Fluorescence Labeling Of Cells Research Articles

Single-cell adhesive profiling in an optofluidic device elucidates CD8+ T lymphocyte phenotypes in inflamed vasculature-like microenvironments

Tissue infiltration by circulating leukocytes occurs via adhesive interactions with the local vasculature, but how the adhesive quality of circulating cells guides the homing of specific phenotypes to different vascular microenvironments remains undefined. We developed an optofluidic system enabling fluorescent labeling of photoactivatable cells based on their adhesive rolling velocity in an inflamed vasculature-mimicking microfluidic device under physiological fluid flow. In so doing, single-cell level multidimensional profiling of cellular characteristics could be characterized and related to the associated adhesive phenotype. When applied to CD8+ Tcells, ligand/receptor expression profiles and subtypes associated with adhesion were revealed, providing insight into inflamed tissue infiltration capabilities of specific CD8+ T lymphocyte subsets and how local vascular microenvironmental features may regulate the quality of cellular infiltration. This methodology facilitates rapid screening of cell populations for enhanced homing capabilities under defined biochemical and biophysical microenvironments, relevant to leukocyte homing modulation in multiple pathologies.

Combination Therapy of Bone Marrow Mesenchymal Stem Cell Transplantation and Electroacupuncture for the Repair of Intrauterine Adhesions in Rats: Mechanisms and Functional Recovery.

Transplantation of bone marrow mesenchymal stem cells (BMSCs) has demonstrated promising clinical utility in the treatment of endometrial injury and the restoration of fertility. However, since the efficacy of BMSCs after transplantation is not stable, it is very important to find effective ways to enhance the utilisation of BMSCs. Electroacupuncture (EA) has some positive effects on the chemotaxis of stem cells and diseases related to uterine injury. In this study, we established the intrauterine adhesion (IUA) model of the Sprague-Dawley rat using lipopolysaccharide infection and mechanical scratching. Phosphate-buffered saline, BMSCs alone, and BMSCs combined with EA were randomly administered to the rats. Fluorescent cell labelling showed the migration of transplanted BMSCs. H&E staining, Masson staining, Western blot, immunohistochemistry, ELISA, and qRT-PCR were utilised to detect changes in endometrial morphology and expressions of endometrial receptivity-related factors, endometrial pro-inflammatory factors, and fibrosis factors. Finally, we conducted a fertility test to measure the recovery of uterine function. The results showed that EA promoted transplanted BMSCs to migrate into the injured uterus by activating the SDF-1/CXCR4 axis. Endometrial morphology showed the most significant improvement in the BMSC + EA group. The expressions of endometrial pro-inflammatory factors and fibrosis indexes in the BMSC + EA group were lower than those in the model and BMSC groups. Further studies revealed that the expression of endometrial receptivity-related factors and the number of embryos implanted on day 8 of gestation increased in the BMSC + EA group compared with the model group and the BMSC group.

BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis.

In this work, we synthesized a green fluorescent dye derivative, 1,3,5,7-tetramethyl-BODIPY, with a heptyl substituent at the 8-position. The obtained highly hydrophobic compound was able to rapidly and irreversibly bind to eukaryotic cells. Incubation of cells with the dye over different periods of time or at different concentrations allowed us to control the degree of cell labeling and the level of fluorescence. This made it possible to modulate the fluorescence level of different eukaryotic cell cultures and then distinguish them by their level of fluorescence signal in the green channel in cytometric experiments. The labeled cells can be combined and further analyzed in the same test tube under identical conditions using the channels in which the dye does not fluoresce. This approach has been tested on a number of tumor cell cultures containing the HER2 receptor on their surface. The representation of the receptor in these cells was analyzed in one test tube in one run using a HER2-specific ligand based on the hybrid protein DARPin9_29-mCherry, which fluoresces in the red region of the spectrum.

Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea.

Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms.

Effect of photoconversion conditions on the spectral and cytotoxic properties of photoconvertible fluorescent polymer markers.

Fluorescence labeling of cells is a versatile tool used to study cell behavior, which is of significant importance in biomedical sciences. Fluorescent photoconvertible markers based on polymer microcapsules have been recently considered as efficient and perspective ones for long-term tracking of individual cells. However, the dependence of photoconversion conditions on the polymeric capsule structure is still not sufficiently clear. Here, we have studied the structural and spectral properties of fluorescent photoconvertible polymeric microcapsules doped with Rhodamine B and irradiated using a pulsed laser in various regimes, and shown the dependence between the photoconversion degree and laser irradiation intensity. The effect of microcapsule composition on the photoconversion process was studied by monitoring structural changes in the initial and photoconverted microcapsules using X-ray diffraction analysis with synchrotron radiation source, and Fourier transform infrared, Raman and fluorescence spectroscopy. We demonstrated good biocompatibility of free-administered initial and photoconverted microcapsules through long-term monitoring of the RAW 264.7 monocyte/macrophage cells with unchanged viability. These data open new perspectives for using the developed markers as safe and precise cell labels with switchable fluorescent properties.

Synthesis and Application of a Near-Infrared Light-Emitting Fluorescent Probe for Specific Imaging of Cancer Cells with High Sensitivity and Selectivity.

The preclinical diagnosis of tumors is of great significance to cancer treatment. Near-infrared fluorescence imaging technology is promising for the in-situ detection of tumors with high sensitivity. Here, a fluorescent probe was synthesized on the basis of Au nanoclusters with near-infrared light emission and applied to fluorescent cancer cell labeling. Near-infrared methionine-N-Hydroxy succinimide Au nanoclusters (Met-NHs-AuNCs) were prepared successfully by one-pot synthesis using Au nanoclusters, methionine, and N-Hydroxy succinimide as frameworks, reductants, and stabilizers, respectively. The specific fluorescence imaging of tumor cells or tissues by fluorescent probe was studied on the basis of SYBYL Surflex-DOCK simulation model of LAT1 active site of overexpressed receptor on cancer cell surface. The results showed that Met-NHs-AuNCs interacted with the surface of LAT1, and C_Score scored the conformation of the probe and LAT1 as five. Characterization and in vitro experiments were conducted to explore the Met-NHs-AuNCs targeted uptake of cancer cells. The prepared near-infrared fluorescent probe (Met-NHs-AuNCs) can specifically recognize the overexpression of L-type amino acid transporter 1 (LAT1) in cancer cells so that it can show red fluorescence in cancer cells. Meanwhile, normal cells (H9c2) have no fluorescence. The fluorescent probe demonstrates the power of targeting and imaging cancer cells.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell–cell interactions in metastatic niches

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell–cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Semiconducting Polymer Dots Directly Stabilized with Serum Albumin: Preparation, Characterization, and Cellular Immunolabeling.

Semiconducting polymer dots (Pdots) are brightly fluorescent nanoparticles of growing interest for bioanalysis and imaging. A recurring challenge with these materials is obtaining robust physical and colloidal stability and low nonspecific binding. Here, we prepared and characterized Pdots with bovine serum albumin (BSA) as the stabilizing agent (BSA-Pdots) instead of a more conventionally used amphiphilic polymer, both without and with cross-linking of the protein using glutaraldehyde (BSA(GA)-Pdots) or disuccinimidyl glutarate. Characterization included fluorescence properties; colloidal stability as a function of pH, ionic strength, and solvent perturbation; shape retention and hardness; and nonspecific binding with common assay substrates, fixed cells, and live cells. These properties were contrasted with the same properties for amphiphilic polymer-stabilized Pdots and silica-coated Pdots. On balance, the BSA-stabilized Pdots were similar or more favorable in their properties, with BSA(GA)-Pdots being especially advantageous. Bioconjugation of the BSA-stabilized Pdots was possible using amine-reactive active-ester chemistry, including biotinylation and bioorthogonal functionalization for immunoconjugation via tetrazine-strained-alkene click chemistry. These approaches were used for selective fluorescent labeling of cells based on ligand-receptor and antibody-antigen binding, respectively. Overall, direct BSA stabilization is a very promising strategy for preparing Pdots with improved physical and colloidal stability, reduced nonspecific interactions, and utility for in vitro diagnostics and other bioanalyses and imaging.

Gelatin methacryloyl and Laponite bioink for 3D bioprinted organotypic tumor modeling

Three-dimensional (3D) in vitro tumor models that can capture the pathophysiology of human tumors are essential for cancer biology and drug development. However, simulating the tumor microenvironment is still challenging because it consists of a heterogeneous mixture of various cellular components and biological factors. In this regard, current extracellular matrix (ECM)-mimicking hydrogels used in tumor tissue engineering lack physical interactions that can keep biological factors released by encapsulated cells within the hydrogel and improve paracrine interactions. Here, we developed a nanoengineered ion-covalent cross-linkable bioink to construct 3D bioprinted organotypic tumor models. The bioink was designed to implement the tumor ECM by creating an interpenetrating network composed of gelatin methacryloyl (GelMA), a light cross-linkable polymer, and synthetic nanosilicate (Laponite) that exhibits a unique ionic charge to improve retention of biological factors released by the encapsulated cells and assist in paracrine signals. The physical properties related to printability were evaluated to analyze the effect of Laponite hydrogel on bioink. Low GelMA (5%) with high Laponite (2.5%–3.5%) composite hydrogels and high GelMA (10%) with low Laponite (1.0%–2.0%) composite hydrogels showed acceptable mechanical properties for 3D printing. However, a low GelMA composite hydrogel with a high Laponite content could not provide acceptable cell viability. Fluorescent cell labeling studies showed that as the proportion of Laponite increased, the cells became more aggregated to form larger 3D tumor structures. Reverse transcription-polymerase chain reaction (RT-qPCR) and western blot experiments showed that an increase in the Laponite ratio induces upregulation of growth factor and tissue remodeling-related genes and proteins in tumor cells. In contrast, cell cycle and proliferation-related genes were downregulated. On the other hand, concerning fibroblasts, the increase in the Laponite ratio indicated an overall upregulation of the mesenchymal phenotype-related genes and proteins. Our study may provide a rationale for using Laponite-based hydrogels in 3D cancer modeling.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

DeepIFC: Virtual fluorescent labeling of blood cells in imaging flow cytometry data with deep learning.

Imaging flow cytometry (IFC) combines flow cytometry with microscopy, allowing rapid characterization of cellular and molecular properties via high-throughput single-cell fluorescent imaging. However, fluorescent labeling is costly and time-consuming. We present a computational method called DeepIFC based on the Inception U-Net neural network architecture, able to generate fluorescent marker images and learn morphological features from IFC brightfield and darkfield images. Furthermore, the DeepIFC workflow identifies cell types from the generated fluorescent images and visualizes the single-cell features generated in a 2D space. We demonstrate that rarer cell types are predicted well when a balanced data set is used to train the model, and the model is able to recognize red blood cells not seen during model training as a distinct entity. In summary, DeepIFC allows accurate cell reconstruction, typing and recognition of unseen cell types from brightfield and darkfield images via virtual fluorescent labeling.

Experimental investigation of effects of indentation depth and probe tip radius on apparent young's modulus of Xenopus laevis oocytes

Throughout their lifespan cells are constantly subjected to mechanical forces. Numerous studies have been conducted to measure mechanical properties of animal cells and understand their effects of the cells’ biological processes. However, the range of the apparent Young's moduli reported in the literature spans three orders of magnitude from 0.1 kPa to 100 kPa, and no clear explanation is given for the source of variation. One of the commonly used experimental techniques to measure material properties at nano- and microscale is nanoindentation. Nanoindentation works by applying a force on a sample via a small probe and recording a force-displacement curve. Probes with different tip shapes and sizes are available. However, there is little guidance on the appropriate choice of probe geometry and size for measurement of properties of animal cells. Eukaryotic cells have a complex structure including a membrane, an actin cortex, a cytoplasm, a nucleus and a cytoskeleton which consists of microfilaments, intermediate filaments and microtubules. These components have different mechanical properties contributing to the overall properties of cells. This work focuses on the effect of tip size and indentation depth on the apparent Young's modulus of Xenopus laevis oocytes measuring approximately 1 mm in diameter. Live cell fluorescent labeling using Lifeact-EGFP and confocal microscopy was used to measure the thickness of the oocyte actin cortex which is expected to have a significant contribution to the cell's stiffness. Apparent mechanical properties were measured using the JPK NanoWizard 4 BioAFM system. Indentations 5, 100 and 400 nm deep using probes with spherical tips of 0.1, 1, and 10 μm radii were performed. Contributions of the membrane and the actin cortex on the overall mechanical properties of the oocytes are analyzed.

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Water-actuated reversible shape-memory polydimethylsiloxane for potential biomedical applications.

Shape memory polymers (SMPs) show great potential in biomedical fields. However, most of the SMPs are not suitable for use in the human body due to their deleteriousness and harsh actuation conditions. It is important to diversify SMPs that could be actuated in the human body environment. Herein, we construct a reversible shape-memory polydimethylsiloxane (RSMPDMS) based on a feasible strategy by deposing the PDMS-salt layer with dynamic micro-creases on the pure PDMS layer. Testing results reveal that it equips with self-expanding, bio-compatibility, drug storage-release and good mechanical toughness. The RSMPDMS could be variously shaped, such as ring, coil, and spiral. The prepared samples present efficient deformation-recovery with high mechanical stability during water absorption-desorption cycles. Moreover, the RSMPDMS is confirmed biocompatible by cell viability analysis and cell fluorescent labeling method, accompanied with efficient drug storage-release. The novel-designed RSMPDMS may contribute to the development of new shape memory biomedical materials.

Perfusion-Based Fluorescent Dye Labeling to Sort Cancer Cells Based on Their Distance from Blood Vessels.

Tumor vasculature is the major extrinsic factor that shapes Intra-tumoral heterogeneity (ITH). Non-uniform exposure of microenvironmental cues greatly impacts cancer cell phenotypes leading to ITH, which exacerbates therapy resistance. This raises a need to study the influence of non-uniform perfusion patterns and the resulting heterogeneity that persists within the tumor microenvironment (TME). A method was developed to identify cancer cells based on their proximity to functional blood vessels (BVs) called perfusion-based fluorescent dye labeling of cells (PFDLC). PFDLC works on the principle of perfusion, where a freely diffusible nuclear binding fluorescent dye (Hoechst 33342) is injected intravenously (i.v.) through a tail vein into atumor-bearing mice. The tumors are retrieved post dye perfusion, dissociated into single cells, and sorted based on their dye uptake proportional to their distance from the nearest blood capillary. This method is amenable to multi-omics as well as functional assays.

Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach

BackgroundEndothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as “no-option,” due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied.MethodsIn the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI.ResultsThe optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs.ConclusionOverall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.

Preparation of layering-structured magnetic fluorescent liposomes and labeling of HepG2 cells.

At present, surgical resection and chemotherapy are still the main treatments for hepatocellular carcinoma and other cancers, but the curative effect and survival rate are not ideal. In this study, we aim to prepare a carrier with low toxicity, high biocompatibility and targeted transport for the treatment of hepatocellular carcinoma. CdSe quantum dots (QDs) modified with oleic acid were synthesized. Then hydrophobic CdSe QDs and hydrophilic super-paramagnetic Fe3O4 particles were encapsulated into different layers of liposomes to form magnetic fluorescent liposomes (MFLs). MFLs in the aqueous would quickly drift towards the external magnet and the entire process was clearly observed with fluorescence microscope. The fluorescence spectra revealed that the fluorescence properties of MFLs were similar to that of CdSe QDs. QDs had an average size of 3.32 nm with good fluorescence properties. The size of MFLs was about 100 nm (transmission electron microscopy (TEM) analysis showed the average size of MFLs was about 82.8 nm and dynamic light scattering (DLS) detection showed 111.9 nm). After being cultured with MFLs for 8 h, HepG2 cells were labeled by MFLs, and good fluorescence images were obtained. MTT analysis also expressed their good biocompatibility. The prepared MFLs had multi-function and could be used as ideal drug carriers.

Water‐Dispersing Perovskite Probes for the Rapid Imaging of Glioma Cells

AbstractReal‐time in vitro detection of glioma cells facilitates precise tumor removal. However, the fluorescent labeling of tumor cells in clinical practice is limited by many factors, including the time consumed, low recognition efficiency, and fluorescence quenching. Here, a general strategy for building perovskite quantum dot (PQD)‐based biological probes utilizing the attraction between positive and negative electric charges is reported. Poly (lactic‐co‐glycolic acid) (PLGA) is chosen for encapsulating PQDs to completely prevent their aggregation, decomposition, or release in water or oxygen. The carboxyl group of PLGA has anchoring coordination with the PQDs, which reduces the surface defects. Moreover, it causes PQD‐based nanocrystals (P‐PNCs) to be surrounded by a positively charged layer in water. Given the specific recognition of chlorotoxin for the channels, rapid imaging of glioma cells is successfully performed in 15 min using P‐PNCs modified with chlorotoxin via charge attraction. The photoluminescence quantum yield of P‐PNC probes reached 87% and remained at 93% after 30 days of dispersion in water, while maintaining a much longer fluorescence lifetime of 15 µs. Therefore, this promising biological probe will be a general nanoplatform for identifying distinct cellular compartments using different biomarker imaging methods.

Investigation into the effect of the core structure, core-doped, core-doped/shell on the photoluminescence of nanoparticles applied to the detection of E. coli O 157:H7 and methicillin-resistant S. aureus (MRSA)

In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using 3-Mercaptopropionic Acid as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The etection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.