Concentration Of Fluorescent Dye Research Articles

FABRICATION OF SILK FIBROIN AND FLUORESCENT SILK FIBROIN FIBERS VIA ELECTROSPINNING

Fibroin, derived from cocoon, is a natural polymer used widely and increasingly in various fields. In recent years, the development of fluorescent fibroin has gained attention in the field of tissue engineering, because of its biocompatibility, oxygen and water vapor permeability, biodegradability, and ability to be tested non-invasively. In this work, silk fibroin and fluorescent silk fibroin fibers were fabricated via electrospinning, a simple process through which continuous submicron fibers can be produced. Degummed silk was extracted by dissolving in calcium chloride solution. The fibroin powder was dissolved in formic acid to form 10, 12, and 14 wt.% solutions. Fluorescent fibroin solutions were achieved with addition of fluoresceine sodium (FS) as fluorescent dye. The fluorescent solutions were prepared at 10 wt.% fibroin with varying FS concentration at 0.1, 0.3, and 0.5 wt.%, and at 0.5 wt.% FS with varying fibroin concentration at 10, 12, and 14 wt.%. The fibroin solutions were fabricated into fibers via electrospinning using an applied voltage of 20 kV. The surface morphology of silk fibroin fibers and fluorescent fibroin fibers, observed by scanning electron microscope (SEM), showed long continuous fibers. The average fiber diameter, in sub-micron range, was found to increase with fibroin concentration. The fluorescence intensity observed by fluorescence spectrophotometer was found to increase with decreasing fluorescent dye concentration. The functional groups and chemical structure of silk fibroin fibers analyzed by infrared spectroscopy (IR) were not affected by the addition of the fluorescent dye.

Influence of focused ultrasound on locoregional drug delivery to the brain: Potential implications for brain tumor therapy

IntroductionEfficient delivery of therapeutics across the blood-brain barrier (BBB) for the treatment of central nervous system (CNS) tumors is a major challenge to the development of safe and efficacious therapies. Locoregional drug delivery platforms offer an improved therapeutic index by achieving high drug concentrations in the target tissue with negligible systemic exposure. Intrathecal (intraventricular) [IT] and convection-enhanced delivery [CED] are two clinically relevant methods being employed for various CNS malignancies. Both of these standalone platforms suffer from passive post-administration distribution forces, sometimes limiting the desired distribution for tumor therapy. Focused ultrasound and microbubble-mediated blood-brain barrier opening (FUS-BBBO) is a recent modality used for enhanced drug delivery. It is postulated that coupling of FUS with these alternative delivery routes may provide benefits. Multimodality FUS may provide the desired ability to increase the depth of parenchymal delivery following IT administration and provide a means for contour directionality with CED. Further, the transient enhanced permeability achieved with FUS-BBBO is well established, but drug residence and transit times, important to clinical dose scheduling, have not yet been defined. The present investigation comprises two discrete studies: 1. Conduct a comprehensive quantitative evaluation to elucidate the effect of FUS-BBBO as it relates to varying routes of administration (IT and IV) in its capacity to facilitate drug penetration within the striatal-thalamic region. 2. Investigate the impact of combining FUS-BBBO with CED on drug distribution, with a specific focus on the temporal dynamics of drug retention within the target region. MethodsFirstly, we quantitatively assessed how FUS-BBBO coupled with IT and IV altered fluorescent dye (Dextran 2000 kDa and 70 kDa) distribution and concentration in a predetermined striatal-thalamic region in naïve mice. Secondly, we analyzed the pharmacokinetic effects of using FUS mediated BBB disruption coupled with CED by measuring the volume of distribution and time-dependent concentration of the dye. ResultsOur results indicate that IV administration coupled with FUS-BBBO successfully enhances delivery of dye into the pre-defined sonication targets. Conversely, measurable dye in the sonication target was consistently less after IT administration. FUS enhances the distribution volume of dye after CED. Furthermore, a shorter time of residence was observed when CED was coupled with FUS-BBBO application when compared to CED alone. Conclusion1. Based on our findings, IV delivery coupled with FUS-BBBO is a more efficient means for delivery to deep targets (i.e. striatal-thalamic region) within a predefined spatial conformation compared to IT administration. 2. FUS-BBBO increases the volume of distribution (Vd) of dye after CED administration, but results in a shorter time of residence. Whether this finding is reproducible with other classes of agents (e.g., cytotoxic agents, antibodies, viral particles, cellular therapies) needs to be studied.

SURG-04. INFLUENCE OF LOW-INTENSITY FOCUSED ULTRASOUND ON LOCOREGIONAL DRUG DELIVERY TO THE BRAIN

Abstract INTRODUCTION Efficient delivery of therapeutic drugs across the blood-brain barrier (BBB) for the treatment of central nervous system (CNS) tumors is a major challenge to the development of safe and efficacious therapies. Among the different approaches developed to circumvent these limitations to improve drug delivery to a target tissue, low-intensity focused-ultrasound (LIFU) is a particularly appealing strategy as it will transiently disrupt the blood-brain barrier. Locoregional (intrathecal [IT] and convection-enhanced delivery [CED]) are clinically relevant routes of drug delivery that bypass the BBB and avoid systemic exposure. These routes of administration are known to achieve otherwise unobtainable drug concentration in specified target tissue. However, it remains unknown how locoregional routes of delivery coupled with FUS could change the drug targeting and pharmacokinetics. METHODS In the present study, we quantitatively assessed how FUS coupled with IT, IV, or CED altered fluorescent dye (Dextran 2000kD) distribution and concentration in a predetermined neurotomical region in a naïve murine model. We then analyzed the pharmacokinetic effects of using FUS mediated BBB disruption coupled with CED by measuring the volume of distribution and time-dependent concentration of the dye. RESULTS Our results indicate that IV administration coupled with LIFU will successfully cause diffusion of dye into the pre-defined sonication targets. Alternatively, measurable dye in the sonication target was hardly detected when administered IT in comparison to IV delivery (VIT = 1.25mm3; VIV = 36.20mm3). In addition, our preliminary qualitative analysis suggests that LIFU coupled with CED shows shorter time of residence of the dye in the parenchyma when compared to CED alone. This suggests that increased BBB permeability from FUS leads to faster drug clearance. CONCLUSION Our results highlight the distributive and pharmacokinetic influence of FUS on various locoregional drug delivery routes.

Binding, brightness, or noise? Extracting temperature-dependent properties of dye bound to DNA

We present a method for extracting temperature-dependent thermodynamic and photophysical properties of SYTO-13 dye bound to DNA from fluorescence measurements. Together, mathematical modeling, control experiments, and numerical optimization enable dye binding strength, dye brightness, and experimental noise (or error) to be discriminated from one another. By focusing on the low-dye-coverage regime, the model avoids bias and can simplify quantification. Utilizing the temperature-cycling capabilities and multi-reaction chambers of a real-time PCR machine increases throughput. Significant well-to-well and plate-to-plate variation is quantified by using total least squares to account for error in both fluorescence and nominal dye concentration. Properties computed independently for single-stranded DNA and double-stranded DNA by numerical optimization are consistent with intuition and explain the advantageous performance of SYTO-13 in high-resolution melting and real-time PCR assays. Distinguishing between binding, brightness, and noise also clarifies the mechanism for the increased fluorescence of dye in a solution of double-stranded DNA compared to single-stranded DNA; in fact, the explanation changes with temperature.

Label-free fluorescent aptasensor based on exonuclease I for the determination of ochratoxin A

Ochratoxin A (OTA) is a common foodmycotoxin that is widely distributed and highly toxic. The establishment of a sensitive, stable, and rapid OTA detection method is important to protect human health and food safety. In this study, a label-free aptamer fluorescent sensor was developed for the rapid determination of OTA by optimizing the concentration of fluorescent dyes, the addition of exonuclease I, and the reaction time. Under the optimal experimental conditions, the fluorescence intensity of SYBR Gold showed a linear relationship with the OTA concentration in the range of 5–500 ng/mL with a correlation coefficient of 0.9966 and a detection limit of 1.49 ng/mL, indicating good selectivity. The method was applied to determine OTA in red wine, with recovery rates ranging from 89.0% to 113.6% and relative standard deviations ranging from 4.3% to 6.2%. Compared with other aptamer-based OTA detection methods, the proposed method has higher sensitivity and selectivity and lower cost. Thus, the proposed method has good application prospects in OTA determination.

Microrobotic Swarms for Intracellular Measurement with Enhanced Signal-to-Noise Ratio.

In cell biology, fluorescent dyes are routinely used for biochemical measurements. The traditional global dye treatment method suffers from low signal-to-noise ratios (SNR), especially when used for detecting a low concentration of ions, and increasing the concentration of fluorescent dyes causes more severe cytotoxicity. Here, we report a robotic technique that controls how a low amount of fluorescent-dye-coated magnetic nanoparticles accurately forms a swarm and increases the fluorescent dye concentration in a local region inside a cell for intracellular measurement. Different from existing magnetic micromanipulation systems that generate large swarms (several microns and above) or that cannot move the generated swarm to an arbitrary position, our system is capable of generating a small swarm (e.g., 1 μm) and accurately positioning the swarm inside a single cell (position control accuracy: 0.76 μm). In experiments, the generated swarm inside the cell showed an SNR 10 times higher than the traditional global dye treatment method. The high-SNR robotic swarm enabled intracellular measurements that had not been possible to achieve with traditional global dye treatment. The robotic swarm technique revealed an apparent pH gradient in a migrating cell and was used to measure the intracellular apparent pH in a single oocyte of living C. elegans. With the position control capability, the swarm was also applied to measure calcium changes at the perinuclear region of a cell before and after mechanical stimulation. The results showed a significant calcium increase after mechanical stimulation, and the calcium increase was regulated by the mechanically sensitive ion channel, PIEZO1.

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules.

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R 2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

Preliminary measurements of the thermal field in a rectangular channel by the Planar Laser-Induced Fluorescence method

This paper describes the preliminary measurements of the temperature field in water channel by the Planar Laser-Induced Fluorescence (pLIF) method in the one-colour mode using commercial LaVision system. The aim of the work is to find suitable parameters of the experiment (concentration of fluorescent dye, laser power, arrangement of the experiment, etc.) for measuring the temperature field in a closed glass channel of rectangular cross-section with a heated bottom-wall and to verify an applicability of this method in general. The intention is to carry out measurements in the transverse plane of the duct such that the specific thermos-convective structures of the Rayleigh-Bénard-Poiseuille convection, i.e. mixed convection, are detected. For simplicity, the initial measurements were performed in the natural convection regime, i.e. the water in the channel was not driven by the circulation pump. In this case, a larger temperature gradient was created near the wall compared to the force convection case when a cold liquid flows continuously into the experimental area. The presented work serves as a methodological guide for future measurements and provides valuable experience for the design of a new channel for investigation of the thermal flow instabilities.

Concentration, cellular exposure and specificity of organelle selective fluorescent dyes in fungal cell biology

This technical focus article discusses the importance of concentration, cellular exposure and specificity for the application of organelle selective fluorescent dyes in fungi using DNA, membrane and cell wall stains as examples. Nonetheless, the presented considerations are generally applicable to all fluorescent dyes applied to living cells.The association of a fluorescent dye with its target molecule generally impairs molecule and consequently organelle function. Effective dye concentration, cellular exposure time and specificity to the target molecule are key factors that influence the biocompatibility of any fluorescent dye. Prominent molecules frequently used as fluorescent staining targets in fungal cell biology are: (i) DNA for nuclear labelling, (ii) α-/β-glucans and chitin for cell wall labelling, and (iii) phospholipids for plasma membrane and endomembrane labelling. In combination with live-cell imaging settings that reduce light stress, i.e. excitation intensities and exposure times set to the minimum that still achieves good signal-to-noise ratios, is the low dosage application of fluorescent markers as so called “vital dyes” essential for visualising cellular processes in an artefact-free fashion.

Fluorescence Correlation Spectroscopy Measurement Based on Fiber Optics for Biological Materials

A robust fluorescence correlation spectroscopy system called fiber-optic based fluorescence correlation spectroscopy (FB-FCS) was developed; this system enables the measurement of diffusion dynamics and concentration of fluorescent molecules based on the principle of fluorescence correlation spectroscopy without any mechanical adjustment of the experimental setup. The system consisted of fiber optics and a water-immersion objective lens. The hydrodynamic diameters and concentrations of organic fluorescent dyes and fluorescently labeled proteins were successfully measured. Because of the fiber-optic-based setup, the FB-FCS system is compact and inexpensive. We expect FB-FCS to be suitable for use in laboratories, medical diagnosis, and environmental measurements.

Tracking Fluorescent Dye Dispersion from an Unmanned Aerial Vehicle

Commercial unmanned aerial vehicles continue to gain popularity and their use for collecting image data and recording new phenomena is becoming more frequent. This study presents an effective method for measuring the concentration of fluorescent dyes (fluorescein and Rhodamine WT) for the purpose of providing a mathematical dispersion model. Image data obtained using a typical visible-light camera was used to measure the concentration of the dye floating on water. The reference measurement was taken using a laboratory fluorometer. The article presents the details of three extensive measurement sessions and presents elements of a newly developed method for measuring fluorescent tracer concentrations. The said method provides tracer concentration maps presented on the example of an orthophoto within a 2 × 2 m discrete grid.

Highly emissive fluorescent silica-based core/shell nanoparticles for efficient and stable luminescent solar concentrators

Luminescent solar concentrators (LSCs) represent a viable spectral conversion technology to maximize sunlight harvesting while promising to reduce the current gap for integration of solar cells into the built environment. In this work, novel highly emissive and photostable core-shell nanostructured luminescent species were synthesized and used in LSC devices. Such systems were obtained by entrapping Lumogen F Red 305 in silica-based shells by means of the sol-gel process using a combination of hydrophilic (tetraethyl orthosilicate) and hydrophobic (phenyltriethoxysilane) silica precursors. By tuning their relative proportions during the synthetic process, fine control over the characteristic dimensions and morphology of the obtained dye-doped core-shell nanoparticles could be achieved, leading to a maximum concentration of non-covalently entrapped fluorescent dye of ~ 1 wt%. Optical characterization showed that the newly synthetized nanoparticles displayed markedly improved photoluminescence quantum yield in solid state (~ 95%) with respect to the non-encapsulated dye (~ 2%), as a result of an effective suppression of dye aggregation and fluorescence quenching phenomena. In addition, their excellent photostability under harsh UV light exposure was demonstrated, resulting from the protective action of the encapsulating hybrid silica-based shell, which can effectively limit photobleaching of the luminescent core. Their incorporation in thin-film LSCs led to a maximum internal photon efficiency as high as ~ 29%, thus providing evidence of their suitability as highly performing luminescent species with superior optical response and excellent photostability. Also, a maximum external photon efficiency as high as 2.12% was achieved. This work provides the first demonstration of silica-based encapsulation of Lumogen F Red 305 and of the application of silica-based core-shell nanoparticles in LSCs, thus paving the path to the development of a new class of highly efficient and stable nanostructured luminophores for the photovoltaic field. The first demonstration of silica-based encapsulation of Lumogen F Red 305 and of the application of silica-based core-shell luminescent species in LSCs is given, thus opening the path to the development of highly efficient and stable nanostructured luminophores for the photovoltaic field. • Novel nanostructured luminescent species are synthetized. • The nanostructured luminophores are based on hybrid core-shell silica nanoparticles. • Such nanoparticles exhibit enhanced luminescence quantum yield and photostability. • Their use as luminophores in luminescent solar concentrators (LSCs) is demonstrated. • The obtained LSCs exhibit high efficiency and prolonged stability.

Rapid Target Binding and Cargo Release of Activatable Liposomes Bearing HER2 and FAP Single-Chain Antibody Fragments Reveal Potentials for Image-Guided Delivery to Tumors.

Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20–30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32–48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins.

Simultaneous Determination of Trace Mefenamic Acid in Pharmaceutical Samples via Flow Injection Fluorometry

Mefenamic acid belongs to non-steroidal anti-inflammatory drugs that are used widely for the treatment of analgesia. Our aim from this study is to establish a new assay for the quantitative determination of mefenamic acid (MFA) in the pharmaceutical sample by two sensitive and rapid flow injection-fluorometric methods. A homemade fluorometer was used in fluorescence measurements, which using solid-state laser diode 405 and 532 nm as a source, combined with a continuous flow injection technique. The first method depends on the effect of MFA on calcein blue (CLB) fluorescence at 405 nm. Another method is a study of rhodamine-6G (Rh-6G) fluorescence after adding MFA, and recording at 532 nm. Optimum parameters as fluorescent dye concentration, basic medium, flow rate, sample volume, purge time, and delay coil have been investigated. The dynamic range of MFA was 0.2 to 2 mmol.L-1; 0.5 to 2.3 mmol.L-1 with linearity percentage (% r2) 98.92 and 99.83%, for Rh-6G and CLB, respectively. Limit of detection at a minimum concentration in calibration curve 189.34 and 199.89 ng/sample, for Rh-6G and CLB, respectively. The comparison of developed methods with the classical method (UV-vis spectrophotometry) was achieved. The proposed methods were successfully applied for the determination of MFA in the pharmaceutical samples and can be used as an alternative method.

Snapshot 3D reconstruction of liquid surfaces.

In contrast to static objects, liquid structures such as drops, blobs, as well as waves and ripples on water surfaces are challenging to image in 3D due to two main reasons: first, the transient nature of those phenomena requires snapshot imaging that is fast enough to freeze the motion of the liquid. Second, the transparency of liquids and the specular reflections from their surfaces induce complex image artefacts. In this article we present a novel imaging approach to reconstruct in 3D the surface of irregular liquid structures that only requires a single snapshot. The technique is named Fringe Projection - Laser Induced Fluorescence (FP-LIF) and uses a high concentration of fluorescent dye in the probed liquid. By exciting this dye with a fringe projection structured laser beam, fluorescence is generated primarily at the liquid surface and imaged at a backward angle. By analysing the deformation of the initial projected fringes using phase-demodulation image post-processing, the 3D coordinates of the liquid surface are deduced. In this article, the approach is first numerically tested by considering a simulated pending drop, in order to analyse its performance. Then, FP-LIF is applied for two experimental cases: a quasi-static pending drop as well as a transient liquid sheet. We demonstrate reconstruction RMS errors of 1.4% and 6.1% for the simulated and experimental cases respectively. The technique presented here demonstrates, for the first time, a fringe projection approach based on LIF detection to reconstruct liquid surfaces in 3D. FP-LIF is promising for the study of more complex liquid structures and is paving the way for high-speed 3D videography of liquid surfaces.

Targeting the Tumor Microenvironment with Fluorescence-Activatable Bispecific Endoglin/Fibroblast Activation Protein Targeting Liposomes.

Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.

Effect of fluorescent dye positioning and concentration on the growth parameters and lipid content of Chlorella sp. in a flat panel photobioreactor.

In this study, two different concentrations of fluorescent dye Rhodamine 6G (R6G) with two different strategies were used in a double layer flat panel photobioreactor (PBR) for investigation of its effect on growth parameters of microalgae Chlorella sp. Results showed that in the first strategy, when the light passed through the dye before reaching the broth, biomass productivity rate (P) and maximum specific growth rate (µmax) relative to control case, were increased up to 60 and 23% respectively. Increasing in these parameters were more, for the low dye concentration. Also, in the second strategy, when the light passed through the microalgae before reaching the dye solution, P and µmax for lower concentration increased about 9 and 15%, respectively. But using high dye concentration growth of the algae was decreased. Furthermore, using R6G caused increase in the lipid and chlorophyll content of the microalgae Chlorella sp.

Quantitative analysis of Lactobacillus delbrueckii subsp. bulgaricus cell division and death using fluorescent dye tracking

Cell growth of lactic acid bacteria is of vital importance in starter culture manufacture in the dairy industry. However, one of the major stumbling blocks in the understanding of bacterial cell growth relates to challenges in the quantification of the cell division or death. To overcome these shortcomings, the intracellular fluorescent cell tracking assay was implemented to monitor Lactobacillus delbrueckii subsp. bulgaricus sp1.1 cell division and death. Optimization of fluorescent dye concentration suggested it could be applied in the tracking of cell division without cytotoxicity. Technical validation of the fluorescent tracking demonstrated this assay was accurate for quantitatively analyzing cell division. Furthermore, the cell death was quantified using the precursor cohort distribution of the time-series fluorescence data in batch culture. The results indicated a dynamic and unbalanced relationship between bacterial cell division and death after exponential phase in the batch culture. These findings suggested that fluorescent dye tracking is a powerful tool for monitoring L. bulgaricus sp1.1 cell division and death and can provide valuable information for bacterial growth behavior in batch culture.

Enhanced fluorescence quenching rate of Coumarin 102 in stimulated emission depletion

The fluorescence quenching rate (KQ) of fluorescence dye in continuous-wave stimulated emission depletion (CW-STED) system is experimentally investigated by utilizing a commercial dye (Coumarin 102). With the plotting of a power-dependence curve, it is found that the quenching rate is increasing rather than staying steady with the raising of depletion beam power in CW STED. Moreover, increasing fluorescence dye concentration and excitation power could also result in the increasing of KQ, which gives rise to the negative influence on spatial resolution of CW STED microscope. The results are helpful in understanding the influence of quenching on CW STED, improving the performance of CW STED microscope and developing new STED-compatible fluorescent dye.

Facile microfluidic fabrication of monodispersed self-coupling microcavity with fine tunability.

Whispering gallery mode (WGM) resonators have received extensive attention because of their nonlinear optical application in lasers and sensors. Optical microcavities are excellent candidates for constructing powerful microlasers and label-free biosensors, owing to their low optical losses and small size. However, most of these microcavity syntheses rely on sophisticated fabrication methods and cannot be manipulated easily. To achieve facile and versatile microcavity fabrication, we present a robust microfluidics method for monodispersed self-coupling optical microcavity fabrication with a fine tunability. The microcavity polydispersity was less than 3%. The optical microcavity size could be varied from 10 to 30µm with a steady quality factor (Q) of approximately 1000. The lowest laser threshold that we obtained was 0.82 µJ with a microcavity size of 20µm. The doped fluorescent dye concentration can be tuned precisely from 0.001 to 0.05 wt% to explore an optimized fluorescent background. The experimental results and theoretical simulation match well in terms of Q and the electrometric resonance field intensity. Compared with previous precise and practical fabrication methods, we have demonstrated a facile approach for versatile optical microcavity fabrication. This method can vary the microcavity materials, size, doped fluorescent dye concentration, WGM resonance spectrum, Q factor, and laser threshold easily to adapt to various circumstances and specific applications.