期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：47
DOI：10.1073/pnas.2115323118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
We propose the integration of chip-based optical nanoscopy with high spatially sensitive quantitative phase microscopy to obtain three-dimensional (3D) morphology of liver sinusoidal endothelial cells (LSEC). LSEC contain large numbers of transcellular nanopores —“fenestrations”—in the plasma membrane, typically clustered in groups of 10 to 50 within areas called sieve plates. Determining the diameter and the height of fenestrated regions is an important indicator of a cell’s functionality, and these dimensions can be influenced by agents such as drugs. Our proposed multimodal microscope offers a solution for 3D nanoscale characterization of fenestration diameter and measurement of the optical thickness of the sieve plates.
Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.
