Abstract
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.
Highlights
The field of cell biology emerged in the 17th century, when van Leeuwenhoek used a light microscope to observe microscopic objects including bacteria and human cells
As discussed above Spatial Light Interference Microscopy (SLIM) measures the complex field at each z-position the intensity distribution in far field or the scattering plane may be determined by calculating its Fourier Transform, a technique known as Fourier Transform Light Scattering (FTLS) 57]
Comparing the scattering maps obtained from the SLIM and deconvolved data, it is clear that there is more information available at higher scattering angles or spatial frequencies corresponding to smaller structures
Summary
The field of cell biology emerged in the 17th century, when van Leeuwenhoek used a light microscope to observe microscopic objects including bacteria and human cells. Since its inception, cell microscopy has contended with two major issues: lack of contrast, due to the thin and optically transparent nature of cells, and diffraction limited resolution. The key development that essentially combined the intrinsic and exogenous contrast imaging fields is to genetically engineer the cell to express green fluorescent protein (GFP) [4]. The advent of this technology made it possible to genetically modify a cell such that it naturally expresses GFP and binds it to prescribed cellular structures, allowing the imaging of living cells
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