- Use a microloader to load the microinjection capillary with
3–5μL of the diluted target RNA solution. This volume is
sufficient for injection of several hundreds of cells. (seeNote 21). - Attach the filled microinjection capillary to the microscope
capillary holder. - Position the microinjection capillary with the joystick so that the
tip is at the center of the field of the view on the microscope. - Retract the capillary by pressing the “Home” button on the
microinjection system. - Place the glass bottom dish containing cells microporated with
MBs on the microscope stage. - Find the focal plane.
- Press the “Home” button to return the tip to the original
position at the center of the field of view on the microscope.
Slowly lower the microinjection capillary to immerse the tip in
the media (if not already immersed) but not yet in contact with
any cells. - Adjust the compensation pressure to be at least 15 psi (seeNote
22 ). - Adjust the injection parameters to Pi (injection pres-
sure)¼100 psi and Ti (injection time)¼1s(seeNote 23). - Press “Quick Clean” several times to remove the residual air in
the tip of the capillary. - Use the joystick to move the tip over the top of a cell.
- Set the Z limit (injection level).
(a) Focus on a cell.
(b) Lower the tip to gently touch the cell membrane until a
gentle wave traverses through the cell from the site of
injection.
(c) Press the “Limit” key on the InjectMan NI 2 control
board to set the height as the Z limit. - Raise the capillary approximately 20–30μm above the cell so
that it can be moved freely without touching the cells, with the
tip still visible. This height corresponds to the search level. - Move the microinjection capillary out of the field of view using
the joystick. - Focus on a cell to be injected.
- Acquire fluorescent images of the cell using the Cy5 filter set.
- Use the joystick to move the capillary over the top of the
imaged cell.
252 Mingming Chen et al.