Fig. 2: Cryo-CLEM workflow using ELI-TriScope.

a, Cells labeled by fluorophores are cultured on an OD grid (T11012SS, TIANLD), loaded into an FEI AutoGrid (Thermo Fisher Scientific), vitrified by plunge freezing and then loaded onto a cryo-holder. b, Screening of cryo-vitrified cells using cryo-FM (optional). The ice thickness and the fluorescence signal are checked at this step. The bright-field atlas merged with fluorescence images of the grid can be acquired, and the positions of cells with good fluorescence signals (orange and red arrows) are recorded. c, Real-time fluorescence signal-navigated cryo-FIB milling by ELI-TriScope. During the cryo-FIB process, the cryo-fluorescence intensity of the target is monitored in real time. In the SEM and fluorescence images from left to right, three different stages during cryo-FIB milling are presented: the beginning stage (left), the middle stage with one side rough milled (middle) and the final stage with two sides fine milled (right). The fluorescence signals from two centrioles are indicated by red and orange arrows and monitored in real time. A line profile of the intensity of the fluorescence signal is correspondingly plotted below for the three stages. A schematic diagram of the thickness of the cryo-lamella at different stages is shown at the bottom. d, The prepared cryo-lamella is transferred into a cryo-transmission electron microscope (TEM) (cryo-TEM) for cryo-ET data collection. Top, cryo-EM micrographs of the cryo-lamella at low (×3,600, left) and high (×33,000, right) magnifications. Bottom, a zoomed-in view of the cryo-EM micrograph at high magnification to show the centrioles, which are labeled with orange and red arrows. e, Statistics of our whole cryo-CLEM workflow from grid vitrification to cryo-ET tilt series data collection.

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