We examined two-dimensional (2D) optical feedback control of phototaxis flagellate Euglena cells confined in closed-type microfluidic channels (microaquariums), and demonstrated that the 2D optical feedback enables the control of the density and position of Euglena cells in microaquariums externally, flexibly, and dynamically. Using three types of feedback algorithms, the density of Euglena cells in a specified area can be controlled arbitrarily and dynamically, and more than 70% of the cells can be concentrated into a specified area. Separation of photo-sensitive/insensitive Euglena cells was also demonstrated. Moreover, Euglena-based neuro-computing has been achieved, where 16 imaginary neurons were defined as Euglena-activity levels in 16 individual areas in microaquariums. The study proves that 2D optical feedback control of photoreactive flagellate microbes is promising for microbial biology studies as well as applications such as microbe-based particle transportation in microfluidic channels or separation of photo-sensitive/insensitive microbes.

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We demonstrate on-chip gas/liquid sensing by using the chemotaxis of live bacteria (Euglena gracilis) confined in an isolated micro-aquarium, and gas/liquid permeation through porous polydimethylsiloxane (PDMS). The sensing chip consisted of one closed micro-aquarium and two separated bypass microchannels along the perimeter of the micro-aquarium. Test gas/liquid and reference samples were introduced into the two individual microchannels separately, and the gas/liquid permeated through the PDMS walls and dissolved in the micro-aquarium water, resulting in a chemical concentration gradient in the micro-aquarium. By employing the closed micro-aquarium isolated from sample flows, we succeeded in measuring the chemotaxis of Euglena for a gas substance quantitatively, which cannot be achieved with the conventional flow-type or hydro-gel-type microfluidic devices. We found positive (negative) chemotaxis for CO2 concentrations below (above) 15%, with 64 ppm as the minimum concentration affecting the cells. We also observed chemotaxis for ethanol and H2O2. By supplying culture medium via the microchannels, the Euglena culture remained alive for more than 2 months. The sensing chip is thus useful for culturing cells and using them for environmental toxicity/nutrition studies by monitoring their motion.

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We found that the transient freezing behavior in photophobic responses of Euglena gracilis is a good indicator of the metabolic status of the cells. The transient blue light photophobic responses of E. gracilis cells were investigated on-chip using a new measurement, 'trace momentum' (TM), to evaluate their swimming activity quantitatively in real time. When blue light of intensity >30 mW cm(-2) was repeatedly switched on and off, a large negative spike in the TM was observed at the onset of the 'blue-light-off' phase. Single-cell trace analysis at a blue light intensity of 40 mW cm(-2) showed that 48% (on average, n = 15) of tumbling Euglena cells ceased activity ('freezing') for 2-30 s at the onset of blue-light-off before commencing forward motion in a straight line (termed 'straightforward swimming'), while 45% smoothly commenced straightforward swimming without delay. The proportion of freezing Euglena cells depended on the blue light intensity (only 20% at 20 mW cm(-2)). When the cells were stimulated by four blue light pulses at the higher intensity, without pre-exposure, the transient freezing behavior was more prominent but, on repeating the stimuli after an 80 min interval in red light, the same cells did not freeze. This shows that the metabolism of the cells had changed to anti-freezing during the interval. The relationship between the interval time with/without light irradiation and the blue light adaptation was elucidated experimentally. The origin of the freezing behavior is considered to be a shortage of a metabolic substance that promotes smooth switching of flagellum movement from in situ rotation mode to a straightforward swimming mode.

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