Abstract
This 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.
Highlights
Dynamic observation and analysis of the motions of biological samples are important for investigating the functions of their specific body parts, such as the flagella of microorganisms, which help us to understand the locomotion mechanisms in many biological processes observed in the life sciences and to develop artificial microswimmers and bioinspired systems[1,2,3,4,5]
We have previously developed hybrid subtractive and additive femtosecond laser microfabrication (fs laser-assisted wet etching (FLAE) of glass followed by water-assisted fs laser modification combined with electroless metal plating), which enables the selective metal deposition of microfluidic structures from the inside out without requiring any complicated photolithography, alignment, or bonding processes[36]
Dynamic observation of flagellar motions of microorganisms Observation of flagellar motions of Euglena cells in electrofluidic devices was performed under a differential interference contrast (DIC) microscopy system (IX71, Olympus Inc.) equipped with a CCD camera (FASTCAM SA3 Model 120K-C3, 500–1000 fps, shutter speed: 1 ms; Photron Limited, Tokyo, Japan)
Summary
Dynamic observation and analysis of the motions of biological samples (cells, microorganisms, and so on) are important for investigating the functions of their specific body parts, such as the flagella of microorganisms, which help us to understand the locomotion mechanisms in many biological processes observed in the life sciences and to develop artificial microswimmers and bioinspired systems[1,2,3,4,5]. To perform a flexible observation, including frontview observation, rapidly and with high efficiency, 3D nondestructive control of microorganism motion along a designable direction in microfluidic environments is highly desirable. Electrical manipulation is an effective approach to controlling the motions of biological samples in microfluidic devices that offers many advantages, such as low cost, ease of operation, high controllability, and high efficiency. It enables a broad range of lab-on-a-chip applications in biological and medical research[11,12,13,14,15,16]. We demonstrate the fabrication of new electrofluidic devices in which designable geometries of electrodes are integrated at the desired positions in
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