Methods and Applications in Fluorescence最新文献

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τ_r) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τ_r) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%,τ_rincreases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Photoluminescent perovskite nanocrystals are mostly used along with base materials such as polymers for material processing and large-scale production purpose. However, the role of polymer in crystal structure engineering and thereby dictating the emission properties of lead halide perovskite nanocrystals has been poorly understood. First, we have developed a polymer-directed antisolvent method for synthesis of halide perovskite crystals at room temperature and observed that the thermodynamic stabilities of crystals drive the formation of perovskite composite crystal of orthorhombic Cs₄PbBr₆and monoclinic CsPbBr₃. Surprisingly, hydrophobic polyvinylidene fluoride (PVDF) can reduce the size of perovskite crystals to nano dimensions even at room temperature. On the other hand, perovskite nanocrystals, CsPbBr₃synthesized by modified hot-injection method undergo rapid encapsulation in PVDF matrices. The size of the encapsulated nanocrystal in PVDF matrices ranges in 88 ± 32 nm. We have illustrated that there are three types of radiative recombination predominantly operative in nanocrystals-doped polymer- (i) surface defect caused radiative recombination (0.6-3 ns), (ii) exciton recombination (3-15 ns), and (iii) shallow trap assisted recombination (10-50 ns). The interface created at nanocrystal and polymer plays a decisive role in populating the shallow trap states in perovskite-polymer nanocomposite. These nanocomposites undergo fast halide exchange in aqueous hydroiodic acid solution and possess remarkable enhancement of water-/photo-stability. This research would pave way for their greater use in hydrogen production and light-emitting devices.

Correlative imaging methods can provide greater information for investigations of cellular ultra-structure, with separate analysis methods complementing each other's strengths and covering for deficiencies. Here we present a method for correlative applications of super resolution and atomic force microscopies, optimising the sample preparation for correlative imaging of the cellular cytoskeleton in COS-7 cells. This optimisation determined the order of permeabilisation and fixation, the concentration of Triton X-100 surfactant used and time required for sufficient removal of the cellular membrane while maintaining the microtubule network. Correlative SMLM/AFM imaging revealed the different information that can be obtained through each microscopy. The widths of microtubules and microtubule clusters were determined from both AFM height measurements and Gaussian fitting of SMLM intensity cross sections, these were then compared to determine the orientation of microtubules within larger microtubule bundles. The ordering of microtubules at intersections was determined from the AFM height profiles as each microtubule crosses the other. The combination of both microtubule diameter measurements enabled greater information on their structure to be found than either measurement could individually.

Optical temperature sensing is widely realized by using upconversion (UC) emission in lanthanide-doped phosphors. There are various parameters that are responsible for UC intensity of the phosphor like particle shape and size, type of symmetry that exist at the site position, distribution of lanthanide ions in the phosphor, and so on. However, a comparative study of the bulk and nanostructure on the temperature sensing ability of such phosphor is rare. In the present work, we have taken Ca_0.79Er_0.01Yb_0.2MoO₄phosphors as a model system and synthesized its bulk (via solid-state reaction method, named SCEY) and nanostructures (via solution combustion route, named CCEY). We further studied their phase, crystal structure, phonon frequency, optical excitation, and emission (upconversion & downshifting) properties. Finally, the optical temperature sensing behavior of SCEY and CCEY, in the range 305 K-573 K, have been compared. The maximum relative sensitivity of the phosphor SCEY and CCEY are 0.0061 K^-1at 305 K and 0.0094 K^-1at 299 K, respectively, while, the maximum absolute sensitivities are 0.0150 K^-1at 348 K, and 0.0170 K^-1at 398 K, respectively. We thus conclude that the temperature sensing ability of nanoparticle-based Ca_0.79Er_0.01Yb_0.2MoO₄phosphor is better compared to its bulk phosphor.

A simple, sensitive, and selective first derivative synchronous fluorimetric method was developed and optimized to track the influence of caffeine content in beverages on the pharmacokinetic parameters of three pharmaceuticals used in relieving headache namely, aspirin (ASP), ibuprofen (IBU), and ergotamine tartrate (ERG). A full validation procedure was carried out to impart validity to the proposed method to apply it to biological fluids. The unique dissolving power of micellar solutions was utilized to avoid multiple extraction steps for both thein vitroandin vivoexperiments, aiming to obtain acceptable recoveries and to accomplish sustainability, where 0.1 M sodium dodecyl sulphate (SDS) was used for this purpose. Moreover, the developed bioanalytical method was subjected to full validation to avoid interferences emerging from biological matrices. The greenness of the proposed method was assessed according to the Analytical Eco-Scale and proved to be excellent green carrying a score of 98%.

Creatinine (Crn) is an important excretory product of the human body. Medical laboratory technology has improved over years and brought many advancements in clinical diagnostics equipment, and testing techniques and made the tests more efficient. Yet, the quantitative analysis of Crn is still carried out by the classical Jaffe's reaction (using Picric acid (PA) with NaOH) method. Since PA is hazardous to human health, alternative solutions such as; nanoparticles and surface-modified nanoparticles can be used. Exploring the optoelectronic properties of carbon-based quantum dots for biomolecule sensing is of current interest among researchers. Nitrogen functionalized graphene quantum dots (Alk-NGQDs) measured featured Crn easier and reduced the time taken for the test carried out in laboratories. The synthesized Alk-NGQDs optical, structural, morphological properties, surface and compositions are studied through XPS, HRTEM, XRD, FTIR, and spectroscopic techniques. Alk-NGQDs at alkaline conditions (pH 9.5) form a stable complex with Crn through intermolecular charge transfer (ICT). The fluorescence titration method is used to sense Crn in commercial Crn samples and human blood serum. To understand the efficacy of sensing creatinine using Alk-NGQDs, working concentration, fluorescence quantum yield, the limit of detection, and quenching constant are calculated using the Stern-Volmer plot. The emission property of Alk-NGQDs is aimed to bring an alternative to the traditional colorimetric Jaffe's reaction.

The research in developing a single ingredient phosphor for white-light emission is progressively increasing. It is well known that the⁴F_9/2 → ⁶H_13/2(yellow) and⁴F_9/2 → ⁶H_15/2(blue) transitions of Dy³⁺ions give near-white light emission. The white light emission of Dy³⁺ions can be enhanced via improving the crystallinity of the host phosphor via co-doping of transition metal ions. In this paper, we report a significant improvement in the white light emission of Dy³⁺doped CaMoO₄by co-doping Zn²⁺ions. The x-ray diffraction pattern confirms the tetragonal phase of pure and doped CaMoO₄phosphor. The peak broadening and a red-shift in the absorption peak are observed by UV-vis absorption analysis of Zn²⁺/Dy³⁺doped CaMoO₄. From Photoluminescence studies, we have observed that in Dy³⁺doped CaMoO₄, the 4% Dy³⁺doped CaMoO₄exhibits maximum emission. The Zn²⁺ions are co-doped to further increase the luminescence intensity of CaMoO₄:4%Dy³⁺and the maximum luminescence is obtained for 0.25% Zn²⁺concentration. Two intense emission peaks centered at 484 nm and 574 nm related to transitions⁴F_9/2 → ⁶H_15/2and⁴F_9/2 → ⁶H_13/2of Dy³⁺ion are observed for Dy³⁺doped phosphor. The⁴F_9/2 → ⁶H_13/2transition is the forced electric dipole transition which is affected by its chemical environment. After Zn²⁺co-doping, the⁴F_9/2 → ⁶H_13/2transition is affected due to a change in asymmetricity around the Dy³⁺ions. The 0.25% co-doping of Zn²⁺gives 34% enhancement in luminescence emission of 4% Dy³⁺doped CaMoO₄. As a result, the CIE coordinates of chromaticity diagram and the color purity of the 0.25% Zn²⁺co-doped CaMoO₄:4Dy³⁺show improvement in the overall white light emission. We have shown that with Zn²⁺co-doping, the non-radiative relaxations are reduced which results in improved white light emission of Dy³⁺ions.

Creatinine level in biological fluids is a clinically relevant parameter to monitor vital functions and it is well assessed that measuring creatinine levels in the human body can be of great utility to evaluate renal, muscular, or thyroid dysfunctions. The accurate detection of creatinine levels may have a critical role in providing information on health status and represents a tool for the early diagnosis of severe pathologies. Among different methods for creatinine detection that have been introduced and that are evolving with increasing speed, fluorescence-based and colorimetric sensors represent one of the best alternatives, thanks to their affordability, sensitivity and easy readability. In this work, we demonstrate that the fluorescein-Au³⁺complex provides a rapid, selective, and sensitive tool for the quantification of creatinine concentrations in ranges typical of sweat and urine. UV-visible absorption, diffuse reflectance spectroscopy, steady state and time resolved fluorescence spectroscopy were used to shed light on the molecular mechanisms involved in the changes of optical properties, which underlie the multiplexed sensor analytical reply. Interestingly, sensing can be performed in solution or on solid nylon support accessing different physiological concentrations from micromolar to millimolar range. As a proof-of-concept, the nylon-based platform was used to demonstrate its effectiveness in creatinine detection on a solid and flexible substrate, showing its analytical colorimetric properties as an easy and disposable creatinine point-of-care test.

Thein vitropanel of technologies to address biomolecular interactions are in play, however microscale thermophoresis is continuously increasing in use to represent a key player in this arena. This review highlights the usefulness of microscale thermophoresis in the determination of molecular and biomolecular affinity interactions. This work reviews the literature from January 2016 to January 2022 about microscale thermophoresis. It gives a summarized overview about both the state-of the art and the development in the field of microscale thermophoresis. The principle of microscale thermophoresis is also described supported with self-created illustrations. Moreover, some recent advances are mentioned that showing application of the technique in investigating biomolecular interactions in different fields. Finally, advantages as well as drawbacks of the technique in comparison with other competing techniques are summarized.

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions.