongly promotes the ability of Coccidioides arthroconidia to transition to spherules at conditions that would otherwise promote formation of hyphae. In wells with macrophages, arthroconidia produced more spherules, and these spherules were generally larger in size than those produced in vitro in the absence of host cells. The presence of macrophages also delayed the time that hyphae first appeared. Using a transwell system, we found that physical contact with macrophages was necessary to promote spherule development and reduce hyphae formation. We discovered that Coccidioides arthroconidia are phagocytosed by macrophages and spherules can develop within macrophages. RNAseq of Coccidioides arthroconidia cultured with macrophages show enrichment of transcripts associated with spherule growth in vitro as well as those previously identified from Coccidioides-infected murine or human lungs. Conclusion Macrophages strongly promote the ability of Coccidioides arthroconidia to transition to the parasitic form of Coccidioides in a contact-dependent manner. This work creates a foundation for better understanding initial critical interactions between macrophages and Coccidioides. Disclosures All Authors: No reported disclosures