Label-free characterisation of amyloids and alpha-Synuclein polymorphs by exploiting their intrinsic fluorescence property

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime represented by model-free phasor plots, as a label-free assay to characterise amyloid structure. Intrinsic amyloid fluorescence arises from structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids (i.e., α-Synuclein (αS), β-Lactoglobulin and TasA) and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of pre-formed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct to those of their fibrillar counterpart. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathology), and for testing small molecule inhibitory compounds.

Energy Metabolism and Intracellular pH Alteration in Neural Spheroids Carrying Down syndrome

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.

Evaluating the impact of doping concentration on the performance of in-band pumped thulium-doped fiber amplifiers

This study is to investigate the impact of thulium concentration on the performance of in-band pumped thulium-doped silica fiber amplifiers with considering ion–ion interactions. Due to the fluorescence quenching in silica glass, the fluorescence lifetimes are required to compute at every Tm concentration. The theoretical model of fluorescence decay curves at in-band pumped thulium-doped silica fiber is used to determine the fluorescence lifetimes of the 3F4 and 3H4 levels. The calculated lifetimes of the commercially available thulium-doped silica fiber are 650 µs for 3F4 level and 14 µs for 3H4 level and these results are consentient well with the experimental reported results. The theoretical evaluating of the amplifier performance shows that the gain amplifier reduces with concentration increase because of the impact of both fluorescence quenching and the reverse cross-relaxation process. Thus, in contrast to pumping wavelength at 790 nm, there are negative effects of the high doping concentration of Tm ions on the amplifier performance at the in-band pumping scheme.

Utilizing selective chlorination to synthesize new triangulenium dyes

Access to functionalization of new sites on the triangulenium core structure has been achieved at an early stage by chlorination with N-chlorosuccinimide (NCS), giving rise to two new triangulenium dyes (1 and 2). By introducing the chlorine functionalities in the acridinium precursor, positions complementary to those previously accessed by electrophilic aromatic substitution of the final dyes can be accesed. The chlorination is selective, giving only one regioisomer for both mono- and dichlorination products. For the monochlorinated acridinium compound a highly selective ring-closing reaction was discovered to generate only a single regioisomer of the cationic [4]helicene product. This discovery aspired further investigations into the mechanism of [4]helicene formation and to the first isolation of the previously proposed intermediate of the two-step SNAr reaction, key to all aza-bridged triangulenium and helicenium systems. A late stage functionalization of DAOTA+ with NCS gave rise to a different dichlorinated compound (2). The fully ring closed chlorinated triangulenium dyes 1, 2 and 3 show a redshift in absorption and emission relative to the non-chlorinated analogues, while still maintaining relatively high fluorescence quantum yields of 36%, 26%, and 41%, and long fluorescence lifetimes of 15 ns, 12.5 ns and 16 ns, respectively. Cyclic voltammetry shows that chlorination of the triangulenium dyes significantly lowers reduction potentials and thus allows for efficient tuning of redox and photo-redox properties.

Fluorescence signatures of SARS CoV-2 spike S1 proteins and an human ACE-2: excitation-emission maps and fluorescence lifetimes

Fast and reliable detection of infectious virus loads of the SARS CoV-2 virus is still an important issue even after more than one year of the pandemic's outbreak. The spike protein's S1 subunit (including its receptor-binding domain) and human angiotensin-converting enzyme 2 (hACE2) are known as key players in the reproduction mechanism of the SARS CoV-2 virus. Because of its high sensitivity and simple application, fluorescence spectroscopy is promising to meet the sensitivity requirements for a virus detection in a clean environment. In such highly complex protein systems, a comprehensive knowledge of fluorescence data is presumed in order to evaluate the specificity of the spectra with respect to a possible detection. This includes full featured information on the fluorescence process, i. e. wavelength and time-dependent data. In this work, fluorescence spectral excitation-emission maps of the involved proteins are presented, namely the S1 part of the spike protein and its receptor-binding domain as well as the hACE2 enzyme. In addition, measurements of fluorescence lifetimes of the proteins are presented and analyzed by a bi-exponential kinetic approach.

Low-temperature and low-pressure effective fluorescence lifetimes and spectra of gaseous anisole and toluene

Fluorescence spectra and lifetimes of anisole and toluene vapor in nitrogen have been measured at conditions below ambient (257–293 K and 100–2000 mbar) upon excitation with 266-nm laser light to expand the applicable range of anisole and toluene laser-induced fluorescence (LIF) for conditions below room temperature that occur in expanding flows and cases with strong evaporative cooling. Anisole fluorescence spectra broaden with decreasing pressure while fluorescence lifetimes decrease simultaneously. This is consistent with a more pronounced effect of internal vibrational redistribution on the overall fluorescence signal and can be explained by significantly reduced collision rates. In the case of toluene, the transition from photo-induced heating to photo-induced cooling was observed for the first time for 266 nm. The data confirm predictions of earlier work and is particularly important for the advancement of the available photo-physical (step-ladder) models: since those transitions mark points where the molecules are already thermalized after excitation (i.e., no vibrational relaxation occurs during deactivation), they are important support points for fitting empirical parameters and allow analytical determination of the ground state energy transferred to the excited state. The data enable temperature and/or pressure sensing, e.g., in accelerating cold flows using laser-induced fluorescence of both tracers.

Exfoliation and Optical Properties of Near-Infrared Fluorescent Silicate Nanosheets

<div><div><div><p>The silicates Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP) emit in bulk bright and stable fluorescence in the near-infrared (NIR), which is of high interest for (bio)photonics due to minimal scattering, absorption and phototoxicity in this spectral range. So far the optical properties of nanosheets (NS) of these silicates are poorly understood. Here, we exfoliate them into nanosheets and report their physicochemical properties. The approach uses ball milling followed by tip sonication and centrifugation steps to exfoliate the silicates into NS with a lateral size ≈ 16-27 nm and thickness ≈ 1-4 nm. They emit at ≈ 927 nm (EB-NS), 953 nm (HB-NS) and 924 nm (HP-NS) and single NS can be resolved in the NIR. Fluorescence lifetimes decrease from ≈ 30-100 μs (bulk) to 17 μs (EB- NS), 8 μs (HB-NS) and 7 μs (HP-NS). NS of different composition/size can be imaged by fluorescence lifetime imaging, which enables lifetime-encoded multicolor imaging both on the microscopic and the macroscopic scale. Finally, remote imaging through tissue phantoms reveals the potential for bioimaging. In summary, we report a procedure to gain NIR fluorescent silicate nanosheets, characterize their photophysical properties and show their potential for NIR photonics.</p></div></div></div>