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Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

Recently, the up-converting (UC) materials, containing lanthanide ions (Ln³⁺)have attracted considerable attention because of the multitude of their potential applications. The most frequently investigated are UC systems based on the absorption of near-infrared (NIR) radiation by Yb³⁺ ions at around 975–980 nm and emission of co-dopants, usually Ho³⁺, Er³⁺ or Tm³⁺ ions. UC can be observed also upon excitation with irradiation with a wavelength different than around 980 nm. The most often studied systems capable of UC without the use of Yb³⁺ ion are those based on the properties of Er³⁺ ions, which show luminescence resulting from the excitation at 808 or 1532 nm. However, also other Ln³⁺ ions are worth attention. Herein, we focus on the investigation of the UC phenomenon in the materials doped with Ho³⁺ ions, which reveal unique optical properties upon the NIR irradiation. The SrF₂ NPs doped with Ho³⁺ ions in concentrations from 4.9% to 22.5%, were synthesized by using the hydrothermal method. The structural and optical characteristics of the obtained SrF₂:Ho³⁺ NPs are presented. The prepared samples had crystalline structure, were built of NPs of round shapes and their sizes ranged from 16.4 to 82.3 nm. The NPs formed stable colloids in water. Under 1156 nm excitation, SrF₂:Ho³⁺ NPs showed intense UC emission, wherein the brightest luminescence was recorded for the SrF₂:10.0%Ho³⁺ compound. The analysis of the measured lifetime profiles and dependencies of the integral luminescence intensities on the laser energy allowed proposing the mechanism, responsible for the observed UC emission. It is worth mentioning that the described SrF₂:Ho³⁺ samples are one of the first materials for which the UC luminescence induced by 1156 nm excitation was obtained.

Since the intracellular pH plays an important role in the physiological and pathological processes, however, the probes that can be used for monitoring pH fluctuation under extreme acidic conditions are currently rare, so it is necessary to construct fluorescent probes for sensing pH less than 4. In this work, we developed a new near-infrared (NIR) fluorescent probe Cy-SNN for sensing pH fluctuation under extremely acidic conditions. For the preparation of this probe, benzothiozolium moiety was chosen as lysosomal targeting unit and NIR fluorophore, and barbituric acid moiety was fused in the polymethine chain of probe to introduce protonation center. Surprisingly, on the basis of the balance of quaternary ammonium salts and free amines, the pk_a value of Cy-SNN was calculated as low as 2.96, implying that Cy-SNN can be used in acidic conditions with pH < 4. Moreover, Cy-SNN exhibited highly selective response to H⁺ over diverse analytes in real-time with dependable reversibility. Importantly, Cy-SNN can be used to specifically target lysosome, providing potential tools for monitoring the function of lysosome in autophagy process.

A near-infrared (NIR) light-triggered release method for nitric oxide (NO) was developed utilizing core/shell NaYF₄: Tm/Yb/Ca@NaGdF₄: Nd/Yb up-conversion nanoparticles (UCNPs) bearing a mesoporous silica (mSiO₂) shell loaded with the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP). To avoid overheating in biological samples, Nd³⁺ was chosen as a sensitizer, Yb³⁺ ions as the bridging sensitizer, and Tm³⁺ ions as UV-emissive activator while co-doping with Ca²⁺ was done to enhance the luminescence of the activator Tm³⁺. NO release from SNAP was triggered by an NIR-UV up-conversion process, initiated by 808 nm light absorbed by the Nd³⁺ ions. NO release was confirmed by the Griess method. Under 808 nm irradiation, the viability of the liver cancer cell line HepG2 significantly decreased with increasing UCNPs@mSiO₂-SNAP concentration. For a UCNPs@mSiO₂-SNAP concentration of 200 μg ml⁻¹, the cell survival probability was 47%. These results demonstrate that UCNPs@mSiO₂-SNAP can induce the release of apoptosis-inducing NO by NIR irradiation.

Anisotropic rare earth ion (RE³⁺) doped fluoride upconversion particles are emerging as potential candidate in diverse areas, ranging from biomedical imaging to photonics. Here, we develop a facile strategy to synthesize NaYF₄: Yb, Gd, Er, and NaYF₄: Yb, Gd, Tm upconversion nanorods via microwave synthesis route by controlling the synthesis time and compared the optical properties similar nanorods prepared via solvothermal technique. With the increase in synthesis time, the phase of the particle found to change from mixed phase to purely hexagonal and morphology of the particles change mixed phase of spherical and rod-shaped particles to completely nanorods for a synthesis time of 60 min. Further, the intrinsically hydrophobic particles changed to hydrophilic by removal of oleic capping via acid treatment and the amine functionalized silica coating. The upconversion luminescence as well as laser power dependent emission properties of the surface modified particles elucidate that surface modification route influence the upconversion luminescence as well as solvent dependent emission properties. Moreover, the laser power dependent studies elucidate that the upconversion process in a multi-photon process.

Upconversion materials have several advantages for many applications due to their great potential in converting infrared light to visible. For practical use, it is necessary to achieve high intensity of UC luminescence, so the studies of the optimal synthesis parameters for upconversion nanoparticles are still going on. In the present work, we analyzed the synthesis temperature effect on the efficiency and luminescence decay of β-NaGd_0.78Yb_0.20Er_0.02F₄ (15–25 nm) upconversion nanoparticles with hexagonal crystal structure synthesized by anhydrous solvothermal technique. The synthesis temperature was varied in the 290 °C–320 °C range. The synthesis temperature was shown to have a significant influence on the upconversion luminescence efficiency and decay time. The coherent scattering domain linearly depended on the synthesis temperature and was in the range 13.1–22.3 nm, while the efficiency of the upconversion luminescence increases exponentially from 0.02 to 0.10% under 1 W cm⁻² excitation. For a fundamental analysis of the reasons for the upconversion luminescence intensity dependence on the synthesis temperature, it was proposed to use the maximum entropy method for luminescence decay kinetics processing. This method does not require a preliminary setting of the number of exponents and, due to this, makes it possible to estimate additional components in the luminescence decay kinetics, which are attributed to different populations of rare-earth ions in different conditions. Two components in the green luminescence and one component in the red luminescence decay kinetics were revealed for nanoparticles prepared at 290 °C–300 °C. An intense short and a weak long component in green luminescence decay kinetics could be associated with two different populations of ions in the surface quenching layer and the crystal core volume. With an increase in the synthesis temperature, the second component disappears, and the decay time increases due to an increase in the number of ions in the crystal core volume and a more uniform distribution of dopants.

We studied room temperature phosphorescence of tryptophan (TRP) embedded in poly (vinyl alcohol) films. With UV (285 nm) excitation, the phosphorescence spectrum of tryptophan appears at about 460 nm. We also observed the TRP phosphorescence with blue light excitation at 410 nm, well outside of the S₀ →S₁ absorption. This excitation reaches the triplet state of tryptophan directly without the involvement of the singlet excited state. The phosphorescence lifetime of tryptophan is in the sub-millisecond range. The long-wavelength direct excitation to the triplet state results in high phosphorescence anisotropy which can be useful in macromolecule dynamics study via time-resolved phosphorescence.

Quantum dots (QDs) have stood out as nanotools for glycobiology due to their photostability and ability to be combined with lectins. Mannose-binding lectin (MBL) is involved in the innate immune system and plays important roles in the activation of the complement cascade, opsonization, and elimination of apoptotic and microbial cells. Herein, adsorption and covalent coupling strategies were evaluated to conjugate QDs to a recombinant human MBL (rhMBL). The most efficient nanoprobe was selected by evaluating the conjugate ability to label Candida albicans yeasts by flow cytometry. The QDs-rhMBL conjugate obtained by adsorption at pH 6.0 was the most efficient, labeling ca. 100% of cells with the highest median fluorescence intensity. The conjugation was also supported by Fourier transform infrared spectroscopy, zeta potential, and size analyses. C. albicans labeling was calcium-dependent; 12% and <1% of cells were labeled in buffers without calcium and containing EDTA, respectively. The conjugate promoted specific labeling (based on cluster effect) since, after inhibition with mannan, there was a reduction of 80% in cell labeling, which did not occur with methyl-α-D-mannopyranoside monosaccharide. Conjugates maintained colloidal stability, bright fluorescence, and biological activity for at least 8 months. Therefore, QDs-rhMBL conjugates are promising nanotools to elucidate the roles of MBL in biological processes.

The use of phasors to analyze fluorescence data was first introduced for time-resolved studies for a simpler mathematical analysis of the fluorescence-decay curves. Recently, this approach was extended to steady-state experiments with the introduction of the spectral phasors (SP), derived from the Fourier transform of the fluorescence emission spectrum. In this work, we revise key mathematical aspects that lead to an interpretation of SP as the characteristic function of a probability distribution. This formalism allows us to introduce a new tool, called multi-dimensional spectral phasor (MdSP) that seize, not only the information from the emission spectrum, but from the full excitation-emission matrix (EEM). In addition, we developed a homemade open-source Java software to facilitate the MdSP data processing. Due to this mathematical conceptualization, we settled a mechanism for the use of MdSP as a tool to tackle spectral signal unmixing problems in a more accurate way than SP. As a proof of principle, with the use of MdSP we approach two important biophysical questions: protein conformational changes and protein-ligand interactions. Specifically, we experimentally measure the EEM changes upon denaturation of human serum albumin (HSA) or during its association with the fluorescence dye 1,8-anilinonaphtalene sulphate (ANS) detected via tryptophan-ANS Förster Resonance Energy Transfer (FRET). In this sense, MdSP allows us to obtain information of the system in a simpler and finer way than the traditional SP. Specifically, understanding a protein's EEM as a molecular fingerprint opens new doors for the use of MdSP as a tool to analyze and comprehend protein conformational changes and interactions.

Semiconductor quantum dots (QDs) have significant advantages over more traditional fluorophores used in fluorescence microscopy including reduced photobleaching, long-term photostability and high quantum yields, but due to limitations in light sources and optics, are often excited far from their optimum excitation wavelengths in the deep-UV. Here, we present a quantitative comparison of the excitation of semiconductor QDs at a wavelength of 280 nm, compared to the longer wavelength of 365 nm, within a cellular environment. We report increased fluorescence intensity and enhanced image quality when using 280 nm excitation compared to 365 nm excitation for cell imaging across multiple datasets, with a highest average fluorescence intensity increase of 3.59-fold. We also find no significant photobleaching of QDs associated with 280 nm excitation and find that on average, ∼80% of cells can tolerate exposure to high-intensity 280 nm irradiation over a 6-hour period.