Engineering approaches for three dimensional single cell imaging

Progress in understanding the inner structure of cells, as well as the cell-matrix structures, is driven by advances and crossover of many disciplines – from physics and engineering to biology. A number of high resolution imaging methods have been evolved to investigate the fine details of biological cells by implementing photons, electrons, X-rays and physical probes, yet proper strategies remain in demand to obtain three dimensional structural and chemical information. The aim of the proposed research is to develop novel approaches for trapping and imaging of biological cells by strategically deploying a set of nanoengineering tools. Firstly, two potential cell isolation methods were developed. Using ion implantation in biocompatible polydimethylsiloxane (PDMS) material with varied ion fluence, accelerating voltage and ion incidence angle, Young’s modulus of PDMS could be regulated from tens of MPa to 2 GPa for controlled cell growth. Additionally, scale-like micro cantilevers on silicon nitride membrane were also fabricated with Focused Ion Beam (FIB) milling, which effectively trapped bacterial cells in the designated suspension flow. In the second phase of the research, FIB tomography was employed to image and assess the detailed 3D ultrastructures of single Klebsiella pneumoniae cells treated with antibiotics polymyxin B. Significant reduction in volume ratio (cytoplasm to cell envelop) and increase in cell length in the treated group were revealed, implying transforming of cytoplasm regions. Finally, Klebsiella pneumoniae cells were probed at nanometer resolution by combining FIB and Atomic Force Microscopy (AFM), and the intracellular mechanics and hydrophobicity properties were acquired by AFM force spectroscopy for structural and chemistry analysis. With a range of functionalized AFM tips currently available, the proposed approach will allow localization of various proteins and drugs to provide unique mechanistic insights into microbial systems.