Improved system for capillary microinjection into living cells.

Abstract A technique for microinjection into cells is described, with several simplifications as to preparation and handling of micropipettes, sample filling and injecting system. The simplified procedure enables us to pull and fill the capillaries in an easy, reproducible and fast way, requiring only about 1 min. The injecting system uses three positive pressure levels (high, injecting, holding) controlled from one button. It provides for quick and reproducible changes among the three pressures. Furthermore, it limits the chances for clogging the microcapillary by elimination of the temporary negative pressure (suction at its tip), which would normally occur when the pressure is reduced from the injecting to a zero (atmospheric) pressure value. The estimated sample ejection rate which is achieved with this system could correspond to 10−11–10−13 ml/sec, according to [6]. Compared with other known techniques using pressurized gas in bottles, this ejected volume is three orders of magnitude smaller and makes the system of interest for work with cells smaller than 50 μm. Whereas the sample volume injected into cells is reproducible, further improvement in the control of the amount actually injected is desirable. The manipulation of the sample ejection system is very easy and no time is needed to learn it. The user can inject the sample volume either in the cytoplasm or the nucleus, as desired, by manipulating a single button. Operational aspects and problems with capillary clogging are discussed. The reliability of the whole system and technique is shown in the results obtained on microinjection of living cells. Cells could be injected at an initial speed of about 600–800 cells per hour, the actual number of cells injected in 1 h being about 400. After injection of SV40DNA, 70–80% of the injected cells have synthesized the T-antigen. The above improvements make the injection technique easily accessible to molecular biologists who need to screen for expression of the recombinants made in vitro, or to study the distribution of fluorescently labelled structural proteins. The pressure, the valves and the penetration of the cell (piezo driver) may be regulated electronically. The system would be of interest for any automated injection process. When the common pressure regulator (from 150 to 1 bar) is available, the cost of the additional equipment shown in fig. 2b is as low as 500 DM.