Living Cell Manipulation and In Situ Nanoinjection Based on Frequency Shift Feedback Using Cantilevered Micropipette Probes

This paper presents a method based on frequency shift feedback for living cell manipulation and in situ nanoinjection with a cantilevered micropipette probe (CMP). This method can detect tip–cell interactive forces at the piconewton level by measuring the frequency shift of the rigid CMP oscillated in the first bending eigenmode (amplitude: ~10 nm). Different interaction states throughout the process of manipulation and injection, including the contact, gripping, detaching, and cell (nuclear) membrane penetration, can be well detected and thereby controlled. In addition, the cell adhesion can be quantified by the integral of the frequency shift during the detachment process. Manipulation and nanoinjection are automatically performed using two types of CMPs with different apex aperture diameters (manipulation: Ø $4~\mu \text{m}$ ; nanoinjection: Ø200 nm), under the control of the dynamic force and microscope vision feedback. The proposed method can control the contact force of ~ 300 pn for nondestructive cell manipulation and can detect cell membrane (~250 pn) and nuclear membrane (~400 pn) penetration forces in nanoinjection. The versatility and robustness of the proposed method are further demonstrated by quantifying the cell-substrate adhesion, the building of cell patterns, and automated cell nanoinjection. Note to Practitioners—Cell manipulation and injection are the essential processes for most cell-based bioengineering applications. Cell manipulation enables not only cell transport and pattern building but also adhesion measurement both between cells and the external matrix. On the other hand, in situ cell injection is a significant process for cell-based medicine development and genetics research. Automated force-controlled cell manipulation and injection will greatly reduce the requirements of the user’s experience and increase the efficiency and consistency of the experiment. This paper introduces a method based on frequency shift feedback for obtaining highly accurate monitoring of the interactions (at the piconewton level) between cells and developing tools for automated and nondestructive manipulation and injection. The research outcome provides a unique solution to achieve high-precision cell manipulation and in situ nanoinjection.