This study demonstrates the capability of Raman microscopy for detecting structural differences in Giardia cells exposed to different drugs and incubation times. While metronidazole (MTZ) visibly affects the cells by inducing extracellular vesicle releases of toxic iron intermediates and modified triple-bond moieties, oseltamivir (OSM) alters the phenylalanine and lipid structures. Modifications in the heme protein environment and the transformation of iron from ferric to ferrous observed for both drug treatments are more notable for MTZ. Different contents and amounts of vesicle excretion are detected for 24 h or 48 h with MTZ incubation. At a shorter drug exposure, releases of altered proteins, glycogen, and phospholipids dominate. Agglomerates of transformed iron complexes from heme proteins and multiple-bond moieties prevail at 48 h of treatment. No such vesicle releases are present in the case of OSM usage. Drug incorporations into the cells and their impact on the plasma membrane and the dynamics of lipid raft confirmed by confocal fluorescence microscopy reveal a more destructive extent by OSM, corroborating the Raman results. Raman microscopy provides a broader understanding of the multifaceted factors and mechanisms responsible for giardiasis treatment or drug resistance by enabling a label-free, simultaneous monitoring of structural changes at the cellular and molecular levels.

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, a closed-loop optoelectronic control system is developed that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical processes or control the activation of photoswitchable inhibitors at desired targets. This technology is fully compatible with laser scanning confocal fluorescence microscopes. The authors demonstrate selective interactions of a 405 nm laser with targeted organelles and simultaneous monitoring of cell responses by fluorescent protein signals. Notably, blue laser interaction with the endoplasmic reticulum leads to a more pronounced reduction in cytosolic green fluorescent protein signals in comparison to that with nuclei and lipid droplets. Moreover, when combined with a photoswitchable inhibitor, microtubule polymerization is selectively inhibited within the subcellular compartments. This technology enables subcellular spatiotemporal optical manipulation over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.

Life on the molecular scale is based on a versatile interplay of biomolecules, a feature that is relevant for the formation of macromolecular complexes. Fluorescence-based two-color coincidence detection is widely used to characterize molecular binding and was recently improved by a brightness-gated version which gives more accurate results. We developed and established protocols which make use of coincidence detection to quantify binding fractions between interaction partners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsically related to single-molecule detection, the concentration of diffusing molecules for confocal detection is typically in the low picomolar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliability of our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity at different temperatures, we were able to determine the thermodynamic parameters of the binding interaction. The results show that the ultra-tight binding is dominated by entropic contributions.

The sole determination of volumetric bone mineral density (vBMD) is insufficient to evaluate overall bone integrity. The accumulation of advanced glycation endproducts (AGEs) stiffens and embrittles collagen fibers. Despite the important role of AGEs in bone aging, the relationship between AGEs and vBMD is poorly understood. We hypothesized that an accumulation of AGEs, a marker of impaired bone quality, is related to decreased vBMD.
Prospectively collected data of 127 patients undergoing lumbar fusion were analyzed. Quantitative computed tomography (QCT) measurements were performed at the lumbar spine. Intraoperative bone biopsies were obtained and analyzed with confocal fluorescence microscopy for fluorescent AGEs, both trabecular and cortical. Spearman\'s correlation coefficients were calculated to examine relationships between vBMD and fAGEs, stratified by sex. Multivariable linear regression analysis with adjustments for age, sex, body mass index (BMI), race, diabetes mellitus and HbA1c was used to investigate associations between vBMD and fAGEs.
One-hundred and twenty-seven patients (51.2% female, 61.2 years, BMI of 28.7 kg/m2) with 107 bone biopsies were included in the final analysis, excluding patients on anti-osteoporotic drug therapy. In the univariate analysis, cortical fAGEs increased with decreasing vBMD at (r = -0.301; p = 0.030), but only in men. In the multivariable analysis, trabecular fAGEs increased with decreasing vBMD after adjusting for age, sex, BMI, race, diabetes mellitus and HbA1c (β = 0.99;95%CI=(0.994,1.000); p = 0.04).
QCT-derived vBMD measurements were found to be inversely associated with trabecular fAGEs. Our results enhance the understanding of bone integrity by suggesting that spine surgery patients with decreased bone quantity may also have poorer bone quality.

Lysosomes play key roles in different cellular processes such as autophagy, phagocytosis, and apoptosis. Lysosomal dysfunction is related to many diseases. Fluorescence lysosome staining strategy is valuable for the researches on the lysosome involvement in different pathological diagnosis. Here we describe fluorescence lysosome staining methods with carbon dots for the identification of lysosomes in living and fixed cells.

Plasmodesmata (PD) provide interconnectivity between plant cells to enable the intercellular transport and communication that is requisite to multicellularity. Being at the interface of the apoplast, plasma membrane (PM), endoplasmic reticulum (ER), and symplast, PD are uniquely positioned to integrate exogenously and endogenously derived signals with plant developmental and physiological responses. The distinct membrane curvature and composition of PD allow them to function as microdomains to facilitate dynamic protein-protein interactions. Förster resonance energy transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropic decay measurements provides valuable tools to analyze these interactions in vivo and in planta. Here we describe a detailed methodology to perform FRET-FLIM and fluorescence anisotropy measurements to analyze protein-protein interactions at PD in a transient expression system using Nicotiana benthamiana; however this can be adapted to other plant species and subcellular compartments.

Nuclear entry of cAMP-dependent protein kinase catalytic subunits is typically inferred from changes in net protein amount or kinase activity in the nucleus. Previous methods to directly assess nuclear entry require kinase subunit overexpression and/or supraphysiological cAMP elevation. We describe a method to detect nuclear entry of catalytic subunits expressed at an endogenous level in living cells, stimulated by cAMP in a physiological range, and in real time.

Oncology research uses different imaging techniques to provide information about the spatial distribution of the chemotherapy drugs used for the targeted tissues. Among them, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is increasingly being used to track the spatial distribution of metal-based chemotherapeutics in different tissue samples. In this investigation, instrumental parameters were optimized for the bioimaging of Pt in HT29 tumour spheroids treated with cisplatin (CDDP) or Texas Red cisplatin (TR-CDDP) using LA-ICP-MS. A high spatial resolution, using pixel dimensions of 2.0 μm × 2.5 μm, and a high sensitivity, with the limits of detection (LOD) better than 0.78 mg kg-1 Pt, was achieved. Matrix-matched gelatine standards and/or isotope dilution (ID) analyses were used to quantify the amount of Pt. Differences between the results of the Pt concentrations determined by the two quantification were less than 4%. The results of the LA analysis revealed that the Pt in the CDDP-treated tumour spheroids was localized primarily in the outer rim of the spheroids and to a lesser extent in the intermediary layer and the necrotic core. Due to the steric effects, significantly lower Pt concentrations were accumulated in the spheroids treated with TR-CDDP (2.2 times lower than in CDDP-treated spheroids, normalized to the spheroid volume), while the Pt was mostly distributed in the areas of the outer rim. Finally, imaging with confocal fluorescence microscopy, which is commonly used in oncology research, was compared with that by LA-ICP-MS. The results of the two complementary techniques demonstrated good agreement in terms of the spatial distribution of the TR-CDDP, while the intensity of the fluorescence matched well with the concentrations of Pt determined with LA-ICP-MS.

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.