From static to dynamic:live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy

Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

A LASER-SCANNING NON-CONTACT MEASURING METHOD FOR ROUNDNESS ERRORS

利用激光狭缝扫描原理和光电传感技术，提出了一种用于测量大型回转体类零件圆度误差的激光扫描非接触测量方法和测量系统。本文详细论述了测量系统及其测量原理、工件安装偏心误差的分离技术和计算机实时数据处理系统，实验结果证实了这种测量方法的可行性。

The Measurement and Control of Diameter in Large-Scale Part Processing

Based on laser-scanned measuring technology, a met ho d of on-line dynamic non-contact measurement and feedback control of processin g dimension, i.e. the double edges laser-scanned large diameter on-line dynami c measurement and control system is presented, which can be used to measure diam eter in large-scale machine part processing. In this paper, the working princip le, overall structure and microcomputer real-time control and data processing s ystem of the system are discussed in detail, the method of double edges scanned large diameter dimension measurement and control is theoretically analyzed, its possibility has been verified by experiments of lathing large diameters machine parts by a vertical lathe. The system adopts the measuring scheme of double edge s laser-scanned combined with grating displacement measurement. The two edges c haracteristic information of the measured diameter is given by the double edges laser-scanned measuring system, the non-contact measurement of large diameter dimension is realized to combine with the grating displacement measuring systems . The main controller gives out feedback control signal by means of measured res ults, and controls advance and retreat of lathe tool by the servo-control syste m of a vertical lathe to realize on-line dynamic non-contact measurement and c ontrol in processing.

Involvement of VKORC1 in the Inhibition of Calcium Oxalate Crystal Formation in HK-2 Cells

Summary： The vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate （CaOx） crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORClshRNA-2 as a PGPU6/GFP/Neo-VKORClshRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC 1 transfection group than in the pFLAG-CMV-7.1 control group （P〈0.01）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate （COM） crystal medium for 48 h was 14±4 per field （100×） in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field （100×） in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC 1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group （P〈0.05）. The expression levels of VKORC 1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field （100x） in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group and 24±6 per field （100×） in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORClshRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. These findings suggested that the VKORC 1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx. Key words： calcium oxalate crystals; kidney stone; vitamin K epoxide reduetase complex subunit 1; laser-scanning confocal microscopy

Development of real-time laser-scanning system to detect tree canopy characteristics for variable-rate pesticide application

Improving pesticide application efficiency is increasingly important in orchard spraying.In this study,a laser-scanning system was designed to acquire gridding volumes of a tree to quantify the geometry characteristics of the tree canopy in real-time.A laser-scanning sensor mounted on a linear guide was utilized to measure the structure of a target tree canopy.A computer was used to receive measurement data from the laser scanner and obtain the movement distance of the laser-scanning sensor from a controller.An algorithm written with VC#program was designed to calculate gridding volumes of trees by recognizing valid measurement data from the laser scanner.Laboratory evaluations were conducted on three kinds of regular objects,and the maximum relative errors of section volumes of the cuboid,triangular prism and cylinder objects were 3.3%,7.9%and 9.4%,respectively,which illustrated that the algorithm could calculate the section volumes in different parts of the objects with high accuracy.A conifer tree and an apple tree were chosen to verify detecting accuracy of the laser-scanning system at variable speeds and grid sizes.The variation coefficients of total volumes for each kind of the tree were 0.078 and 0.041,respectively,which indicated that the laser-scanning system could be applied to provide the gridding volumes of different canopy densities in real-time with good reliability for guiding a variable-rate sprayer.

Design and performance evaluation of a variable-rate orchard sprayer based on a laser-scanning sensor

To improve the precision spraying strategy and reduce excessive pesticide application in orchards,an air-assisted sprayer integrated with a laser-scanning system was developed to realize the toward-target variable-rate spraying.In the spray control system,a method of calculating canopy gridding volumes was designed to ensure that the canopy was divided into a uniform grid size,a variable-rate spray model was used in the flow rate decision software to control the spray output according to the canopy gridding volumes and travel speed,and a method of saving and accessing spray data was used to control the spray delay.The effects of different grid sizes and travel speeds on the spray performance were evaluated by quantifying spray coverage uniformity inside tree canopies.The results showed that spray coverage uniformity declined with increasing grid width from 0.14 to 0.28 m although the mean spray coverage on each target location showed no significant differences.Additionally,there were no significant variations in mean spray coverage at speeds of 1.0,1.2 and 1.4 m/s for a tested tree of 1.6 m width and at any experimental speeds for a tested tree of 1.3 m width,which indicated that the variable-rate sprayer could provide good spray coverage uniformity under various travel speeds with a canopy size limitation.Compared with the same sprayer without the variable-rate spray function,the intelligent sprayer prototype realized effective toward-target spraying and avoided overspraying while providing sufficient spray coverage.

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2+) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .

Recording of calcium transient and analysis of calcium removal mechanisms in cardiac myocytes from rats and ground squirrels

With confocal microscopy, we recorded calcium transients and analyzed calcium removal rate at different temperatures in cardiac myocytes from the rat, a non-hibernator, and the ground squirrel, a hibernator. The results showed a remarkable increase of the diastolic level of calcium transients in the rat but no detectable change in the ground squirrel. Calcium transient of the ground squirrel, compared with that of the rat at the same temperature, had a shorter duration and showed a faster calcium removal. As indicated by the pharmacological effect of cyclopiazonic acid, calcium uptake by sarcoplasmic reticulum (SR) was the major mechanism of calcium removal, and was faster in the ground squirrel than in the rat. Our results confirmed the essential role of SR in hypothermia-tolerant adaptation, and negated the importance of Na-Ca exchange. We postulated the possibility to improve hypothermia-tolerance of the cardiac tissue of non-hibernating mammals.

Diffusion growth of Φ ternary intermetallic compound in the Mg-Al-Zn alloy system:In-situ observation and modeling

Study on the diffusion growth of ternary intermetallic compounds in Mg-Al-Zn based light-weight alloys is important due to its close interrelation with alloy property.However,there is a very lack of existing data due to difficulties in both experimental and computational aspects.The current work aims at presenting the experimental observation on the diffusion growth behavior of Φ phase at 360℃ as well as calculating its composition-dependent interdiffu sion coefficients.We designed and succes s fully fabricated four Mg-τ ternary diffusion couples annealed at 360℃ for different times,where the diffusion path goes across the Φ phase region and the diffusion growth of ternary intermetallic compound can be solely detected.In-situ observation of the time-dependent growth of Φ phase was performed to accurately determine the parabolic growth constant.The experimental data were then subjected to a numerical inverse method to generate a set of self-consistent interdiffusivities of the ternary intermetallic compounds,which can reproduce the presently observed diffusion growth behavior of Φ ternary intermetallic compound in Mg-τ diffusion couples.