Lipid Droplets In Living Cells Research Articles

Polarity-Driven Two-Photon Fluorescent Probe for Monitoring the Perturbation in Lipid Droplet Levels during Mitochondrial Dysfunction and Acute Pancreatitis.

Lipid droplets (LDs) act as an energy reservoir in cancer cells; on the other hand, mitochondria are hyperactive to fulfill the energy demand to accelerate cell proliferation. We are interested in unfolding the relationship between the cellular energy reservoir and energy producer through fluorescence labeling. Thus, a dual organelle-targeted fluorescent probe MLD-1 has been rationally developed. It visualized the crosstalk between mitochondrial dysfunction and the fluctuation of LDs in live cells. Its two-photon ability allowed us to acquire deep tissue images. For the first time, we have shown that the probe has the ability to track the accumulation of LDs in different mouse organs during pancreatic inflammation. MLD-1, being a selectively polarity-driven, chemo- and photostable LD probe, may offer great possibilities for studying LD-associated biology in due course.

Thiophene-conjugated benzothiazole derivatives as versatile skeletons for staining subcellular organelles in living cells

A series of hydroxy thiophene-conjugated benzothiazole derivatives (HTBs) was prepared to investigate their fluorescence emission behaviors and capability to selectively stain subcellular organelles for fluorescence imaging. The HTB derivatives exhibited excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) behaviors, according to their molecular structure. Subtle changes in the molecular structure of the HTB influenced its response to changes in the surrounding environment, such as the solvent polarity and/or pH. In vitro cellular imaging experiments revealed that HTBA (5-(benzo[d]thiazol-2-yl)-[2,2′-bithiophen]-4-ol) could selectively stain mitochondria. In contrast, HTBB-CO2Et (ethyl 5'-(benzo[d]thiazol-2-yl)-3′-hydroxy-[2,2′-bithiophene]-5-carboxylate), which contains a regioisomeric hydroxy thiophene unit with an electron-withdrawing ethyl ester group, showed the capability to stain lipid droplets (LDs) in living cells. When the hydroxyl group in HTBB-CO2Et was protected with a benzyl group, effective staining of LDs in live cells was achieved. Thus, we successfully obtained an effective fluorescent probe. Collectively, the results indicated that small structural changes resulted in substantial changes in the intracellular localization and in vitro imaging ability of the prepared compounds.

Chemists Focus on Probes, Biologists on Cells—But Who Talks about Probe-Cell Interactions? A Critical Account of the Suboptimal Reporting of Novel Fluorescent Imaging Probes, Using Lipid Droplet Stains as a Case Study

Many current reports in the scientific literature describe novel fluorescent probes intended to provide information on various structures or properties of live cells by using microscopic imaging. Unfortunately, many such reports fail to provide key information regarding the staining process. It is often the case that neither the necessary minimum technical detail (probe concentration, solvent and cosolute, temperature and time of staining, and details of post-staining washes) nor a discussion of the proposed staining mechanism are provided. Such omissions make it unnecessarily difficult for biomedical end-users to try out reported novel probes in their own laboratories. The validity of these criticisms is explored and demonstrated by a detailed analysis of 75 non-cherry-picked articles describing novel fluorescent probes for the detection of lipid droplets in live cells. This dataset also suggests that papers from journals with high journal impact factors or from better-known research groups are no more likely to provide better protocol information or discussion of the mechanism than papers from less prestigious sources. Comments on possible reasons for this suboptimal reporting are offered. The use of a suitable information/feature checklist, following best practice in many leading chemical and biological journals, is suggested as a mechanism for ameliorating this situation, with a draft checklist being provided.

Unraveling Molecular Assembly and Tracking Lipid Droplet Dynamics Using Fluorescent Phenanthroimidazole Derivatives

Small strategic perturbations of the molecular structures impart significant variation in the evolution dynamics and properties of supramolecular self-assembled architectures. However, probing the in situ evolution dynamics of molecular assembly remains a challenge. Herein, we unraveled the real-time, early stage environment-sensitive dynamic molecular self-assembly processes and stimuli-induced reversible morphological transformation between supramolecular fibers and spherical nanoaggregates of alkyl chain-substituted phenanthroimidazole-based luminogen (BPIB1) through fluorescence lifetime imaging microscopy (FLIM). The presence of an octyl chain and basic nitrogen centers in BPIB1 led to a distinct self-assembly pattern. The gradual progression in the fluorescence lifetime provides a unique strategy for elucidating the dynamics of the self-assembly process leading to distinct nano/microarchitectures. Among the varied self-assembled structures, the smaller-sized (diameter ∼ 20–30 nm), highly photostable, water-dispersible, and biocompatible fluorescent nanoaggregates of BPIB1 were employed for tracking the dynamics of lipid droplets in live cells and a model organism, C. elegans, using fluorescence correlation spectroscopy and FLIM. Thus, a combined microscopic and spectroscopic approach demonstrated in the present study opens up new avenues to explore the formation pathways of diverse molecular aggregates and their use to decipher complex organelle dynamics.

Advances in small molecule two-photon fluorescent trackers for lipid droplets in live sample imaging.

Two-photon fluorescent trackers for monitoring of lipid droplets (LDs) would be highly effective for illustrating the critical roles of LDs in live cells or tissues. Although a number of one-photon fluorescent trackers for labeling LDs have been developed, their usability remains constrained in live sample imaging due to photo damage, shallow imaging depth, and auto-fluorescence. Recently, some two-photon fluorescent trackers for LDs have been developed to overcome these limitations. In this mini-review article, the advances in two-photon fluorescent trackers for monitoring of LDs are summarized. We summarize the chemical structures, two-photon properties, live sample imaging, and biomedical applications of the most recent representative two-photon fluorescent trackers for LDs. Additionally, the current challenges and future research trends for the two-photon fluorescent trackers of LDs are discussed.

One-step assembly of Pd-Keggin-polyoxometalates for catalytic benzothiadiazole generation and derived cell-imaging probe application

One-step assembly of organic-ligand modified Pd-Keggin-POMs has been rarely reported, so as for their applications in catalytic benzothiadiazole generation and derived cell-imaging probing. Herein, three Pd-Keggin-POMs (compounds 1–3) have been successfully synthesized via a one-step assembly strategy. Thus-obtained Pd-Keggin-POMs with well-defined structures and heterogeneous properties enable highly efficient catalytic benzothiadiazole generation. Specifically, compound 3 showed outstanding catalytic activities in Suzuki-Miyaura coupling reactions for the generation of benzothiadiazole derivatives (yields, 90%-97%) and was represented as one of the best catalysts reported to date. Consequently, the obtained benzothiadiazoles were used as the bio-probe for tracking lipid droplets in living-cells and exhibited large Stokes shifts (130 nm), low cytotoxicity and good targeting, which could be also applied to mark the distribution of LDs in living HeLa cells. Systematic investigations clearly decipher the functions of Pd-Keggin-POMs toward finding novel bio-probe materials, highlighting a new insight into the generation of sustainable materials in life-science.

Lipophilic Red-Emitting Carbon Dots for Detecting and Tracking Lipid Droplets in Live Cells.

Lipid droplets (LDs), a dynamic organelle, are of vital importance in regulating the storage of neutral lipids and energy homeostasis. The aberrant expression of LDs is found to be highly associated with diverse metabolic diseases. Thus, detecting and monitoring LDs are essential to study the pathological and physiological processes of LDs in living bodies. However, it remains challenging to obtain suitable imaging probes to track LDs in vivo. Fortunately, the emergence of carbon dots (CDs), which are fluorescent nanomaterials with good biocompatibility and high stability, has provided us an unprecedented choice. In this work, CDs were synthesized via a solvothermal treatment of commercial reagents, 3-dimethylaminophenol. Interestingly, the prepared CDs show an intense red emission in non-hydrogen-bonding solution and have strong LD-targeting ability without any postmodification of ligands. Moreover, due to their low phototoxicity and excellent photostability, CDs were successfully applied to track the dynamics of LDs in live cells and image LDs in different cell lines and lipid-rich tissues. Overall, this work here proposed an LD-specific red-emitting CD probe, which will be of great value for learning more about LD-associated behaviors and diseases.

Label-Free Live-Cell Imaging of Internalized Microplastics and Cytoplasmic Organelles with Multicolor CARS Microscopy.

As the bioaccumulation of microplastics (MPs) is considered as a potential health risk, many efforts have been made to understand the cellular dynamics and cytotoxicity of MPs. Here, we demonstrate that label-free multicolor coherent anti-Stokes Raman scattering (CARS) microscopy enables separate vibrational imaging of internalized MPs and lipid droplets (LDs) with indistinguishable shapes and sizes in live cells. By simultaneously obtaining polystyrene (PS)- and lipid-specific CARS images at two very different frequencies, 1000 and 2850 cm-1, respectively, we successfully identify the local distribution of ingested PS beads and native LDs in Caenorhabditis elegans. We further show that the movements of PS beads and LDs in live cells can be separately tracked in real time, which allows us to characterize their individual intracellular dynamics. We thus anticipate that our multicolor CARS imaging method could be of great use to investigate the cellular transport and cytotoxicity of MPs without additional efforts for pre-labeling to MPs.

Orange/red dual-emissive boron- and nitrogen-codoped carbon dots for wash-free and selective staining of lipid droplets in live cells

Lipid droplet (LD) is an organelle that stores lipids and plays crucial roles in many metabolic processes and diseases. However, developing a method for long-term tracing of LDs and monitoring LD movement remains a challenge. Here, by using a solvent-free carbonization method, we prepare a kind of boron- and nitrogen-codoped carbon dots (BNCDs) from 3-aminophenylboronic acid. The as-synthesized BNCDs have an average size of ∼6.9 nm, long-wavelength fluorescence emission, and excitation wavelength-independent fluorescence emission, and can realize wash-free LD imaging without the introduction of LD-targeting ligands. Besides, the BNCDs have the characteristics of good biocompatibility, simple preparation, and low cost. More importantly, the BNCDs can effectively track the dynamic changes of LDs (including lipophagy) in live animal cells. This work shows that the developed BNCDs can be used as an ideal probe for nondestructively visualizing LDs, which may have implications for the future studies on LDs-related metabolism and diseases.

Photostable fluorescent probes based on multifunctional group substituted naphthalimide dyes for imaging of lipid droplets in live cells.

We designed and synthesized multifunctional group substituted naphthalimide (MFGNI) dyes by introducing glycine ethyl ester and azetidine on 1,8-naphthalimide. With different azetidine substituents, the emission of the MFGNI dyes was shifted from blue to green. These MFGNI dyes exhibited high photoluminescence quantum yields (61% to 85%) and large Stokes shifts (67 nm). The amides and hydroxyl groups improved the photostability of the MFGNI dyes. Due to the small molecular weight and lipophilic properties, these MFGNI dyes specifically stained lipid droplets in living cells.

Fluorescent probes based on multifunctional encapsulated perylene diimide dyes for imaging of lipid droplets in live cells.

Due to their strong hydrophobicity and the aggregation-caused quenching effect, the application of perylene diimide (PDI) dyes in biological and medicinal fields lags far behind that of other dyes. Based on a multifunctional encapsulation strategy, we prepared isopropylphenyl sulfone encapsulated PDI dyes (SFPDIs). The four hydrophilic sulfone groups on the bay position of the PDIs not only effectively inhibit the fluorescence quenching caused by π-aggregation but also endow the SFPDIs with good live-cell permeability. The six lipophilic isopropylphenyl groups encapsulate PDI emitters and confer SFPDIs with excellent lipid droplet-targeting ability. Furthermore, the strong electron-withdrawing sulfone groups give the PDIs excellent anti-oxidation ability.

D-π-A structure fluorophore: NIR emission, response to viscosity, detection cyanide and bioimaging of lipid droplets

Intracellular viscosity, an important microenvironment factor, is closely involved in various cell processes as well as diseases. On the other hand, cyanide is one of the most hazardous chemicals for human health and environments. However, a NIR fluorescent probe for both response to viscosity and detection of cyanide remains vacant. Herein, we reported a D-π-A structure fluorophore (named CTR) which exhibited NIR emission and fluorescent enhancement response to viscosity via the molecular rotor strategy. Furthermore, CTR displayed fluorescent and colorimetric response to cyanide. Notably, test strips stained with CTR were fabricated, which could serve as an efficient and suitable cyanide test kit. Moreover, CTR could selectively accumulate in lipid droplets and visualize the metabolism of lipid droplets in live cells. These findings would provide new avenue to design fluorescent probe for effective response to viscosity, detection of cyanide, and bioimaging of lipid droplets in live cells.

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.

Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.

Lipid droplets are dynamic subcellular organelles that participate in a range of physiological processes including metabolism, regulation and lipid storage. Their role in disease, such as cancer, where they are involved in metabolism and in chemoresistance, has emerged over recent years. Thus, the value of lipid droplets as diagnostic markers is increasingly apparent where number and size of droplets can be a useful prognostic. Although diverse in size, LDs are typically too small to be easily enumerated by conventional microscopy. The advent of super-resolution microscopy methods offers the prospect of detailed insights but there are currently no commercial STED probes suited to this task and STED, where this method has been used to study LDs it has relied on fixed samples. Here, we report a pyrene-based ceramide conjugate PyLa-C17Cer, that stains lipid droplets with exceptionally high precision in living cells and shows excellent performance in stimulated emission depletion microscopy. The parent compound PyLa comprises a pyrene carboxyl core appended with 3,4-dimethylaminophenyl. The resulting luminophore exhibits high fluorescent quantum yield, mega-Stokes shift and low cytotoxicity. From DFT calculations the Stokes shifted fluorescent state arises from a dimethylaminophenyl to pyrene charge-transfer transition. While the parent compound is cell permeable, it is relatively promiscuous, emitting from both protein and membranous structures within the living mammalian cell. However, on conjugation of C17 ceramide to the free carboxylic acid, the resulting PyLa-C17Cer, remains passively permeable to the cell membrane but targets lipid droplets within the cell through a temperature dependent mechanism, with high selectivity. Targeting was confirmed through colocalisation with the commercial lipid probe Nile Red. PyLa-C17Cer offers outstanding contrast of LDs both in fluorescence intensity and lifetime imaging due to its large Stokes shift and very weak emission from aqueous media. Moreover, because the compound is exceptionally photochemically stable with no detectable triplet emission under low temperature conditions, it can be used as an effective probe for fluorescence correlation spectroscopy (FCS). These versatile fluorophores are powerful multimodal probes for combined STED/FCS/lifetime studies of lipid droplets and domains in live cells.

Multifunctional fluorescent leucomalachite green derivatives for chemodosimetric detection of Fe3+, specific imaging of lipid droplets and intracellular pH monitoring

Fluorescent leucomalachite green derivatives detect micromolar concentrations of Fe3+, specifically stain lipid droplets in live cells and monitor intracellular pH variations in microorganisms.

Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore.

Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiological processes as well as diseases. Herein we synthesized a push-pull molecule named DAF (Dimethyl Aniline Furaldehyde) that possesses a strong positive solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.

Quinone-Derived π-Extended Phenazines as New Fluorogenic Probes for Live-Cell Imaging of Lipid Droplets.

We describe a new synthetic methodology for the preparation of fluorescent π-extended phenazines from the naturally-occurring naphthoquinone lapachol. These novel structures represent the first fluorogenic probes based on the phenazine scaffold for imaging of lipid droplets in live cells. Systematic characterization and analysis of the compounds in vitro and in cells led to the identification of key structural features responsible for the fluorescent behavior of quinone-derived π-extended phenazines. Furthermore, live-cell imaging experiments identified one compound (P1) as a marker for intracellular lipid droplets with minimal background and enhanced performance over the lipophilic tracker Nile Red.

Specific Two-Photon Imaging of Live Cellular and Deep-Tissue Lipid Droplets by Lipophilic AIEgens at Ultralow Concentration

Lipid droplets are highly associated with obesity, diabetes, inflammatory disorders, and cancer. A reliable two-photon dye for specific lipid droplets imaging in live cells and live tissues at ultralow concentration has rarely been reported. In this work, four new aggregation-induced emission luminogens (AIEgens) based on the naphthalene core were designed and synthesized for specific two-photon lipid droplet staining. The new molecules, namely, NAP AIEgens, exhibit large Stokes shift (>110 nm), high solid-state fluorescence quantum yield (up to 30%), good two-photon absorption cross section (45–100 GM at 860 nm), high biocompatibility, and good photostability. They could specifically stain lipid droplets at ultralow concentration (50 nM) in a short time of 15 min. Such ultralow concentration is the lowest value for lipid droplets staining in live cells reported so far. In vitro and ex vivo two-photon imaging of lipid droplets in live cells and live mice liver tissues were successfully demonstrated. In ad...

Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues.

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M-1 cm-1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.

Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes.

Lipid droplets (LDs) are subcellular organelles with important roles in lipid storage and metabolism and involved in various diseases including cancer, obesity, and diabetes. Conventional methods, however, have limited ability to provide quantitative information on individual LDs and have limited capability for three-dimensional (3-D) imaging of LDs in live cells especially for fast acquisition of 3-D dynamics. Here, we present an optical method based on 3-D quantitative phase imaging to measure the 3-D structural distribution and biochemical parameters (concentration and dry mass) of individual LDs in live cells without using exogenous labelling agents. The biochemical change of LDs under oleic acid treatment was quantitatively investigated, and 4-D tracking of the fast dynamics of LDs revealed the intracellular transport of LDs in live cells.