Optimizing the Fluorescence Properties of Nanoemulsions for Single Particle Tracking in Live Cells.

Nanoemulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly(ethylene glycol), through the use of nonionic surfactants, confers high stealth in biological medium with reduced nonspecific interactions. Tracking individual NE by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new dioxaborine barbituryl styryl (DBS) dyes that displayed high molar extinction coefficients (up to 120 000 M-1 cm-1) with relatively low quantum yields in solvents and impressive fluorescence enhancement when dissolved in viscous oils (up to 0.98). The reported screening of nine different oils allowed disclosing a range of efficient "oil/dye" couples and understanding the main parameters that lead to the brightest NEs. We determine vitamin E acetate/DBS-C8 as the representative most efficient couple, combining high dye loading capabilities and low aggregation-induced quenching, leading to <50 nm ultrabright NEs (with brightness as high as 30 × 106 M-1 cm-1) with negligible dye leakage in biological media. Beyond a comprehensive optical and physicochemical characterization of fluorescent NEs, cellular two-photon excitation imaging was performed with polymer-coated cell penetrating NEs. Thanks to their impressive brightness and photostability, NEs displaying different charge surfaces were microinjected in HeLa cells and were individually tracked in the cytosol to study their relative velocity.

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.

Recent Advances in Fluorescent Probes for Lipid Droplets.

Lipid droplets (LDs) are organelles that serve as the storage of intracellular neutral lipids. LDs regulate many physiological processes. They recently attracted attention after extensive studies showed their involvement in metabolic disorders and diseases such as obesity, diabetes, and cancer. Therefore, it is of the highest importance to have reliable imaging tools. In this review, we focus on recent advances in the development of selective fluorescent probes for LDs. Their photophysical properties are described, and their advantages and drawbacks in fluorescence imaging are discussed. At last, we review the reported applications using these probes including two-photon excitation, in vivo and tissue imaging, as well as LDs tracking.

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.