Detected Fluorescence Intensity Research Articles

Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication.

Pseudorabies is an acute and febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3-3937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.

Fluorescence of Single Conjugated Chains of MEH‐PPV Dispersed in Polymer Matrix: What Do We See in the Microscope?†

Comprehensive SummaryIt has been a mystery that the detected fluorescence intensity of single MEH‐PPV molecules is much lower than expected based on their chain length. In this review, after re‐evaluating of the literature data in the light of new specially designed experiments, we present a comprehensive explanation of this issue: The actual size of MEH‐PPV molecules at single molecule level is much smaller than expected due to de‐aggregation and chain scission, while static quenching ("dark matter") also exists for large molecules, further reducing their brightness.

Optical fluorescent sensor based on perovskite QDs for nitric oxide gas detection.

In this paper, a new, to the best of our knowledge, optical fluorescent sensor for the sensing of nitric oxide (NO) gas is developed. The optical NO sensor based on C s P b B r 3 perovskite quantum dots (PQDs) is coated on the surface of filter paper. The C s P b B r 3 PQD sensing material can be excited with a UV LED of a central wavelength at 380nm, and the optical sensor has been tested in regard to monitoring different NO concentrations from 0-1000ppm. The sensitivity of the optical NO sensor is represented in terms of the ratio I N2/I 1000p p m N O , where I N2 and I 1000p p m N O represent the detected fluorescence intensities in pure nitrogen and 1000ppm NO environments, respectively. The experimental results show that the optical NO sensor has a sensitivity of 6. In addition, the response time was 26s when switching from pure nitrogen to 1000ppm NO and 117s when switching from 1000ppm NO to pure nitrogen. Finally, the optical sensor may open a new approach for the sensing of the NO concentration in the harsh reacting environmental applications.

Delivery of Corn-Derived Nanoparticles with Anticancer Activity to Tumor Tissues by Modification with Polyethylene Glycol for Cancer Therapy.

We recently reported that intratumoral injection of corn-derived nanoparticles (cNPs) affords anticancer activity in tumor-bearing mice. To increase their applicability in cancer therapy, we examined the tissue distribution of cNPs after intravenous injection in mice, modified their surface with polyethylene glycol (PEG) to improve tumor delivery, and examined tissue distribution and anticancer activity of PEG-cNPs in tumor-bearing mice. N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) was added to cNPs by sonication to obtain PEG-cNPs, and the ratio of DSPE-PEG to cNPs was optimized by evaluating the modification efficiency. cNPs and PEG-cNPs were labeled with fluorescent dyes DiO or DiR, and their tissue distribution was subsequently examined after intravenous administration to mice. Finally, we determined the anticancer activity and toxicity of PEG-cNPs. No detectable fluorescence intensity was observed in mouse serum after intravenous DiR-cNP injection. DSPE-PEG was successfully modified into cNPs, and a PEG:cNPs ratio of 50 was determined as optimal for preparing PEG-cNPs, based on their size and zeta potential. DiO-PEG-cNPs exhibited significantly higher serum concentrations and lower liver accumulation than DiO-cNPs. Moreover, DiR-PEG-cNPs accumulated in tumor tissues of colon26 tumor-bearing mice. Repeated intravenous PEG-cNP injections significantly retarded tumor growth, with no significant hepatotoxicity or nephrotoxicity. Overall, these results indicate that controlling the tissue distribution of cNPs via PEG modification on their surface can be a valuable strategy for developing intravenously injectable cNPs for cancer therapy.

A novel CRISPR/Cas14a1-Exo III aptasensor for melamine detection coupled with systematically studied binding mechanism of truncated aptamer

In this work, the aptamer of melamine was truncated and mutated. Based on isothermal titration calorimetry, circular dichroism and molecular dynamics, the thermodynamic information, structural changes and binding pocket of the truncated aptamer SJs32 with 32 bases to melamine were systematically studied. Meanwhile, a novel aptasensor coupled with CRISPR/Cas14a1 and Exo III was developed for melamine detection. SJs32 was first hybridized with the complementary DNA (cDNA) to form SJs32-cDNA. In the presence of melamine, SJs32 recognized melamine to form SJs32-melamine and performed structural transformation, which resulted in the releasing of cDNA into the solution. Exo III digested the SJs32-melamine and the released melamine led to the dissociation of more cDNA from SJs32-cDNA complex. Finally, the combination of cDNA and sgRNA activated the trans-cleavage activity of Cas14a1. According to the change of fluorescence intensity, the sensitive and rapid detection of melamine was realized with the linear detection range of 4 nmol/L-1 µmol/L. The developed strategy broadened the application of melamine aptamer in food safety and provided a new insight for the development of CRISPR/Cas14a1 aptasensor.

Effect of erythromycin on the ultrastructure of human macrophages exposed to cigarette smoke extract in vitro

ABSTRACT Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P < .05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P < .05) and CSE+EM 48 h groups (P < .05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress.

C-Met specific CAR-T cells as a targeted therapy for non-small cell lung cancer cell A549

ABSTRACT Non-small cell lung cancer (NSCLC) is considered to be one of the most prevalent and fatal malignancies, with a poor survival rate. Chimeric antigen receptor T cell (CAR-T) cell therapy is one of the most exciting directions in the field of Cellular immunotherapy. Therefore, CAR-T cells that target c-Met have been developed for use in NSCLC therapy and might be a potential therapeutic strategy. The anti c-Met ScFv structure was fused with the transmembrane and intracellular domains. Using a lentiviral vector to load the c-Met CAR gene, then transfected the c-Met CAR lentiviral into human T cells to obtain the second generation c-Met CAR-T expressing CARs stably. In vitro co-culture, experiments revealed that CAR-T cells have high proliferative activity and the potential to secrete cytokines (IL-2, TNF-α, and IFN-γ). c-Met CAR-T cells showed special cellular cytotoxicity in LDH release assay. A subcutaneous tumor model in nude mice was used to test the anticancer effectiveness of c-Met CAR-T cells in vivo. For c-Met positive NSCLC tissue, according to tumor volume, weight, fluorescence intensity, and immunohistochemical detection, c-Met CAR-T cells had stronger tumor growth suppression compared to untransduced T cells. HE staining revealed that c-Met CAR-T cells did not produced side effects in nude mice. Taken together, we provided useful method to generate c-Met CAR- T cells, which exhibit enhanced cytotoxicity against NSCLC cells in vitro and in vivo. Thus, providing a new therapeutic avenue for treating NSCLC clinically. Highlights (1) c-Met CAR-T capable of stably expressing c-Met CARs were constructed. (2) c-Met CAR-T have strong anti-tumor ability and proliferation ability in vitro. (3) c-Met CAR-T can effectively inhibit the growth of A549 cells subcutaneous xenografts.

Technical Note: Measuring the thickness of histological sections by detecting fluorescence intensity of embedding foam

Fluorescence intensity of embedding foam in paraffin blocks can be used to measure the thickness of histological microsections. We embedded samples of embedding foam and produced several microsections of varying thicknesses using routine processing and staining. Fluorescence intensity in the blue area of the embedding foam detected with a slide scanner was compared to absolute thickness as measured using confocal microscopy. Correlation analysis displayed a clear linear correlation with convincingly low prediction interval. The concept of measuring thickness of histological microsections by detecting fluorescence intensity of embedding foam is suggested as an approach to high-throughput measuring of histological sections applicable for a fully digitized pathology department. No acquisition of dedicated equipment is required and the method can be applied as a fully automated technique requiring no time consumption.

Effects of different solvents on ultraviolet absorption and fluorescence intensity of 2,4-dichlorophenol, 2,4,6-trichlorophenol and Pentachlorophenol

In order to study the influence of the UV-visible absorption spectra and fluorescence spectrum from 2,4-dichlorophenol (2,4-dcp), 2,4,6-trichlorophenol (2,4,6-tcp) and pentachlorophenol (ptcp) in different solvents. Using methanol, tetrahydrofuran (THF), ethanol, dimethyl sulfoxide (DMSO) and acetone solvent models, computer simulation prediction and analysis of the three chlorophenols were carried out. It provides a reference for the choice of solvents in the determination of chlorophenols by the Ultraviolet-Vis absorption spectrophotometry and fluorescence intensity detection.

Glass capillary assembled microfluidic three-dimensional hydrodynamic focusing device for fluorescent particle detection

A simple fabrication and reusable microfluidic three-dimensional hydrodynamic focus device is proposed in this study, which can be used for fluorescent particle counting and fluorescence intensity detection. The three-dimensional hydrodynamic focus microfluidic device offers numerous advantages over devices with a two-dimensional focus function. However, the fabrication process of most such devices is relatively complicated. In this study, a three-dimensional hydrodynamic focus device is fabricated by assembling commercially available square, round, and tapered glass capillaries. The inner diameter of the square capillary matches the outer diameter of the circular capillary to ensure that the centers of different capillaries are on the same axis. Thus, the device can provide a symmetric three-dimensional hydrodynamic focus flow. The proposed device can control the diameter of the focused flow by changing the flow rate of the sheath and sample flow. Standard fluorescent beads and fluorescent cells are passed through this device to accurately count the number of fluorescent particles and detect the intensity of fluorescence.

A novel ratiometric fluorescent probe for differential detection of HSO3- and ClO- and application in cell imaging and tumor recognition.

By connecting 1,8-naphthalimide and indole sulfonate, a ratio fluorescent probe capable of differential detection of hydrogen sulfite and hypochlorite was synthesized for the first time. It was able to achieve the qualitative detection of HSO3- and ClO- with high sensitivity and selectivity, respectively. It provides a multi-purpose probe and is based on different emission channels without mutual interference. The probe has the advantages of larger Stokes shift (ClO-: 115nm, HSO3-: 88nm), longer λem (ClO-: 515nm, HSO3-: 548nm) and better water solubility (DMF/PBS = 1:99, v/v). In addition, the probe is a ratio fluorescence probe, which can detect fluorescence intensity with two different emission waves. It provides internal self-calibration, reduces interference from the background and increases detection accuracy. In vitro cytotoxicity and imaging experiments show that the probe can effectively perform the detection of exogenous HSO3- and ClO- in cells. It can also achieve the detection of HSO3- and ClO- in the plasma environment. Because the probe can detect endogenous ClO-, it also has a good prospect for biological application in identifying tumor cells. Graphical abstract.

Multiresonant plasmonic nanostructure for ultrasensitive fluorescence biosensing

Abstract A novel metallic nanostructure for efficient plasmon-enhanced fluorescence readout of biomolecular binding events on the surface of a solid sensor chip is reported. It is based on gold multiperiod plasmonic grating (MPG) that supports spectrally narrow plasmonic resonances centered at multiple distinct wavelengths. They originate from diffraction coupling to propagating surface plasmons (SPs) forming a delocalized plasmonic hotspot associated with enhanced electromagnetic field intensity and local density of optical states at its surface. The supported SP resonances are tailored to couple with the excitation and emission transitions of fluorophores that are conjugated with the biomolecules and serve as labels. By the simultaneous coupling at both excitation and emission wavelengths, detected fluorescence intensity is enhanced by the factor of 300 at the MPG surface, which when applied for the readout of fluorescence immunoassays translates to a limit of detection of 6 fM within detection time of 20 min. The proposed approach is attractive for parallel monitoring of kinetics of surface reactions in microarray format arranged on a macroscopic footprint. The readout by epi-fluorescence geometry (that inherently relies on low numerical aperture optics for the imaging of the arrays) can particularly take advantage of the reported MPG. In addition, the proposed MPG nanostructure can be prepared in scaled up means by UV-nanoimprint lithography for future practical applications.

Evaluation of cell metabolic adaptation in wound and tumour by Fluorescence Lifetime Imaging Microscopy

Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. The pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of NADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer.

The feasibility of locating the affected bone of BRONJ with indocyanine green.

The morbidity of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is also increasing with the use of bisphosphonates (BPs). Removing affected bone accurately is the most effective treatment. This study aimed to explore the feasibility of Indocyanine green (ICG)-based Near-Infrared fluorescence (NIF) imaging to remove BRONJ affected bone. Firstly, the rat model of BRONJ was constructed. And 5mg/kg ICG were injected via tail vein, after 12hr, the affected and healthy bone were dissected for ICG-based NIF imaging and quantification detection of fluorescence intensity. Finally, all the bone samples were sent for further pathological examination. All the affected bone tissues in rat BRONJ model were fluorescence developed with ICG. And the fluorescence developed regions were further confirmed as affected bone tissues with pathological examination. The fluorescence intensity in affected bone tissues, adjacent, and opposite bone tissues was 1.93*107±2.08*106, 1.19*106±2.33*105, and 1.24*106±1.57*105, respectively (p<.05). Conclusions It was feasible that the extent of affected bone in rat model with BRONJ could be estimated intraoperative via ICG-based NIF imaging. This novel approach would become an auxiliary method in the treatment of patients with BRONJ in the future.

Rapid detection of Helicobacter pylori using cytotoxin-associated gene A based on loop-mediated isothermal amplification assay and magnetic nanoparticles

The current methods for detecting Helicobacter pylori infection are time-consuming and have relatively low sensitivity. More appropriate tests are needed. A rapid, specific, and sensitive method was presently developed to detect the cytotoxin-associated gene A (cagA) of H. pylori. Genomic DNA was extracted using magnetic nanoparticles and then amplified by the loop-mediated isothermal amplification (LAMP) reaction using primers we designed. To assess the diagnostic value of the LAMP assay in detecting H. pylori cagA, agarose gel electrophoresis as well as detection of fluorescence intensity after adding fluorescent dye were done. Specificity analysis showed that 11 pathogenic bacterial strains common in human gut were negative for cagA, with a positive result obtained only for H. pylori. Sensitivity analysis demonstrated a cagA detection limit of 100 fg. The results were consistent with that of the 3C-urea breath test. The novel LAMP assay can directly identify H. pylori cagA in the gastric juice of clinical patients with high sensitivity and specificity. The comparatively more rapid and more sensitive method may be valuable for clinical applications.

Droplet-Based Immunosensor for Simultaneous Immunoassays of Multiplex Histidine-Tagged Proteins.

This paper presents a droplet-based immunoassay chip allowing each droplet to be positioned in a passive droplet-positioning cavern under continuous flow. In addition, the chip surface can immobilize any kind of histidine-tagged capture agents for performing simultaneous multiplex immunoassays. Distinct families of monodispersed droplets were generated since a diaphragm, which is a thin elastomeric flap film suspended from the top of the main channel, forms a double T junction for shearing the aqueous liquids by the carrier flow. These two types of monodispersed droplets traverse the main channel to the downstream detection area and enter the passive positioning caverns for further immunoassay. A layer of Ni-Co film was coated on the substrate by electrodeposition in order to immobilize the multiplex histidine-tagged capture molecules. In this study, the tumor suppressor protein p53 and the extracellular signal-related kinase 1 (ERK1) were used as the capture agents. Then, both histidine-tagged proteins p53 and ERK1 were immobilized by the Ni-Co layer in a microarray format for subsequent immunoassay and fluorescence detection. The experimental results show that the detected fluorescence intensity is proportioned to the concentration of the encapsulated content in a small droplet. This proposed droplet-based immunoassay chip can immobilize multiplex histidine-tagged proteins, irrelevant to the species of proteins, to carry out simultaneous immunoassays and allow the operation sequence to be conducted automatically through the manipulation of droplets.

MiRNA143 Downregulated the Expression of Cyclophilin A to Inhibit the Progression of Atherosclerosis

To investigate the influence of microRNA (miR)-143 on atherosclerosis and its working mechanism via cyclophilin A. Human acute monocytic leukemia cells (human acute monocytic leukemia cells, human THP-1 monocytes) were treated with ox-LDL 80 mg/L for 48 h to establish a foam cell model. The modeling was evaluated by oil red O staining. The expressions levels of miRNA-143 and CyPA were detected by q-PCR. RNA extracted was confirmed by SiO2 nano magnetic bead. Dual-luciferase reporter gene assay was used to verify the targeted regulatory relationship between miRNA-143 and CyPA. Next, the cells were transfected with miR-143 mimics and treated with a CyPA inhibitor (cyclophilin A). The expression levels of CyPA and downstream matrix metalloproteinases-9 (MMP-9) in the downstream were detected by q-PCR and western blot, respectively. No or few lipid droplets were observed in the normal THP-1 cells. After treatment with ox-LDL for 48 h, a large amount of red-stained lipid was observed in the cytoplasm, indicating that the THP-1 derived foam cell models were successfully constructed. q-PCR results indicated that miRNA-143 was significantly downregulated in the THP-1-derived foam cell model, whereas CyPA was upregulated (P &lt; 0.05); the dual luciferase reporter gene assay was performed for miRNA-143 and 3'-UTR of CyPA, and the fluorescence intensity of the 3'-UTR vector group was significantly lower than that of the empty vector group (P &lt; 0.05). Fluorescence intensity detection of mutations at the target site showed that it had no effect was produced on the fluorescence intensity. This implied a negative regulatory effect conferred of miRNA-143 on CyPA; As compared to the model group, transfection with miRNA-143 mimics led to a significant downregulation of CyPA mRNA (P &lt; 0.05), which was accompanied by significant downregulation of both MMP-9 mRNA and protein (P &lt; 0.05). MMP-9 was also significantly downregulated upon the addition of the CyPA inhibitor cyclosporine A (P &lt; 0.05).

High-efficiency fluorescence collection for NV- center ensembles in diamond.

The negatively charged nitrogen vacancy (NV-) center ensembles in diamond have been demonstrated to be a promising platform for quantum metrology, but the poor fluorescence collection efficiency of a microscope objective limits the sensitivity of the NV- based sensors. Here we present a method for increasing the collected fluorescence intensity with a total internal reflection (TIR) lens. The detected fluorescence intensity is increased by approximately a factor of 56 compared with detection using a microscope objective with NA = 0.55, leading to a collection efficiency of 47.7% ± 3.1%. The signal-to-noise ratio is improved by a factor of 7.6 using the TIR lens. The proposed method is of great significance for collecting fluorescence from NV- centers in a large volume and can be used in weak fluorescence detection systems.

Single-particle characterization of theranostic liposomes with stimulus sensing and controlled drug release properties

Stimuli-responsive nanotheranostic systems, integrated with diagnosis and treatment features, have recently emerged and attracted much interest. However, most of the research mainly focuses on the novelty of nanomaterials, and undervalues the significance of single-particle characterization which can provide detailed physical and biochemical information for performance evaluation and heterogeneity assessment. Due to the small particle size and low content of functional modules, high throughput and multiparameter analysis of individual stimuli-responsive nanoparticles still remains challenging. Here, fabricating a reactive oxygen species (ROS)-responsive liposome (Lipo@BODIPY11) and taking it as an example, we report the development of a strategy for theranostic nanoparticle characterization by a laboratory-built nano-flow cytometer (nFCM). Coincident detection of light scatter and fluorescence intensity provided a measure for liposome quality assessment. Theranostic performance referred to stimuli-responsive capability and drug release behavior upon ROS treatment were obtained in minutes. Besides, the dissociation of functional modules from liposomes and the formation of aggregates under high modification degree were revealed, which was otherwise masked by ensemble-averaged methods. At last, consistent results were also observed in intracellular studies. This nFCM-based method provides a comprehensive approach for the proof-of-principle study, heterogeneity assessment and quality control of biochemical nanosensors and theranostic nanomaterials.

Track-etched membrane as fluorescence-based pH biosensor

Robust and cost-effective stimuli-responsive polymers show prominent advantages to be integrated into detection devices. In addition, modified track-etched membranes with chemical-sensing polymers possess additional robustness features such as including the sensing material into sub-micron pores. In this study, we report the preparation of track-etched PET membranes with fluorescent response in direct relation to changes in the pH of the environment. Immobilised Fluorescein and Green Fluorescent Protein have been used as pH-sensor elements. The former was not sensitive to pH, while the latter had a similar pH sensitivity to that of the free protein. Modifications of track-etched membranes were carried out by grafting polymerisation initiated by the remnant radicals, a straightforward technique for selective modification of the inner wall of pores. The biosensor prepared with the fluorescent protein was able to sense the pH of a buffer solution in the range 4 to 8. Furthermore, this membrane evidenced capacity to sense the pH of the cell growth by in situ fluorescence intensity detection during E.coli cell culture in microwells.