Characterization of the livestock production system and potential for enhancing productivity through improved feeding in Amoni Division, Mweiga District, Central Kenya, May 2010

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

Fluorene Analogues of Prodan with Superior Fluorescence Brightness and Solvatochromism

In a search for environmentally sensitive (solvatochromic) dyes with superior properties, we extended the electronic conjugation of one of the best solvatochromic dyes, Prodan, by substituting its naphthalene core with fluorene. The newly synthesized fluorene derivatives bearing strong electron-donor (dialkylamino) and -acceptor (carbonyl) groups at the 2 and 7 positions showed red-shifted absorption (close to 400 nm), twice as large of a absorption coefficient (43 000 M⁻¹ cm⁻¹), and a manifold larger two-photon absorption cross section (∼400 GM) compared to Prodan. Studies in solvents revealed much stronger fluorescence solvatochromism of the new dyes, which is connected with their twice as large transition dipole moment (14.0 D). Similarly to Prodan, they exhibit high fluorescence quantum yields, while their photostability is largely improved. Thus, substitution of the naphthalene core in Prodan with fluorene resulted in new fluorophores with superior spectroscopic and solvatochromic properties. We expect them to find a variety of applications as environmentally sensitive probes and labels in biology

Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles

Ultrasmall polymer nanoparticles are rapidly gaining importance as nanocarriers for drugs and contrast agents. Here, a straightforward modular approach to efficiently loaded and stable sub-20-nm polymer particles is developed. In order to obtain ultrasmall polymer nanoparticles, we investigated the influence of one to two charged groups per polymer chain on the size of particles obtained by nanoprecipitation. Negatively charged carboxylate and sulfonate or positively charged trimethylammonium groups were introduced into the polymers poly(d,l-lactide-<i>co</i>-glycolide) (PLGA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA). According to dynamic light scattering, atomic force and electron microscopy, the presence of one to two charged groups per polymer chain can strongly reduce the size of polymer nanoparticles made by nanoprecipitation. The particle size can be further decreased to less than 15 nm by decreasing the concentration of polymer in the solvent used for nanoprecipitation. We then show that even very small nanocarriers of 15 nm size preserve the capacity to encapsulate large amounts of ionic dyes with bulky counterions at efficiencies >90%, which generates polymer nanoparticles 10-fold brighter than quantum dots of the same size. Postmodification of their surface with the PEG containing amphiphiles Tween 80 and pluronic F-127 led to particles that were stable under physiological conditions and in the presence of 10% fetal bovine serum. This modular route could become a general method for the preparation of ultrasmall polymer nanoparticles as nanocarriers of contrast agents and drugs

Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix

Fluorescent nanoparticles (NPs) help to increase spatial and temporal resolution in bioimaging. Advanced microscopy techniques require very bright NPs that exhibit either stable emission for single-particle tracking or complete on/off switching (blinking) for super-resolution imaging. Here, ultrabright dye-loaded polymer NPs with controlled switching properties are developed. To this aim, the salt of a dye (rhodamine B octadecyl ester) with a hydrophobic counterion (fluorinated tetraphenylborate) is encapsulated at very high concentrations up to 30 wt % in NPs made of poly­(lactic-<i>co</i>-glycolic acid) (PLGA), poly­(methyl methacrylate) (PMMA), and polycaprolactone (PCL) through nanoprecipitation. The obtained 35 nm NPs are nearly 100 times brighter than quantum dots. The nature of the polymer is found to define the collective behavior of the encapsulated dyes so that NPs containing thousands of dyes exhibit either whole particle blinking, for PLGA, or stable emission, for PMMA and PCL. Fluorescence anisotropy measurements together with small-angle X-ray scattering experiments suggest that in less hydrophobic PLGA, dyes tend to cluster, whereas in more hydrophobic PMMA and PCL, dyes are dispersed within the matrix, thus altering the switching behavior of NPs. Experiments using a perylene diimide derivative show a similar effect of the polymer nature. The resulting fluorescent NPs are suitable for a wide range of imaging applications from tracking to super-resolution imaging. The findings on the organization of the load innside NPs will have impact on the development of materials for applications ranging from photovoltaics to drug delivery

Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix

Fluorescent nanoparticles (NPs) help to increase spatial and temporal resolution in bioimaging. Advanced microscopy techniques require very bright NPs that exhibit either stable emission for single-particle tracking or complete on/off switching (blinking) for super-resolution imaging. Here, ultrabright dye-loaded polymer NPs with controlled switching properties are developed. To this aim, the salt of a dye (rhodamine B octadecyl ester) with a hydrophobic counterion (fluorinated tetraphenylborate) is encapsulated at very high concentrations up to 30 wt % in NPs made of poly­(lactic-<i>co</i>-glycolic acid) (PLGA), poly­(methyl methacrylate) (PMMA), and polycaprolactone (PCL) through nanoprecipitation. The obtained 35 nm NPs are nearly 100 times brighter than quantum dots. The nature of the polymer is found to define the collective behavior of the encapsulated dyes so that NPs containing thousands of dyes exhibit either whole particle blinking, for PLGA, or stable emission, for PMMA and PCL. Fluorescence anisotropy measurements together with small-angle X-ray scattering experiments suggest that in less hydrophobic PLGA, dyes tend to cluster, whereas in more hydrophobic PMMA and PCL, dyes are dispersed within the matrix, thus altering the switching behavior of NPs. Experiments using a perylene diimide derivative show a similar effect of the polymer nature. The resulting fluorescent NPs are suitable for a wide range of imaging applications from tracking to super-resolution imaging. The findings on the organization of the load innside NPs will have impact on the development of materials for applications ranging from photovoltaics to drug delivery

Exploiting Fast Exciton Diffusion in Dye-Doped Polymer Nanoparticles to Engineer Efficient Photoswitching

Photoswitching of bright fluorescent nanoparticles opens new possibilities for bioimaging with superior temporal and spatial resolution. However, efficient photoswitching of nanoparticles is hard to achieve using Förster resonance energy transfer (FRET) to a photochromic dye, because the particle size is usually larger than the Förster radius. Here, we propose to exploit the exciton diffusion within the FRET donor dyes to boost photoswitching efficiency in dye-doped polymer nanoparticles. To this end, we utilized bulky hydrophobic counterions that prevent self-quenching and favor communication of octadecyl rhodamine B dyes inside a polymer matrix of poly­(d,l-lactide-<i>co</i>-glycolide). Among tested counterions, only perfluorinated tetraphenylborate that favors the exciton diffusion enables high photoswitching efficiency (on/off ratio ∼20). The switching improves with donor dye loading and requires only 0.1–0.3 wt % of a diphenylethene photochromic dye. Our nanoparticles were validated both in solution and at the single-particle level. The proposed concept paves the way to new efficient photoswitchable nanomaterials

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

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

Quantification of Local Hydration at the Surface of Biomolecules Using Dual-Fluorescence Labels

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H₂O]_L (or partial volume of water: <i>W</i>_A = [H₂O]_L/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, <i>N</i>*/<i>T</i>*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log­(<i>N</i>*/<i>T</i>*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W_A values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that <i>W</i>_A values for the label at the peptide N-terminus are lower (0.63–0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (<i>W</i>_A = 0–0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure

Turn-on Fluorene Push–Pull Probes with High Brightness and Photostability for Visualizing Lipid Order in Biomembranes

The rational design of environmentally sensitive dyes with superior properties is critical for elucidating the fundamental biological processes and understanding the biophysical behavior of cell membranes. In this study, a novel group of fluorene-based push–pull probes was developed for imaging membrane lipids. The design of these fluorogenic conjugates is based on a propioloyl linker to preserve the required spectroscopic features of the core dye. This versatile linker allowed the introduction of a polar deoxyribosyl head, a lipophilic chain, and an amphiphilic/anchoring group to tune the cell membrane binding and internalization. It was found that the deoxyribosyl head favored cell internalization and staining of intracellular membranes, whereas an amphiphilic anchor group ensured specific plasma membrane staining. The optimized fluorene probes presented a set of improvements as compared to commonly used environmentally sensitive membrane probe Laurdan such as red-shifted absorption matching the 405 nm diode laser excitation, a blue-green emission range complementary to the red fluorescent proteins, enhanced brightness and photostability, as well as preserved sensitivity to lipid order, as shown in model membranes and living cells