J. Exp. Theor. Anal., Volume 1, Issue 2 (December 2023) – 3 articles

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 µL and 100 µL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze–thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements’ accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems. Full article

Correlative light and electron microscopy (CLEM) is an advanced imaging approach that faces critical challenges in the analysis of both materials and biological specimens. CLEM integrates the strengths of both light and electron microscopy, in a hardware and software correlative environment, to produce a composite image that combines the high resolution of the electron microscope with the large field of view of the light microscope. It enables a more comprehensive understanding of a sample’s microstructure, texture, morphology, and elemental distribution, thereby facilitating the interpretation of its properties and characteristics. CLEM has diverse applications in the geoscience field, including mineralogy, petrography, and geochemistry. Despite its many advantages, CLEM has some limitations that need to be considered. One of its major limitations is the complexity of the imaging process. CLEM requires specialized equipment and expertise, and it can be challenging to obtain high-quality images that are suitable for analysis. In this study, we present a CLEM workflow based on an innovative sample holder design specially dedicated to the examination of thin sections and three-dimensional samples, with a particular emphasis on geosciences. Full article

In this study, the microstructural properties of selective laser melted 316L stainless steel were investigated using optical, scanning and transmission electron microscopy as well as X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy. The results show a very fine microstructure with visible melt pool boundaries and austenite as the predominant phase. Extremely fine sub-grain structures can be found within the grains, consisting of colonies of round or elongated cellular structures depending on orientations. Due to the prevailing cooling and solidification conditions, micro-segregations occur, leading to enrichment of the sub-grain boundaries with alloying elements such as silicon, chromium, manganese and molybdenum. The presence of ferrite could be detected in this area using TEM analysis. Full article