Files

Action Filename Description Size Access Comment License License Resource Version
Show more files...

Abstract

The development of atomic force microscopy (AFM) has enabled a major breakthrough in the study of individual biological objects, such as nucleic acids, proteins and protein complexes. More recently the use of AFM to investigate eukaryotic cells has been explored. In one approach, the AFM probe can be used as a needle that delivers material into a single living cell while the AFM microscope controls precisely the interactions between the probe and the biological sample. The work presented here was dedicated to the development of a microinjection system for single cells based on atomic force microscopy. Demonstration experiments of liquid delivery into cells were also performed in order to characterize the system, its potential and its limits. As the injection of liquid into a cell requires the insertion of the tip into a cell, a detailed study of AFM probe-cell interactions was carried out. In the introduction microinjection into adherent cells, its applications and limitations are described. The main limitation of this method is lack of control over the cell penetration. Since atomic force microscope (AFM) offers this possibility, a novel microinjection tool for liquid delivery into single adherent cells based on the AFM is proposed in this work. A case study examines the specifications of an AFM-based microinjection system, such as control of delivered volume and control of AFM-probe cell interactions. Given the specifications, a detail design of the system is proposed with an AFM probe with microfluidic channels (NADIS) as a core component. In next two chapters, the fabrication and characterization of the system is presented including the flow of liquids through the NADIS probes. Some limitations of the system are discussed together with possible approaches to improvement. Further, an in depth analysis of cell indentation is undertaken. Aspects such as determination of tip insertion and factors influencing the probability of cell membrane penetration by an AFM tip are discussed. Cell membrane rupture with an AFM probe is described with a simple mechanical model. Biophysical analysis of the tip insertion is presented followed by development of a five parameter analysis of force-separation curves. In addition the effect of tip penetration on cell viability is addressed. Finally, the AFM-based microinjection system is used to deliver liquids into individual adherent cells. Microinjection into the cytoplasm, but not into the nucleus is demonstrated. The experiments study possible system leakage, clogging of the tip opening with cell residues and injection parameters. Finally the probe-cell interactions during the injections are analysed.

Details

Actions

Preview