AbstractThis paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional (3D) manipulation of microorganisms by hybrid subtractive and additive femtosecond (fs) laser microfabrication (fs laser-assisted wet etching of glass followed by water-assisted fs laser modification combined with electroless metal plating). The technique enables the formation of patterned metal electrodes in arbitrary regions in closed glass microfluidic channels, which can spatially and temporally control the direction of electric fields in 3D microfluidic environments. The fabricated electrofluidic devices were applied to nanoaquariums to demonstrate the 3D electro-orientation of Euglena gracilis (an elongated unicellular microorganism) in microfluidics with high controllability and reliability. In particular, swimming Euglena cells can be oriented along the z-direction (perpendicular to the device surface) using electrodes with square outlines formed at the top and bottom of the channel, which is quite useful for observing the motions of cells parallel to their swimming directions. Specifically, z-directional electric field control ensured efficient observation of manipulated cells on the front side (45 cells were captured in a minute in an imaging area of ~160×120 μm), resulting in a reduction of the average time required to capture the images of five Euglena cells swimming continuously along the z-direction by a factor of ~43 compared with the case of no electric field. In addition, the combination of the electrofluidic devices and dynamic imaging enabled observation of the flagella of Euglena cells, revealing that the swimming direction of each Euglena cell under the electric field application was determined by the initial body angle.