P2211/B529
Observation and AI supported analysis of Lipid Droplet Dynamics in Live Organoid using Label-Free 4D High-resolution Holotomography.
H.-J. Kim1 , H. Lee1 , J. Lee1 , M. Kim1 , S. Lee1 , Y. Park 1,2; 1 Tomocube, Inc., Daejeon, Korea, Republic of, 2 Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, Republic of

Abstract: 

Holotomography imaging allows for label-free, 4-dimensional (X,Y,Z, and Time) observation of biological processes in organoids, which is crucial given the 3-dimensional structure of organoids that mimics actual organs. This structure reflects cellular interactions and provides deeper insights into the mechanisms of biological processes. Due to these features, acquiring the 4-dimensional behavior of organoids is essential for understanding biomolecular mechanisms. However, existing imaging techniques, such as fluorescence microscopy, have limitations, including phototoxicity and the need for pre-processing of biological samples like immunostaining. In this research, we successfully visualized the dynamics of lipid droplets in a hepatosteatosis model using mouse hepatic organoids. The hepatosteatosis models were induced by treatment with multiple combinations of free fatty acids, and accumulation of lipid droplets was observed for more than 48 hours without fluorescence staining. Holotomographic imaging visualizes biological samples based on the refractive index, and the refractive index of lipid droplets is higher than that of other cellular components, making them ideal for label-free visualization through HT imaging. Not only obtaining image data of biological processes but also analyzing the massive amount of data is important to interpret and gain more insight into these processes. As the performance of artificial intelligence models improves, it becomes possible to apply them across various fields, including the analysis and annotation of biological images. In this study, we employed an AI model to segment lipid droplets from organoid cell layers and conducted quantitative analysis using TomoAnalysis software, which offers customized image analysis processes. This approach provided automated and accurate annotations of subcellular components, enhancing our ability to analyze complex multimodal images.





P1149/B152
Real-Time Visualization and Analysis of Mitochondrial Fission and Fusion with Holotomography imaging.
H.-J. Kim1 , J. Lee1 , H. Hoang1 , M. Kim1 , S. Lee1 , Y. Park1,2; 1 Tomocube, Inc., Daejeon, Korea, Republic of, 2 KAIST, Daejeon, Korea, Republic of

Abstract:

The balance between mitochondrial fission and fusion is essential for cellular homeostasis, with imbalances implicated in numerous diseases, making these dynamics essential biomarkers. However, morphological analysis of mitochondria using fluorescence and electron microscopy is limited by phototoxicity, photobleaching, and an inability to capture live cell dynamics. This study utilizes Holotomography (HT) imaging with the HT-X1 system for non-invasive, real-time, and 3D analysis of mitochondrial morphology without the need for fluorescent staining. We specifically examine mitochondrial structural changes in Hep3B cells following MPP+ and Mdivi-1 treatments, capturing dose-dependent and temporal mitochondrial dynamics. Through HT, we observed significant morphological changes, quantitatively assessed using TomoAnalysis software enhanced with machine learning for accurate mitochondrial segmentation and length measurement. Importantly, utilizing a 96-well format, this methodology allows for simultaneous imaging and analysis of multiple wells under various conditions, significantly enhancing throughput and experimental efficiency. Through this methodology, we achieved detailed mitochondrial segmentation from HT images, enabling the measurement of individual mitochondrial lengths and the quantitative assessment of their distribution changes in response to drug dosage and over time. These sophisticated methodological approaches substantially advance mitochondrial research, providing reliable tools for biomarker identification and a more detailed understanding of the cellular processes involved in disease pathogenesis.





P2285/B606
PAK1-Mediated Upregulation of Macropinocytosis and Vacuole Accumulation in Senescent Cells.
K. Kozik, K. Kono; Okinawa Institute of Science and Technology, Okinawa, Japan

Abstract: 
Cellular senescence, a sustained cell cycle arrest, has been implicated in various age-related diseases, including frailty, dementia, diabetes, and cancer. As organisms age, senescent cells accumulate, and their removal ameliorates various age-associated disorders. Senescent cells remain metabolically active, exhibiting changed morphology, secretion of pro-inflammatory cytokines, and cytoplasmic vacuolization. However, the mechanisms underlying the accumulation of cytoplasmic vacuoles in senescent cells are poorly understood. Here, we show that macropinocytosis, an endocytic process that enables cells to internalize extracellular material through the formation of large vesicles, explains the vacuole accumulation in senescent normal human fibroblast cells. Using holotomography, we observed pronounced plasma membrane ruffling and an increased formation of vacuoles in senescent cells. Specifically, we showed a significant increase in the number, area, and volume of cytoplasmic vacuoles in senescent human WI-38 and BJ fibroblasts, as well as senescent human osteosarcoma U2OS cells. Furthermore, we detected an upregulation of macropinocytosis in senescent cells, evidenced by increased uptake of high molecular weight dextran. Senescent cells induced by distinct stimuli, such as DNA damage, plasma membrane damage, or telomere shortening, uniformly showed upregulated macropinocytosis. siRNA-mediated knockdown of PAK1, a critical regulator of actin cytoskeleton POSTERS-776 dynamics, significantly reduced macropinocytosis, decreased vacuole accumulation, and altered gene expression profiles. Our findings identify PAK1 as a key regulator of macropinocytosis in senescent cells, providing new insights into the mechanisms of vacuole accumulation in cellular senescence and and potential therapeutic targets for age-related diseases.