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We performed in situ penetration of single wild-type yeast cell (W303) using nano-pipette inside an environmental scanning electron microscope (E-SEM). An E-SEM nanorobotic manipulator has been constructed with three units and seven degrees of freedom (DOFs) in total. Micro pulling and FIB etching were used to control the tip size of nano-pipette accurately. Cells were fixed on the cooling stage in a perpendicular direction with the nano-pipette. Nano-pipette was assembled on the nanomanipulator to apply penetration force on a single cell. The E-SEM can provide the real-time observation with nanoscale resolution during the experiment. Materials of pipette, translational speed, and tip diameter were three important factors to the successful penetration of yeast cell. We discussed these factors and pointed out the suitable parameters for yeast penetration.