Microalgae are photosynthetic organisms that appeared on Earth more than three billion years ago. Diatoms, Euglena and other members of this family typically inhabit in the sea or fresh water, and possess a very simple, unicellular form. Some of them are even able to move using tiny appendages known as flagella. Perhaps the easiest and simplest example for use in scientific experiments for school children, algae are also in high demand for next-generation industrial research and development as a raw material for the production of biofuel.
At RIKEN, researchers are unraveling a variety of hidden functions of single-cell, flagellated algae that swim in fresh water using a newly developed ‘nano-aquarium’. Far from an ordinary fish tank for ornamental purposes, the nano-aquarium is actually a tiny microchip; a glass plate just five square millimeters in size embedded with flow channels and micro-devices. The algae, which normally swim around at lightning speed, move within the tightly controlled channels, and sometimes are given physical stimuli using a movable micro-needle in order to observe their response. Such nano-manipulation techniques provide great assistance for analyzing the detailed mechanisms of algae using RIKEN’s cutting-edge optical microscopes, opening up a new realm of biological and evolutional research on these ancient microorganisms.
The great potential of algae
The nano-aquarium project was initiated by Hiroyuki Kawano, a research scientist specialized in laser physics in the Laboratory for Cell Function Dynamics of the RIKEN Brain Science Institute in Wako, Saitama. Kawano joined the laboratory in 2003 to take advantage of his expertise gained at the Laser Technology Laboratory (headed by Katsumi Midorikawa) of the RIKEN Advanced Science Institute, also in Wako. The Laboratory for Cell Function Dynamics, headed by Atsushi Miyawaki, is at the world’s forefront in the development of fluorescence proteins and related optical technologies.
In 2005, Miyawaki’s laboratory created a high-performance video microscope for in vivo observation of neurons in collaboration with Olympus and Kinki University. Ikuko Ishikawa, a former professor of Tokyo Gakugei University and specialist in algal physiology, was invited to act as a research scientist on the project with the aim of utilizing micro-organisms as a sample to maximize the performance of the new device. However, “soon after we started experiments we found it very difficult to capture clear images of microalgae even with our cutting-edge microscope because some algae move around too fast to be observed, and others make unexpected movements,” Ishikawa says. “So, I consulted with my colleagues to see whether we can control their behavior,” Kawano adds.
The result of those consultations was the nano-aquarium project, which was awarded funding under the category of ‘challenging research’ from the President’s Fund. The project was kicked off in 2006 by four researchers—Kawano and Ishikawa from the Laboratory for Cell Function Dynamics, and Koji Sugioka, a senior research scientist, and Yasutaka Hanada, a special postdoctoral researcher, from the Laser Technology Laboratory.
Direct fabrication of three-dimensional voids using femtosecond lasers
The development of the nano-aquarium microchip would not have been achieved without Sugioka’s femtosecond laser expertise. Ultrashort light pulses can be focused into a spot with a diameter of just 0.3 micrometers, equivalent to the length of a virus. These ultrashort pulses of light make it possible to achieve the processing precision necessary to fabricate the fine channels and voids that make up the nano-aquarium. Longer pulses of light, such as those produced by conventional lasers, cause heating in the materials leading to thermal expansion and cracking. Femtosecond lasers are therefore becoming known as the next-generation technology for shaping glass, semiconductor and even diamond with nanoscale precision.
Around 2002, Sugioka started creating three-dimensional tunnels inside a glass plate by shifting the focal point of laser beam a fraction at a time. The fabrication of voids is completed by immersing the patterned glass plate in acid solvent, which etches away the glass around the tunnels. The manufacturing process for the nano-aquarium differs from the process used to fabricate common microfluidic chips, which are fabricated using semiconductor-etching technology and are used to control and analyze the biological and chemical properties of a tiny amount of fluid sample.
Based on his laser-processing expertise, Sugioka joined hands with Hanada to develop the nano-aquarium. Sugioka recalls that one of the greatest technical challenges was to create channels with a square cross-section in order to give the observer a clearer view of a sample movement under the microscope. Hanada also developed a built-in, movable needle that researchers can use to stimulate the algae samples. “By trial and error I adjusted the beam strengths, exposure time and pulse length to process the nano-device in just the right way,” he says. Sugioka adds that they can even make a micro-wheel to generate water flow in the channel.
Unraveling the secrets of algae
Kawano’s team has been developing a number of different microchips, each tailored to a different sample, because the design of channels and microdevices determines the angle of observation. “It was fascinating to see directly and clearly how chloroplasts get assembled in the middle of algae when stimulated with a micro-needle,” Ishikawa says. Kawano has recently discovered that another type of algae uses its flagella in a different manner to what had been long believed. “We could confirm the finding because we made it swim vertically in the H-shaped channel. Other researchers would have no choice but to observe it two-dimensionally in a petri dish,” Kawano notes.
The team published a joint paper1 in 2008, and although the support of the President’s Fund ended in March 2009, the researchers continue to work together to polish the quality of the microchips and uncover more truths about algae using their own research funding. “I am grateful for our team’s enthusiasm for collaboration,” Ishikawa says. “Thanks to them, we have now entered a new stage in the dynamic and beautiful world of colorful microorganisms.”