Application In Live 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.

Synthesis and Characterization of a Novel Intrinsically Fluorescent Analog of Cholesterol with Improved Photophysical Properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.

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.

Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

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.

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.

Highly Selective Drug-Derived Fluorescent Probes for the Cannabinoid Receptor Type 1 (CB1R).

The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.

A dual responsive bis-thiophene affixed thiosemicarbazide based chemosensor for colorimetrically Hg2+ and fluorometrically Cu2+ ions and their applications in live cell imaging

In this work, we developed a fast and straightforward colorimetric and photoluminescent chemosensor probe (P1), featuring bis-thiophene-thiosemicarbazide moieties as its signaling and binding unit. This probe exhibited rapid sensitivity to Hg2+ and Cu2+ ions in a semi-aqueous medium, resulting in distinct colorimetric and photoluminescent changes. In the presence of Cu2+, P1 displayed an impressive 50-fold increase in photoluminescence (PL) at 450 nm (with excitation at 365 nm). The probe P1 formed a 1:1 complex with Hg2+ and Cu2+ ions, featuring association constant values of 4.04 × 104 M−1 and 1.25 × 103 M−1, respectively. P1 has demonstrated its efficacy in the analysis of real samples, yielding promising results. Additionally, the probe successfully visualized copper ions on a mouse fibroblast cell line (NIH3T3), highlighting its potential as an intracellular probe for copper ion detection.

A highly selective fluorescent sensor with aggregation-induced emission for detection of H2S and its application in live cells imaging

Hydrogen sulfide (H2S) is traditionally considered as an ecologically toxic gas, has been proven to be the third endogenous signalling gasotransmitter and plays important roles in physiological and pathophysiological conditions. Herein, we designed and synthesized a novel “turn off” fluorescent probe for the detection of hydrogen sulfide(H2S) in vitro. Probe A could detect H2S in CH3CN/PBS system with significant fluorescence quenching. Probe A also showed obviously aggregation-induced emission (AIE) effects. Fluorescence titration experiments revealed probe A combine with H2S in a 1: 1 ratio with a detection limit of 6.54 μM. Cell imaging results demonstrated the potential applicability of probe A for H2S detection in living cells. The supramolecular assembly of probe A and β-cyclodextrin further revealed the interaction with H2S.

NCS-catalyzed synthesis of 2,4,5-triaryl-1H-imidazole and its selective detection of iron ions

In this study, an efficient synthesis of 2,4,5-trisubstituted imidazoles was developed via cyclocondensation starting from o-dione, substituted aromatic aldehyde and ammonium acetate using catalyst NCS, and was characterized by HR-MS, FT-IR, 1H and 13C NMR spectroscopy. A novel fluorescence sensor 4-(1H-phenanthro[9,10-d]imidazol-2-yl)-N, N-diphenylaniline (3bd) was discovered for the detection of Fe3+ ions. The probe 3bd exhibited strong blue radiation in solution. In the presence of Fe3+ ions, a significant fluorescence change of blue light to green light was observed in the EtOH/H2O (4/1, v/v) solution of 3bd (Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+, Pb2+, Sr2+, Zn2+). The limits of detection of probe 3bd for Fe3+ ions was calculated to be as low as 0.70 μM, and it had excellent anti-interference ability. The probe 3bd complexed with Fe3+ ions at 1:1, and the complexation constant was 3.93 × 104 M−1. The probe 3bd realized the detection of Fe2+ oxidation to Fe3+, and the pH showed fine stability in the environment between 4.0–7.0, which ensured its potential application prospects. It also showed good reversibility upon addition of EDTA. Furthermore, the viability of 3bd to Fe3+ had practical application in live cell imaging.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so scanning the sample in order to build a full, meaningful image, takes time. Parallelization of the illumination is the only way to make these microscopy techniques more suitable for real-time live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields. Parallelized illumination with both Gaussian and doughnut beams will be then used to acquire super-resolution images.

Sustainable carbon dots from Borreria hispida: enhanced colorimetric sensing of Fe3+ ions and biological applications in live cell imaging.

This study presents the synthesis of advanced nanomaterials derived from the hedge-grown herbal plant, Borreria hispida, and explores their environmental and biological applications. Using a one-step hydrothermal synthesis method, carbon dots derived from Borreria hispida (BHCD) were fabricated and thoroughly characterized through XRD, TEM, FTIR, CHNS, UV-visible, and PL spectroscopy analyses. Under UV illumination, these plant-based carbon dots demonstrated exceptional water solubility, notable photo stability, and a high quantum yield of 40.8%. The average particle size of BHCD was absorbed around 0.5 to 3.5 nm, contributing to superior selectivity and sensitivity in detecting Fe3+ ions, with a limit of detection of 1.2 × 10-6 M. Investigation into the sensing mechanism revealed a binding model wherein two carbon atom molecules bind to one Fe3+ atom in a 2 : 1 ratio for BHCDs and Fe3+ interactions. Additionally, the effectiveness of the developed fluorescent probe for Fe3+ detection was validated using real water samples from ponds and lakes, highlighting its potential for environmental monitoring applications. Furthermore, the biological effects of BHCD were evaluated through cytotoxic assays, demonstrating significant inhibitory effects on MCF7 breast cancer cell lines, with a maximum cell viability of 60%. This research underscores the multifaceted potential of BHCD in environmental monitoring and biomedical applications.

Label-Free Interference Imaging of Intracellular Trafficking.

Intracellular cargo trafficking is a highly regulated process responsible for transporting vital cellular components to their designated destinations. This intricate journey has been a central focus of cellular biology for many years. Early investigations leaned heavily on biochemical and genetic approaches, offering valuable insight into molecular mechanisms of cellular trafficking. However, while informative, these methods lack the capacity to capture the dynamic nature of intracellular trafficking. The advent of fluorescent protein tagging techniques transformed our ability to monitor the complete lifecycle of intracellular cargos, advancing our understanding. Yet, a central question remains: How do these cargos manage to navigate through traffic challenges, such as congestion, within the crowded cellular environment? Fluorescence-based imaging, though valuable, has inherent limitations when it comes to addressing the aforementioned question. It is prone to photobleaching, making long-term live-cell imaging challenging. Furthermore, they render unlabeled cellular constituents invisible, thereby missing critical environmental information. Notably, the unlabeled majority likely exerts a significant influence on the observed behavior of labeled molecules. These considerations underscore the necessity of developing complementary label-free imaging methods to overcome the limitations of fluorescence imaging or to integrate them synergistically.In this Account, we outline how label-free interference-based imaging has the potential to revolutionize the study of intracellular traffic by offering unprecedented levels of detail. We begin with a brief introduction to our previous findings in live-cell research enabled by interferometric scattering (iSCAT) microscopy, showcasing its aptitude and adeptness in elucidating intricate nanoscale intracellular structures. As we delved deeper into our exploration, we succeeded in the label-free visualization of the entire lifespan of nanoscale protein complexes known as nascent adhesions (NAs) and the dynamic events associated with adhesions within living cells. Our continuous efforts have led to the development of Dynamic Scattering-particle Localization Interference Microscopy (DySLIM), a generalized concept of cargo-localization iSCAT (CL-iSCAT). This label-free, high-speed imaging method, armed with iSCAT detection sensitivity, empowers us to capture quantitative and biophysical insights into cargo transport, providing a realistic view of the intricate nanoscale logistics occurring within living cells. Our in vivo studies demonstrate that intracellular cargos regularly contend with substantial traffic within the crowded cellular environment. Simultaneously, they employ inherent strategies for efficient cargo transport, such as collective migration and hitchhiking, to enhance overall transport rates─intriguingly paralleling the principle and practice of urban traffic management. We also highlight the synergistic benefits of combining DySLIM with chemical-selective fluorescent methods. This Account concludes with a "Conclusions and Outlook" section, outlining promising directions for future research and developments, with a particular emphasis on the functional application of iSCAT live-cell imaging. We aim to inspire further investigation into the efficient transport strategies employed by cells to surmount transportation challenges, shedding light on their significance in cellular phenomena.

Turn-on fluorescent probe for ultrafast detection of phosgene in the environment and household bleach; its applications in live cell imaging

Phosgene is a vital industrial gas that might substantially imperil public safety in the case of an accident or terrorist attack due to its extreme toxicity and history of use as a chemical warfare agent. Herein we built a simple photoinduced electron transfer-intramolecular charge transfer based turn-on fluorescent probe for ultrafast detection of phosgene within ≤ 3 s. The probe provides low detection limit (0.01 ppm) with distinct colour and fluorescence changes only in the presence of phosgene. With the help of a readily available colour smartphone app, the probe-loaded paper strips allowed for the direct visualization and quantitative measurement of phosgene vapour. Using the probe-coated strips to detect phosgene that evolved from common household bleach. Finally, the probe was utilized to detect phosgene in living cells.

A Ratiometric Fluorescent Detection Platform Using G‐CDs@[Ru(bpy)3]2+ for the Specific Detection of Hypochlorite and Live Cell Imaging

AbstractHypochlorite (ClO−), an essential reactive oxygen species (ROS) in physiological processes, is identified to be closely connected with oxidative stress and related diseases. Meanwhile, ClO− is a commonly‐used disinfector for water treatment, and in public places, under acidic conditions, it's easily decomposed into hypertoxic chlorine gas. Since the strong oxidizing property of ClO−, many oxidizing agents may disturb the ClO− detection. Specific and accurate detection of ClO− with superior sensitivity is a challenge. In this work, a sensing platform for rapid, sensitive, and specific ClO− detection is constructed using green fluorescent carbon dots (G‐CDs), with a linear detection range of 0.5–11 µm and a detection limit of 0.233 µm. Moreover, introducing a red fluorescent tripyridinium ruthenium ([Ru(bpy)3]2+) as a reference, a ratiometric fluorescence nanoprobe G‐CDs@[Ru(bpy)3]2+ is prepared and shows favorable intracellular imaging of exogenous and endogenous ClO−. With G‐CDs@[Ru(bpy)3]2+‐based test paper microarrays and a color recognition APP, a smartphone‐based sensing system for point‐of‐care testing of ClO− is also fabricated. In summary, this work proposed a versatile and economical smartphone‐based sensing system that featured reliability and simplicity, and suggested its potential applications in environmental water quality monitoring and live cell imaging.

A pyridyl-benzimidazole based ruthenium(II) complex as optical sensor: Targeted cyanide detection and live cell imaging applications

An extreme toxicity of cyanide (CN−) ion in diverse environmental media has encouraged significant attention for scheming well-organised molecular probes for its selective and sensitive detection. Keeping in mind, we present here a monometallic Ru(II) complex (Ru-1) based on 2-(pyridin-2-yl)-1H-benzo[d]imidazole moiety acting as a highly selective luminescent probe for CN− recognition in pure water. Besides, Ru-1 also acted as an efficient sensor for F−, AcO− and H2PO4− ions along with CN− when acetonitrile was chosen as solvent system. The binding constant (Kb) and detection limit (LoD) for CN− have been depicted as 3.05 × 106 M−1 and 12.8 nM, respectively, in water. The close proximity of N-H site with Ru(II) centre along with its remarkable acidity were identified as mainly responsible for the high selectivity of Ru-1 toward CN− in water. Job’s plot and DFT analyses were carried out to support the anion binding mechanism. Furthermore, the time-resolved fluorescence (TRF) spectroscopy was performed to assess the cyanide-induced emission lifetime change of Ru-1 in aqueous medium. In order to investigate applied potential, the probe Ru-1 was notably developed into paper-based strips that could readily detect CN− ion in mM range via naked eye under 365 nm light illumination, and also adequately employed to detect CN− in human breast cancer MCF-7 cell lines and natural food sources (such as apple seeds and sprouting potatoes).

Synthesis and Photophysical Characterization of a pH-Sensitive Quadracyclic Uridine (qU) Analogue.

Fluorescent base analogues (FBAs) have become useful tools for applications in biophysical chemistry, chemical biology, live-cell imaging, and RNA therapeutics. Herein, two synthetic routes towards a novel FBA of uracil named qU (quadracyclic uracil/uridine) are described. The qU nucleobase bears a tetracyclic fused ring system and is designed to allow for specific Watson-Crick base pairing with adenine. We find that qU absorbs light in the visible region of the spectrum and emits brightly with a quantum yield of 27 % and a dual-band character in a wide pH range. With evidence, among other things, from fluorescence lifetime measurements we suggest that this dual emission feature results from an excited-state proton transfer (ESPT) process. Furthermore, we find that both absorption and emission of qU are highly sensitive to pH. The high brightness in combination with excitation in the visible and pH responsiveness makes qU an interesting native-like nucleic acid label in spectroscopy and microscopy applications in, for example, the field of mRNA and antisense oligonucleotide (ASO) therapeutics.

Tri-armed Schiff base fluorescent sensor for the rapid recognition of Zn(II): application in live cell imaging, test strips and TLC.

Various metal ions exist in nature and human beings and play limitless vital roles in both the atmosphere and biology. A fundamental and useful aspect is the qualitative and quantitative assessment of Zn(II) at concentration levels as low as parts per billion (ppb). Thus, the design and development of novel fluorescent turn-on receptors have gained significant interest because of their potential for use in live cell imaging to detect biologically relevant metal ions with high selectivity and sensitivity. The present research illustrates the design and synthesis of a novel fluorescent sensor [(1,3,5-triazine-2,4,6-triyl)tris(hydrazine-2-yl-1-ylidene)tris(methaneylylidene)]tris(2,4-di-tert-butylphenol) (THDBP) for the selective and sensitive probing of Zn(II). The sensor exhibited a fluorescence turn-on mechanism upon treatment with Zn(II) ions at λemi. 503 nm in aq. acetonitrile. The formation of a 1 : 3 complex between THDBP and Zn(II) is confirmed from the Job plot and ESI-MS spectrum. The evaluated limit of detection (LOD) and association constant (Ka) of the sensor THDBP for Zn(II) were found to be 1.03 × 10-10 M and 2.33 × 108 M-1, respectively. Further research demonstrates the practical application of the sensor for the detection of Zn(II) ions in live cells. The sensing ability of the sensor THDBP was also explored through inexpensive test strips and TLC sheets.

Salicylidene-based dual responsive ̀turn on ́ fluorometric chemosensors for selective detection of Zn2+, Al3+ and F ̅ ions: Theoretical investigation, and applications in live cell imaging of zebrafish larvae and molecular logic gate operation

Four salicylidene-based dual responsive chemosensors 1,5-bis(5-bromosalicylaldehyde)carbohydrazone (R1), 1,5-bis(5-bromosalicylaldehyde) thiocarbohydrazone (R2), 1,5-bis(3-ethoxysalicylaldehyde)carbohydrazone (R3) and 1,5-bis(3-ethoxysalicylaldehyde) thiocarbohydrazone (R4) were synthesized and characterized. The molecular structures of R1 and R3 were confirmed by...