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Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2-3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Monitoring the concentration of Fludarabine phosphate, a standard chemotherapeutic drug widely used in cancer treatment, is vital for ensuring the drug's safety and effectiveness, tailoring treatments to individual needs, and consequently improving overall patient outcomes. Regarding the limitations of conventional techniques in terms of complexity, large time measurements, and a high cost, there is an urgent need to develop simple, rapid, and cost-effective devices. In this paper, we report the design of an aptasensor for the specific and selective detection of Fludarabine. Systematic evolution of ligands by exponential enrichment (SELEX) protocol was performed to select a specific aptamer for Fludarabine. Eleven rounds were carried out, and nine sequences were selected. Based on the dissociation constant (Kd), Ful-3, exhibiting the highest affinity (18.86 nM), was chosen and integrated into a simple electrochemical aptasensing platform for Fludarabine detection. Electrochemical results demonstrated good performance of the selected aptamer in detecting Fludarabine within the analytical range of 1 to 150 pg/mL and with LOD and LOQ in the order of 0.11 pg/mL (0.31 fM) and 0.39 pg/mL (1.06 fM), respectively. The developed platform also showed good selectivity against different analogous molecules and high applicability in serum-spiked samples, with recovery percentages ranging from 96.81 to 99.04%. Considering the encouraging results, this research presents an excellent alternative in terms of simplicity, stability, ease of use, reduction of sample volume, and low cost., (© 2024. The Author(s).)

Broadband Coherent anti-Stokes Raman (BCARS) microscopy is an imaging technique that can acquire full Raman spectra (400-3200 cm -1 ) of biological samples within a few milliseconds. However, the CARS signal suffers from an undesired non-resonant background (NRB), deriving from four-wave-mixing processes, which distorts the peak line shapes and reduces the chemical contrast. Traditionally, the NRB is removed using numerical algorithms that require expert users and knowledge of the NRB spectral profile. Recently, deep-learning models proved to be powerful tools for unsupervised automation and acceleration of NRB removal. Here, we thoroughly review the existing NRB removal deep-learning models (SpecNet, VECTOR, LSTM, Bi-LSTM) and present two novel architectures. The first one combines convolutional layers with Gated Recurrent Units (CNN + GRU); the second one is a Generative Adversarial Network (GAN) that trains an encoder-decoder network and an adversarial convolutional neural network. We also introduce an improved training dataset, generalized on different BCARS experimental configurations. We compare the performances of all these networks on test and experimental data, using them in the pipeline for spectral unmixing of BCARS images. Our analyses show that CNN + GRU and VECTOR are the networks giving the highest accuracy, GAN is the one that predicts the highest number of true positive peaks in experimental data, whereas GAN and VECTOR are the most suitable ones for real-time processing of BCARS images., (© 2024. The Author(s).)

Direct laser acceleration (DLA) of electrons in plasmas of near-critical density (NCD) is a very advancing platform for high-energy PW-class lasers of moderate relativistic intensity supporting Inertial Confinement Fusion research. Experiments conducted at the PHELIX sub-PW Nd:glass laser demonstrated application-promising characteristics of DLA-based radiation and particle sources, such as ultra-high number, high directionality and high conversion efficiency. In this context, the bright synchrotron-like (betatron) radiation of DLA electrons, which arises from the interaction of a sub-ps PHELIX laser pulse with an intensity of 10 19 W/cm 2 with pre-ionized low-density polymer foam, was studied. The experimental results show that the betatron radiation produced by DLA electrons in NCD plasma is well directed with a half-angle of 100-200 mrad, yielding (3.4 ± 0.4)·10 10 photons/keV/sr at 10 keV photon energy. The experimental photon fluence and the brilliance agree well with the particle-in-cell simulations. These results pave the way for innovative applications of the DLA regime using low-density pre-ionized foams in high energy density research., (© 2024. The Author(s).)

Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel., (© 2024. The Author(s).)

Here, we report on the development and application of a compact multi-core fiber optical probe for multimodal non-linear imaging, combining the label-free modalities of Coherent Anti-Stokes Raman Scattering, Second Harmonic Generation, and Two-Photon Excited Fluorescence. Probes of this multi-core fiber design avoid moving and voltage-carrying parts at the distal end, thus providing promising improved compatibility with clinical requirements over competing implementations. The performance characteristics of the probe are established using thin cryo-sections and artificial targets before the applicability to clinically relevant samples is evaluated using ex vivo bulk human and porcine intestine tissues. After image reconstruction to counteract the data's inherently pixelated nature, the recorded images show high image quality and morpho-chemical conformity on the tissue level compared to multimodal non-linear images obtained with a laser-scanning microscope using a standard microscope objective. Furthermore, a simple yet effective reconstruction procedure is presented and demonstrated to yield satisfactory results. Finally, a clear pathway for further developments to facilitate a translation of the multimodal fiber probe into real-world clinical evaluation and application is outlined., (© 2023. Springer Nature Limited.)

The intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging., (© 2023. The Author(s).)

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner., (© 2022. The Author(s).)

In the present paper we report the development of the Continuous Motion scanning technique and its implementation for a new generation of scanning systems. The same hardware setup has demonstrated a significant boost in the scanning speed, reaching 190 cm 2 /h. The implementation of the Continuous Motion technique in the LASSO framework, as well as a number of new corrections introduced are described in details. The performance of the system, the results of an efficiency measurement and potential applications of the technique are discussed.

Acclimation and adaptation, which are key to species survival in a changing climate, can be observed in terms of membrane lipid composition. Remodelling membrane lipids, via homeoviscous adaptation (HVA), counteracts membrane dysfunction due to temperature in poikilotherms. In order to assess the potential for acclimation and adaptation in the honeycomb worm, Sabellaria alveolata, a reef-building polychaete that supports high biodiversity, we carried out common-garden experiments using individuals from along its latitudinal range. Individuals were exposed to a stepwise temperature increase from 15 °C to 25 °C and membrane lipid composition assessed. Our results suggest that S. alveolata was able to acclimate to higher temperatures, as observed by a decrease in unsaturation index and 20:5n-3. However, over the long-term at 25 °C, lipid composition patterns are not consistent with HVA expectations and suggest a stress response. Furthermore, unsaturation index of individuals from the two coldest sites were higher than those from the two warmest sites, with individuals from the thermally intermediate site being in-between, likely reflecting local adaptation to temperature. Therefore, lipid remodelling appears limited at the highest temperatures in S. alveolata, suggesting that individuals inhabiting warm environments may be close to their upper thermal tolerance limits and at risk in a changing climate.