Wash-free Imaging Research Articles

AIE-active cyclometalated iridium(III) complexes for the detection of lipopolysaccharides and wash-free imaging of bacteria.

Infectious diseases caused by bacteria pose a major threat to human health and are currently one of the leading causes of mortality worldwide. Therefore, the development of probes for rapid detection of bacteria and their pathogenic components is highly essential. Aggregation-induced emission (AIE)-active compounds have shown great promise for the diagnosis of bacterial infections. In this study, we have synthesized three cationic AIE-active cyclometalated iridium(III) polypyridyl complexes viz., [Ir(C^N)2(N^N)]Cl2 (Ir1-Ir3), where C^N is a cyclometalating ligand such as pq = 2-phenylquinoline (in Ir1), pbt = 2-phenylbenzothiazole (in Ir2), and dfppy = 2-(2,4-difluorophenyl)pyridine (in Ir3), and N^N is a 2,2'-bipyridine derivative, for detection of lipopolysaccharide (LPS) in aqueous solution and wash-free imaging of bacteria. These complexes exhibit rapid sensing of LPS also known as endotoxin released by the bacteria, with a detection limit in the nanomolar range being determined by fluorescence spectroscopy within 5 min. Detection of both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria by the complexes is visible to the naked eye and was also observed by fluorescence microscopy imaging. The above features of the complexes make them a promising scaffold for the detection of bacterial contamination in aqueous samples.

Dual microenvironmental parameter-responsive lysosome-targeting carbon dots for the high contrast discrimination of a broad spectrum of cancer cells

The development of new carbon dots (CDs) for fluorescence-based cancer diagnosis has recently attracted extensive attention. Diagnosis methods based on ligand-receptor fluorescence suffer from the heterogeneity of receptor expression. Changes in the microenvironments of cancer cells provide opportunities for accurate and broad-spectrum cancer diagnosis. The lysosomes in cancer cells have lower polarity and higher viscosity than normal cells. Based on these two key microenvironmental parameters, dual-responsive CDs with inherent lysosome-targeting ability were synthesized via one-step hydrothermal treatment. The CDs exhibit many advantageous properties including facile synthesis, good water solubility, pH-independent emission, excellent photostability, good biocompatibility, and wash-free imaging ability. The CDs were successfully employed in the fluorescence-based discrimination of a broad spectrum of cancer cells from normal cells with high contrast. The CDs are promising candidates for use in the field of cancer diagnosis. By taking advantage of lower polarity and higher viscosity of cancerous lysosomes, polarity and viscosity dual microenvironmental parameter-responsive lysosome-targeting CDs were rationally prepared and showed great potential in high contrast discrimination of a broad spectrum of cancer cells from normal cells.

Stable Super-Resolution Imaging of Lipid Droplet Dynamics through a Buffer Strategy with a Hydrogen-Bond Sensitive Fluorogenic Probe.

Although super-resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better-performing and more photostable probes for long-term super-resolution imaging. Herein, we report a probe, LD-FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD-FG allows wash-free imaging of LDs, owing to a hydrogen-bond sensitive fluorogenic response. The replacement of photobleached LD-FG by intact probe molecules outside the LDs ensures the long-time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e., a fast LD coalescence followed by a slow ripening step. The excellent performance of LD-FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging.

A water-soluble AIEgen for ultrafast and wash-free imaging of plasma membranes in biosystems

Visualizing the plasma membrane (PM) in biological systems is in a front field of life science research. Herein, a rotor-based compound Car-py with aggregation-induced emission (AIE) activity has been designed and synthesized. Car-py can selectively light-up the PM through binding to the lipid bilayer structure owing to a restriction of intramolecular motions (RIM). Importantly, compared with commercial PM dyes, Car-py exhibits many advantages, including wash-free procedure, good photostability, ultrafast staining process, long-term tracking, excellent plasma membrane specificity, and favorable biocompatibility, which were confirmed by the fluorescence image in vitro and in vivo. Such PM-specific probe may extend the arsenal of currently available fluorescent agents to visualize the plasma membrane.

A new class of nitrobenzoic acid-based AIE photosensitizers for highly efficient photodynamic antibacterial therapy

Photodynamic therapy (PDT) has been drawing more and more attention in the antibacterial field. Traditional photosensitizers (PSs) tend to aggregate in aqueous media, which reduces the generation of reactive oxygen species (ROS) and seriously affects the photodynamic efficacy. Many efforts have been made to prevent aggregation of traditional PSs. By contrast, aggregation-induced emission PSs (AIE-PSs) take advantage of aggregation to boost ROS generation and fluorescence intensity. However, the efficacies of the reported antibacterial AIE-PSs are poor. Herein, we report a new class of highly effective antibacterial AIE-PSs based on nitrobenzoic acid structure. TTVBA, a negatively charged AIE-PS, can not only selectively kill spherical bacteria (Staphylococcus aureus (S. aureus)) rather than rod-shaped bacteria (Escherichia coli (E. coli)), but also be easily extended to several AIE-PSs (TTVBP1-3) with positive charges and broad-spectrum antibacterial activity. We demonstrate that TTVBP2 can kill 3.0 log10 of S. aureus at very low concentration (125 nmol L−1), TTVBP3 can kill 4.7 log10 of Staphylococcus epidermidis (S. epidermidis) at a concentration of 1 µmol L−1 and 3.8 log10 of E. coli at 5 µmol L−1, thus enabling them among the most effective antibacterial AIE-PSs reported so far. Meanwhile, these AIE-PSs exhibit excellent wash-free imaging ability for bacteria by simple mixing with bacteria. We thus envision that TTVBA, a nitrobenzoic acid-based extendable AIE PS, provides a new route for the design of AIE-PSs in antibacterial treatment.

A dibenzothiophene core-based small-molecule AIE probe for wash-free and selective staining of lipid droplets in live mammalian and fungal cells

Lipid droplets (LDs) are spherical dynamic subcellular organelles, and play crucial roles in a number of cellular functions, such as lipid metabolism, protein degradation, membrane formation, energy storage, and signal transduction. It is widely recognized that the dysfunction of LDs can lead to many diseases, and hence it is important to monitor the size, distribution, and movement of LDs in living cells. Herein, we report a dibenzothiopene core-based fluorescent probe named triphenylamine dibenzothiophene-S,S-dioxide (TPA-DBTD), which can exhibit aggregation-induced emission (AIE) characteristic and realize selective and wash-free imaging of LDs in live cells. Most importantly, this probe can efficaciously track the dynamic changes of LDs including lipophagy (an autophagic process that is responsible for the degradation of lipids) in live mammalian cells and stain LDs in fungi. Hence, we believe that TPA-DBTD with high photostability, good biocompatibility, AIE characteristic, and LD staining ability in diverse biological systems (mammalian cells and microorganisms) can find practical applications in the fields of biomedical science, cell biology, and diagnosis of LDs-associated diseases.

Nitrogen-doped carbon dots for wash-free imaging of nucleolus orientation.

Carbon dots (CDs) are a rising star in the field of cellular imaging, especially cytoplasmic imaging, attributing to the super-stable optical performance and ultra-low biological toxicity. Nucleolus can accurately reflect the expression state of a cell and is strongly linked to the occurrence and development of many diseases, so exploring bran-new CDs for nucleolus-orientation imaging with no-wash technology has important theoretical value and practical significance. Herein, nitrogen-doped carbon dots (N-CDs) with green fluorescence (the relative fluorescence quantum yield of 24.4%) was fabricated by the hydrothermal treatment of m-phenylenediamine and p-aminobenzoic acid. The N-CDs possess small size, bright green fluorescence, abundant surface functional groups, excellent fluorescence stability and good biocompatibility, facilitating that the N-CDs are an excellent imaging reagent for cellular imaging. N-CDs can particularly bind to RNA in nucleoli to enhance their fluorescence, which ensures that the N-CDs can be used in nucleolus-orientation imaging with high specificity and wash-free technique. This study demonstrates that the N-CDs have a significant feasibility to be used for nucleolus-orientation imaging in biomedical analysis and clinical diagnostic applications.

Rapid membrane-specific AIEgen featuring with wash-free imaging and sensitive light-excited killing of cells, bacteria, and fungi

C6-BD equipped with AIE and ESIPT characteristics was used to visualize the cell membrane with high selectivity, stability, and wash-free operation. It also universal for imaging of bacteria and fungi with light-excited killing via ROS generation.

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes.

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

Towards tetrazine-based near-infrared fluorogenic dyes: Is there a wavelength limit?

Tetrazine-based fluorogenic dyes have been broadly applied in the studies of many biomolecules in live cells via wash-free imaging. While near-infrared (NIR) dyes are much preferred in these scenarios, few tetrazine-based NIR fluorogenic dyes have been reported to date. In this work, we developed three tetrazine-based cyanine dyes with emission wavelengths of up to ~790 nm and fluorescence turn-on ratios of ~4. We also performed quantum chemical calculations to rationalize the low turn-on ratio in these dyes. Our results show that based on the dark-state quenching mechanism, the tetrazine moiety is intrinsically not suitable for developing NIR fluorogenic dyes with emission wavelengths of >~710 nm.

Cationic Organic Nanoaggregates as AIE Luminogens for Wash-Free Imaging of Bacteria and Broad-Spectrum Antimicrobial Application.

The increase in the use of bactericides is a matter of grave concern and a serious threat to human health. The present situation demands rapid and efficient detection and elimination of antibiotic-resistant microbes. Herein, we report the synthesis of a simple C3-symmetric molecular system (TGP) with an intrinsic positive charge through a single-step Schiff base condensation. In a water-dimethyl sulfoxide (DMSO) solvent mixture (80:20 v/v), TGP molecules self-aggregate to form spherical nanoparticles with a positively charged surface that displays efficient fluorescence owing to the aggregation-induced emission (AIE) phenomenon. Both Gram-positive and Gram-negative bacteria could be effectively detected through "turn-off" fluorescence spectroscopy as the electrostatic interaction of the resultant nanoaggregates with the negatively charged bacterial surface induced quenching of fluorescence of the nanoparticles. The fluorescence analysis and steady-state lifetime studies of TGP nanoparticles suggest that a nonradiative decay through photoinduced electron transfer from the nanoparticles to the bacterial surface leads to effective fluorescence quenching. Further, the TGP nanoaggregates demonstrate potent antimicrobial activity against microbes such as multidrug-resistant bacteria and fungi at a concentration as low as 74 μg/mL. A combination of factors including ionic surface characteristics of the nanoparticles for strong electrostatic binding on the bacterial surface followed by possible photoinduced electron transfer from the nanoaggregates to the bacterial membrane and enhanced oxidative stress in the membrane resulting from reactive oxygen species (ROS) generation is found accountable for the high antimicrobial activity of the TGP nanoparticles. The effective disruption of membrane integrity in both Gram-positive and Gram-negative bacteria upon interaction with the nanoaggregates can be observed from field emission scanning electron microscopy (FESEM) studies. The development of simple pathways for the molecular design of multifunctional broad-spectrum antimicrobial systems for rapid and real-time detection, wash-free imaging, and eradication of drug-resistant microbes might be crucial to combat pathogenic agents.

TICT based fluorescent probe with excellent photostability for real-time and long-term imaging of lipid droplets

Lipid droplets (LDs) are dynamic organelles and involve in various physiological processes by regulation of the storage and metabolism of lipid molecules. The real-time and long-term imaging of LDs’ distribution and movement is critical for investigation of their biological functions. However, current LDs-targeted fluorescent probes suffer from low photostability and high background noise. To tackle these challenges, we herein reported that the red-emissive fluorescent probe DCQTB with twisted intramolecular charge transfer (TICT) characteristics can be used for wash-free imaging of LDs with advantages of fast cell penetration ability, high specificity, excellent photostability, and low phototoxicity. This LDs-specific fluorescent probe is thus promising for investigation of the biological functions of LDs.

Synthesis of a series of ethylene glycol modified water-soluble tetrameric TPE-amphiphiles with pyridinium polar heads: Towards applications as light-up bioprobes in protein and DNA assay, and wash-free imaging of bacteria

Available online Development of water soluble AIE-active “light-up” bioprobes for the detection of biomacromolecules has drawn huge research interests in recent past. In this study, a series of ethylene glycol modified water soluble tetrameric tetraphenylethylene amphiphiles with pyridinium polar heads (TPE-xEG-Py, x = 3, 4, 6 or 1a-c) have been synthesized by varying the ethylene glycol spacer. Their unique structure allows them to form vesicles and other nanoaggregates in aqueous solutions. These amphiphiles were successfully utilized for fluorimetric detection and quantitation of BSA and DNA based on the electrostatic interactions to trigger AIE-emission from the TPE moiety. The electrostatic interaction was also proved very effective in wash-free imaging of both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria with up to 92 folds increase in fluorescence response within bacterial concentration 0–12 × 108 CFU mL−1. The strategy is advantageous due to cost-effective and easy synthesis, high water solubility, and fast response.

A water-soluble AIE-gen for organic-solvent-free detection and wash-free imaging of Al3+ ions and subsequent sensing of F− ions and DNA tracking

A water-soluble TPE-based AIEgen (TPE-diBuS) was developed for organic-solvent-free detection of Al3+ ions and its wash-free cell imaging. The TPE-diBuS-Al ensemble was used for the detection of F− ions and DNA tracking.

Highly selective and wash-free visualization of resistant bacteria with a relebactam-derived fluorogenic probe.

Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine β-lactamases (SBLs), which are the major causes of bacterial resistance to β-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.

Fluorescence Resonance Energy Transfer-Based Wash-Free Bacterial Imaging and Antibacterial Application Using a Cationic Conjugated Polyelectrolyte.

The increase in bacterial infection and antibiotic resistance has posed a severe threat to the human health. This threat has warranted an imperative demand for the development of a new and effective bactericidal material to eradicate the antibiotic-resistant pathogenic bacteria. In this work, we report the wash-free imaging of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) bacteria using cationic conjugated polyelectrolyte[9,9-bis(6'-methylimidazoliumbromide)hexyl-fluorene- co-4,7-(2,1,3-benzothiadiazole)] (PFBT-MI) based on aggregation-induced fluorescence resonance energy transfer (FRET). Cationic imidazolium group strapped on the polymer side chain not only increases its solubility in water but also helps in binding with the negatively charged bacterial membrane via electrostatic interactions to turn on its bright yellow emission. The change in the fluorescence color of conjugated polyelectrolyte in the presence of bacteria could be visualized very easily via naked eyes under a UV lamp (365 nm). Furthermore, the antibacterial activity of PFBT-MI against both S. aureus and E. coli was observed because of the amphiphilic nature of the conjugated polyelectrolyte which in turn is due to the presence of ionic functionality and conjugated polymer backbone that can intercalate very proficiently into the bacterial membrane, which disrupts the membrane integrity and thus results in toxicity. Morphologically, the membrane damage was perceived via field emission scanning electron microscopy (FESEM) images, which clearly indicated the disruption of cell membrane upon exposure to PFBT-MI. The PFBT-MI acts as an effective antibacterial agent, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of (30 μM or 23.7 μg/mL) and (60 μM or 47.7 μg/mL) for S. aureus and for E. coli (60 μM or 47.7 μg/mL) and (100 μM or 79 μg/mL), respectively. Moreover, PFBT-MI shows less cytotoxicity against mammalian cells at concentration greater than MIC.

An Easily Accessible Ionic Aggregation-Induced Emission Luminogen with Hydrogen-Bonding-Switchable Emission and Wash-Free Imaging Ability.

Ionic fluorophores are powerful tools for the study of environmental science and bio-imaging. However, traditional ionic dyes usually require long synthetic steps and suffer from a quenching effect caused by aggregation. A water-soluble ionic aggregation-induced emission luminogen called DBTA is presented, which is readily accessed by a one-step reaction. The switchable emission manipulated by hydrogen bonding provided solid evidence for the restriction of intramolecular motions as the mechanism of aggregation-induced emission. DBTA can not only differentiate solvents with different H-bond donor acidities but also capable of wash-free imaging in living HeLa cells and fish larva.

Photoluminescence Lifetime Imaging of Synthesized Proteins in Living Cells Using an Iridium-Alkyne Probe.

Designing probes for real-time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near-infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium-alkyne complex. This probe offers the benefits of deep-red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash-free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C2-BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Bright and Photostable Fluorophores for Advanced Fluorescence Microscopy

Advanced fluorescence microscopy, including single-molecule and super-resolution imaging, demands bright and photostable fluorophores. We have recently reported a general approach to improve fluorophores brightness in living cells by substituting the N,N-dimethylamino groups found in classic dyes with four-membered azetidine rings (Nature Methods 12, 244250 (2015)). In an unpublished work we have synthesized new derivatives containing substituents on the azetidine ring. Using this approach we were able to fine tune the wavelength and fluorogenecity of the fluorophore without affecting brightness. Here, we report that several of these novel substituted-azetidine fluorophores, as well as the substituted-xanthene ones, exhibit substantial improvements in photostability in living cells. These fluorophores enable robust multi-color, wash-free imaging with a large photon budget. We are investigating their phototoxicity and mechanism in order to maximize the photostability, to generalize this approach to different fluorophores, and to apply these fluorophores to diverse biological settings, including living cells, tissues, and animals. We freely share our fluorophores with the academic community. This work is supported by HHMI and NIH (U01 EB 021236).