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Transparent three-dimensional microstructures for the trapping of living cells

The simultaneous manipulation and observation of morphological features of single living cells along with the recording of functional changes related to cellular metabolism, interaction and communication in real time are of growing interest. With the advent of atomic force microscopy (AFM) structural studies of native cells became possible which in combination with adequate light microscopy give a much better resolution than light microscopy alone. However, the motion of the cell, the softness of the cell membrane and the two-dimensional growth of cells in culture limit applicability and resolution of this technique. A good mechanical fixation of living cells in a structure could be achieved by embedding cells into partially covered grooves produced in Si/SiO2. However, for additionally optical microscopy studies a transparent material is essential. In this study we present the fabrication of different transparent three-dimensional structures in a three- layer system (Si3N4, SiO2, Si3N4) on quartz specially sized for the trapping of living neural cells under physiological conditions. For the fabrication of the structures we utilized a combination of e-beam lithography (EBL), laser ablation, reactive ion beam etching (RIBE) and different wet etching techniques. The structures consist of a nano-net of Si3N4 covering a micro-cavity in SiO2 confining enough to prevent the cell body from escaping, but not so constraining that it hinders normal growth and development. Another type of presented structures consists of cavities which are connected by covered micro-channels, hence, an observation of cell-cell interactions is also possible. Advantages of these microstructures are the trapping of cells, the stabilization of the cell membrane and the precise placement of the cells for a multitude of biological investigations.