The sub-project will focus on the development of the new alternatives of the micro-patterned support surfaces for selective adhesion of cells and reproducible deposition of such multi-material films on the 3mm TEM grids. The sub-project will thus deliver 3 mm TEM grid which will help to increase the throughput of cellular lamella preparation by cryo-FIB. The project will include several sub-tasks: (I) development of multilayer nano-fabricated surfaces with general pattern for cell adhesion and testing of the selective adhesion of the cells; (II) optimization of the protocol for transfer of the surfaces onto the various types of the TEM grids; (III) development of surfaces allowing adhesion of the cells into specific shape; (IV) testing applicability of the developed support films on different cell lines. We plan to deliver the protocols for the preparation of a multi-layered surface with spatially distinct properties deposited on the 3mm TEM grid which will be used for focused ion beam micromachining of cellular lamellas. Our industry partner will test suitability of such a design for utilization with the Autogrid and its potential for increasing the automation level of the lamella preparation process by cryo-FIB. Cryo-ET has developed to the structural biology technique capable of delivering structural information about molecular sociology in situ. However, the preparation of the suitable sample for cryo-ET is still very challenging primarily due to the low yield of the high-quality cellular lamellas transferred to the transmission electron microscope for tilt series acquisition. In case of adherent mammalian cells, the sample preparation comprises adhesion of the cells onto the sample carrier (typically carbon film on the TEM grid), sample vitrification, and preparation of thin lamella (<300nm) using FIB under cryo-conditions. The main objective is the development of the support film for selective adhesion of cells which will be deposited on the TEM grid. While there is enormous development of the instrumentation for cellular lamella preparation by cryo-FIB, the quality of the samples introduced into FIB instrument and reproducibility of the sample preparation is currently the major limiting step efficient utilization of the FIB instrumentation.

Klíčová slova
precise targeting