Probes For Live Cell Imaging Research Articles

“AIE+ESIPT” based naphthothiazole fluorescent probes for live cell imaging

Dual-functional organic fluorophores with aggregation induced emission (AIE) behavior and excited-state intramolecular proton transfer (ESIPT) behavior are of great significance to the design of imaging probes to bioimage systems. However, the pursuit of probes exhibiting dual AIE+ESIPT effects for live cell imaging remains an ongoing research challeng. Herein, we have successfully developed naphthothiazole fluorescent probes, HNBT-H and HNBT-G, based on the "AIE+ESIPT" effect. These probes were designed by incorporating responsive units for β-galactosidase (β-gal) and H2O2 into the pre-existing "AIE+ESIPT" HNBT fluorophore framework. The HNBT-H probe demonstrated the capability to distinguish various levels of H2O2. Furthermore, the applicability of HNBT-H and HNBT-G have been examined in living systems. These probes exhibit a good biocompatibility and low toxicity, the cell imaging experiments demonstrate the capability of HNBT-H and HNBT-G for fluorescence visualization in response to β-gal and H2O2 in RAW264.7 cells and PC12 cells.

A β-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific β-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of β-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of β-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function.

For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800-2800 cm-1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.

Dicyanostilbene‐Derived Highly Sensitive Two‐Photon Fluorescence Temperature Probe for Live Cell Imaging

AbstractA novel two‐photon fluorescence temperature‐sensitive probe (SP) with the push‐pull electronic architecture (Donor‐π‐Acceptor, D‐π‐A) derived from dicyanostilbene was developed. Its linearly dependent coefficients (R2) between the emission intensity of SP and temperature reached 0.998 and 0.999 in one‐ and two‐photon fluorescence (OPF and TPF), respectively, and corresponding function expressions were IF=−0.0144T+1.2751 (OPF) and IF=−0.0152T+1.2970 (TPF). Its maximum two‐photon excitation wavelength (λmax2) is 750 nm, and the corresponding maximum two‐photon absorption cross‐section (δmax) is 1550 GM. DFT (Density Functional Theory) Calculation demonstrated that the electron cloud densities of two nitrogen atoms (N29 and N31) on two cyano groups are 15.9 and 16.0 times higher than that of nitrogen atom on 9‐carbazolyl group (N13), respectively. This fully indicates that the electron cloud of nitrogen atom on 9‐carbazolyl group is highly delocalized to the fluorophore matrix. SP can be used for live cell imaging and detect cell temperature to diagnose disease within physiological temperature range with excellent photostability and without cytotoxicity.

Recent advances in Si-rhodamine-based fluorescent probes for live-cell imaging.

Fluorescence imaging is a powerful technique for visualizing biological events in living samples with high temporal and spatial resolution. Fluorescent probes emitting far-red to near infrared (NIR) fluorescence are particularly advantageous for in vivo imaging due to their high tissue permeability and low autofluorescence, as well as their suitability for multicolor imaging. Among the far-red to NIR fluorophores, Si-rhodamine is one of the most practical fluorophores for the development of tailor-made NIR fluorescent probes because of the relative ease of synthesis of various derivatives, the unique intramolecular spirocyclization behavior, and the relatively high water solubility and high photostability of the probes. This review summarizes these features of Si-rhodamines and presents recent advances in the synthesis and applications of far-red to NIR fluorescent probes based on Si-rhodamines, focusing on live-cell imaging applications such as fluorogenic probes, super-resolution imaging and dye-protein hybrid-based indicators.

Divinylanthracene-Containing Tetracationic Organic Cyclophane with Near-Infrared Photoluminescence

Near-infrared (NIR) light is known to have outstanding optical penetration in biological tissues and to be non-invasive to cells compared with visible light. These characteristics make NIR-specific light optimal for numerous biological applications, such as the sensing of biomolecules or in theranostics. Over the years, significant progress has been achieved in the synthesis of fluorescent cyclophanes for sensing, bioimaging, and making optoelectronic materials. The preparation of NIR-emissive porphyrin-free cyclophanes is, however, still challenging. In an attempt for fluorescence emissions to reach into the NIR spectral region, employing organic tetracationic cyclophanes, we have inserted two 9,10-divinylanthracene units between two of the pyridinium units in cyclobis(paraquat-p-phenylene). Steady-state absorption, fluorescence, and transient-absorption spectroscopies reveal the deep-red and NIR photoluminescence of this cyclophane. This tetracationic cyclophane is highly soluble in water and has been employed successfully as a probe for live-cell imaging in a breast cancer cell line (MCF-7).

Open questions in plant cell wall synthesis.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support the cell while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of many model and crop plants have facilitated cataloging and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

Highly stable multi-encapsulated red-emitting cesium lead halide nanocrystals for efficient copper ion detection and imaging in live cells

Recently, cesium lead halide (CsPbX3: X = Cl, Br, I) perovskite nanocrystals (NCs) have shown potentiality in the detection of different heavy metal ions and bioimaging applications for their smaller particle size, tunable emission spectra, high luminescent intensity, narrow emission spectra, multiphoton absorption property, etc. CsPbBr3 NCs are explored extensively in bioimaging applications for their comparatively higher stability in water. A longer wavelength excitation light source is recommended due to less scattering of light results in deeper penetration in living cells. However, imaging in live cells with red-emitting perovskite NCs is not suitable because of their poor structural stability, low emission intensity, and complex synthesis process. To tackle these issues, we introduce a specific halide exchange to the NCs and then multi-encapsulation shelling techniques that eventually improve the structural and environmental stability of the red-emitting NCs. Here, we synthesized Zn-doped CsPbBr3 NCs via the ligand-assisted reprecipitation (LARP) synthesis method in an open atmosphere. Then the highly luminescent red emitting CsPbBrxI3−x NCs with Zn doping were grown via the exchange of halide ions from pre-synthesized Zn-doped CsPbBr3 NCs to maintain the stable perovskite crystal structure in the nanoscale regime. Such NCs were further encapsulated with different shelling materials and then compared their photophysical properties. Among them, n-isopropyl acrylamide passivated Zn-doped CsPbBrxI3−x/SiO2/polyvinylpyrrolidone core/shell/shell NCs showed the highest stability against water and UV-irradiation. These NCs efficiently detect Cu2+-ions in water and achieved a detection limit of around 9.68 µM. The NCs were also tested for their compatibility as a fluorescent probe for live cell imaging using mammalian Chinese Hamster Ovary cells. As a proof-of-principle, we demonstrate both the efficient uptake and biocompatibility of the perovskite NCs. This work provides insight for expanding research activities on red-emitting perovskite NCs by developing a new strategy to improve the stability of the NCs, which is beneficial for both cost-effective bioimaging applications and heavy metal ion detection in water.

Highly water-stable, luminescent, and monodisperse polymer-coated CsPbBr3 nanocrystals for imaging in living cells with better sensitivity.

Recently, CsPbX3 (X= Cl, Br, I) nanocrystals (NCs) have evolved as a potential contender for various optoelectronic applications due to some of their excellent photophysical properties. Their superior non-linear optical properties enable them to take part in bioimaging applications due to their longer penetration depth and less scattering effect in living cells. However, the poor stability of perovskite NCs in aqueous media still remains a great challenge for practical usage. Comparatively stable silica-coated NCs have a tendency to agglomerate among other NCs and transform into bigger particles. Such big particles clog the inside of narrow channels during the uptake and can't effectively reach the targeted cells. To tackle such issues, we introduce a fast and reproducible synthesis process of CsPbBr3 NCs that are coated with different long-chained organic ligands/polymers and compared their photophysical properties. Among them, polyvinylpyrrolidone (PVP) encapsulated NCs are highly luminescent in the green spectral region and showed a maximum photoluminescence quantum yield (PLQY) of up to 84%. The incorporation of n-isopropyl acrylamide (NIPAM) along with PVP further improves the stability of the PVP-coated NCs against heat and moisture. These NCs exhibit higher water stability compared to silica-coated NCs and maintained their emission properties for about one week in DI water. The smaller particle size, uniform size distribution, higher structural stability, and better dispersivity of polymer-coated NCs in the aqueous media enable them to perform as fluorescent probes for live cell imaging in mammalian Chinese Hamster Ovary (CHO-K1) cells. There is no adverse affect in the cells' viability and morphology even after long incubation periods (∼72 hours). The dosage of Pb-ions contained in the polymer-coated NCs is calculated as below 5 μg mL-1, which is suitable for live cell imaging. This work provides insight for expanding the use of these NCs significantly into bioimaging applications with higher sensitivity.

Visualization of the dynamic interaction between nucleosomal histone H3K9 tri-methylation and HP1α chromodomain in living cells

Histone lysine methylation is an epigenetic mark that can control gene expression. In particular, H3K9me3 contributes to transcriptional repression by regulating chromatin structure. Successful mitotic progression requires correct timing of chromatin structure changes, including epigenetic marks. However, spatiotemporal information on histone modifications in living cells remains limited. In this study, we created an FRET-based probe for live-cell imaging based on the HP1α chromodomain (HP1αCD), which binds to H3K9me3. The probe was incorporated into chromatin and the emission ratio decreased after treatment with histone methyltransferase inhibitors, indicating that it successfully traced dynamic changes in H3K9me3. Upon entry into mitosis, the probe's emission ratio transiently increased with a concomitant increase in H3K9me3, then exhibited a stepwise decrease, probably due to loss of HP1αCD binding caused by phosphorylation of H3S10 and demethylation of H3K9me3. This probe will be a useful tool for detecting dynamic changes in chromatin structure associated with HP1α.

Systematic Exploration of Furoindolizine-Based Molecular Frameworks towards a Versatile Fluorescent Platform.

The photophysical behaviors of fluorescent molecules largely determine their major utility in biological studies. Despite their well-defined characteristics, classical fluorophores have often been challenged by their limited synthetic methodology and tunability in adjusting intrinsic optical properties. A novel heterocyclic core equipped with modular functional groups could offer the flexibility to control its photophysical properties with a minimum synthetic effort. By conducting a systematic analysis guided by quantum calculations, we proposed the furoindolizine-based molecular framework as a unique fluorescent platform capable of providing versatile photophysical properties with minimal structural modification. A broad tunability of furoindolizine derivatives' photophysical properties such as emission wavelength, Stokes shift, fluorescent brightness, and charge transfer characteristics was achieved through synergistic interaction between two functional moieties. Furthermore, this modular platform led to live-cell imaging probes with two distinct optical features simply by reorganizing a pair of functional moieties.

Nitrobenzoxadiazole derivatives of the rat selective toxicant norbormide as fluorescent probes for live cell imaging

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant’s pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Silicon functionalization expands the repertoire of Si-rhodamine fluorescent probes.

Fluorescent dyes such as rhodamines are widely used to assay the activity and image the location of otherwise invisible molecules. Si-rhodamines, in which the bridging oxygen of rhodamines is replaced with a dimethyl silyl group, are increasingly the dye scaffold of choice for biological applications, as fluorescence is shifted into the near-infrared while maintaining high brightness. Despite intense interest in Si-rhodamines, there has been no exploration of the scope of silicon functionalization in these dyes, a potential site of modification that does not exist in conventional rhodamines. Here we report a broad range of silyl modifications that enable brighter dyes, further red-shifting, new ways to modulate fluorescence, and the introduction of handles for dye attachment, including fluorogenic labeling agents for nuclear DNA, SNAP-tag and HaloTag labeling. Modifications to the bridging silicon are therefore of broad utility to improve and expand the applications of all Si-dyes.

Bio-orthogonally activated tetraphenylene-tetrazine aggregation-induced emission fluorogenic probes.

Tetrazine-based bio-orthogonally activated fluorogenic probes have drawn great attention due to their excellent performance in bioimaging; however, most of them suffer from aggregation-caused quenching (ACQ) problems. Herein, we developed a set of novel tetrazine-modified tetraphenylenes (TPEs) as bio-orthogonally activated aggregation-induced emission (AIE) fluorogenic probes. Both the fluorescence and AIE features are quenched by tetrazine, which is mediated by the through-bond energy-transfer (TBET) mechanism, and are activated upon converting tetrazine to pyridazine via the inverse electron-demand Diels-Alder (iEDDA) reaction. The activated cycloadducts displayed a notable fluorescence enhancement, a large Stokes shift, a high fluorescence quantum yield, and evident AIE-active features. Manipulating the length and position of the π-linker enables fine-tuning of the photophysical properties of the probes, while an overlong planar π-linker leads to AIE-to-ACQ transformation. We also designed bi-tetrazyl-substituted probes, which exhibited a higher turn-on ratio than the mono-tetrazyl analogs owing to the 'double-quenched' function. When they reacted with double-clickable linkers, fluorescent macrocycles were obtained because of the restriction of the free rotation of the phenyl rings of TPE. Using an organelle-pretargeting strategy, we succeeded in applying these probes for mitochondria-specific bio-orthogonal imaging in live cells under no-wash conditions, which is expected to provide a powerful tool for biomedical applications.

Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.

SUMMARYAssembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase β (Polβ) and XRCC1 to DNA lesion sites identifies a role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polβ’s dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

Live-cell imaging probes to track chromatin modification dynamics.

The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized by both small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind weakly to specific modifications, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformational changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.

An AIE triggered fluorescence probe with three-photon absorption and its biological applications

Three-photon absorption (3 PA) in the near IR region is among the most prominent nonlinear optical (NLO) effects and has attractive applications in chemical/biological sensing and imaging. Yet, rationally constructed molecules with small molecular weight and reasonable 3 PA cross-section has been rarely reported. Herein, we designed a novel three-photon absorption photostable luminogen (namely X1) with enhanced aggregation induced emission (AIE) and the ability to achieve multi-photon imaging with femtosecond laser excitation. X1 was constructed from diaminobenzene and diethylamino salicylaldehyde forming a novel di-Schiff base. It possesses a large conjugated delocalization which exhibits large three-photon absorption (3 PA) cross-section values. We also showed that by using a suitable delivery vector, X1 compound could applied as a live cell imaging probe thus providing a valuable tool to study lipid droplets/lysosome interaction in depth tissues.

A sustainable green synthesis of functionalized biocompatible carbon quantum dots from Aloe barbadensis Miller and its multifunctional applications

Herein, we demonstrated a sustainable green approach for the preparation of fluorescent biocompatible carbon quantum dots by microwave-assisted reflux synthesis from Aloe barbadensis Miller (Aloe vera) extract. The Transmission Electron Microscopic images reveal that the as-prepared CQDs are spherical with less than 5nm in size. The CQDs are amorphous, showed an excitation-independent behaviour, emitted blue fluorescence and have a fluorescence quantum yield of 31%. The presence of -OH groups contributed to the blue emission and helped CQDs to disperse uniformly in an aqueous solution. The prepared CQDs were employed as a photocatalyst for the environmental remediation to degrade the anionic dye, eosin yellow under visible light irradiation. The results showed that the CQDs exhibited excellent photocatalytic efficiency of 98.55% within 80min and a 100% efficiency within 100min. Further, the cytotoxic properties of as-prepared CQDs are investigated in the MCF-7 breast cancer cell line using MTT assay. The results demonstrated a notable reduction in cell viability in a dose-subjected manner, and the cell viability decreased to 50% (IC50) at a concentration of 52.2±1.35μg/mL. Furthermore, cellular internalization of CQDs in breast cancer cells is studied. As expected, CQDs are found to internalize by the cancer cells with blue emission as revealed by fluorescence microscope. In the end, CQDs in human breast cancer cells demonstrate the anti-proliferative effect and are found to be an impressive fluorescent probe for live-cell imaging, paving a path for its potential biomedical applications.

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Silafluorene as a promising core for cell-permeant, highly bright and two-photon excitable fluorescent probes for live-cell imaging

In this study, we report the synthesis, linear and non-linear photophysical studies and live-cell imaging of two two-photon activatable probes based on a silafluorene core: SiFluo-V and SiFluo-L. Thanks to their quadrupolar (A-π-D-π-A) design, these probes exhibit respectively good to impressive two-photon cross-sections (from 210 GM to 2150 GM). TD-DFT calculations support the experimental evidence that SiFluo-L displays far better two-photon absorption properties than SiFluo-V. Moreover, SiFluo-L possesses all requirements for bioimaging as it is water soluble, cell-permeant and presents a low cytotoxicity (IC80 ≥ 10 μM). It labels mitochondria in live-cell imaging at low laser power with high brightness, contrast and photostability. This study demonstrates that silafluorene is a promising core to develop new two-photon fluorophores for live-cell imaging.