Q150 coater

After time-lapse imaging, the cell culture medium in the dish was removed, and the platform was washed twice with 1% PBS for 5 min each time. Then, the cells were fixed with 4% paraformaldehyde for 15 min. After cell fixation, the platform was washed with 1% and 0.25% PBS for 5 min each, followed by rinsing twice in DI water for 10 min each time. Subsequently, cells were dehydrated for 5 min each time using a series of increasing ethanol concentrations (30%, 50%, 70%, 80%, 95%, and 100%). Cells were dried using a critical point dryer, in which CO₂ was the transitional medium. A thin layer of gold was then sputter-coated on the platform using a thin film coater (Q150 coater, Quorum Technologies Ltd., UK). High-resolution images of the fixed cells were captured using a field emission scanning electron microscope (SEM) (SU5000 FE-SEM, Hitachi, Japan) with a 10 kV electron beam. These images were used to analyze the number and length of filopodia and long protrusions of cells. Typical filopodia were narrow with widths of 200–400 nm and lengths of 4–30 μm. Long protrusions had widths >400 nm, and protrusion length was defined as the distance between the edge of the cell membrane and the protrusion tip, which could be 5–50 μm long.

Cells on platforms were washed twice with PBS and fixed for 15 min with 4% paraformaldehyde. The cells were then treated with ethanol at concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100% for 5 min each, and then dried in a critical point dryer (EM CPD300, Leica, Germany) for 4 h. To prevent charging during imaging in the scanning electron microscope (SEM, SU5000 FE-SEM, Hitachi, Japan), a thin layer of Au was coated onto the dried samples using a thin film coater (Q150 coater, Quorum Technologies, Lewes, UK). The coated samples were then mounted onto a 51 mm stub using conductive tape. Samples without and with cells were captured using a secondary electrons detector with a 10 kV accelerating voltage to achieve high-resolution images.

After time-lapse imaging, cells were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, USA) for 15 min. The platform was then washed with 1% and 0.25% PBS for 5 min each, and then rinsed twice in DI water for 10 min each. Subsequently, cells were dehydrated using a series of increasing ethanol concentrations (30%, 50%, 70%, 80%, 95%, and 100%) for 5 min at each concentration. Cells were dried using a critical point dryer. A thin layer of gold was then sputter-coated on the platform using a thin film coater (Q150 coater, Quorum Technologies Ltd., UK), which provided a conductive surface for imaging. High-resolution images of the fixed cells were captured using a field emission scanning electron microscope (SEM, SU5000 FE-SEM, Hitachi, Japan) with a 10 kV electron beam. These images were used to analyze the cell morphology, providing insight into the structure and organization of the cells on the platform.