By: Koji Sugioka, Yasutaka Hanada, Katsumi Midorikawa, Ikuko Shihira Ishikawa, Hiroyuki Kawano, Atsushi Miyawaki

RIKEN – Advanced Science Institute, Brain Science Institute

It is becoming increasingly important to observe and analyze the dynamics and functions of microorganisms both for fundamental investigations (such as elucidating the functions of biological cells) and for applications to biomicro systems and medicine. We used femtosecond (fs) laser 3D micromachining to fabricate microfluidic chips (which we term nanoaquariums) for observing microorganisms. Nanoaquariums enable us to drastically reduce the observation time relative to that for the conventional observation method using Petri dishes. Furthermore, they can be used to perform highly functional analysis, which biologists have long desired to realize. We have developed a technique for fabricating nanoaquariums that involves directly forming 3D hollow microstructures with smooth internal surfaces in photostructurable glass by fs laser direct writing followed by annealing and wet etching in dilute hydrofluoric acid (see Fig. 1). This technique permits rapid prototyping of 3D microfluidic systems with different structures, which is greatly desired by biologists for observing different microorganisms. Furthermore, functional microelements such as micromechanical elements and micro optical elements can be easily integrated into the microfluidic structure, permitting more functional observation and analysis to be performed.

Using Nanoaquariums with a simple 3D microfluidic structure (see Fig. 2(a)), we analyzed for the first time the continuous 3D motion of the flagellum of Euglena gracilis from the front of its body (see Fig. 2(b)).

We recently used nanoaquariums to investigate the functions of the cyanobacterium Phormidium. Phormidium is an endosymbiotic bacterium that glides to a seedling root in soil and accelerates its growth. It is important to understand the gliding mechanism to increase vegetable production.

With this goal, we fabricated nanoaquariums with various microfluidic structures embedded in photostructurable glass. These embedded microfluidic structures permitted easy and efficient observation of Phormidium gliding to a seedling root. Integration of optical waveguides and filters in the nanoaquariums by fs laser processing enabled the gliding mechanism to be clarified. We found that Phormidium is attracted by CO2 released from the seedling root. In addition, light with an intensity of over 2380 lx is required. Additionally, only red light is effective for gliding since it efficiently induces photosynthesis by chlorophyll a in Phormidium. We thus conclude that photosynthesis induced by red light drives the gliding of Phormidium.

These results demonstrate the effectiveness of fs laser 3D micromachining for rapid prototyping nanoaquariums with various microfluidic structures and for integrating functional microelements, which enable us to perform highly functional observation and analysis of the dynamics of aquatic microorganisms.

Fig. 1. Schematic illustrations of fabrication procedure of 3D microfludic structures inside the photostructurable glass. (1) 3D latent images are written inside the photostructurable glass by the fs laser direct writing. (2) The samples are conducted to programmed heat treatment to develop the modified regions. (3) The samples are soaked in a solution of 10% hydrofluoric (HF) acid diluted with water in an ultrasonic bath to selectively etch the laser-exposed regions. Finally 3D microfludic structures are formed inside the glass.

Fig. 2. (a) Schematic illustration of nanoaquarium for observation of the continuous 3D motion of the flagellum of Euglena gracilis. The scheme (1) permits side-view observation of Euglena gracilis swimming in the microchannel, while the observation from the front of its body is realized by using the scheme (2). (b) Optical microscope image of front view of Euglena gracilis swimming upward in the reservoir.