Label_Free Optical Diffraction Tomography를 통한 다양한 세포의 물리학적 특성 및 세포 내 조성 변화 비교 분석

Abstract

Label-free optical diffraction tomography (ODT) enables the observation of cells and cellular organelles without the need for fluorescent labels or other preprocessing. It overcomes the constraints associated with conventional cell imaging techniques like fluorescence microscopy or electron microscopy. In the case of electron microscopy or fluorescence microscopy, which provide 2D images, a preprocess of the cell is necessary, which can induce cell invasion and cause changes in the physical and chemical composition inside the cell compared to live cells. However, ODT provides 3D images of cells and cellular organelles, as well as physical parameters such as refractive index, volume, and dry mass, allowing for the characterization of microenvironment changes within cells and internal biological properties without any preprocess applying on cell. In addition, when observing cells with ODT, Various preprocess on cell such as cell fixation, gene modification, immunofluorescence, and various cell trackers for biomarker can be applied to observe cells in different condition. With these preprocess, physical parameters obtained from ODT distinguish various types of cells under different conditions. Label-free cells and cells subjected to various manipulations undergo changes in both morphology and internal composition, which can be quantitatively evaluated through RI rendering by converting these changes into numerical values. Utilizing the advantages of ODT, observation of cells enables the discrimination of physical characteristics between similar cell categories in a live state. Additionally, specific markers within cells can be targeted through labeling, resulting in internal changes that can be quantitatively evaluated. In this study, numeric parameters such as the refractive index and volumes of human stem cells, fibroblasts cell, and HeLa cells were measured using ODT. Differences in refractive index caused by various physical stimuli, such as temperature, cell fixation, GFP protein tagging, and organelle tracking, were also compared. Three types of stem cells (hLD-SCs, hUCM-MSCs, and hiPSC) were campared to fibroblasts. The results revealed that stem cells exhibited widely distributed vesicles with larger volumes and higher mean RI values compared to fibroblasts. After cell fixation and an increase in temperature, a significant overall decrease in the refractive index (RI) value of organelles, such as the nucleus and cytoplasm, was observed. Interestingly, while the RI values of cell nuclei were affected, the RI values of the cytoplasm were barely detected after membrane permeation. Moreover, the expression of GFP and GFP-tagged proteins was found to markedly increase the RI values of organelles in live cells, demonstrating more effective changes in RI compared to chemical fluorescence staining for cell organelles. This method enables the identification of differences between cells without the need for specific biological markers and indirectly assesses the impact of external stimuli on cellular organelles and this information can serve as a valuable reference for future studies that employ other conventional microscopy techniques.