Probes For Live Cell Imaging Research Articles

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.

Structure of the Lifeact-F-actin complex.

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

A Rapid Synthesis of Nuclear-Staining Small Fluorescent Molecules for Brain Imaging

Summary Fluorescent probes are important tools in cellular imaging. However, many readily available small-molecule imaging probes are either cytotoxic or easily photobleached and thus not compatible with lengthy live cell or tissue-imaging experiments. One particularly limited field of application is neuroscience, in which small-molecule probes for live cell imaging at the brain tissue level are highly scarce. Here, we describe a rapid synthetic route to generate a series of nuclear stains that enable experiments with live brain cells (i.e., water solubility, cell permeability, high quantum yield, high photostability, and most important, negligible cytotoxicity). As a proof of principle, we demonstrate that these probes are compatible with ex vivo and in vivo nuclear labeling experiments, including retrograde transport to monitor and map certain brain regions of the mouse model. The structural versatility and superior non-cytotoxic properties of these probes may suggest their potential use in labeling and tracing of neuronal populations.

A Near-Infrared Fluorogenic Pyrrole-Imidazole Polyamide Probe for Live-Cell Imaging of Telomeres.

Telomeres are closely associated with cellular senescence and cancer. Although some techniques have been developed to label telomeres in living cells for study of telomere dynamics, few biocompatible near-infrared probes based on synthetic molecules have been reported. In this study, we developed a near-infrared fluorogenic pyrrole-imidazole polyamide probe (SiR-TTet59B) to visualize telomeres by conjugating a silicon-rhodamine (SiR) fluorophore with a tandem tetramer pyrrole-imidazole polyamide targeting 24 bp in the telomeric double-stranded (ds) DNA. SiR-TTet59B was almost nonfluorescent in water but increased its fluorescence dramatically on binding to telomeric dsDNA. Using a peptide-based delivery reagent, we demonstrated the specific and effective visualization of telomeres in living U2OS cells. Moreover, SiR-TTet59B could be used to observe the dynamic movements of telomeres during interphase and mitosis. This simple imaging method using a synthetic near-infrared probe could be a powerful tool for studies of telomeres and for diagnosis.

Fluorescent Benzoselenadiazoles: Synthesis, Characterization, and Quantification of Intracellular Lipid Droplets and Multicellular Model Staining.

In this work, we described the synthesis of 10 new fluorescent 2,1,3-benzoselenadiazole small-molecule derivatives and their chemical- and photocharacterizations. The new derivatives could, for the first time, be successfully applied as selective live cell imaging probes (at nanomolar concentrations) and stained lipid-based structures preferentially. Density functional theory (DFT) calculations were used to help in understanding the photophysical data and the intramolecular charge-transfer (ICT) processes of the synthesized dyes. Some derivatives showed impressive cellular responses, allowing them to be tested as probes in a complex multicellular model (i.e., Caenorhabditis elegans). When compared with the commercially available dye, the new fluorescent compounds showed far better results both at the cellular level and inside the live worm. Inside the multicellular complex model, the tested probes also showed selectivity, a feature not observed when the commercial dye was used to carry out the bioimaging experiments.

A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation.

Conjugation of proteins to AMP (AMPylation) is a prevalent post‐translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chemical proteomics profiling of AMPylated proteins. Using straightforward strain‐promoted azide–alkyne click chemistry, the probe provides stable fluorescence labelling in living cells.

Self-Assembly of Highly Stable Zirconium(IV) Coordination Cages with Aggregation Induced Emission Molecular Rotors for Live-Cell Imaging.

The self-assembly of highly stable zirconium(IV)-based coordination cages with aggregation induced emission (AIE) molecular rotors for in vitro bio-imaging is reported. The two coordination cages, NUS-100 and NUS-101, are assembled from the highly stable trinuclear zirconium vertices and two flexible carboxyl-decorated tetraphenylethylene (TPE) spacers. Extensive experimental and theoretical results show that the emissive intensity of the coordination cages can be controlled by restricting the dynamics of AIE-active molecular rotors though multiple external stimuli. Because the two coordination cages have excellent chemical stability in aqueous solutions (pH stability: 2-10) and impressive AIE characteristics contributed by the molecular rotors, they can be employed as novel biological fluorescent probes for in vitro live-cell imaging.

Novel near-infrared fluorescent probe for live cell imaging.

Near infrared (NIR) fluorescent probes play a crucial role in biological system imaging. A novel NIR fluorescent probe, IR787, was designed in the present study. Compared with indocyanine green (ICG), IR787 showed lower background fluorescent interference and higher fluorescence enhancement. Fluorescence intensities were detected by a Cary Eclipse fluorescence spectrophotometer. The interference of intracellular ions (Cu2+, Ca2+, Mg2+ and Zn2+) on the measurement was negligible, which indicated a good photostability of IR787. MTT assay demonstrated that cell viability of human lung adenocarcinoma epithelial cell line A549 was not significantly affected by the use of the IR787 probe compared with the ICG probe. This result suggested that the IR787 probe was safe for in vitro cell imaging. In vitro NIR optical imaging experiments further revealed cellular uptake and strong intracellular NIR fluorescence of the IR787 probe in A549 cells. The excitation wavelength was 787 nm for IR787. Compared with the previously reported NIR fluorescent probe ICG, the IR787 NIR fluorescent probe had improved prospects for intracellular imaging. IR787 may play a pivotal role in the understanding cell biology, pharmacology and disease diagnosis.

BODIPY-Labeled Cyclobutanes by Secondary C(sp3 )-H Arylations for Live-Cell Imaging.

Arylated cyclobutanes were accessed by a versatile palladium-catalyzed secondary C(sp3 )-H activation, exploiting chelation assistance by modular triazoles. The C-H arylation led to cyclobutane natural product derivatives in a highly regioselective fashion, setting the stage for the easy access to novel fluorogenic boron-dipyrrin (BODIPY)-labeled probes for live-cell imaging.

Holding-Oriented versus Gating-Oriented Live-Cell Distinction: Highlighting the Role of Transporters in Cell Imaging Probe Development.

Small molecule imaging probes are powerful tools to understand complex biological systems. The mainstreams of imaging probe developments have been focused on the target holding of the probes; the holding targets are often cell-type-specific biomarkers. This type of the probe mechanism can be designated as holding-oriented live-cell distinction (HOLD). Our group has worked on the development of cell-type-selective probes using a diversity-oriented fluorescence library approach (DOFLA), where unbiased phenotypic screening is employed using fluorescent library compounds. Through the conventional target identification methods such as an affinity-based analysis, we elucidated that some of the probe mechanisms are HOLD. However, we also realized that sometimes there is no specific holding target for probes or the holding targets are ubiquitous. The observation led us to test an alternative mechanism of cell-type-specific probes as gating-oriented live-cell distinction (GOLD). We started to examine the gating mechanism of probes, which is mainly based on transporters but which does not necessarily require probe holding to cellular targets. Transporters can control the in and out movement of various nutrients and chemicals. Different expression levels of transporters in various cell types could provide the molecular mechanism of differential staining of cells by regulating the intracellular accumulation of a certain specific probe. A number of GOLD probes have been developed by modifying or mimicking endogenous substrates of transporters such as inorganic ions, glucose, amino acids, or neurotransmitters, utilizing broad substrate specificity of transporters. The radiolabeled or fluorophore-conjugated substrate mimetics have been widely used for live cell distinction and various applications such as disease-related cell or tissue imaging. In humans, there are about 400 solute carrier (SLC) transporters and 50 ATP-binding cassette (ABC) transporters. Since some transporters have broad substrate specificity, they can transport not only derivatives of endogenous natural substrates but also totally synthetic diverse imaging probes, such as DOFLA probes. Without preconsidering the structure of endogenous substrates, we recently demonstrated a series of live-cell imaging probes and elucidated their molecular mechanism as a gating one, either by SLC or ABC transporters. Transporter inhibitor panel and CRISPR-based transporter libraries could provide a systematic gating target elucidation platform. Considering the generality of DOFLA and the CRISPR-based genomic tool for transporter systems (>450 in humans), the GOLD approach will offer new insight and promise for unprecedented levels of novel cell imaging probe development.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA's life cycle. Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Photoluminescence properties of gadolinium phosphate nanoprisms doped with lanthanide ions for multicolor live cell imaging

Lanthanide fluorescence nanoprobe is versatile in multicolor cell imaging, although it still faces a great challenge in improving its luminescent efficiency. Herein, we validated the feasibility of lanthanide ions co-doped gadolinium phosphate nanocrystals serving as a safe, effective and color-tunable fluorescence probe for live cell imaging. Well-crystallized hexagonal phase GdPO4 nanoparticles with multi-morphologies such as nanowires, nanorods, tetragonal nanocubes and hexagonal nanoprisms were synthesized via a typical hydrothermal method. We have systematically investigated the synthesis of different morphologies of GdPO4 nanoparticles through adjusting their processing parameters. The nanoparticles can gain a bright color-tunable photoluminescence from red, orange, yellow to green region when the GdPO4 nanoparticles are co-doped with Eu3+ and Tb3+ ions. The Tb3+ doping significantly enhances the characteristic 4f → 4f transitions of Eu3+ owing to a specific cross-relaxation process from Tb3+ to Eu3+ ions; the mechanism underlying the cross relaxation was exposited in detail. Further biological experiments in vitro proved that the prepared nanoprisms could exhibit a favourable biocompatibility with negligible toxicity. The current investigation manifests a high potential of the nanoparticles applicable to fluorescence imaging probe for live cells.

A general modular approach for the solubility tagging of BODIPY dyes

We describe a general and practical strategy for the direct one-step incorporation of a tunable solubility module at the boron atom of F-BODIPY dyes. The tethering reaction uses easy-to-handle reagents, has broad functional group compatibility and proceeds under mild conditions without requiring any pre-functionalization of the starting F-BODIPY to yield the solubility-tagged O-BODIPY derivative in 51–86% yield. The module can be introduced at the end of the synthetic route without perturbing the fluorophore scaffold, thus minimizing difficulties in product isolation. Its orthogonal geometrical arrangement with respect to the plane of the BODIPY chromophore hampers intermolecular aggregation processes that quench fluorescence, while the covalent attachment to the boron atom has a minimal effect on the absorption properties. Fully water-soluble and hydrolytically stable BODIPYs were prepared by incorporating either neutral (tetra- and octaethylene glycol chains) or zwitterionic (sulfobetaine) hydrophilic tags in the module. The new dyes are valuable live cell imaging probes that ameliorate the undesired partitioning into lipophilic compartments that is often observed for standard BODIPYs. This strategy can be readily adapted to the general and highly practical post-synthetic introduction of new functionalities into F-BODIPY dyes, including phase-tagging, by appropriately choosing the nature of the chemical tags attached to the module.

“Vigna radiata” based green C-dots: Photo-triggered theranostics, fluorescent sensor for extracellular and intracellular iron (III) and multicolor live cell imaging probe

Fluorescent C-dots have drawn increasing attention due to their superior properties such as high aqueous solubility, optical absorptivity, favorable biocompatibility and appreciable photoluminescence. Nature offers various precursors for the synthesis of green C-dots like fruits, vegetables, flowers, animal derivatives and many more. Herein, nitrogen doped C-dots (N@VRCD) were developed by economical, one step hydrothermal method using “Vigna radiata” sprouts as sole carbon precursor and ethylenediamine as the nitrogen source, which was optimized for enhanced optical properties. To the best of our knowledge, N@VRCD shows excellent quantum yield ∼58%. Importantly, N@VRCD also exhibits significant light induced phototoxicity to cancerous cells which enable the possibility of using it as a potential theranostics agent. Further, the N@VRCD displays highly sensitive and selective sensing behaviour towards Fe3+ ions by getting fluorescence “turn-off” with the limit of detection as low as 140 nM. Moreover, N@VRCD shows biocompatible and hemocompatible nature with a potential of multicolor live cell imaging and intracellular Fe3+ detection which was validated by confocal microscopy and flow cytometry.

Monitoring Lipid Droplet Dynamics in Living Cells by Using Fluorescent Probes.

We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LD-specific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and Lipi-Green allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.

Cucurbit[7]uril-conjugated dyes as live cell imaging probes: investigation on their cellular uptake and excretion pathways.

Here we report the endocytosis and excretion pathways of two different dye-conjugated cucurbit[7]urils, (cyanine 3-conjugated CB[7] and rhodamine X-conjugated CB[7]), which have great potential as molecular probes for live cell imaging. The dye-CB[7]s are translocated into live cells (human breast carcinoma cells, MCF-7) via multiple pathways, predominantly by clathrin-mediated endocytosis, and excreted from cells via lysosome-associated exocytosis. Interestingly, the CB[7] moiety has a substantial influence on the uptake and excretion pathways. These findings may widen the applications of the dyes conjugated to CB[7] and assist in the design of new molecular probes for live cell imaging.

Nucleic Acid Strand Displacement with Synthetic mRNA Inputs in Living Mammalian Cells.

Strand displacement reactions are widely used in DNA nanotechnology as a building block for engineering molecular computers and machines. Here, we demonstrate that strand displacement-based probes can be triggered by RNA expressed in mammalian cells, thus taking a step toward adapting the DNA nanotechnology toolbox to a cellular environment. We systematically compare different probe architectures in order to identify a design that works robustly in living cells. Our optimized strand displacement probe combines chemically modified nucleic acids that enhance stability to degradation by cellular nucleases with structural elements that improve probe retention in the cytoplasm. We visualize probe binding to individual mRNA carrying 96 repeats of a target sequence in the 3'UTR. We find that RNA counts based on live cell imaging using a strand displacement probe are comparable to counts from independent measurement based on fluorescence in situ hybridization experiments. We used probes with scrambled toeholds and scrambled binding domains to demonstrate that target recognition indeed occurs through toehold-mediated strand displacement. Our results demonstrate that strand displacement probes can work reliably in mammalian cells and lay the groundwork for future applications of such probes for live-cell imaging and molecular computing.

“Probe, Sample, and Instrument (PSI)”: The Hat-Trick for Fluorescence Live Cell Imaging

Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.

Multiple Multicomponent Reactions: Unexplored Substrates, Selective Processes, and Versatile Chemotypes in Biomedicine.

Multiple multicomponent reactions rapidly assemble complex structures. Despite being very productive, the lack of selectivity and the reduced number of viable transformations restrict their general application in synthesis. Hereby, we describe a rationale for a selective version of these processes based in the preferential generation of intermediates which are less reactive than the initial substrates. In this way, applying the Groebke-Blackburn-Bienaymé reaction on a range of α-polyamino-polyazines, we prepared a family compact heterocyclic scaffolds with relevant applications in medicinal and biological chemistry (live cell imaging probes, selective binders for DNA quadruplexes, and antiviral agents against human adenoviruses). The approach has general character and yields complex molecular targets in a selective, tunable and direct manner.

Mitochondrial Specific H2S n Fluorogenic Probe for Live Cell Imaging by Rational Utilization of a Dual-Functional-Photocage Group.

Reactive sulfur species play a very important role in modulating neural signal transmission. Hydrogen polysulfides (H2S n, n > 1) are recently suggested to be the actual signaling molecules. There are still few spatiotemporal controllable-based probes to detect H2S n. In this work, for the first time, we proposed the photocleavage product of the common photoremovable protecting group (2-nitrophenyl moiety) capable of trapping H2S n. Taking advantage of this, we constructed the probe H1 containing a photocontrollable group, a mitochondrial directing unit and a signal reporter fluorescein dye. H1 exhibited excellent fluorescence enhancement (50-fold) in response to H2S n under the aqueous buffer only after UV irradiation. H1 also showed high selectivity and sensitivity for H2S n over other reactive sulfur species, reactive oxygen species, and other analytes, especially biothoils including hydrogen sulfide, cysteine, homocysteine, and glutathione. We showed the utility of H1 to image H2S n in living cells with high spatiotemporal resolution.