Visualization Of Tracers Research Articles

An Optimized Mouse Brain Atlas for Automated Mapping and Quantification of Neuronal Activity Using iDISCO+ and Light Sheet Fluorescence Microscopy

In recent years, the combination of whole-brain immunolabelling, light sheet fluorescence microscopy (LSFM) and subsequent registration of data with a common reference atlas, has enabled 3D visualization and quantification of fluorescent markers or tracers in the adult mouse brain. Today, the common coordinate framework version 3 developed by the Allen’s Institute of Brain Science (AIBS CCFv3), is widely used as the standard brain atlas for registration of LSFM data. However, the AIBS CCFv3 is based on histological processing and imaging modalities different from those used for LSFM imaging and consequently, the data differ in both tissue contrast and morphology. To improve the accuracy and speed by which LSFM-imaged whole-brain data can be registered and quantified, we have created an optimized digital mouse brain atlas based on immunolabelled and solvent-cleared brains. Compared to the AIBS CCFv3 atlas, our atlas resulted in faster and more accurate mapping of neuronal activity as measured by c-Fos expression, especially in the hindbrain. We further demonstrated utility of the LSFM atlas by comparing whole-brain quantitative changes in c-Fos expression following acute administration of semaglutide in lean and diet-induced obese mice. In combination with an improved algorithm for c-Fos detection, the LSFM atlas enables unbiased and computationally efficient characterization of drug effects on whole-brain neuronal activity patterns. In conclusion, we established an optimized reference atlas for more precise mapping of fluorescent markers, including c-Fos, in mouse brains processed for LSFM.

MagC, magnetic collection of ultrathin sections for volumetric correlative light and electron microscopy.

The non-destructive collection of ultrathin sections on silicon wafers for post-embedding staining and volumetric correlative light and electron microscopy traditionally requires exquisite manual skills and is tedious and unreliable. In MagC introduced here, sample blocks are augmented with a magnetic resin enabling the remote actuation and collection of hundreds of sections on wafer. MagC allowed the correlative visualization of neuroanatomical tracers within their ultrastructural volumetric electron microscopy context.

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer’s disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.

Investigation of hydrodynamic characteristics of laminar flow condition around sphere using PIV system

This paper aims to determine the hydrodynamic characteristics of flow around the sphere in unsteady state condition. An experimental test-rig was designed and constructed for this purpose with the application of an adjusted laser optics system. It is based on the technology of pulsed particle visualization of micro tracers in the cross section per unit time interval. Visualization with Particle Image Velocimetry (PIV-system) is used to study the properties of the flow such as its structure. The PIV-system is the most accepted technique allowed one to measure the instantaneous velocity distribution in fluid applications. In this experimental study, o-ring is used to simulate turbulence on the sphere surface and creates very high-level fluctuations, which creates the flow undergoing a laminar-to-turbulent transition. This transition leads to a delay of the separation point of flow from the sphere surface causing a significant reduction in the drag coefficient, reaching 45%. New results obtained can be useful in the development of numerical validation as well as in design processes.

Investigation of the effect of passive vortex inserts of different geometrical shapes on the vibrations reduction efficiency in pipelines with two-phase flow

This paper describes the experimental test bench used in coolant flow research with application of adjusted optical laser system. It is based on the technology of pulsed particle visualization of micro tracers, i.e. Particle Image Velocimetry (PIV system) that allows for the analysis of the microstructure of the hydrodynamic liquid flow passed through the vortex inserts. Optical measurement (PIV-method) of hydro- and aerodynamic characteristics of the coolant flow allowed obtaining the velocity distribution in straight and various curved sections of the pipeline in order to reliably define the vortex formation condition and the occurrence of the pressure gradient on the outer and inner walls of the curved sections. This gives us the opportunity to verify the calculation models describing the problem of passive control (decrease) of the displacement of pipelines of power equipment, and thus to improve the reliability and safe operation of the system in general.

Visualization of Mixing Processes in a Heterogeneous Sand Box Aquifer

Mixing is increasingly recognized as a critical process for understanding and modeling reactive transport. Yet, mixing is hard to characterize because it depends nonlinearly on concentrations. Visualization of optical tracers in the laboratory at high spatial and temporal resolution can help advance the study of mixing processes. The solute distribution is obtained by analyzing the relationship between pixel intensity and tracer concentration. The problem with such techniques is that grain borders, light fluctuations, and nonuniform brightness contribute to produce noisy images of concentrations that cannot be directly used to estimate mixing at the local scale. We present a nonparametric regression methodology to visualize local values of mixing from noisy images of optical tracers that minimizes smoothing in the direction of concentration gradients. This is achieved by weighting pixel data along concentration isolines. The methodology is used to provide a full visualization of mixing dynamics in a tracer experiment performed in a reconstructed aquifer consisting of two materials with contrasting hydraulic properties. The experiment reveals that mixing is largest at the contact area of regions with different permeability. Also, the temporal evolutions of mixing and dilution rates are significantly different. The mixing rate is more persistent than the dilution rate during tracer invasion, and the opposite is true during flushing, which helps in understanding the complementary nature of these two measures.

Evaluation of [ 11C]hemicholinium-15 and [ 18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[ 11C]Hemicholinium-15 ([ 11C]HC-15) and [ 18F]hemicholinium-15 ([ 18F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [ 11C]HC-15 was prepared by N-[ 11C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [ 11C]CH 3OTf in 55–70% radiochemical yield decay corrected to end of bombardment (EOB) and 2–3 Ci/μmol specific activity at end of synthesis (EOS). [ 18F]HC-15 was prepared by N-[ 18F]fluoromethylation of the precursor using [ 18F]FCH 2OTf in 20–30% radiochemical yield decay corrected to EOB and >1.0 Ci/μmol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20 min post-intravenous injection, and the results show the heart region uptakes 1.32 ± 0.75%ID/g in R-ventricle for [ 11C]HC-15 and 1.28 ± 0.81%ID/g in L-ventricle for [ 18F]HC-15, respectively. The dynamic PET imaging studies of [ 11C]HC-15 in rats were acquired 60 min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0 mg/kg of unlabeled HC-15 prior to [ 11C]HC-15 injection. [ 11C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [ 13N]NH 3, [ 11C]HC-15, and [ 18F]HC-15. PET studies in dogs of both [ 11C]HC-15 and [ 18F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [ 11C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.

1108 色層を用いた可視化によるエンジン内ガス流動の計測 : 空気流動計測への改良と検討

1108 色層を用いた可視化によるエンジン内ガス流動の計測 : 空気流動計測への改良と検討

Measurement of in-cylinder flow by visualization of tracers in colour layer: examination of principle and measurement of three dimensional position of tracer

Measurement of in-cylinder flow by visualization of tracers in colour layer: examination of principle and measurement of three dimensional position of tracer

Identification and distribution of neuroendocrine gonadotropin-releasing hormone neurons in the ewe.

The final common pathway controlling reproductive function in vertebrates is the GnRH neuron and its projection to the median eminence (ME), site of peptide release into the pituitary portal system. GnRH neurons are widely distributed; therefore we sought to test the hypothesis that those projecting to the ME are located in specific regions. We used as a model the sheep, a species in which a great deal of information regarding the physiology of GnRH secretion is known. To identify cells projecting to the ME (i.e., neuroendocrine neurons), ewes (n = 10) received injections into the ME of neuronal tract-tracing compounds: cholera toxin-beta subunit (CT-beta) or one of two fluorescent compounds (rhodamine isothiocyanate or fluorescein-conjugated dextran). Forty-eight h later, animals were perfused intracranially and their brains were processed for immunocytochemical localization of GnRH and CT-beta using a dual-immunofluorescent procedure or by single-label immunofluorescent visualization of GnRH combined with direct visualization of fluorescent tracers. Small, well-circumscribed injections into the ME were made successfully in 6 of 10 animals, and these overlapped the location of GnRH terminals and fibers. Neuroendocrine GnRH neurons (those GnRH neurons containing retrogradely transported tracer) were identified throughout their previously reported range: within the diagonal band of the Broca/medial septal region, medial preoptic area (MPOA), anterior hypothalamic area, and medial basal hypothalamus. Although the absolute number of neuroendocrine GnRH neurons varied by region, the percentage of the total GnRH population within each of these areas that was retrogradely labeled did not differ (p > 0.05). Injections placed unilaterally within the ME labeled a similar proportion of GnRH cells both ipsilateral and contralateral to the injection site in all areas except the MPOA, where ipsilaterally labeled cells were approximately twice as numerous as those labeled contralaterally. Injections that missed the ME and were placed either into the third ventricle or into the arcuate nucleus labeled only 0.5% and 4-11% of GnRH neurons, respectively. These results do not support the hypothesis that in the ewe, GnRH neurons projecting to the ME are localized to specific regions. Thus, we postulate that GnRH release into the hypophyseal portal system reflects the output of GnRH neurons located in multiple areas.

Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.

Development of a Method- to Measure Flow Field by Flow Visualization and Image Processing Techniques

A computerized system for flow measurement is aimed to be developed which can afford the velocity measurement of multiple points at the same time. The system consists complimentarily of image processing of visualized flows and numerical computations of the continuity and Navier-Stokes equations ; first the velocity field of a scanned plane is measured by digitizing the path of tracers through image processing techniques, and then numerical computa tions are invoked to make the scanned plane flows three-dimensional and.to fill up unmeasu rable points by the image processing.In the present paper, the first part is mainly studied on plane flows ; a method of visualization of tracers and an algorithm to determine the velocity on a plane. The basic idea is to analyze three images of a tracer on successive frames which is crossing the slit beam with a finite thickness. They make it possible to distinguish the actual positions of the tracer. The slit beam is scanned to three-dimensionize of the plane flows. This idea is realized by an algorithm on a micro-computer which is applied to measure uniform flows, wake flow of a circular cylinder and unsteady channel flow.Although several points are still left to be improved for higher accuracy, the present system is proved to work basically well and the extension is confirmed to three-dimensional velocity measurements. It is primarily important to acquire stable pictures of tracers independent of the direction to camera, time and position.