Red Fluorescence Intensity Research Articles

Effects of mixing of europium oxide in resin composites on the fluorescence characteristics and mechanical properties

ObjectiveThe objective of this study is to disperse europium oxide (Eu2O3) in a resin composite (RC) using a planetary centrifugal mixer and assess its effects on photoluminescence and mechanical properties.Materials and methodsA commercially available RC was mixed with Eu2O3 at various concentrations using a planetary centrifugal mixer. The fabricated samples were evaluated using scanning electron microscopy and a spectrofluorometer to assess their crystal structures, particle sizes, and photoluminescence properties. Vickers microhardness measurements were performed, along with a three-point bending test. Statistical analyses were performed to assess the mechanical properties.ResultsThe intensity of red fluorescence increased with the increase of europium oxide concentration. The fluorescence spectra at 613 and 620 nm exhibited higher intensities under excitation at 254 nm. Eu2O3 was dispersed in RC regardless of the Eu2O3 concentration, and no aggregation was observed. Regarding the mechanical properties, there were no significant differences in the flexural strength or modulus, and the Vickers hardness gradually increased with increasing Eu2O3 concentration.ConclusionTo mix Eu2O3 with RC, visible fluorescence emission was observed even with increasing the Eu2O3 concentration, and the mechanical properties of RC were unaffected. Based on our study, a 15 wt% concentration of Eu₂O₃ is the appropriate concentration, as it achieves strong fluorescence emission without compromising the mechanical properties or color tone of the RC.

The Circadian Rhythm of Intracellular Protoporphyrin IX Accumulation Through Heme Synthesis Pathway in Bladder Urothelial Cancer Cells Exposed to 5-Aminolevulinic Acid.

Background/Objectives: The standard recommendation for patients with non-muscle invasive bladder cancer is 5-aminolevulinic acid-mediated photodynamic diagnosis. The intensity of the fluorescence caused by the intracellular accumulation of protoporphyrin IX (PPIX) varies among tumors and patients. This study investigated the circadian rhythm of intracellular PPIX accumulation in bladder urothelial cancer cells exposed to 5-aminolevulinic acid. Methods: The expression of two clock genes, PER2 and BMAL1, and their impact on intracellular PPIX accumulation were evaluated in two bladder cancer cell lines, UM-UC-3 and J82, and mouse xenograft models. We evaluated the enzymes involved in the heme synthesis pathway that potentially affect the circadian rhythm of intracellular PPIX accumulation. The red fluorescence intensity of the images captured during photodynamic diagnosis-assisted transurethral resection of bladder tumors was quantified and compared among the four groups according to surgery start time: 9 a.m.-11 a.m., 11 a.m.-1 p.m., 1-3 p.m., and 3-5 p.m. Results: We observed the circadian rhythm of intracellular PPIX accumulation, which was potentially regulated by the clock genes PER2 and BMAL1. Two enzymes involved in the heme synthesis pathway, coproporphyrinogen oxidase and ferrochelatase, exhibit a circadian rhythm. The fluorescence intensity started gradually increasing at 12 p.m., and the highest level was observed in patients who underwent surgery between 3 and 5 p.m. Conclusions: Our findings suggest that it may be possible to optimize the timing of the photodynamic diagnosis in photodynamic diagnosis-assisted transurethral resection of bladder cancer based on the circadian rhythm to improve tumor detection and treatment outcomes.

Antibacterial Effects of Paeonia lactiflora Extract on Oral Microcosm Biofilms

The aim of this study was to evaluate the antibacterial effects of Paeonia lactiflora (PL) extract on oral microcosm biofilms and determine its usefulness as a natural alternative to current antimicrobial agents. Oral biofilms were cultivated on hydroxyapatite disks using human saliva for 2 days, following which they were treated with 1.5 mL of distilled water (DW), 30 μg/mL of PL extract, or 0.12% chlorhexidine gluconate (CHX) once daily for 3 days. Antibacterial effects were assessed by measuring red fluorescence intensity (RatioR/G), bacterial viability (RatioG/G+R), and aciduric bacterial counts. RatioR/G was evaluated using quantitative light-induced fluorescence digital imaging. RatioR/G and RatioG/G+R in the PL group were 0.94-fold and 0.73-fold lower than those in the DW group (p < 0.001 and p = 0.011, respectively) and comparable to those in the CHX group (p = 0.356 and p = 0.964, respectively). Aciduric bacterial counts in the PL group were 0.97-fold lower than those in the DW group (p < 0.001), with no significant difference from those in the CHX group (p = 0.892). Thus, the antibacterial effect of PL extract was comparable to that of CHX; this suggests its potential as a natural alternative for managing oral biofilm-associated diseases.

Highly sensitive, environmental-friendly and rapid visual detection of uranium using a novel fluorescent fiber-optical sensing system based on aggregation-induced emission with bovine serum albumin as sensitizing agent

In order to achieve the on-site rapid detection of trace uranium under excitation with visible light and avoid the harmful effect of toxic organic solvent to human body and organisms, in this work, a novel water-soluble, environmental-friendly and biocompatible aggregation-induced emission (AIE) fluorescence probe was fabricated by introducing the pyridine quaternary ammonium cation and amide group into the propeller structure of triphenylamine (TPA). And by creatively using bovine serum albumin (BSA) in PBS buffer as the sensitizing agent to replace the toxic organic solvent, the probe can emit intense red fluorescence under excitation with visible light (470 nm), exhibiting very low cytotoxicity and great anti-interference ability against large equivalent of different metal ions and anions over wide pH range. After further integration with the self-constructed portable fiber optical sensing system, the satisfying detection limit as low as 7.44 nM (1.77 ppb) can be achieved. The established method has been applied successfully for sensing uranium in complex real samples such as uranium leachate, seawater and cancer cells, demonstrating excellent analytical performance and great potential for in-situ rapid detection and timely evaluation of the uranium contamination in vivo and in field.

Intense Red Fluorescence From Surface Traps in BaSO4:V5+, Eu3+ and Its Applications.

BaSO4:V5+ synthesized at 1000°C in air exhibits an intense emission band peaking at 495 nm from surface traps assisted by atmospheric oxygen on excitation at 350 nm due to charge transfer from O2- to V5+ in the (VO4)3- tetrahedral complex. BaSO4:Eu synthesized under similar conditions exhibits both Eu2+ (375 nm) and Eu3+ (619 nm) fluorescence. Vanadium codoping quenches the Eu2+ fluorescence but enhances the Eu3+ fluorescence in BaSO4:V, Eu due to charge compensation. However, Eu codoping quenches the vanadium fluorescence by diffusion of vanadium into the crystal, and V2O5 also serves as a flux enhancing Eu3+ doping efficiency in BaSO4 lattice. The 619nm Eu3+ PL emission intensity in BaSO4:V5+, Eu3+ is comparable to 592nm emission from commercial (Y,Gd) BO3:Eu3+. However, V5+ emission shows strong thermal quenching, while Eu3+ emission shows little thermal quenching up to 210°C, making BaSO4:V5+, Eu3+ a promising new red phosphor for improving the color rendering of blue light-based white LED. The ratio of Eu3+ to V5+ fluorescence increases with excitation temperature (30°C°C-180°C) enabling BaSO4:V5+, Eu3+ to be used as a non-contact luminescence thermometer.

Rational design of an ultrabright quinolinium-fused rhodamine turn-on fluorescent probe for highly sensitive detection of SO2 derivatives: Applications in food safety and bioimaging

Sulfur dioxide (SO2) is an essential signaling molecule involved in various physiological processes within living organisms. Bisulfite (HSO3−) possesses antioxidant, antimicrobial, and preservative properties, making it a common food additive. However, elevated levels of SO2 or excessive HSO3− intake can lead to a range of diseases, highlighting the importance of detecting SO2 and its derivatives (HSO3−/SO32−). This study presents a quinolinium-fused rhodamine fluorogenic probe (RQB-R) for ultrafast, highly selective, and sensitive detection of HSO3−. The probe operates via a dual-response mechanism, exhibiting a visible color change and a transition from nonemissive to intense red fluorescence upon interaction with HSO3−. The detection mechanism involves a 1,4-nucleophilic addition reaction of HSO3− at the 4-position of the quinolinium unit, which bypasses the photoinduced electron-transfer fluorescence quenching pathway and activates the intramolecular charge transfer mechanism, thereby enhancing fluorescence emission. Practical applications of the RQB-R probe include rapid quantification of HSO3− levels in sugar samples and integration into smartphone-assisted detection platforms. This method demonstrates excellent biocompatibility and enables visualization of both exogenous and endogenous HSO3− within MCF-7 cells, with a specific focus on targeting mitochondria.

Antibacterial Effects of Black Cumin Seed Oil on Oral Microcosm Biofilms.

Interest in natural extracts for managing oral biofilms is increasing, with black cumin seed oil (BCSO) demonstrating efficacy against Streptococcus mutans. The effectiveness of antibacterial agents should be evaluated using multi-species oral biofilm models that closely mimic actual conditions. This study aimed to compare the antibacterial effects of BCSO and chlorhexidine gluconate (CHX) on oral microcosm biofilms. Biofilms using human saliva as the inoculum were cultured for 2 days and subsequently treated with 0.5% dimethyl sulfoxide, 0.5% BCSO, or 0.12% CHX once daily for 6 days. Following treatment, the red fluorescence intensity (RatioR/G) of the oral biofilm; biomass, including extracellular polymeric substance (EPS) levels and live bacteria counts; and colony-forming units (CFUs) of aciduric bacteria were evaluated. RatioR/G after BCSO treatment (1.26 ± 0.03) was not significantly different from that after CHX treatment (p = 0.552). The EPS levels were also not significantly different between the two groups (p = 0.743). The live bacteria count was 0.55 times lower in the BCSO-treated group than in the CHX-treated group (p = 0.018). No significant between-group difference was observed in the CFUs of aciduric bacteria (p = 0.935). These results suggest that BCSO exhibits antibacterial effects similar to those of CHX, highlighting its potential as an effective alternative.

Unveiling dynamic alterations of lipid droplet polarity during NAFLD-triggered lipophagy utilizing a far-red fluorescent probe with large stokes shift

Lipophagy as a form of autophagy, can degrade lipid droplets, thereby acting as a critical regulator of cellular lipid metabolism, helping maintain the intracellular lipid homeostasis. In this study, we developed LP-SCUN, a far-red fluorescent probe for pinpointing lipid droplets with high sensitivity to solvent polarity. This probe allows for in situ imaging of lipophagy, tracking how lipid droplets move from non-polar to polar environments within lysosomes, leading to noticeable changes in fluorescence signals. The triphenylamine and triethylene glycol monomethyl ether groups of LP-SCUN facilitated its selective binding toward lipid droplets. Significantly, lipophagy and subsequent formation of autolysosomes decreased the environmental polarity, leading to a significant decrease in red fluorescence intensity (λex = 500 nm and λem = 643 nm). As such LP-SCUN was suitable for the in situ and real-time tracking of the cellular lipophagic processes. With this research we used LP-SCUN to elucidate the mechanism of lipid metabolism in liver cells of palmitate-induced hyperlipidemia cell models and high-fat diet-induced hyperlipidemia animal models. The results indicated that non-alcoholic fatty liver disease (NAFLD) can trigger an increase in cell lipophagy levels, while the inhibition of cell lipophagy can effectively alleviate the damage caused by NAFLD to cells and liver tissue in mice.

Self-Internal Standard Fluorescence for Ultrasensitive Detecting of mtDNA to Evaluate Matrilineal Genetic Defect Levels.

Mitochondrial DNA (mtDNA) is a unique genetic material characterized by maternal inheritance. It possesses a circular structure devoid of histone protection and exhibits low cellular abundance, which poses great challenges for its sensitive and selective detection at the living cell level. Herein, we have designed three bis-naphthylimide probes with varying linker lengths (NANn-OH, n = 0, 2, 6), facilitating the formation of distinct twisted or folded molecular conformations in the free state. These probes emit the red fluorescence around 627 nm with different fluorescence quantum yields (ΦNAN0-OH = 0.0016, ΦNAN2-OH = 0.0136, and ΦNAN6-OH = 0.0125). When encountering mtDNA (0.4-3.4 μg/mL), these probes undergo conformational changes depending on the length of the attached C-strand and exhibit a gradually increasing fluorescence signal around 453 nm. The fluorescence intensity increased to 13.5-fold, 1.9-fold, and 8.2-fold, respectively. Notably, the red fluorescence intensities around 627 nm remain constant throughout this process, thus serving as an inherent correction mechanism for proportional fluorescence signal enhancement to improve selectivity and sensitivity. NAN0-OH, NAN2-OH, and NAN6-OH showed good linearity for mtDNA in the range of 0.4-3.4 μg/mL with detection limits of LODNAN0-OH = 1.04 μg/mL, LODNAN2-OH = 1.10 μg/mL, and LODNAN6-OH = 1.15 μg/mL. Cellular experiments reveal that NAN6-OH effectively monitors curcumin-induced mtDNA damage in HepG-2 cells while enabling monitoring of genetic mtDNA damage. We anticipate that this tool holds significant potential for the precise evaluation of maternal genetic defects, thereby enhancing hypersensitive assessment in clinical medicine.

Synthesis, Crystal Structure, and Photophysical Properties of Bromothiophene-Functionalized BF2-Curcuminoid as a Versatile Building Block.

A novel bromothiophene-functionalized BF2-curcuminoid (BTC-BF2) is synthesized by Knoevenagel condensation reaction. The structure of BTC-BF2 is determined by 1H-nuclear magnetic resonance (1H NMR), 13C-nuclear magnetic resonance (13C NMR), and high-resolution mass spectrometry (HRMS). Moreover, a nearly coplanar single crystal structure is successfully obtained and form a mesh structure through intermolecular multiple C─H···F hydrogen bond interactions. As expected, as-prepared BTC-BF2 exhibits solvent-dependent photophysical properties in solvents with different polarity and an intense red solid-state fluorescence. Density functional theory calculations further verify the relationships between its intrinsic electronic features and the photophysical properties. For its potential application aspect, BTC-BF2 shows a certain ability to generate singlet oxygen under irradiation with 530nm green light. Moreover, BTC-BF2 can be utilized as versatile building block to construct novel far-red or NIR BF2-curcuminoid complexes for widely biological applications.

A novel low-background nitroreductase fluorescent probe for real-time fluorescence imaging and surgical guidance of thyroid cancer resection

Thyroid cancer always appears insidiously with few noticeable clinical symptoms. Due to its limitations, conventional ultrasound imaging can lead to missed or misdiagnosed cases. Surgery is still the primary treatment method of thyroid cancer, but removal of surrounding healthy tissues to minimize recurrence leads to overtreatment and added patient suffering. To address this challenge, herein, a nitroreductase (NTR) fluorescent probe, Ox-NTR, has been developed for detecting thyroid cancer and tracking the surgical removal of thyroid tumors by fluorescence imaging. The conjugated structure of oxazine 1 was disrupted, significantly reducing the issue of high background signals, thus effectively achieving low background fluorescence. Under hypoxic conditions, the nitro group of Ox-NTR can be reduced to an amine and subsequently decomposed into oxazine 1, emitting intense red fluorescence. Ox-NTR has a low detection limit of 0.09 μg/mL for NTR with excellent photostability and selectivity. Cellular studies show that Ox-NTR can effectively detect NTR levels in hypoxic thyroid cancer cells. Moreover, the ability of Ox-NTR of rapid response to thyroid cancer in vivo is confirmed by fluorescence imaging in mice, distinguishing tumors from normal tissues due to its superior low background fluorescence. Utilizing this fluorescence imaging method during surgical resection can guide the removal of tumors, preventing both missed tumor tissues and accidental removal of healthy tissue. In summary, the novel Ox-NTR offers precise detection capabilities that provide significant advantages over traditional imaging methods for thyroid cancer diagnosis and treatment, making it a valuable tool to guide tumor removal in surgical procedures.

An ESIPT + AIE based dual-response fluorescent probe for continuous detection of PhSH and HClO and visualization of PhSH-induced oxidative stress in living cells

As a valuable industrial chemical, thiophenol (PhSH) is poisonous, which can be easily absorbed by the human body, leading to many serious health issues. In addition, PhSH-triggered oxidative stress is considered to be related with the pathogenesis and toxicity of PhSH. Therefore, efficient methods for monitoring PhSH and ROS production induced by PhSH in living systems are very meaningful and desired. Herein, we reasonably developed a facile dual-response fluorescent probe (HDB-DNP) by incorporating the dinitrophenyl (DNP) group into a novel methylthio-substituted salicylaldehyde azine (HDB) with AIE and ESIPT features. The probe itself was non-fluorescent owing to the strong quenching effect of DNP group. In the presence of PhSH, HDB-DNP gave an intense red fluorescence (610 nm), which can rapidly switch to green fluorescence (510 nm) upon further addition of HClO, allowing the successive detection of PhSH and HClO in two well-separated channels. HDB-DNP proved to be a very promising dual-functional probe for rapid (PhSH: < 17 min; HClO: 10 s) and selective detection of PhSH and HClO in physiological conditions with low detection limit (PhSH: 13.8 nM; HClO: 88.6 nM). Inspired by its excellent recognition properties and low cytotoxicity, HDB-DNP was successfully applied for monitoring PhSH and PhSH-induced HClO generation in living cells with satisfactory results, which may help to better understand the pathogenesis of PhSH-related diseases.

Quantification and visualization of penicillin G residues in milk using a ratiometric fluorescent nanoprobe based on carbon dots and gold nanoclusters

Developing an effective assay for monitoring penicillin G (PNG) is important for safeguarding ensuring public health and food safety. This work details the application of carbon dots (CDs) emitting blue light and gold nanoclusters (GNCs) with red emission as ratiometric fluorescence (RF) probes for the sensitive detection of PNG. The RF probe under a single excitation of 360 nm wavelength displayed two discrete emission peaks at 444 and 660 nm related to CDs and GNCs, respectively. Following the introduction of PNG, the red fluorescence intensity of GNCs was found to be reduced through the inner filter effect (IFE); meanwhile, the blue fluorescence of CDs remained unchanged under optimal conditions. The quantification of PNG can be achieved by this RF nanoprobe by following the I660/I444 ratio in two ranges of 0.1–0.8 ppm and 0.8–30.0 ppm and with a limit of detection (LOD) as low as 30 ppb. Furthermore, the RF probe has been successfully employed to determine PNG in milk samples with acceptable recoveries and results were validated by HPLC. Finally, the visual capability of this RF probe can greatly enhance and facilitate on-site determination process of PNG.

Influence of Biofilm Maturity on the Antibacterial Efficacy of Cold Atmospheric Plasma in Oral Microcosm Biofilms.

As biofilms mature, biomass and extracellular polysaccharide (EPS) content increases, enhancing pathogenicity. Therefore, this study aimed to evaluate the antibacterial efficacy of cold atmospheric plasma (CAP) against oral microcosm biofilms and the influence of biofilm maturity on treatment. Oral microcosm biofilms were cultured on hydroxyapatite disks for 2 and 6 days. Based on the treatment and biofilm maturity, these were subsequently allocated into six groups (N = 19 each): Groups 1 and 2 were incubated with distilled water for 1 min; Groups 3 and 4 were treated with CAP for 2 min, and Groups 5 and 6 were treated with 0.12% chlorhexidine gluconate for 1 min. Groups 1, 3, and 5 represent 2-day biofilms, and Groups 2, 4, and 6 represent 6-day biofilms. Treatments were repeated daily for 5 days. Antibacterial efficacy was analyzed by measuring oral biofilms' red fluorescence intensity (RatioR/G) and quantifying EPS content and bacterial viability. The RatioR/G was 1.089-fold and 1.104-fold higher in Groups 4 and 6 than in Groups 3 and 5 following antibacterial treatment, respectively (p < 0.001). EPS content increased by 1.71-fold in Group 6 than in Group 5 (p < 0.001). Bacterial survival rate was the lowest in Group 3 (p = 0.005). These findings underscore the relevance of CAP treatment in maintaining antibacterial efficacy regardless of the biofilm development stage, highlighting its potential utility in oral care.

Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration

In this work, two neolignans – dehydrodieugenol (1) and dehydrodieugenol B (2) – were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 μM) whereas compound 2 displayed IC50 values of 169 and 174 μM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 μM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) – 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

Design a Friendly Nanoscale Chemical Sensor Based on Gold Nanoclusters for Detecting Thiocyanate Ions in Food Industry Applications.

The surfactant cetyltrimethylammonium bromide (CTAB) induces the aggregation of gold nanoclusters (GNCs), leading to the development of a proposed fluorometric technique for detecting thiocyanate (SCN-) ions based on an anti-aggregation mechanism. This approach is straightforward to execute, highly sensitive, and selective. A significant quenching effect occurs in fluorescence upon using the aggregation agent CTAB in GNCs synthesis, resulting in a transition from intense red fluorescence to dim red. The decrease in fluorescence intensity of GNCs in the presence of CTAB is caused by the mechanism of fluorescence quenching mediated by aggregation. As the levels of SCN- rise, the fluorescence of CTAB-GNCs increases; this may be detected using spectrofluorometry or by visually inspecting under UV irradiation. The recovery of red fluorescence of CTAB-GNCs in the presence of SCN- enables the precise and discerning identification of SCN- within the concentration range of 2.86-140 nM. The minimum detectable concentration of the SCN- ions was 1 nM. The selectivity of CTAB-GNCs towards SCN- ions was investigated compared to other ions, and it was demonstrated that CTAB-GNCs exhibit exceptional selectivity. Furthermore, we believe that CTAB-GNCs have novel possibilities as favorable sensor candidates for various industrial applications. Our detection technique was validated by analyzing SCN- ions in milk samples, which yielded promising results.

Abstract 4224: The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment

Abstract We developed an in vitro protocol to generate tumor-specific cytotoxic lymphocytes (CTL) by priming T cells from healthy donors on HER2+ breast cancer cell line SKBR-3. Subsequently, these primed CTLs were tested in a 3D immune cell killing assays in a life cell imaging device. An image analysis workflow was applied to understand the T cell movement in spatial relation to the tumor and the kinetic of tumor cell killing. The activity of the CTLs was compared with non-activated CD8+T cells, staurosporin and CD8+ T cells, unspecifically activated by αCD2/αCD3/αCD28 beads. The tumor cells were expressing mKate whereas the T cells were label-free. By analyzing a combination of different masks, we could computationally measure tumor spheroid characteristics and dynamic attributes such as changes in shape, percent of cells expressing red fluorescentce, and relative average distance of T cells from the spheroid in each timelapse assay. This process was applied at each frame to determine the average T cell distance from its corresponding spheroid center and subsequently calculate the average T cell flux over the course of each assay by linking data between image frames. Additional measurements allowed for the quantification of tumor debris accumulation over time both relative to the primary tumor mass and in absolute terms. Our analyses revealed that bead-activated T cells approached the tumor spheroid with the highest speed and infiltrated the spheroid homogeneously. In contrast the CTLs approached the spheroid slower and started tumor cell killing from the rim of the spheroid towards the center. The reduction of spheroid area over time displayed a very similar kinetic when co-cultured with CTL or bead-activated T cells. At the end of the experiment the CTLs induced a more pronounced shrinkage of spheroid size. Interestingly the size of the spheroid and the red fluorescence intensity were not necessarily aligned. The positive control Staurosporin induced a fast reduction of the mKate signal, whereas the spheroid area did not change over time. In summary, the bead-activated T cell approached the spheroid fast, infiltrated the complete area and induced tumor cell killing homogenously across the tumor area. The CTL approached the spheroids slower but started killing earlier from the outside towards the center of the spheroid. The observed differences in infiltration and killing characteristics of the investigated T cell types lead to a better understanding of T cell biology in the context of the tumor biology, which can be translated into applications during dug development. Taken together, with the applied image analysis algorithm we were able to deconvolute the biological processes in the assay in much more detail. The additional read-outs increase the translational value of the assay tremendously in the context of drug development as well as precision medicine. Citation Format: Ryan Kerwin, Ina Rohleff, Martin Blanchard, Kanstantsin Lashuk, Bryan Walker, Julia B. Schueler. The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4224.

Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography.

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.

Time-traceable micro-taggants for anti-counterfeiting and secure distribution of food and medicines.

This study presents an innovative solution for the enhanced tracking and security of pharmaceuticals through the development of microstructures incorporating environmentally responsive, coded microparticles. Utilizing maskless photolithography, we engineered these microparticles with a degradable masking layer with 30 μm thickness that undergoes controlled dissolution. Quantitative analysis revealed that the protective layer's degradation, monitored by red fluorescence intensity, diminishes predictably over 144 h in phosphate-buffered saline under physiological conditions. This degradation not only confirms the microparticles' integrity but also allows the extraction of encoded information, which can serve as a robust indicator of medicinal shelf life and a deterrent to tampering. These findings indicate the potential for applying this technology in real-time monitoring of pharmaceuticals, ensuring quality and authenticity in the supply chain.

Single Fluorescent Probe for Multiple Tasks: Illuminating Lipid Droplets and Lysosomes in Dual Channels and Distinguishing Autophagy and Apoptosis.

Lipid droplets (LDs) and lysosomes play key roles in autophagy and cell apoptosis, and the discriminative visualization of the two organelles and simultaneously of autophagy and apoptosis is very helpful to understand their internal relationships. However, fluorescent probes that can concurrently achieve these tasks are not available currently. Herein, we delicately fabricate a robust probe CAQ2 for multiple tasks: illumination of LDs and lysosomes in dual emission colors as well as discriminative visualization of cell apoptosis and autophagy. The probe exhibited both lipophilic and basic properties and displayed different emission colors in neutral and protonated forms; thus, LDs and lysosomes emitted blue and red fluorescence colors, respectively. Because of the lysosomal acidification during autophagy, CAQ2 detected autophagy with evidently enhanced red emission. Because of the lysosomal alkalization during apoptosis, CAQ2 imaged apoptosis with a drastically decreased red fluorescence intensity. With the robust probe, the autophagy under starvation and lipidless conditions was visualized, and the apoptosis induced by H2O2, ultraviolet (UV) irradiation, and rotenone treatment was successfully observed. The efficient detoxification of Na2S against rotenone treatment was successfully revealed.