Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response

Objective We investigated whether it is possible to induce a state of “LPS-sensitization” in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. Methods DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001–0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. Results Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. Conclusion Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Correcting Artifacts in Ratiometric Biosensor Imaging; an Improved Approach for Dividing Noisy Signals

The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of the method first by examining two noninteracting fluorophores distributed evenly in cells. This generated a uniform ratio that could provide a ground truth. We then analyzed actual protein activities reported by a single chain biosensor for the guanine exchange factor (GEF) Asef, and a dual chain biosensor for the GTPase Cdc42. The reduction of edge artifacts revealed persistent Asef activity in a narrow band (∼640 nm wide) immediately adjacent to the cell edge. For Cdc42, the NCF method revealed an artifact that would have been obscured by traditional background subtraction approaches.

Three-dimensional temperature and velocity measurements in fluids using thermographic phosphor tracer particles

Many flows of technical and scientific interest are intrinsically three-dimensional. Extracting slices using planar measurement techniques allows only a limited view into the flow physics and can introduce ambiguities while investigating the extent of 3D regions. Nowadays, thanks to tremendous progress in the field of volumetric velocimetry, full 3D-3C velocity information can be gathered using tomographic PIV or PTV hence eliminating many of these ambiguities (Discetti and Coletti, 2018; Westerweel et al., 2013). However, for scalar quantities like temperature, 3D measurements remain challenging. Previous approaches for coupled 3D thermometry and velocimetry combined astigmatism PTV with encapsulated europium chelates particles (Massing et al., 2018) or tomographic PIV with thermochromic liquid crystals particles (Schiepel et al., 2021). Here we present a new technique based on solid thermographic phosphor tracer particles, which have been extensively used for planar fluid temperature and velocity measurements (Abram et al., 2018) and are applicable in a wide range of temperatures. The particles are seeded into a gas flow where their 3D positions are retrieved by triangulation from multiple views and their temperatures are derived from two-colour luminescence ratio imaging. In the following, the experimental setup and key processing steps are described before a demonstration of the concept in a turbulent heated jet is shown.

Correcting artifacts in ratiometric biosensor imaging; an improved approach for dividing noisy signals

The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of the method first by examining two noninteracting fluorophores distributed evenly in cells. This generated a uniform ratio that could provide a ground truth. We then analyzed actual protein activities reported by a single chain biosensor for the guanine exchange factor Asef, and a dual chain biosensor for the GTPase Cdc42. The reduction of edge artifacts revealed persistent Asef activity in a narrow band (∼640 nm wide) immediately adjacent to the cell edge. For Cdc42, the NCF method revealed an artefact that would have been obscured by traditional background subtraction approaches.

Fabrication and Characterization of Simple Structure Fluidic-Based Memristor for Immunosensing of NS1 Protein Application

Non-structural protein 1 (NS1 protein) is becoming a commonplace biomarker for the diagnostic of early detection of dengue. In this study, we sought to use a label-free approach of detecting NS1 protein by harnessing fluidic-based memristor sensor. The sensor was fabricated using sol-gel spin coating technique, by which TiO2 thin film is coated on the surface of Indium tin oxide (ITO) and a glass substrate. The sensor was then functionalized with glycidoxypropyl-trimethoxysilane (GPTS), acting as antibody for NS1. The addition of the target NS1 formed an antibody-antigen complex which altered the physical and electrical properties in sensing region. Sensing of the sensor is incumbent upon the measurement of Off-On resistance ratio. Imaging with Field Emission Scanning Electron Microscope (FESEM) evinced the successful immobilization of the antibody and the subsequent capture of the NS1 protein by the immobilized antibody. The detection limit actualized by the developed sensor was 52 nM and the diameter of 2 mm gives the most optimal measurement. The developed sensor demonstrated an immense potential towards the development of label-free diagnostic of early dengue infection.