Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

Predictors and clinical outcomes of permanent pacemaker implantation after transcatheter aortic valve implantation.

OBJECTIVE: This study aimed to identify the incidence, risk factors, and outcomes of permanent pacemaker (PPM) implantation after transcatheter aortic valve implantation (TAVI) procedures. METHODS: A retrospective analysis was conducted on 70 patients who underwent TAVI at the Department of Cardiology, Fujian Provincial Hospital, from January 2018 to March 2022. Based on whether a new PPM was implanted after TAVI, all patients were divided into two groups: NEW PPM and NO PPM. Baseline characteristics and clinical data were compared between the two groups. Univariate analysis was used to analyze different variables between the two groups. A binary logistic regression analysis was used to evaluate independent correlates for PPM implantation after TAVI. RESULTS: The mean age of the 70 patients was 73.1 ± 8.8 years. The incidence of PPM implantation was 17.1%. Patients with diabetes and chronic kidney disease were more likely to require PPM (50% vs. 20.7%, p = 0.042, 25% vs. 5.2%, p = 0.042). Our study did not identify any significant differences in the incidence of electrocardiographic conduction disturbances except for the previous right bundle branch block (RBBB) (NO PPM 6.9% vs. NEW PPM 33.3%, p < 0.05). We found that prosthesis size, implantation depth, procedural duration, and length of hospital and intensive care unit (ICU) stays were comparable between the two groups. The leading independent predictors of PPM implantation were previous RBBB (odds ratio 10.129, p = 0.034). CONCLUSION: The previous RBBB was the leading independent predictor of PPM implantation. New PPM was not associated with significantly new-onset left BBB, extended post-procedure hospitalization, ICU stay, or procedural duration.

Pharmacokinetic and excretion study of eight active constituents in rat by LC-MS/MS after oral administration of the Toddalia asiatica extract.

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.

Absorption characteristics of ilexgenin A and ilexsaponin B1 in human umbilical vein endothelial cells after administration of the total triterpenoid saponins from Ilex pubescens.

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.

Relationship between components of metabolic syndrome and arterial stiffness in Chinese hypertensives.

Background: Subjects with metabolic syndrome showed increased risk of cardiovascular events. We investigated the relationship between components of metabolic syndrome and arterial stiffness in Chinese hypertensives.Method: 680 subjects (aged 58.44 ± 11.67 years, male 63.53%, hypertension 65.00%) were divided into five groups based on the number of known components of metabolic syndrome (MSCs) according to the criteria of 2010 Chinses Guidelines for Prevention and Management of Hypertension (0MSCs: n= 86; 1MSCs: n= 153; 2MSCs: n= 201; 3MSCs: n= 148; 4/5MSCs: n= 92.). Body weight, height, waist circumference, blood pressure and clinical biochemical tests were measured. Carotid-femoral pulse wave velocity (cfPWV) was measured using a non-invasive automatic device (Complior Analysis, France).Results: The level of cfPWV was significantly increased with the increasing number of MSCs (8.20 ± 1.54 vs 8.72 ± 1.48 vs 9.34 ± 1.77 vs 9.64 ± 1.86 vs 9.91 ± 2.19 m/s, P<0.05). In subjects with hypertension (n= 442), cfPWV was higher than those without hypertension (n= 238) (9.59 ± 1.90 vs 8.49 ± 1.50 m/s, P<0.05) . Stepwise multiple regression analysis revealed that age, gender, the number of MSCs, heart rate as well as serum uric acid level were determinants for cfPWV (P<0.05). In the subgroups stratified by age, systolic blood pressure correlated with cfPWV in hypertensives under 55 years old, while in non-hypertensives the correlation was found after 60 years old.Conclusion: The arterial stiffness became significant with the increasing of the metabolic components numbers, which was independent of age, gender and blood pressure. And the presence of hypertension played the most important role in the progress of arterial stiffness even compared with age.

Robust hollow-fiber-pigtailed 930 nm femtosecond Nd:fiber laser for volumetric two-photon 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.

910nm femtosecond Nd-doped fiber laser for in vivo two-photon microscopic imaging.

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.

Core-pumped femtosecond Nd:fiber laser at 910 and 935 nm.

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.

Adaptive Optics in an Oblique Plane Microscope.

Adaptive optics (AO) can restore diffraction limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both the illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.

Adaptive optics in an oblique plane microscope.

Adaptive optics (AO) can restore diffraction-limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such, is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.

Application of Electrospun Drug-Loaded Nanofibers in Cancer Therapy.

In the 21st century, chemotherapy stands as a primary treatment method for prevalent diseases, yet drug resistance remains a pressing challenge. Utilizing electrospinning to support chemotherapy drugs offers sustained and controlled release methods in contrast to oral and implantable drug delivery modes, which enable localized treatment of distinct tumor types. Moreover, the core-sheath structure in electrospinning bears advantages in dual-drug loading: the core and sheath layers can carry different drugs, facilitating collaborative treatment to counter chemotherapy drug resistance. This approach minimizes patient discomfort associated with multiple-drug administration. Electrospun fibers not only transport drugs but can also integrate metal particles and targeted compounds, enabling combinations of chemotherapy with magnetic and heat therapies for comprehensive cancer treatment. This review delves into electrospinning preparation techniques and drug delivery methods tailored to various cancers, foreseeing their promising roles in cancer treatment.

Electrical Conductivity Characteristic of Polyimide under Long-Term Combined Electron Irradiation and Thermal Cycle in Vacuum.

During long-term operation, low-earth-orbit spacecraft are exposed to a severe environment of electron irradiation and thermal cycle. This affects the electric properties of polyimide, an essential insulation material for spacecraft electrical transmission equipment, particularly the conductivity characteristic. Therefore, this paper investigates the conductivity and its evolution of polyimide after the combination of 20 keV, 8 nA/cm2 electron irradiation, and 243-343 K, 5 K/min thermal cycle in a vacuum environment for 432 h. The results show that the conductivity increases by about 2 orders of magnitude over 432 h, with the threshold field for electric-field-dependent conductivity decreasing. The conductivity growth rate varies, rising during the first 192 h, then increasing in the midelectric field, and decreasing in the high electric field regions. The thermally stimulated depolarization current method demonstrates that increases in γ, ß1, and ß2 trap densities, associated with enhanced motility of end groups, diamines, and dianhydrides after long-chain breaks, lead to higher conductivity and growth rate. Additionally, increases in ß3 and α trap densities, related to increased C═O bonds and free radicals, reduce the threshold field and the conductivity growth rate in the range of 57.0-100.0 kV/mm after 192 h. These findings provide a reference for the performance evaluation and enhancement of spacecraft polyimide materials.

navigate: an open-source platform for smart light-sheet microscopy.

navigate is a turnkey, open-source software solution designed to enhance light-sheet fluorescence microscopy (LSFM) by integrating smart microscopy techniques into a user-friendly framework. It enables automated, intelligent imaging with a Python-based control system that supports GUI-reconfigurable acquisition routines and the integration of diverse hardware sets. As a comprehensive package, navigate democratizes access to advanced LSFM capabilities, facilitating the development and implementation of smart microscopy workflows without requiring deep programming knowledge or specialized expertise in light-sheet microscopy.

Projective light-sheet microscopy with flexible parameter selection.

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence. Here we demonstrate the power of props by functional imaging within distinct regions of the zebrafish brain, monitoring calcium firing inside muscle cells of moving Drosophila larvae, super-resolution imaging of selected cell layers, and by optically unwrapping the curved surface of a Drosophila embryo. We anticipate that props will accelerate volumetric interrogation, ranging from subcellular to mesoscopic scales.

Mesoscopic Oblique Plane Microscopy via Light-sheet Mirroring.

Understanding the intricate interplay and inter-connectivity of biological processes across an entire organism is important in various fields of biology, including cardiovascular research, neuroscience, and developmental biology. Here, we present a mesoscopic oblique plane microscope (OPM) that enables whole organism imaging with high speed and subcellular resolution. A microprism underneath the sample enhances the axial resolution and optical sectioning through total internal reflection of the light-sheet. Through rapid refocusing of the light-sheet, the imaging depth is extended up to threefold while keeping the axial resolution constant. Using low magnification objectives with a large field of view, we realize mesoscopic imaging over a volume of 3.7×1.5×1 mm3 with ~2.3 microns lateral and ~9.2 microns axial resolution. Applying the mesoscopic OPM, we demonstrate in vivo and in toto whole organism imaging of the zebrafish vasculature and its endothelial nuclei, and blood flow dynamics at 12 Hz acquisition rate, resulting in a quantitative map of blood flow across the entire organism.

Pharmacokinetic and tissue distribution study of six saponins in the rat after oral administration of Ilex pubescens extract using a validated simultaneous UPLC-qTOF-MS/MS assay.

Ilex pubescens Hook. et Arn is a medicinal plant of the Ilex family that is mainly used for the treatment of cardiovascular diseases. Its main medicinal ingredients are total triterpenoid saponins (IPTS). However, the pharmacokinetics and tissue distribution of the main multi-triterpenoid saponins are lacking. This is the first report that demonstrates a sensitive ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-qTOF-MS/MS) method for the quantification of ilexgenin A (C1), ilexsaponin A1 (C2), ilexsaponin B1 (C3), ilexsaponin B2 (C4), ilexsaponin B3 (DC1) and ilexoside O (DC2) in rat plasma and various tissues of the heart, liver, spleen, lungs, kidney, brain, stomach, duodenum, jejunum, ileum, colon and thoracic aorta. The chromatographic separation was carried out on an Acquity HSS T3 UPLC column (2.1 × 100 mm, 1.8 µm, Waters, USA) with a mobile phase consisting of 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B) at a flow rate of 0.25 mL/min. The MS/MS detection was performed by electrospray ionization (ESI) using selected ion monitoring (SIM) in negative scan mode. The developed quantification method showed good linearity over the concentration range of 10-2000 ng/mL for plasma and 25-5000 ng/mL for tissue homogenates with R2 ≥ 0.990. Lower limits of quantification (LLOQ) was 10 ng/mL in plasma and 25 ng/mL in tissue homogenates. The intra- and inter-day precision were less than 10.39%, and the accuracy was between - 1.03% and 9.13%. The extract recoveries, dilution integrity and matrix effect were well within satisfactory limits. Using the validated method, the pharmacokinetic parameters, including half-life, AUC, Cmax, CL, and MRT, of six triterpenoid saponins in rats after oral administration were provided by establishing their plasma concentration-time curves, while their absolute quantification in various tissues after oral administration was also determined at first, which provides a scientific basis for their clinical application.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

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.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

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.

Triterpenoid saponins of Ilex pubescens against TNF-α induced inflammation and apoptosis in human umbilical vein endothelial cells via autophagy pathway.

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.