I. Inoue

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells.

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cells growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.

Simultaneous measurement of sensor-protein dynamics and motility of a single cell by on-chip microcultivation system

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information.

Differential Analysis of Single-Cell Cultivation Using on-Chip Microculture System and Optical Trapping

We developed the system for one cell level differential analysis employing on-chip microculture system and optical trapping. The experimental result showed that system had the ability to reveal the fluctuations in growth rate and division intervals of genetically identical Escherichia coli cells even in a homogeneous condition. This indicates the importance of the new approach for one cell level phenomenon.

Observation of Individual Cell Using on-Chip Culture System

We developed an on-chip continuous culture system for single-cell analysis. The advantages of the system are: (i) contamination-free continuous cultivation of isolated single cells or groups of cells using semipermiable membrane lid on the microchamber wells with biotin-streptavidin attachment; (ii) 0.2-µm resoltion for phase-contrast/fluorecent real time continuous observation using 5–20 µm deep microchambers on 0.2-mm-thick glass slide; (iii) flexible medium buffer exchange; and (iv) flexible cultivation chamber size. Using the system we examined the chamber size dependency of Escherichia coli’s growth, and found no significant difference between 20-µm and 70-µm microchambers.