Fluorescent Labeling Research Articles

Abstract A047: Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique

Abstract There is a continues need to develop technologies to detect and characterize circulating tumor cells that can become aggressive, metastatic, and hard to treat. Among these aggressive CTC are cells known as Hypoxic cell populations that learn to adapt and survive to low oxygen levels. Our project aims to test the applicability of a novel technique to detect Hypoxic Circulating Tumor Cells (H-CTC) that can move from tumors to circulating blood invading other tissues and becoming resistant to chemotherapy and radiation. The proposed assay incorporates a set of patented organic compounds as markers of hypoxic circulating tumor cells (H-CTC) with potential clinical applications. To date, our research has generated preliminary in-vitro studies with a novel NBQ-TOM demonstrating its capacity as markers of hypoxic human cancer cells in vitro. Comparison with the control Pimonidazole demonstrated the advantages of novel NBQ- TOM compounds as hypoxic marker. To determine the capacity of NBQ-TOM as hypoxic fluorescent marker, samples of 15 ml human whole blood were spiked with hypoxic or normoxic colon cancer cells (COLO 205). Spiked hypoxic or normoxic cells are then enriched, recovered, and treated with NBQ-TOM (25uM) for 24 hours. After treatment generated fluorescence is measured with an OPTIMA BMG Fluorimeter to demonstrate the significant formation of a fluorescent metabolite on hypoxic tumor cells in contrast to cells treated under normoxic environment. Also, fluorescence microscopy analysis confirmed the stronger fluorescence generation at hypoxic conditions. These preliminary results strongly support the applicability of these molecules for an in-vitro detection assay of hypoxic CTC in circulating blood that can become metastatic. Additional sensitivity and specificity analysis needs to be implemented. Citation Format: Beatriz Zayas, Karoline Rios, Osvaldo Cox. Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A047.

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus.

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yield viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

A fluorescence-based lateral flow immunoassay using AIEgen-encapsulated nanoparticles to rapidly and sensitively detect pro-gastrin-releasing peptide.

Afluorescence lateral flow immunoassay (F-LFIA) is presentedusing nanoparticles with encapsulated molecules whose emission is caused by aggregation as the fluorescent label to quantitatively detect gastrin-releasing peptide precursor(proGRP) in serum. The detection system was optimized to achieve a broad linear detection range of 10 ~ 5000 pg/mL and a detection limit of 6 pg/mL for F-LFIA. The proposed method exhibited good sensitivity, specificity, and reproducibility. The performance and applicability of the F-LFIA were evaluated in the analysisof 183 human serum samples, and the results were strongly correlated with those of the electrochemiluminescence immunoassay. The proposed F-LFIA can serve as a precise and rapid detection method to detect proGRP and has potential for the early diagnosis of small-cell lung cancer (SCLC).

Energy landscape of conformational changes for a single unmodified protein

Resolving the free energy landscapes that govern protein biophysics has been obscured by ensemble averaging. While the folding dynamics of single proteins have been observed using fluorescent labels and/or tethers, a simpler and more direct measurement of the conformational changes would not require modifications to the protein. We use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein, Bovine Serum Albumin (BSA), and quantify changes in the three-state conformation dynamics with temperature. A Markov model with Kramers’ theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including mapping out the energy landscape of particularly challenging intrinsically disordered proteins.

Simultaneous 2-photon and 3-photon excitation with a red fluorescent protein-cyanine dye probe pair in the 1700-nm excitation window for deep in vivo neurovascular imaging

In vivo imaging of the neurovascular network is considered to be one of the most powerful approaches for understanding brain functionality. Nevertheless, simultaneously imaging the biological neural network and blood vessels in deep brain layers in a non-invasive manner remains to a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2-photon and 3-photon fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum of around 550 nm is used as a 3-photon excited probe to label neurons, and a cyanine derivative dye Q820@BSA has a NIR absorption maximum of 825 nm as a 2-photon excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging with high emission spectral separation (>250 nm). In particular, the 2-photon action cross-section of Q820@BSA was found to be about 2-fold larger than that of indocyanine green (ICG), a commonly used red 2-photon fluorescence labeling agent, at the same excitation wavelength. Benefiting from the long wavelength advantage in reducing scattering in both 2 and 3-photon excitation of the fluorescence pairs, we demonstrated in vivo neurovascular imaging in intact adult mouse brains through white matter and deep into the hippocampus in the somatosensory cortex.

Live imaging of airway epithelium reveals that mucociliary clearance modulates SARS-CoV-2 spread

SARS-CoV-2 initiates infection in the conducting airways, where mucociliary clearance inhibits pathogen penetration. However, it is unclear how mucociliary clearance impacts SARS-CoV-2 spread after infection is established. To investigate viral spread at this site, we perform live imaging of SARS-CoV-2 infected differentiated primary human bronchial epithelium cultures for up to 12 days. Using a fluorescent reporter virus and markers for cilia and mucus, we longitudinally monitor mucus motion, ciliary motion, and infection. Infected cell numbers peak at 4 days post infection, forming characteristic foci that tracked mucus movement. Inhibition of MCC using physical and genetic perturbations limits foci. Later in infection, mucociliary clearance deteriorates. Increased mucus secretion accompanies ciliary motion defects, but mucociliary clearance and vectorial infection spread resume after mucus removal, suggesting that mucus secretion may mediate MCC deterioration. Our work shows that while MCC can facilitate SARS-CoV-2 spread after initial infection, subsequent MCC decreases inhibit spread, revealing a complex interplay between SARS-CoV-2 and MCC.

Ovule abortion accompanied by programmed cell death in Castanea mollissima

Chinese chestnut (Castanea mollissima Blume.) ovary contains 12-18 ovules, but only one can develop and grow normally, which indicates that the abortive rate of ovules can go as high as 94%. In this study, we observed the developmental characteristics and cytological changes of abortive ovules and fertile ovules during development. Morphological analysis showed it would take about 15∼20 days from globular to cotyledon embryo in fertile ovules, which is accompanied with the formation and apoptosis of endosperm. But there were stark differences in the nutrient transport, microstructure and ultrastructure of fertile and abortive ovules. The fluorescence disodium fluorescein and FDA indicated that all ovules were viable at early stages, but some ovules became inactive at different times as the ovary developed. Fluorescence labelling and ultrastructure showed cell nuclei of abortive ovules were disintegrated at 25 days after anthesis (DAA). Compared with the fertile ovules, starch grains were synthesized at earlier periods, but disappeared immediately, and the cells were usually irregular in shape, and had folded cell membranes in the abortive ovules. Furthermore, the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess programmed cell death in fertile and abortive ovules, results showed that DNA fragmentation was occurred in the cells of abortive ovules. In short, these results provide new insights into ovule abortion in the angiosperm.

Optimized Methyl methacrylate embedding of small and large undecalcified bones.

Methyl methacrylate (MMA) plastic embedding has been long established as a technique for the processing and histological assessment of bones. It provides the added benefit over paraffin in that it does not require decalcification of the tissue in order visualize the cellular detail, thus preserving vital information about the amount of unmineralized osteoid present in addition to the degree of mineralization in the bone. It also allows for the incorporation of dynamic histomorphometric analysis through the retention of fluorescent labels incorporated into the bone. Efficient infiltration of hard tissue is essential to the processing of bones and producing quality slides suitable for achieving usable quantifiable histology out the other end. This technique:• Updates previously published MMA embedding protocols to reflect utilization of stabilized acrylamides (over the unstabilized reagents of the past)• Outlines the techniques that are important for embedding both small (mus), medium (rattus), and large (porcine, lagomorph, human) histological samples.• Updates the clearing and infiltration processes utilized and validates quality of the sample preparation though histological staining to confirm preservation of cellular detail, mineralization information, and enzymatic activity

Luminescent Tb3+/Sm3+ co-doped hydroxyapatite nanoparticles as an imaging probe in N2a cells

This work reports the synthesis of hydroxyapatite (HAp) nanoparticles co-doped with trivalent terbium (Tb3+) and samarium (Sm3+) ions by a surfactant free chemical precipitation method. Co-doping enables to combine the optical properties of Tb3+ and Sm3+ ions. Characterization techniques like X-Ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), energy dispersive X-ray spectroscopy (EDX) were used to determine the crystalline and structural properties. These studies confirmed that the lanthanide ions Tb3+ and Sm3+ were successfully doped onto the HAp lattice. The photoluminescence emission spectra exhibited intense emissions from both the lanthanide ions. The Sm3+ photoluminescence spectra showed enhanced emission,indicating that energy is being transferred from the Tb3+ to Sm3+ ions. To examine the cell viability, N2a cellswere subjected to the MTT cytotoxicity assay. As demonstrated by the cell imaging on N2a cells, the synthesised nanoparticles are ideal candidates for fluorescent labelling using lanthanide ions. Furthermore, thestrong cytocompatibility of Tb3+/Sm3+ co-doped hydroxyapatitesuggests that it is a promising nanoscale biomaterial.

Human milk fat globule size distributions: comparison between laser diffraction and 3D confocal laser scanning microscopy

Milk fat globules (MFGs) in human milk provide energy to breastfed infants and support infant development. Accurate measurements of MFG size distributions are important to better understand MFG function and origin, as well as the influence of MFG size on milk composition analysis methods. Nevertheless, commonly used laser diffraction systems have never been thoroughly validated for size distribution measurements in human milk. Here, we introduce a new method for determining the size distribution of milk fat globules in human milk, using 3D confocal laser scanning microscopy (CLSM) in combination with fluorescent labeling of MFGs. We validate and compare 3D CLSM to laser diffraction (Mastersizer 2000, Malvern Panalytical), using polystyrene microsphere size standards. Next, we apply both methods to evaluate MFG size distributions in human milk. We show that 3D CLSM can be used to obtain more accurate size distributions between 500 nm and 10 μm compared to laser diffraction. Importantly, MFG size distributions obtained with 3D CLSM contain no secondary population around 1 μm, in contrast to laser diffraction measurements. This suggests that the bimodal MFG distribution obtained by laser diffraction can be an artifact of the built-in fitting algorithm, instead of an actual feature of human milk. This work demonstrates that care should be taken when interpreting size distributions of MFGs measured with laser diffraction and that 3D CLSM is an accurate alternative for measuring size distributions in lactation and dairy research.

Kinetics of i-motif folding within the duplex context

Non-canonical nucleic acid structures possess an ability to interact selectively with proteins, thereby exerting influence over various intracellular processes. Numerous studies indicate that genomic G-quadruplexes and i-motifs are involved in the regulation of transcription. These structures are formed temporarily during the unwinding of the DNA double helix; and their direct determination is a rather difficult task. In addition, i-motif folding is pH-dependent, with most i-motifs having low stability at neutral pH. However, some genomic i-motifs with long cytosine repeats were shown to be stable at pH 7.3, suggesting their functionality within the nucleus. Here we studied pH-dependent behavior of a model i-motif with flanking sequences that forms a duplex motif. Kinetic studies on bimodular structures with cytosine residues replaced with an environment-sensitive fluorescent label reveal the stabilization of the i-motif structure near the i-motif-duplex junction. These results highlight the importance of the natural environment of i-motifs for the correct assessment of their stability.

Synthesis and Evaluations of Cleavable Triazene‐Modified Nucleotide for DNA Sequencing

AbstractA fluorescence‐labeled nucleotide with a cleavable triazene linker was designed and synthesized as a potential reversible terminator for DNA sequencing by synthesis (SBS). The key intermediate, a triazene‐modified nucleotide, was successfully synthesized through the triazenylation of an amino‐modified nucleotide, followed by fluorescence labeling to yield the desired product. The synthesized triazene‐modified nucleotide can effectively serve as a substrate for Klenow Fragment (exo−) DNA polymerase and be incorporated into DNA strands. Once the first modified nucleotide is incorporated, no further extension is possible, even with an unblocked 3′‐OH group. After the fluorescent label is completely removed under acidic conditions, the triazene reversible terminator can be incorporated into DNA strands again, thereby enabling the DNA sequencing cycles.

Automated production of batched unclonable micro-patterns anti-counterfeiting labels with strong robustness and rapid recognition speed

Anti-counterfeiting technologies are indeed crucial for information security and protecting product authenticity. Traditional anti-counterfeiting methods have their limitations due to their clonable nature. Exploring new technologies, particularly those based on pixel-level textures, is a promising avenue to address the clonable issue due to high encoding capacity. However, research in this field is still in its early stages. This work introduces a new fluorescent anti-counterfeiting label technology with four key characteristics: efficient laser etching, high-throughput fabrication and segmentation, robustness aided by data augmentation, and an exceptionally high recognition speed. Specifically, the etching achieves a speed of 1,200 labels/3s, the high-throughput procedures yield a rate of 2,400 labels/4 mins, and the total count is about 5.2 × 104 labels. The number of labels is further augmented to about 5.2 × 106 by implementing arbitrary rotation and brightness variation to enhance the robustness in the recognition procedure. We divide these labels into 44 categories based on differences in patterns. Utilizing machine learning methods, we have achieved a total recognition (including extraction and search process) time per label averaging 421.96 ms without classification, and 40.13 ms with classification. Specifically, the search process with classification is nearly fiftieth times shorter than the non-classification method, reaching 8.52 ms in average. The overall recognition time is much faster than previous works, and achieves an accuracy of over 98.7%. This work significantly increases the practical significance of pixel-level anti-counterfeiting labels.

Simultaneous dual-colour labelling of mitochondria and lysosomes: An indolium-based approach

Fluorescence is a valuable tool for understanding mitochondria-lysosome interactions, which is central for unveiling their roles in health and disease. Herein, we describe novel indolium-based molecules for labelling mitochondria and lysosomes simultaneously with organelle-specific fluorescence properties. These probes readily enable clear distinction of mitochondria and lysosomes by combining confocal and two-photon fluorescence microscopy. Our results show that this unique behaviour relies on aggregation. The functioning of these probes is not compromised by mitophagy, allowing the visualisation of these organelles in separate emission channels by using a single fluorescent marker under all scenarios. Their enhanced experimental simplicity compared to using multiple dyes makes these novel compounds well-suited for automated screening applications within living cells.

Green Synthesis and Characterization of Hydroxyapatite Nanorods with Enhanced Antibacterial and Anticancer Properties

This study reports the microwave synthesis of hydroxyapatite nanorods using Lantana camara L. flower extract (LCF-HA) and characterized by FTIR, XRD, TEM and SEM-EDX techniques. The extract coating on the hydroxyapatite nanorods improves the antibacterial efficacy against Gram-positive bacteria. The study also evaluates the mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and capacity to induce apoptosis of LCF-HA. It shows that LCF-HA efficiently inhibits the growth of osteosarcoma cells (MG-63) in a dose-dependent manner. The efficacy of LCF-HA in inducing apoptosis in osteosarcoma cells is established by dual acridine orange/ethidium bromide (AO/EB) fluorescence labelling. Additionally, LCF-HA induces an increase in reactive oxygen species (ROS) generation and a reduction in mitochondrial membrane potential (MMP) levels. The findings indicate that derived LCF-HA has significant anticancer efficacy by inducing apoptosis in osteosarcoma cells through enhanced ROS generation, suggesting that derived LCF-HA may serve as a promising therapeutic target for bone cancer cell lines. Furthermore, the study demostrates excellent compatibility with osteoblast cells, highlighting its potential for bone regeneration applications.

Targeted Magnetic Nanoparticles for Beta-Amyloid Detection

Background/Objectivities: The presence of beta-amyloid plaques is a part of the pathogenesis of Alzheimer’s disease, but there is currently no universally accepted method for magnetic resonance (MR) imaging of the disease. However, it is known that magnetic nanoparticles (MNPs) can improve the T2 contrast in MR images of various targets. Methods: We used cubic MNPs, which were produced by thermal decomposition and then it was covalently bonded to a modified fluorescently labeled tetrapeptide, HAEE-Cy5, for visualizing beta-amyloid plaques. The interaction of MNPs-HAEE-Cy5 and beta-amyloid was determinate by confocal microscopy using SH-SY5Y cell line. Results: MNPs exhibit relatively high relaxivity (approximately 200 mM−1s−1), which is crucial for enhancing target visibility in MR imaging. HAEE provides targeted delivery of MNPs by specifically interacting with beta-amyloid, while the fluorescent label Cy5 enables monitoring the efficacy of the interaction through confocal microscopy. Conclusions: The MNPs modified with HAEE-Cy5 demonstrated excellent binding to beta-amyloid plaques in vitro, as shown by experiments on the SH-SY5Y cell line. These results suggest that the proposed method has potential for use in future MR imaging studies of Alzheimer’s disease.

Ultrafast and Ultrasensitive Bacterial Detection in Biofluids: Leveraging Resazurin as a Visible and Fluorescent Spectroscopic Marker.

Here we report the application of chemometric analysis for modeling absorbance spectroscopy and fluorescence emission data from a resazurin-based assay targeting low-level bacterial detection in biofluids. Bacteria spiked samples were incubated with resazurin and absorbance and fluorescence data were collected at 30 min intervals. The absorbance data was subjected to Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) and compared with the univariate fluorescence spectroscopy approach. The analysis demonstrated the multidimensional nature of the absorbance data, highlighting the appearance of the resorufin peak at the 2 h time point with a low bacterial inoculum of 0.01 CFU mL-1 across all the samples tested-water, urine and serum. The PLSR models supported the PCA data and exhibited strong predictive capabilities for water (RC2 = 0.937, RCV2 = 0.934), urine (RC2 = 0.899, RCV2 = 0.880) and serum (RC2 = 0.985, RCV2 = 0.967). Conversely, fluorescence is contingent upon resorufin existence, necessitating a prolonged waiting period postincubation with resazurin to verify the presence of bacteria, especially when contamination levels are low. Given the substantial global impact of bacteria-related infections, this method detects bacteria at low concentrations precisely and rapidly, improving efficiency and adaptability for point-of-care settings, promising swift diagnosis of bacterial infections, environmental monitoring, or food-quality control.

A low-cost device for measuring TEER at cultivation of epithelial/endothelial cells on inserts.

Background: The barrier properties of epithelial and endothelial cells are usually studied in vitro when cells are cultured on mesh inserts in culture plates, and it is necessary to assess the state of the cell monolayer on the membrane of the inserts before the experiment. Typically, monolayer density is analyzed by passing a fluorescent label through the insert with cells or by measuring the transendothelial resistance (TEER) of the cell layer. Many studies use both methods of assessing monolayer integrity in parallel, depending on the purpose of the study. The TEER method also allows to record early changes in the monolayer state under the action of various substances. Aim of this work is to demonstrate the possibility of TEER measurements using the proposed device (conductometer) made from freely available components using endothelial/epithelial cells as an example. Materials and Methods. A device (conductometer) for measuring TEER, created on the basis of easily accessible components, is presented. The device was tested by culturing two cell lines – hybridoma of endothelial origin Ea.hy926 and human colon adenocarcinoma Caco-2. Caco-2 cells were cultured for 22 days and Ea.hy926 cells were cultured for 7 days. The integrity of the cell monolayer and the density of intercellular contacts were evaluated by the TEER value determined by the proposed conductometer, as well as by the fluorescein permeability of the cell monolayer. Results: The results of TEER measurements using the proposed device and at the same time, the fluorescein permeability of the cell monolayer during the cultivation of Caco-2 and EA.hy926 cells are presented. For Caco-2 cells from the moment of 100% confluence the TEER value gradually increased reaching maximum values by 20-21 days, after which it decreased slightly. The permeability values decreased as the cells were cultured, indicating the formation of dense contacts. For EA.hy926 cells the rise in TEER values are observed on day 3 and decrease was observed on day 7. The results of TEER and permeability obtained by the proposed conductometer have a strong inverse correlation for both cell lines and are in good agreement with each other. Conclusions. The presented device, made on the basis of simple and affordable components, is similar to commercially available devices and can be used to study the integrity and density of a monolayer in the cultivation of epithelial/endothelial cells, to study the processes of trans/paracellular transport under the action of various substances, as well as in experiments with the co-cultivation of various cell lines.

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

Triarylmethanol Derivatives with Ultralong Organic Room-Temperature Phosphorescence.

Triphenylmethyl-based compounds such as rhodamines and fluoresceine representing an old and well-known class of triphenylmethane dyes, are widely used in fluorescent labeling of bioimaging. Inspired by ultralong room temperature phosphorescence of triphenylphosphine derivatives, herein we report a methoxy substituted triarylmethanol ((4-methoxyphenyl)diphenylmethanol, LJW-1) exhibits ultralong room temperature phosphorescence (RTP) under ambient condition with afterglows of about 7 seconds. Its multiple C-H···π intermolecular interactions, C-H···O intermolecular interactions, hydrogen bond and π-π interactions are beneficial for forming rigid environment in the aggregated state which is evidently an important factor in the appearance of excellent RTP. Different substituents on triarylmethanol result in compounds with different lifetimes varying from 7 µs to 818 ms. The substituent effects on the phosphorescence of triarylmethanol derivatives provide an efficient method for the design of organic RTP materials and may be enlightening the phosphorescence research of triarylmethanol derivatives.