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Kinematics of the knee during gait has mostly been studied using optical motion capture systems (MCS). The presence of soft tissue artifacts (STA) between the skin markers and the underlying bone presents a major impediment to obtaining a reliable joint kinematics assessment. In this study, we determined the effects of STA on the calculation of knee joint kinematics during walking and running, through the combination of high-speed dual fluoroscopic imaging system (DFIS) and magnetic resonance imaging technique. Ten adults walked and ran while data was collected simultaneously from MCS and high-speed DFIS. The study showed that measured STA underestimated knee flexion angle, but overestimated knee external and varus rotation. The absolute error values of the skin markers derived from knee flexion–extension angle, internal-external rotation, and varus-valgus rotation during walking were −3.2 ± 4.3 deg, 4.6 ± 3.1 deg, and 4.5 ± 3.2 deg respectively, and during running were −5.8 ± 5.4 deg, 6.6 ± 3.7 deg, and 4.8 ± 2.5 deg respectively. Average errors relative to the DFIS for flexion–extension angle, internal-external rotation, and varus-valgus rotation were 78 %, 271 %, 265 % during walking respectively, and were 43 %, 106 %, 200 % during running respectively. This study offers reference for the kinematic differences between MCS and high-speed DFIS, and will contribute to optimizing methods for analyzing knee kinematics during walking and running.

The development of adjacent segment degeneration (ASD) is a major concern after lumbar spinal fusion surgery, but the causative mechanisms remain unclear. This study used a combined in vivo and in silico method to investigate the changes of anatomical dimensions and biomechanical responses of the adjacent segment (L3-4) after spinal fusion (L4-S1) in five patients under weight-bearing upright standing conditions. The in vivo adjacent disc height changes before and after fusion were measured using a dual fluoroscopic imaging system (DFIS), and the measured in vivo intervertebral positions and orientations were used as displacement boundary conditions of the patient-specific three-dimensional (3D) finite element (FE) disc models to simulate the biomechanical responses of adjacent discs to fusion of the diseased segments. Our data (represented by medians and 95% confidence intervals) showed that a significant decrease by − 0.8 (− 1.2, − 0.4) mm (p < 0.05) in the adjacent disc heights occurred at the posterior region after fusion. The significant increases in disc tissue strains and stresses, 0.32 (0.21, 0.43) mm/mm (p < 0.05) and 1.70 (1.07, 3.60) MPa (p < 0.05), respectively, after fusion were found in the posterolateral portions of the outermost annular lamella. The intradiscal pressure of the adjacent disc was significantly increased by 0.29 (0.13, 0.47) MPa after fusion (p < 0.05). This study demonstrated that fusion could cause alterations in adjacent disc biomechanics, and the combined in vivo and in silico method could be a valuable tool for the quantitative assessment of ASD after fusion.

Patellar tendinopathy is prevalent in sports requiring high jumping demands, and understanding the in vivo biomechanical behavior of the patellar tendon (PT) during landing is crucial for developing effective injury prevention and rehabilitation strategies. This study investigates the in vivo biomechanical behavior of the PT during the landing phase of a stop-jump task, integrating musculoskeletal modelling, finite element analysis (FEA), and a high-speed dual fluoroscopic imaging system (DFIS). A subject-specific knee joint model was constructed from CT, MRI, and dynamic X-ray data for a 27-year-old male (178 cm, 68 kg) at six time points during landing. Musculoskeletal simulations were used to estimated knee joint moments and quadriceps muscle forces, which were then applied to the finite element models. DFIS ensured accurate 3D spatial alignment of the models. Ridge regression analysis explored the relationship between applied biomechanical loads and the maximum equivalent (von Mises) stress in the PT. Maximum PT stress was observed at the bone attachment sites, with the highest stress (94.44 MPa) at initial ground contact, decreasing to a minimum of 16.37 MPa during landing. Regression analysis demonstrated a significant correlation (R2 = 0.859, P < 0.001) between knee flexion moments, quadriceps muscle forces, and maximum PT stress, identifying these factors as key determinants of PT loading. This study underscores the importance of knee flexion moments and quadriceps muscle forces in influencing PT stress during landing. Future studies should include larger cohort to validate these results and explore the potential of machine learning for real-time injury risk prediction.

The biomechanical mechanism of postoperative patellofemoral joint (PFJ) complications after open-wedge high tibial osteotomy (OWHTO) has not been investigated. This study was to determine the length changes in the patellar tendon (PT), medial patellotibial ligament (MPTL), medial patellofemoral ligament (MPFL), and quadriceps moment arm (QMA) during staircase motion before and after OWHTO. Computed tomography (CT) scans of 15 patients’ lower extremities were used to reconstruct three-dimensional models, and magnetic resonance imaging (MRI) of the knee and hip joints was used to mark the soft tissue footprints. Then, such soft tissue lengths were quantified by a dual fluoroscopic imaging system (DFIS). Additionally, function scores were used to assess patient outcome changes. The results showed that there was a contraction of the PT after OWHTO due to its adhesion to the osteotomy site, causing PT length to be negatively correlated to the open-wedge angle. In addition, the shortening of the MPTL and QMA caused patellar instability and an imbalance in the strength of the lower extremities. Additionally, most knee function scores improved after OWHTO, except the Feller scores. Multiple methods should be considered to optimize surgical procedures, postoperative rehabilitation, and physical therapy.

Myeloperoxidase (MPO)-mediated oxidative stress has been suggested to play an important role in the pathological dysfunction of epileptic brains. However, there is currently no robust brainimaging tool to detect real-time endogenous hypochlorite (HClO) generation by MPO or a fluorescent probe for rapid highthroughput screening of antiepileptic agents that control the MPO-mediated chlorination stress. Herein, we report an efficient two-photon fluorescence probe (named HCP) for the real-time detection of endogenous HClO signals generated by MPO in the brain of kainic acid (KA)-induced epileptic mice, where HClO-dependent chlorination of quinolone fluorophore gives the enhanced fluorescence response. With this probe, we visualized directly the endogenous HClO fluxes generated by the overexpression of MPO activity in vivo and ex vivo in mouse brains with epileptic behaviors. Notably, by using HCP, we have also constructed a high-throughput screening approach to rapidly screen the potential antiepileptic agents to control MPO-mediated oxidative stress. Moreover, from this screen, we identified that the flavonoid compound apigenin can relieve the MPO-mediated oxidative stress and inhibit the ferroptosis of neuronal cells. Overall, this work provides a versatile fluorescence tool for elucidating the role of HClO generation by MPO in the pathology of epileptic seizures and for rapidly discovering additional antiepileptic agents to prevent and treat epilepsy.

It is highly important and challenging to develop donor-acceptor-donor structured small-molecule second near-infrared window (NIR-II) dyes with excellent properties such as water-solubility and chem/photostability. Here, we discovery an electron acceptor, 6,7-di(thiophen-2-yl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline (TQT) with highest stability in alkaline conditions, compared with conventional NIR-II building block benzobisthiadiazole (BBT) and 6,7-diphenyl-[1,2,5] thiadiazolo[3,4-g]quinoxaline (PTQ). The sulfonated hydrophilic dye, FT-TQT, is further synthesized with 2.13-fold increased quantum yield than its counterpart FT-BBT with BBT as acceptor. FT-TQT complexed with FBS is also prepared and displays a 16-fold increase in fluorescence intensity compared to FT-TQT alone. It demonstrates real-time cerebral and tumor vessel imaging capability with µm-scale resolution. Dynamic monitoring of tumor vascular disruption after drug treatment is achieved by NIR-II fluorescent imaging. Overall, TQT is an efficient electron acceptor for designing innovative NIR-II dyes. The acceptor engineering strategy provides a promising approach to design next generation of NIR-II fluorophores which open new biomedical applications. Small molecule NIR-II fluorophores are of interest for a range of applications but can suffer from chemical and photostability issues. Here, the authors report on the development of an acceptor molecule with improved stability in alkaline conditions expanding the range of possible applications.

Developing high-quality NIR-II fluorophores (emission in 1000–1700 nm) for in vivo imaging is of great significance. Benzothiadiazole-core fluorophores are an important class of NIR-II dyes, yet ongoing limitations such as aggregation-caused quenching in aqueous milieu and non-activatable response are still major obstacles for their biological applications. Here, we devise an activatable nanoprobe to address these limitations. A molecular probe named BTPE-NO 2 is synthesized by linking a benzothiadiazole core with two tetraphenylene groups serving as hydrophobic molecular rotors, followed by incorporating two nitrophenyloxoacetamide units at both ends of the core as recognition moieties and fluorescence quenchers. An FDA-approved amphiphilic polymer Pluronic F127 is then employed to encapsulate the molecular BTPE-NO 2 to render the nanoprobe BTPE-NO 2 @F127. The pathological levels of H 2 O 2 in the disease sites cleave the nitrophenyloxoacetamide groups and activate the probe, thereby generating strong fluorescent emission (950~1200 nm) and ultrasound signal for multi-mode imaging of inflammatory diseases. The nanoprobe can therefore function as a robust tool for detecting and imaging the disease sites with NIR-II fluorescent and multispectral optoacoustic tomography (MSOT) imaging. Moreover, the three-dimensional MSOT images can be obtained for visualizing and locating the disease foci. Fluorescent imaging in the second biological window has advantages for in vivo applications. Here, the authors synthesise a molecular nanoprobe which activates upon binding H 2 O 2 , generating both strong fluorescent NIR-II emission and ultrasound signal for multi-mode imaging of inflammatory diseases.

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. Super-resolution fluorescent imaging typically makes use of intense phototoxic illumination. Here the authors achieve live-cell super-resolution imaging using low-illumination standard microscopes with the aid of a new analytical approach called Super-Resolution Radial Fluctuations (SRRF), provided as an ImageJ plugin.

Sentinel lymph node imaging and biopsy is important to clinical assessment of cancer metastasis, and novel non-radioactive lymphographic tracers have been actively pursued over the years. Here, we develop gold molecular clusters (Au 25 ) functionalized by phosphorylcholine (PC) ligands for NIR-II (1000–3000 nm) fluorescence imaging of draining lymph nodes in 4T1 murine breast cancer and CT26 colon cancer tumor mouse models. The Au-phosphorylcholine (Au-PC) probes exhibit ‘super-stealth’ behavior with little interactions with serum proteins, cells and tissues in vivo, which differs from the indocyanine green (ICG) dye. Subcutaneous injection of Au-PC allows lymph node mapping by NIR-II fluorescence imaging at an optimal time of ~ 0.5 − 1 hour postinjection followed by rapid renal clearance. Preclinical NIR-II fluorescence LN imaging with Au-PC affords high signal to background ratios and high safety and biocompatibility, promising for future clinical translation. Fluorescent tracers facilitate the identification and subsequent collection of tumour draining lymph node biopsies, enabling important clinical assessment. Here, the authors present a molecular gold nanocluster NIR-II fluorescent imaging probe and demonstrate its utility to visualise draining lymph nodes in breast and colon cancer mouse models.

Carbon dots (CDs) possess superior fluorescent properties in that they do not blink, are biocompatible, chemically inert, have small size and well tunable photoluminescence (PL), can be easily functionalized with biomolecules, and can be multi-photon excited to give up-converted PL. This review (with 141 refs.) summarizes recent progress in the field of imaging using carbon dots doped with heteroatoms (X-CDs). Following an introduction, we discuss top-down and bottom-up strategies for synthesis and methods for surface modification. We also compare the differences in synthesis for undoped CDs and X-CDs. Specifically, CDs doped with heteroelemets nitrogen, phosphorus, sulfur, selenium, boron and silicium are treated. We then discuss method for determination of the properties (particle size, ZP), how doping affects fluorescence (spectra, quantum yields, decay times), and how dopants affect upconversion (UC, anti-Stokes luminescence). We finally review the progress made in fluorescent imaging of cells tissue, and other biomatter. This review also gives new hints on how to use synthetic methods for tuning the structure of X-CDs, how doping affects properties, and how to achieve new bioimaging applications. Graphical abstract Carbon dots doped with heteroatoms (X-CDs) are a kind of fluorescent nanomaterials that display bright fluorescence, high quantum yield, photostability, biocompatibility and low toxicity. Hence, they possess large potential for both in-vitro and in-vivo bioimaging.