Examples of incompletely vitrified CDM lift-out lamellae. (A) CDMs were vitrified in cell culture medium without cryoprotectants. (A1) Overview of a whole cryo-lift-out lamella. The lamella covers roughly 15 µm of CDM depth, ranging from proximal to the EM grid substrate (z = 0 µm) to close to the CDM surface (z = 15 µm). Cell bodies are annotated in transparent red color. (A2) Zoom-in of the lamella as annotated with a red rectangle in A1. Areas with reflections caused by incomplete vitrification are marked with a white, dashed line. (A3) Zoom-in of the area annotated with a yellow rectangle in A2, showing high-contrast ECM structures of interest. (B) Example of a lamella with incomplete vitrification, despite the use of cryoprotectant (cell culture medium, with 10% BSA, degassed). (B1) Lamella overview. Strong reflections are observed throughout the area (marked with a white dashed line). (B2) Zoom-in into the lamella, as annotated by a yellow rectangle in B1. The reflections obscure cell and ECM details, while the background would have been acceptable. (C) Example of a lamella with too high background, introduced by the cryoprotection buffer (20% dextran, 5% sucrose in PBS, without degassing). (C1) Lamella overview. Weak reflections can be seen throughout the area (white dashed line), resulting in a categorization of the vitrification status as incomplete. (C2) Zoom-in into the lamella, annotated by a yellow rectangle in C1. The high background reduces the visibility of cellular and ECM structures. Scale bar dimensions are annotated in the figure.