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Microinjection is well recognized as an ideal but technically difficult method for the direct introduction of any molecules into single cells (King, 2004; Matsuoka et al., 2006). The difficulty depends on the cell size, intracellular structure, and the physical properties of the cell surface. Egg cells are an easy target because of their large size of 100–200 μm. Blastocysts are also a large target for the injection of embryonic stem (ES) cells (Fig. 1 (A)). For blastocysts, the tip diameter of an injector is 5 μm or greater. On the other hand, fibroblasts are as small as 20–30 μm but comparatively easy targets because their cell surface appears to be firm. Moreover they appear to be resilient against deep insertion of an injection capillary. By contrast, plant cells are larger than fibroblasts but much more difficult targets. Plant cells usually have large vacuoles in the intracellular space and therefore microinjection should be performed into a thin space between the cell membrane and the vacuole. Typical examples are tobacco cultured cells, BY-2, with a size of 40×80 μm (Fig. 1 (B)) and rice protoplasts with a diameter of 30–50 μm. By comparison, ES cells are extraordinarily difficult targets because of their small size (15–20 μm) (Fig. 1 (C)) and sticky cell surface. In fact, microinjection of a plasmid vector into ES cells was not successfully performed by a microinjection expert until our experiment performed in 2005 (Matsuoka et al., 2005). Before then, microinjection speed was no higher than 10 cells per h and the success rate was 7–8%. In the case of ES cells, the success rate was only 0.2%. Therefore, it was essential to increase the throughput of the method for the purpose of single-cell studies. Thus, we developed a useful robot that could support the microinjection operator; a single-cell manipulation supporting robot (SMSR) (Matsuoka et al., 2005). Using the SMSR, the injection speed increased to 100 cells per h and its success rate reached as high as 10%, even when operated by non-specialist personnel. The quantity of DNA ejected from the injection capillary was estimated to be no greater than 50 fg, and consequently the quantity of DNA actually introduced into a cell was in the fg range. Moreover, when an enhanced green fluorescent protein (EGFP) expression vector was injected into ES cells, the EGFP expression intensity changed in line with the varied concentration of the vector in the injection capillary. This method has been termed femtoinjection because it has enabled semiquantitative injection at the femtogram level (Matsuoka et al., 2007).

Functional Control of Target Single Cells in ES Cell Clusters and Their Differentiated Cells by Femtoinjection