Search Results (3)

Freeze–thaw procedures impair sperm morphology and function, affecting viability, motility, redox balance, and subcellular organization. Although antioxidants may mitigate these effects, their interaction with donor-specific variability remains unclear. We combined quantitative holotomography with conventional physiological assessments within a multivariate framework based on principal component analysis (PCA) and nested cross-validated Linear Discriminant Analysis (LDA) to evaluate donor-specific responses to antioxidant-supplemented cryopreservation. Spermatozoa from ten stallions was analyzed before and after freezing under five conditions: fresh semen; frozen semen with INRA Freeze, frozen semen with HF-20, and HF-20 supplemented with matcha, spirulina, horseradish, or quercetin. For each condition, sperm kinetics, mitochondrial activity, oxidative stress, DNA integrity, and three-dimensional volumetric measurements of whole-cell and subcellular compartments derived from holotomography were integrated into a single dataset. LDA achieved 0.734 cross-validated accuracy for stallion classification, revealing strong donor-specific signatures. In contrast, classification by antioxidant treatment was near chance (0.248). Fresh semen was clearly distinct from all cryopreserved groups. Holotomography showed reduced whole-cell and post-acrosomal/midpiece volumes after freezing, while nuclear volume was unchanged. Antioxidant supplementation produced minor, inconsistent effects, with partial midpiece preservation in some donors but no global pattern. Overall, inter-stallion variability dominates post-thaw sperm phenotype. Antioxidant effects were detectable but modest, supporting individualized strategies to optimize equine semen cryopreservation protocols. Full article

Background/Objectives: Holotomography is an advanced imaging technique that enables high-resolution, three-dimensional visualization of microscopic specimens without the need for fixation or staining. Here we aim to apply holotomography technology to image live Hypsibius exemplaris in their native state, avoiding invasive sample preparation procedures and phototoxic effects associated with other imaging modalities. Methods: We use a low concentration of 7% ethanol for tardigrade sedation and sample preparation. Holotomographic images were obtained and reconstructed using the Tomocube HT-X1 system, enabling high-resolution visualization of tardigrade anatomical structures. Results: We captured detailed, label-free holotomography images of both external and internal structures of tardigrade, including the digestive tract, brain, ovary, claws, salivary glands, and musculature. Conclusions: Our findings highlight holotomography as a complementary high-resolution imaging modality that effectively addresses the challenges faced with traditional imaging techniques in tardigrade research. Full article

One major challenge associated with lung cancer organoids (LCOs) is their predominant derivation from surgical specimens of patients with early-stage lung cancer. However, patients with advanced lung cancer, who are in need of chemotherapy, often cannot undergo surgery. Therefore, there is an urgent need to successfully generate LCOs from biopsy specimens. Conventional lung biopsy techniques, such as transthoracic needle biopsy and forceps biopsy, only yield small amounts of lung tissue, resulting in a low success rate for culturing LCOs from biopsy samples. Furthermore, potential complications, like bleeding and pneumothorax, make it difficult to obtain sufficient tissue. Another critical issue is the overgrowth of normal lung cells in later passages of LCO culture, and the optimal culture conditions for LCOs are yet to be determined. To address these limitations, we attempted to create LCOs from cryobiopsy specimens obtained from patients with lung cancer (n = 113). Overall, the initial success rate of establishing LCOs from cryobiopsy samples was 40.7% (n = 46). Transbronchial cryobiopsy enables the retrieval of significantly larger amounts of lung tissue than bronchoscopic forceps biopsy. Additionally, cryobiopsy can be employed for peripheral lesions, and it is aided via radial endobronchial ultrasonography. This study significantly improved the success rate of LCO culture and demonstrated that the LCOs retained characteristics that resembled the primary tumors. Single-cell RNA sequencing confirmed high cancer cell purity in early passages of LCOs derived from patients with advanced lung cancer. Furthermore, the three-dimensional structure and intracellular components of LCOs were characterized using three-dimensional holotomography. Finally, drug screening was performed using a specialized micropillar culture system with cryobiopsy-derived LCOs. LCOs derived from cryobiopsy specimens offer a promising solution to the critical limitations of conventional LCOs. Cryobiopsy can be applied to patients with lung cancer at all stages, including those with peripheral lesions, and can provide sufficient cells for LCO generation. Therefore, we anticipate that cryobiopsy will serve as a breakthrough strategy for the clinical application of LCOs in all stages of lung cancer. Full article