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Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals., 49 pages, 10 figures

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe a large field of view in a dimensionality reduced format, identify features of interest, and volumetrically image these regions with high spatiotemporal resolution.

Ilex pubescens is a famous Chinese herbal medicine, frequently used to treat cardiovascular disease in South China. In this study, we aim to explore the absorption properties of ilexgenin A (C1) and ilexsaponin B1 (C3) in vascular endothelial cells after administration of the total triterpenoid saponins from Ilex pubescens (IPTS) and clarify the possible transport mechanisms. A UPLC-qTOF-MS/MS system was used to identify the components in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs). Afterwards, a rapid, highly selective and sensitive method was established to simultaneously quantify the concentration of C1 and C3 in HUVECs after administration of IPTS. The results demonstrate that pretreatment with IPTS could promote the survival of HUVECs and reduce the damage caused by TNF-α to HUVECs. Among the main 11 components in IPTS, eight components could be absorbed by HUVECs, including seven triterpenoids and one phenolic acid. The uptake of C1 and C3 by HUVECs occurred in a time-, temperature- and concentration-dependent manner, indicating the participation of passive diffusion and active transportation mechanisms. The two triterpenoid saponins all exhibited rapid absorption and a bimodal phenomenon in their concentration-time profiles, and equilibrium could be achieved after 6 h. Furthermore, C1 and C3 intracellular transportation was regulated by serum proteins, sodium-dependent glucose transporter 1 and P-glycoprotein. The current research for the first time demonstrates the in vitro pharmacokinetics characteristics of C1 and C3 in HUVECs lines, which could supply a new way of understanding the treatment of cardiovascular diseases.

Objectives Triterpenoid saponins of Ilex pubescens (IPTS), the main active components of Ilex pubescens, has a therapeutic effect on atherosclerosis (AS). The ingredients in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs) may play an essential role in AS. This study attempted to explore its mechanism from the perspectives of HUVECs’ inflammation, apoptosis, and autophagy. Methods By using a tumour necrosis factor-α (TNF-α)-induced HUVECs injury model, cell viability and the expression of intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9), cleave-caspase-3 and cleave-caspase-9, in combination with the results of flow cytometry, JC-1 and Hoechst 33258 staining were investigated to evaluate the anti-inflammatory and anti-apoptotic impact effects of IPTS on HUVECs. Afterwards, the expression of microtubule-associated proteins light chain 3II (LC3II) and sequestosome 1 (p62) was determined to test the effect of IPTS on autophagy. Finally, by adding an autophagy inhibitor 3-methyladenine (3-MA), we investigated whether IPTS exerts anti-inflammatory and anti-apoptotic effects through the autophagy pathway. Key findings We firstly demonstrated that pretreatment with IPTS could increase the cell viability, maintain the cell morphology and reduce TNF-α-induced inflammation and apoptosis of HUVECs. Moreover, IPTS pretreatment was proved to raise the expression of LC3II /LC3I while decreasing the expression of p62, which indicated that IPTS could activate HUVECs’ autophagy. IPTS has been shown for the first time to exert anti-inflammatory and anti-apoptotic effects through autophagy and thereby resisting TNF-α-induced inflammatory injury of HUVECs. Conclusions This study preliminarily confirmed that IPTS ameliorated HUVECs’ inflammation and apoptosis by increasing autophagy.

In this paper, the space charge behavior of polyimide at cryogenic temperatures is studied. The pulsed electro-acoustic (PEA) and thermally stimulated current (TSC) methods are exploited to evaluate the space charge distribution, quantification of space charge accumulation and trap characteristics of polyimide. The results show that the change of space charge accumulation at 273 to 123 K is divided into three temperature regimes. At 273–223 K, the space charge accumulation decreases significantly with decreasing temperature, which is mainly due to the suppression of charge generation and migration. At 223–173 K, the negative space charge gradually increases with decreasing temperature and dominates the space charge distribution. Analysis shows that this is due to the significant decrease of trap level in the sample, which improves the carrier mobility and promotes the electron injection and migration. According to quantum chemical calculation and dielectric spectroscopy measurement, the rotational displacement motion of water molecules related to γ relaxation in polyimide produces more shallow electron traps, which may be the main reason for the decrease of trap level. When the temperature is below 173 K, the space charge accumulation decreases with decreasing temperature. This study provides guidance for low temperature insulation design.

Imaging biological dynamics in 3D requires fast, high-resolution volumetric imaging techniques. We present a 3D-from-2D imaging pipeline that permits volumetric reconstruction from 2D projection snapshots, applicable to a range of microscopy image acquisition platforms.

Toddalia asiatica L., a significant medicinal plant in the family Rutaceae, has been applied to treat rheumatoid diseases for decades. Its pharmacological activities are mainly attributed to the existence of generous coumarins and alkaloids; however, the pharmacokinetics of Toddalia asiatica L. remain unclear. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the simultaneous determination of four coumarins, three alkaloids and one flavonoid (hesperidin, nitidine chloride, chelerythrine, toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine) in rat feces as well as four coumarins and one alkaloid (toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine) in rat plasma and urine. Chromatographic separation was accomplished on an Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 5 μm) with acetonitrile (containing 0.1% formic acid) and 5 mmol/L aqueous ammonium formate for gradient elution. A correlation coefficient greater than 0.9925 reflected the excellent linearity of the analytical response. The lower limits of quantification were 30.0, 10.0, 10.0, 30.0, 5.0, 10.0, 2.5 and 2.5 ng/mL for hesperidin, nitidine chloride, chelerythrine, toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine, respectively. The intra- and inter-day precision were less than 12.7%, and the accuracy was between -11.8% and 12.9%. In summary, this study is the first to study the pharmacokinetics and excretion of T. asiatica extract after oral administration, which may provide a scientific basis for its clinical applications.

Funder: MedImmune, and Infinitus (China) Ltd., In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

We present a beam splitter mask that can be easily added to a multiphoton raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of 3D volumetric imaging. Recently, by conjugating either a movable-mirror to the image plane or an electrotuneable lens (ETL) to the back-focal plane respectively, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil based actuators), while ETLs introduce spherical and higher order aberrations, thereby preventing high-resolution imaging. Here, we introduce a novel optical design that can transform a lateral-scan motion into a spherical-aberration-free, high-resolution, rapid axial scan. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in image space. We characterize the optical performance of this remote focusing technique and use it to accelerate axially swept light-sheet microscopy (ASLM) by one order of magnitude, allowing the quantification of rapid vesicular dynamics in 3D.

Charge accumulation is one of the main factors leading to electrostatic discharge and restricting the development of spacecraft. This paper investigates the effect of temperature (243~343 K) on the charging behaviour of polyimide in vacuum using electron beam radiation test system. The surface potential distribution shows that there are two charging modes, "negative-peak" mode and "negative-crater" mode. For investigating the charge accumulation mechanism, the space charge experiments were carried out by the method of pulsed electro-acoustic. The thresholds of injection from the polyimide/electrode interface are affected by temperature. Under high temperature (above 323 K), the hole could be easily injected from the polyimide/electrode interface, with the extremely low injection threshold. The incident electrons and the injected holes co-existence and form the "negative-peak" mode. Under low temperature (below 303 K), the area of hole injection from the electrode decreases with the decrease in temperature. The hole injected locally forms an opposing electric field, which makes the surface potential present the "crater" mode. This work shows that the hole injected from the dielectric/electrode interface play a critical role in the charge accumulation process under electron radiation, which has important significance to avoid the spacecraft electrostatic discharge problem.

The spacecraft often interacts with plasma, high energy particles and magnetic fields on orbit. It causes the charge injected in the insulation materials and forms a high electric field. The discharge phenomenon will happen when the internal electric field reaches the discharge voltage threshold. The insulation material is a key component of electrical equipment on the spacecraft, its electrical properties affects the reliability and lifetime of the whole spacecraft. The insulation material on spacecraft withstands fast thermal cycle during operation on orbit, which causes internal thermal stress in it. The accumulation of internal thermal stress changes the material molecular structure and depolarization characteristics, thus affecting the behavior of the space charge inside the insulation material. In this paper, the temperature environment was achieved by contact heating plant and liquid nitrogen immersion to do accelerated aging test based on the temperature of operating spacecraft. By the mean of this method, 0, 50, 100, 150 and 200 times of the fast thermal cycle was carried out. The molecular structure characteristics, the thermally stimulated current characteristics and the space charge characteristics of the samples with different cycling times were studied through the methods including XPS, XRD, TSC and PEA. The results show that with the increase of fast thermal cycling times, the relative content of the C-N and C=O bonds increase and it also increases the shallow energy level charges. The charges are more easily to move inside the sample. Fast thermal cycle also increases the degree of crystallinity, thus the interface between crystalline region and amorphous region expands. It also increases the charge of deep energy level, which increased the amount of space charge injected in the sample.

Traditionally, conductivity, a key parameter for evaluating the probability of electrostatic discharge, has been subscribed to the belief that it is a constant value. However, in actual working conditions, such as the low earth orbit environment, the conductivity value of dielectrics is not constant due to the variation in temperature. Therefore, it is of great practical significance to investigate the conductivity value of dielectrics in the time-varying field and explore the hidden mechanism of the charging phenomenon. In this paper, we investigated the dynamic conductivity value using polyimide during four temperature cycles. The result shows that there is a positive correlation between conductivity and temperature above the turning point temperature. However, when the temperature is lower than the turning point temperature, the conductivity value hardly changes with temperature. In the four cycles, the conductivity value in the same temperature decreases with the cycles. The space charge distribution test and the thermally stimulated current experiment are carried out to explain the charging phenomenon. The results show that the heterocharge accumulates continually and is captured by deep traps above 0.55 eV. It could be concluded that the thermally stimulated detrapping effect makes the charge trapping difficult, which results in the conductivity value in the same cycle changing with the temperature above the turning point temperature and the heterocharge accumulating. The trap-filling effect inhibits the charge conduction process, which makes the conductivity value in the same temperature to decrease with temperature cycles.

As a new kind of nanocomposite material, CB/LDPE has attracted wide attention in recent years. In this paper, CB/LDPE composites with a small doping content (0.03wt%) were fabricated by using the melt blending method. Conductivity characteristics of LDPE and CB/LDPE films were measured at different temperatures and different electric field. Space charge injection threshold and activation energy at different temperatures were calculated for both films. The experimental results show that, compared with LDPE material, the CB/LDPE material increases the space charge injection threshold and weakens the dependence of the conductivity on temperature. The analysis indicates that the improvement of conductivity characteristics of CB/LDPE is related to the formation of the homo-polarity charge layer on the surface of the sample. This is beneficial to the development and utilization of CB/LDPE materials in the new materials of HVDC cable.

A robust 780 nm femtosecond fiber laser was demonstrated with 191 fs pulse width, 256 MHz repetition rate and 1 W average power. It is ideal for two-photon microscopic imaging.

We demonstrate a robust high power 930 nm femtosecond Nd:fiber laser system with hollow-core photonic bandgap fiber (HC-PBGF) as the output delivery, which can be easily integrated into compact two-photon microscopy system for bio-imaging. The whole laser system can deliver up to 17.4 nJ, 220-fs pulses at 930 nm with repetition rate of 46 MHz. In this paper, this laser was demonstrated as the light source for volumetric imaging of zebrafish blood vessel.

We present an Er-doped fiber (Er:fiber)-based femtosecond laser at 780 nm with 256 MHz repetition rate, 191 fs pulse duration, and over 1 W average power. Apart from the careful third-order dispersion management, we introduce moderate self-phase modulation to broaden the output spectrum of the Er:fiber amplifier and achieve 193 fs pulse duration and 2.43 W average power. Over 40% frequency doubling efficiency is obtained by a periodically poled lithium niobate crystal. Delivering through a hollow-core photonic bandgap fiber, this robust laser becomes an ideal and convenient light source for two-photon autofluorescence imaging.

We demonstrate a chirped pulse amplifier based on a W-type Nd-doped silicate glass fiber. The system delivers 500 mW 220-fs pulses at 930 nm.

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

We optimized pre-chirp in an Nd-doped fiber amplifier to achieve high-quality femtosecond 920 nm pulses. The in vivo zebrafish imaging proves the laser amplifier an ideal source in two-photon microscopic imaging.

We report a core-pumped all-normal dispersion mode-locked Nd-doped fiber laser at 910 and 935 nm. The pulse is compressed to 198 fs, and the pulse energy is 1.3 nJ. The slope efficiency is more than 14%. This laser is tested as the optical source for the two-photon fluorescence imaging of pollen.

We demonstrate a passively all-normal dispersion mode-locked Nd-doped fiber laser at 905-940 nm with high optical to optical efficiency. The central wavelengths can be tunable through tuning birefringent filter.

Pre-chirp technique was used in an Nd-doped fiber amplifier to optimize high-quality 910 nm pulses with the pulses width of 114 fs and pulse energy of 4.4 nJ. The in vivo zebrafish imaging results from our totally home-made microscopy proves our femtosecond Nd fiber laser an ideal source in two-photon microscopic imaging.

When the 3s3p 1 P level of an Mg atom is resonantly excited by a laser, an anomalous emission with a wavelength of 5184 A can be observed, which corresponds to the transition for 3s4s 3 S to 3s3p 3 P 2 . According to the temporal behavior of this emission and its intensity dependence on atom density, it is believed that the experiment seems to show the transition from a spontaneous process to a cooperative process. The population inversion between 3s4s 3 S and 3s3p 3 P 2 is created by inelastic collisions of the lower excited 3s3p 1 P atoms.