In this paper, an automatic system for single cell analysis inside an environmental scanning electron microscopy (ESEM) was proposed. Single yeast cell was put on an tungsten probe substrate inside ESEM. The endeffector for single cell analysis was fixed to an nanorobotic manipulator, which has three degrees of freedom, i.e. X, Y and Z translation. The real time images during the experiment can be observed by ESEM system in realtime. Therefore, the position of the endeffector and the single cell can be recognized by imaging processing. These position information were used as the feedback signal to control the movement of the nanorobotic manipulator. Finally, a single cell cutting experiment was performed to demonstrate the working mechanism of this system. Two types of cell pattern substrates were also designed and fabricated as the cell analysis chips for the automation single cell analysis in the future.

In this paper, a novel oscillating nano knife for single cell cutting at its native state was proposed. The nano knife with a small edge angle 5° was fabricated from a commercial AFM cantilever by focused ion beam (FIB) etching. The spring constant of the nano knife was calibrated by nanorobotic manipulation method. A piezo actuator was employed to generate the vibration of the nano knife. The oscillating amplitude response to the applied voltage was calculated based on the realtime image observation. Single yeast cell (W303) cutting using oscillating nano knife was performed based on the nanorobotic manipulation system inside an environment-SEM. The maximum cutting forces and the slices angle were measured from the deformation of the nano knife beam and the single cell. The result showed the oscillating nano knife was capable and effective for single cell cutting task.

This paper presents the local stiffness measurements of Caenorhabditis Elegans (C. elegans) by buckling nanoprobes through the nanorobotic manipulation system inside an environmental-scanning electron microscope (E-SEM). C. elegans has complex outer and inner structures constructed by approximately one thousand cells. For example, the lateral alae are the surface fine structure by seam cell body. In this paper, their fine structures were observed by E-SEM directly; without any drying or dyeing processes. The observation environments are controlled under different E-SEM chamber pressures for clear observation of C. elegans. The local stiffness of C. elegans was measured by buckling measurement method of the nanoprobe fabricated by focus ion beam (FIB) etching at the tip of atomic force microscope (AFM) cantilever. The measurement position can arbitrarily be controlled by the nanorobotic manipulator inside the E-SEM. In this work, from experimental results, the measured elasticity on lateral alae was 1.6 times larger than it around lateral alae. This local stiffness measurement technique can readily be applied to reveal unknown biological local stiffness, cell health conditions and novel cell diagnosis.

We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM) - nanomanipulator system. Compression experiments to penetrate a cell wall of an individual cell under two difference conditions, i.e. ESEM (600 Pa) and high vacuum (HV) (about 3 mPa) modes have been conducted. Data from the experiments clearly show a significant increase of about twenty five times of penetration force values from ESEM mode to HV mode; i.e. 152 nN and 4 muN for 5 mum cell size with sharp tip indenter. This can be further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e. ESEM mode (2.16 plusmn 1.21 MPa) and HV (20.58 plusmn 2.09 MPa) for all cell sizes with sharp tip indenter. Further analysis also revealed that the size of the cells can influence its mechanical strength. As the size of the cell increases, the compression force needed to penetrate its wall also increases. From these experimental results, we can conclude that the force needed to penetrate a single cell does not only depend on the internal factor, i.e. cell size but also being influenced by the external factors, i.e. the environmental conditions.