Application In Cell Imaging Research Articles

Exploring the Impact of Variability in Cell Segmentation and Tracking Approaches.

Segmentation and tracking are essential preliminary steps in the analysis of almost all live cell imaging applications. Although the number of open-source software systems that facilitate automated segmentation and tracking continue to evolve, many researchers continue to opt for manual alternatives for samples that are not easily auto-segmented, tracing cell boundaries by hand and reidentifying cells on consecutive frames by eye. Such methods are subject to inter-user variability, introducing idiosyncrasies into the results of downstream analysis that are a result of subjectivity and individual expertise. The methods are also susceptible to intra-user variability, meaning findings are challenging to reproduce. In this pilot study, we demonstrate and quantify the degree of intra- and inter-user variability in manual cell segmentation and tracking by comparing the phenotypic metrics extracted from cells segmented and tracked by different members of our research team. Furthermore, we compare the segmentation results for a ptychographic cell image obtained using different automated software and demonstrate the high dependence of performance on the imaging modality they were developed to handle. Our results show that choice of segmentation and tracking methods should be considered carefully in order to enhance the quality and reproducibility of results.

Green synthesis of fluorescent N-doped carbon quantum dots from castor seeds and their applications in cell imaging and pH sensing.

Water-soluble fluorescent N-doped carbon quantum dots (N-CQDs) were hydrothermally prepared through a green synthesis route using castor seeds as a single precursor and a hydrothermal method. Several experimental techniques have been used to characterize synthesized N-CQDs to confirm their structure and to verify their applicability in cell imaging and pH sensing. The synthesized N-CQDs were found to have are characterized by amorphous nature with a spherical shape with an average particle size of 6.57nm as revealed from XRD and TEM measurements. The FTIR results reveal the presence of carboxylic and hydroxyl functional groups on the surface of the CQDs, which was also confirmed by XPS analysis. The fluorescence characterization of the synthesized N-CQDs showed blue emission and excitation dependence with good photostability. It was found that the optimal excitation and emission wavelengths were (λEx = 360) and (λEm = 432) nm, respectively. The fluorescence quantum yield (QY) of about 9.6% at the optimum excitation wavelength 360nm. Moreover, the fluorescence intensity of N-CQDs showed good linear dependence with the pH values in ranges of 3.5 - 7.5 and 8 - 12 as well as high sensitivity for slight changes of pH values. According to these results, two fluorescent pH sensors were created based on acidic and basic media. The obtained N-CQDs have zeta potential of -21.86 mV and thus have excellent stability in water. Moreover, N-CQDs derived from the castor seeds have antimicrobial activity and exhibits low cytotoxicity to WI-13 cells with IC50 = 394.4 ± 13.8µg/mL. The results of this study demonstrated that the synthesized N-CQDs derived from castor seeds can be used as pH sensing and antimicrobial materials. On the other hand, they are also promising in applications in cell imaging, thermo-sensing and optoelectronics.

Metal-Responsive Fluorophore and Amikacin-Conjugated Heparin for Bacterial Cell Imaging and Antibacterial Applications.

The escalating prevalence of bacterial infections presents a formidable challenge to current global healthcare systems. Rapid identification and quantification of bacterial pathogens with anticipated sensitivity and selectivity are crucial for targeted therapeutic interventions to mitigate disease burden, drug resistance, and further transmission. Concurrently, there is a pressing need to innovate novel approaches to combat infections and counter antibiotic resistance. Herein, we demonstrated the development of heparin (HP) conjugates modified with a Zn2+-induced "turn-on" fluorophore, 2-(pyridin-2-yl)-1H-benzo[d]imidazole (PBI), that interacts with bacterial cells via specific binding with the surface-exposed heparin-binding proteins (HPBs), thereby inducing fluorescence signals for rapid and selective sensing of whole bacterial cells. Additionally, amikacin (Amk) antibiotic was integrated into the modified heparin polymer (HP-PBI-Amk) to augment its antibacterial efficacy via reactive oxygen species generation. Despite the nephrotoxicity of only amikacin, its inclusion in the biopolymer retains its antibacterial properties while providing biocompatibility. The outcome of this study demonstrates the development of HP-PBI and HP-PBI-Amk as promising strategies for bacterial detection and eradication, respectively, offering potential avenues for future research and clinical applications.

Heat application in live cell imaging.

Thermal heating of biological samples allows to reversibly manipulate cellular processes with high temporal and spatial resolution. Manifold heating techniques in combination with live-cell imaging were developed, commonly tailored to customized applications. They include Peltier elements and microfluidics for homogenous sample heating as well as infrared lasers and radiation absorption by nanostructures for spot heating. A prerequisite of all techniques is that the induced temperature changes are measured precisely which can be the main challenge considering subcellular structures or multicellular organisms as target regions. This article discusses heating and temperature sensing techniques for live-cell imaging, leading to future applications in cell biology.

An integrated portable system for laser speckle contrast imaging and digital holographic microscopy

A multimodal quantitative phase imaging platform combining digital holographic microscopy (DHM) with laser speckle contrast imaging (LSCI) is demonstrated for imaging weakly scattering and transparent samples in a label-free manner. It uses the principle of coherence of the light source for interferometric detection of phase of transparent and semi-transparent samples. The speckle formation resulting from the coherence property of the laser source is used to track dynamic activities in the regions of interest in the sample. Integration of these two techniques onto a microfluidic chip leads to an optofluidic real-time microscope for live cell imaging applications. In this work, we have developed an integrated multimodal system combining digital holographic microscopy and laser speckle contrast imaging system for Optofluidics and in vitro studies. The sample flowing through the microfluidic channel is imaged to record holograms and video of intensity images at the same time. This enables to map the flow within a microfluidic channel and quantify the channel as well as the particle flow through the channel. The channel morphology along with the particles flowing through the channel are quantified using DHM. The two-dimensional speckle contrast images map the flow of the dynamic microbeads and cells with very high contrast in almost real time. A low-cost portable multimodal quantitative phase microscope combining DHM and LSCI has been demonstrated for real time imaging with applications in optofluidics.

Synthesis of coumarin based Tb3+ complex and its application in cell imaging

Synthesis of coumarin based Tb3+ complex and its application in cell imaging

A turn-on AIE dual-channel fluorescent probe for sensing Cr3+/ClO− and application in cell imaging

A turn-on AIE dual-channel fluorescent probe for sensing Cr3+/ClO− and application in cell imaging

A near infrared fluorescent probe for hypoxia based on dicyanoisophorone and its application in Hela cells imaging

A near infrared fluorescent probe for hypoxia based on dicyanoisophorone and its application in Hela cells imaging

Galla chinensis residue-derived carbon dots for sensitive detection of doxorubicin hydrochloride and cell imaging

As an antibiotic, the imbalance of the concentration of doxycycline hydrochloride (DOX) can cause many harms, so real-time detection of the DOX content is crucial. In this study, Galla chinensis residue (GCR) was used as a carbon source, m-phenylenediamine was used as an N-doping agent, and blue-green fluorescent carbon dots (N-GCRCDs) were prepared by a simple hydrothermal synthesis method, with a high quantum yield of 27.31 %. Under optimized conditions, a specific fluorescent probe was established to detect DOX. The addition of DOX resulted in a significant decrease in fluorescence intensity, with a detection range of 5.7–114.9 μM and the detection limit was 1.38 μM. Finally, the feasibility of this method was validated in human urine and the experimental results exhibited a DOX recovery rate of 97.45–104.41 % and an RSD of 0.91–2.64 %. The quenching mechanism was mainly attributed to the inner filter effect and static quenching. Furthermore, the potential application of N-GCRCDs in cells was studied and biocompatibility was evaluated using HepG2 cells, which showed low toxicity at concentrations of 0–50 μg/mL and successful application for HepG2 cell imaging. This study provides reference for resource utilization of GCR and quantitative detection of antibiotics.

Concentration dependent carbon nanodots: Tunable luminescent color and fluorescence excitation-wavelength dependence

Carbon nanodots (CNDs) exhibiting concentration dependent properties have been synthesized through a one-pot hydrothermal reaction process utilizing diethylenetriamine and l-aspartic acid. At solid-state or high concentrations, the CNDs display excitation-wavelength independent fluorescence (FL) emissions, while at low concentrations, they exhibit excitation-wavelength dependent FL emissions. Detailed characterization of the structure and optical properties reveals that the concentration dependent FL properties can be ascribed to the intrinsic-state luminescence of the CNDs at low concentrations and the assembled-state luminescence at solid-state/high concentrations. With the increase of concentration, the self-assembly behavior of CNDs may cause the transition from intrinsic-state dominant luminescence to assembled-state dominant luminescence, and finally lead to the red-shift of FL color. Furthermore, the CNDs@Urea composites possess adjustable room-temperature phosphorescence (RTP) from turquoise to yellow by controlling the CNDs doping concentration. CNDs with concentration dependent optical properties have shown certain potentials in the fields of cell imaging, fingerprint recognition and anti-counterfeiting applications.

Synthesis of a novel water-soluble pyridine dicarboxylate and its application in fluorescence cell imaging

Abstract A novel water-soluble substituted pyridine dicarboxylate containing a quaternary ammonium ion with potential applications in fluorescence cell imaging was synthesized and characterized. Remarkably, this compound exhibited water solubility in the absence of additional solubilizers, enabling direct dissolution in phosphate-buffered saline for cell studies. No obvious cytotoxicity was observed in 4T1 cells (mouse breast cancer cells) within the concentration range of 0.2–25 μmol L−1, with a cell survival rate above 89%. Furthermore, the compound exhibited a maximum two-photon absorption cross-section of 86 GM at an excitation wavelength of 780 nm, demonstrating high cell permeability, effective distribution in the cytoplasm, and preferential targeting of organelles. Single- and two-photon fluorescence imaging of cells revealed a distinctive blue emission and strong bright green emission, respectively. The newly synthesized substituted pyridine dicarboxylate exhibited low cytotoxicity and strong fluorescence imaging properties, demonstrating its potential in cell imaging applications.

Thiophene appended schiff base probe for selective ‘turn-on’ response for copper (ii) ion and their applications in live cell imaging Thiophene appended schiff base probe for selective ‘turn-on’ response for copper (ii) ion and their applications in live cell imaging

In this research, we have synthesized a thiophene Schiff base from 5-phenyl thiophene-2-carboxaldehyde and 2-thiophene carboxylic acid hydrazide and characterized by using FT-IR NMR, HRMS and SC-XRD analysis techniques. The probe exhibited a selective turn-on fluorescence response for Cu2+ over 20 other common metal ions in a CH3CN (9:1, v/v) solution. The Job’s plot represents a 1:1 binding complexation mode and further DFT study was carried out to aid in understanding the geometric structure and interaction of the ligand and Cu2+. The detection limit (LOD) and limit of quantification (LOQ) were found to be 20 nM and 69 nM respectively. Furthermore, the probe’s sensing ability was tested in HeLa cells and their fluorescence imaging also supported the effective turn-on response towards Cu2+ ion.

Fluorescence and Circular Dichroism Dual-Mode Probe for Chiral Recognition of Tyrosine and Its Applications in Bioimaging.

Chiral amino acids (AAs) are essential in metabolism and understanding physiological processes, and they could be used as biomarkers for the diagnosis of different diseases. In this study, chiral Cdots@Van were prepared by postmodifying an achiral Cdots core with vancomycin for recognizing and determining the enantiomeric excess (ee) of tyrosine (Tyr) enantiomers. The fluorescence response of Cdots@Van is based on an "on-off" strategy, with different quenching percentages for d- and l-tyrosine. Interestingly, the circular dichroism (CD) spectrum of Cdots@Van responded to only one form of Tyr enantiomer, specifically d-Tyr, and remained nearly unchanged upon the addition of l-Tyr. Quantum mechanical (QM) calculations were in excellent agreement with the experimental results, confirming the stronger binding affinity of Cdots@Van for d-Tyr compared to l-Tyr. We further investigated the chiral recognition ability of the interconnected vancomycin particles, which was synthesized using the EDC/NHS coupling reaction between vancomycin molecules without a Cdots core. Surprisingly, unlike free vancomycin molecules, interconnected vancomycin displayed an enantiomeric recognition ability by CD spectroscopy, similar to what was observed for Cdots@Van. Crucially, this chiral probe has been successfully utilized for cell imaging applications.

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

Enhanced adsorptive removal of methylene blue dye from aqueous media by potato peel-derived carbon quantum dots and their application in cell imaging

Enhanced adsorptive removal of methylene blue dye from aqueous media by potato peel-derived carbon quantum dots and their application in cell imaging

Precise determination of human serum albumin levels in blood serum by a D-π-A based near-infrared fluorescent probe via TICT mechanism

Human serum albumin (HSA) levels in the body are crucial for the accurate diagnosis of various health issues, necessitating continuous monitoring and novel detection methods. This study focused on the development and synthesis of two novel D-π-A small molecule near-infrared fluorescent probes, PTPA-B and CPTPA-B. The probes demonstrated selective detection of HSA with a rapid response time of 1 min and high sensitivity, featuring limits of detection (LOD) of 0.102 μM and 1.702 μM. Under physiological conditions, the intrinsic fluorescence of these probes is minimal due to intramolecular charge transfer (TICT). Upon entering the hydrophobic cavity of HSA, the probes exhibit red fluorescence by inhibiting the TICT process. Additionally, the probes exhibited enhanced biocompatibility in living cells and enabled fluorescence imaging of L-02 live cells. Therefore, the PTPA-B fluorescent probe serves as an effective tool for serum detection, revealing its potential applications in live cell imaging and precise clinical diagnosis.

Supramolecular J- and H-Aggregation of Naphthalimide-Conjugated Dipeptide in Mixed-Solvent Systems and its Application in Cell Imaging.

In this work, a core-substituted NMI-conjugated dipeptide (4MNLV) was extensively studied in mixed solvent systems to explore the polarity effect on the self-assembly pattern and their photophysical property. 4MNLV adopted J- or H- type aggregation pattern depending upon the polarity index of the solvent system chosen. The self-assembly process was achieved through the anti-solvent effect. UV-vis study suggested that if the stock solution of 4MNLV was diluted with a relatively more polar solvent (compared to the stock solvent), then the system acquired J- type of aggregation pattern by showing a red-shift in their absorption maxima (λmax). Conversely, when the stock was diluted by a relatively less polar solvent, H-type of aggregation was observed, where blue shift of λmax was noticed. The emission spectra and the lifetime of the self-assembled materials were also influenced by the chosen solvent system. The chirotopic behaviour of these self-assembled materials was studied through CD spectroscopy. Morphological study indicated the formation of helical nanofibrillar structures. The bright green fluorescence of these highly biocompatible naphthalimide-peptide conjugate was used for cell imaging application, indicating its futuristic scope.

Novel benzothiazole-based fluorescent probe for efficient detection of Cu2+/S2− and Zn2+ and its applicability in cell imaging

BackgroundIn recent years, environmental pollution has been increasing due to the excessive emission of toxic ions, which has caused serious harm to human health and ecological environment. There are various methods for detecting Cu2+, S2− and Zn2+, but the traditional ion detection methods have obvious disadvantages, such as poor selectivity and long detection time. Therefore, it is still crucial to develop simple, efficient and rapid detection methods. ResultsA fluorescent probe based on benzothiazole, (E)-N'-(3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzylidene)-3,4,5-tris(benzyloxy)benzohydrazide (BT), was designed and synthesized. It was characterized using ESI-MS, 1H NMR, and 13C NMR. BT can be used as a chemosensor to detect Cu2+, S2− and Zn2+ in CH3CN/H2O (7:3, v/v, pH = 7.4, HEPES buffer: 0.1 M), with detection limits of 0.301 μM, 0.017 μM, and 0.535 μM, respectively. At an excitation wavelength of 320 nm, BT exhibits an “on-off-on” response to Cu2+/S2− and enhanced fluorescence response to Zn2+, with a change in fluorescence color from orange to green. The coordination ratio of ions to the probe was determined to be 1:1 through Job's plot and hydrogen spectral titration. The recognition mechanism was discussed in conjunction with theoretical calculations. Furthermore, the probe has been successfully used in test strips and medical swabs colorimetry, as well as live cell imaging. SignificanceThe probe BT lays the foundation for the design and synthesis of multifunctional fluorescent probes. As a portable detection method, probe BT was used to detect Cu2+, S2− and Zn2+ on strips. Furthermore, the probe was applied to biological cells to detect target ions with low cytotoxicity and excellent cell permeability. This indicating that it can be used as a potential candidate for tracking Cu2+ and S2− in clinical diagnostics and biological systems.

A rhodamine-based AIE chemodosimeter for ratiometric fluorescent sensing Hg2+ and cell imaging application

In this work, a tetraphenyl ethylene functionalized rhodamine chemodosimeter, namely, TRP, has been designed, built, and structurally determined. The chemodosimeter exhibited an AIE effect in an EtOH-H2O mixture with the maximal emission intensity at 463 nm at a water fraction of 70 %. Then TRP was applied for ratiometric monitoring of Hg2+ ions in EtOH/H2O solution (v/v = 3/7), presenting about a 35-fold fluorescence intensity ratio enhancement (F649/F463), visible color changes, and well-resolved emission bands (187 nm). In addition, TRP showed high selectivity and sensitivity in Hg2+ ions sensing and achieved a limit of detection (LOD) of 10.0 nM, which was mainly attributed to the production of an oxadiazole functionalized and spirolactam ring opened rhodamine derivative-induced by Hg2+ ions. Significantly, using TRP as a tracer, ratiometric fluorescent detection of Hg2+ ion has been successfully conducted via living cell imaging, demonstrating its potential for practical applications in biological systems.

Smartphone-Assisted and Optical Quantification of Copper and Glucose Using Palm Wine-Tailored Carbon Dots and Their Multiple Logic Gate Application.

In this work, potassium, sulfur, nitrogen, and chlorine self-doped carbon dots (CDs) were hydrothermally synthesized using palm wine as a carbon source. The palm wine-derived CDs (PW-CDs) are amorphous in nature and displayed an average particle size of 4.19 ± 0.89 nm. The as-synthesized CDs are used to fabricate a photoluminescent sensing probe to simultaneously detect Cu2+ and glucose via the "Turn ON-OFF-ON" mechanism. The PL quenching mechanism of PW-CDs enables the selective and sensitive detection of Cu2+ ions with a detection limit (LOD) of 0.8 ppb (4.7 nM). The sensing probe quantified Cu2+ in tap water, drinking water, and e-waste samples to prove its viability. Using CDs to quantify copper in e-waste leachate samples is a novel approach as no prior instances of such application have been reported. The system's performance is considered to be highly reproducible due to the relative standard deviation being <6.64%, along with excellent recoveries within the range of 93.24-109.86%. The quenched PL can be recovered by introducing glucose into the PW-CD + Cu2+ system; this strategy is employed to quantify glucose with a LOD of 0.11 ppm (0.61 μM). The feasibility of this sensor was confirmed by the determination of glucose in actual human plasma specimens of diabetic patients. It is to be noted that these samples were neither diluted nor spiked with glucose. The developed PW-CD + Cu2+ sensing system yields satisfactory recoveries of 93.45-107.37%. This probe was also incorporated into a smartphone-based sensing platform to detect Cu2+ and glucose with desirable recoveries. The proposed smartphone-based sensing platform is flexible, reliable, and accurate, making it suitable for resource-constrained areas. Furthermore, based on the effect of Cu2+ ions and glucose on the PL response and absorbance spectra of PW-CDs, four logic gates (YES, IMPLICATION, NOT, and OR) were designed, and PW-CDs were also used for cell imaging applications.