Fig. 1: Schematic overview of the IncelluloED experimental workflow.
From: Single-cell structural biology with intracellular electron crystallography
First, intracellular protein crystals are produced and detected inside cells using the InCellCryst pipeline2 (panel 1). Selected cells are collected and plunge-frozen on transmission electron microscope (TEM) grids. The top surface of the frozen samples is coated with a thin platinum layer deposited by sputtering at cryogenic temperatures. The Pt layer enhances surface contrast during subsequent reflected light microscopy, reduces charging, and protects the sample during subsequent imaging in a dual-beam focused ion-beam scanning electron microscope (FIB/SEM). The workflow continues with cryo-light microscopy (panel 2), where regions of interest and the coordinates of crystals relative to the sample surface are determined using combined 3D dual-channel reflected light and negative fluorescence microscopy. The samples are then cryo-transferred into a cryo-FIB/SEM (panel 3), where the top sample surface is imaged. Correlation of the FIB/SEM images with the light microscopy data from panel 2 enables localization of target crystals beneath the surface. Fiducial markers are then generated on the sample using the FIB for subsequent targeted milling. At this point, an additional protective organoplatinum coating is applied using a gas injection system (Pt GIS), and crystal lamellae with a thickness of 250–300 nm are produced by cryo-FIB milling. Finally, the frozen lamellae are transferred into a cryo-TEM (panel 4), where a complete 3D electron diffraction (ED) dataset is collected from a single crystal lamella to determine the protein structure.
