Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2012;7(6):e39816. doi: 10.1371/journal.pone.0039816. Epub 2012 Jun 28.
Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.
研究活细胞的三维亚细胞结构对于我们理解生物功能至关重要。然而，对活细胞进行断层成像具有挑战性，主要是因为它们是透明的，即弱散射结构。因此，这种类型的成像主要使用荧光技术来实现。虽然共聚焦荧光成像是实现切片的常用方法，但它需要荧光探针，而这些探针通常对活标本有害。另一方面，通过利用结构的固有对比度，可以以非侵入性的方式研究活细胞。一种提供有关纳米级结构的高分辨率定量信息的方法是一种称为空间光干涉显微镜（SLIM）的宽带干涉技术。除了提供定量相位信息外，当与高数值孔径物镜结合使用时，SLIM 还提供出色的深度切片能力。然而，与所有线性光学系统一样，SLIM 的分辨率受到衍射的限制。在这里，我们提出了一种新颖的 3D 场反卷积算法，该算法利用相位图像的稀疏性，并提供超出衍射极限的分辨率的图像。我们使用这种称为反卷积空间光干涉层析成像（dSLIT）的无标记方法来可视化大肠杆菌细胞中的螺旋状亚细胞结构，这些结构很可能是细胞骨架 MreB 蛋白和分裂位点调节 MinCDE 蛋白。以前，这些结构仅使用专门的菌株和质粒以及荧光技术观察到。我们的结果表明，dSLIT 可以用于以实际和非侵入性的方式研究这些结构。
