Abstract
Solvatochromic dyes have emerged as a new class of fluorescent probes in the field of lipid membranes due to their ability to identify the lipid organization of biomembranes in live cells by ...changing the colour of their fluorescence. This type of solvatochromic function is useful for studying the heterogeneous features of biomembranes caused by the uneven distribution of lipids and cholesterols in live cells and related cellular processes. Therefore, a variety of advanced solvatochromic dyes have been rapidly developed over the last decade. To provide an overview of the works recently developed solvatochromic dyes have enabled, we herein present some solvatochromic dyes, with a particular focus on those based on pyrene and Nile red. As these dyes exhibit preferable photophysical properties in terms of fluorescence microscopy applications and unique distribution/localization in cellular compartments, some have already found applications in cell biological and biophysical studies. The goal of this review is to provide information to researchers who have never used solvatochromic dyes or who have not discovered applications of such dyes in biological studies.
Graphical Abstract
Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the ...emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.
Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, ...photostability, lack of a convenient spectral range, and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in its push–pull structure raises concerns about its potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push–pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ∼100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400 nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissues–lipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space, and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.
To replace molecular biological and immunological methods, biosensors have recently been developed for the rapid and sensitive detection of bacteria. Among a wide variety of biological materials, ...bacteriophages have received increasing attention as promising alternatives to antibodies in biosensor applications. Thus, we herein present a rapid and highly selective detection method for pathogenic bacteria, which combines dark-field light scattering imaging with a plasmonic biosensor system. The plasmonic biosensor system employs bacteriophages as the biorecognition element and the aggregation-induced light scattering signal of gold nanoparticle-assembled silica nanospheres as a signal transducer. Using Staphylococcus aureus strain SA27 as a model analyte, we demonstrated that the plasmonic biosensor system detects S. aureus in the presence of excess Escherichia coli in a highly selective manner. After the sample and the S. aureus phage S13′-conjugated plasmon scattering probe were mixed, S. aureus detection was completed within 15–20 min with a detection limit of 8 × 104 colony forming units per milliliter.
Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that ...insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.
High‐speed two‐photon microscopy can be used to analyze vascular dynamics in living animals and is essential for the understanding of brain diseases. Recent advances in fluorescent probes/optical ...systems have allowed successful imaging of the hippocampal vasculature in the deep brain of mice (1 mm from the brain surface) under low‐speed conditions (1–2 fps); however, using high‐speed techniques (>30 fps), observation of the deep‐brain vasculature is still challenging. Here, a new nanoemulsion that encapsulates thousands of red‐emissive pyrene dye molecules while maintaining their high two‐photon brightness 1.5 × 102 GM (GM = 10−50 cm4·s·photon−1·molecule−1) at 960 nm excitation and delivers a large amount of such pyrene dyes (65 nmol) into the blood vessels of mice is developed. Remarkably, the nanoprobe is found to exploit the inherent performance of a commonly used Ti:sapphire excitation laser and a sensitive gallium arsenide phosphide nondescanned fluorescence detector to the limit, enabling visualization of the brain vasculature under the cortex region of mice (up to 1.5 mm) under very low‐speed conditions. As a highlight, such a nanoprobe is successfully used to directly observe the blood flow in the hippocampal CA1 region (1.1 mm) through high‐speed resonant scanning (120 fps).
A novel dye‐loaded nanoemulsion is developed for high‐speed two‐photon microscopic imaging of the deep‐brain vasculature in mice. The nanoprobe delivers numerous bright pyrene dyes into the blood vessels of mice, fully exploiting the performance of a Ti:sapphire laser and a gallium arsenide phosphide detector. This approach allows the visualization of hippocampal blood flow in mice at above the video rate.
Currently, drug‐delivery strategies using nanocarriers (NCs) deal with encapsulation of cargo or its covalently modified prodrug. Herein, we propose a concept of reversible pH‐controlled capture and ...delivery of active cargo based on dynamic covalent chemistry inside lipid nano‐droplets (nanoemulsions), coined as “drug sponge”. We designed a highly lipophilic hydrazide (LipoHD) capable of reacting with a free cargo‐ketone (fluorescent dye and doxorubicin drug) directly inside lipid NCs, yielding a lipophilic hydrazone prodrug efficiently captured in the oil core. LipoHD‐loaded NCs spontaneously accumulated cargo‐ketones, yielding formulations stable against cargo leakage at pH 7.4, and further released their dye/drug cargo at low pH range (5.0–6.8) in solution and live cells. Doxorubicin‐loaded drug‐sponge NCs showed cytotoxicity in four cancer cell lines and capacity to inhibit tumor growth in subcutaneous xenografts of mice. Finally, unprecedented extraction of dye/drug cargos directly from cells and tissues (i.e. detoxification) was realized by the drug‐sponge NCs.
A drug‐sponge nanocarrier that reversibly captures and releases drugs or contrast agents under pH control is developed using dynamic covalent chemistry. Inside this lipid droplet nano‐reactor, lipophilic capture molecules transform free cargo‐ketones into encapsulated hydrazone prodrugs, enabling the controlled release of active cargos and also their extraction from cells and tissues for detoxification.
Sweat is an essential protection system for the body, but its failure can result in pathologic conditions, including several skin diseases, such as palmoplantar pustulosis (PPP). As reduced ...intraepidermal E-cadherin expression in skin lesions was confirmed in PPP skin lesions, a role for interleukin (IL)-1-rich sweat in PPP has been proposed, and IL-1 has been implicated in the altered E-cadherin expression observed in both cultured keratinocytes and mice epidermis. For further investigation, live imaging of sweat perspiration on a mouse toe-pad under two-photon excitation microscopy was performed using a novel fluorescent dye cocktail (which we named JSAC). Finally, intraepidermal vesicle formation which is the main cause of PPP pathogenesis was successfully induced using our "LASER-snipe" technique with JSAC. "LASER-snipe" is a type of laser ablation technique that uses two-photon absorption of fluorescent material to destroy a few acrosyringium cells at a pinpoint location in three-dimensional space of living tissue to cause eccrine sweat leakage. These observatory techniques and this mouse model may be useful not only in live imaging for physiological phenomena in vivo such as PPP pathomechanism investigation, but also for the field of functional physiological morphology.
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