Extended Data Fig. 1: Design and construction of ELI-TriScope.

(a) Schematic diagram of the ELI-TriScope system showing two major components, a cryo-holder-based cryo-transfer system and a cryo-STAR system. Along the direction of the optical path of the cryo-STAR system, the excitation light beam coming out of the light source is reflected by a dichroic mirror into the aperture, reflected into the objective lens by an adjustable mirror and then excites the fluorescent molecules in the cryo-specimen. Next, the excited fluorescence is received by the objective lens, is reflected into the aperture by the adjustable mirror, passes through a dichroic mirror, is filtered and is focused on a CMOS camera through a tube lens. The cryo-transfer system is equipped with a 3D motorized device that is adapted to the cryo-holder. (b) Photograph of our built ELI-TriScope system based on FEI Helios NanoLab 600i. A custom-designed cryo-transfer system replaces the original chamber door, and the cryo-STAR system is fixed on the other side of the chamber. (c) Photograph of the internal architecture of ELI-TriScope. The cryo-holder is tilted 30 degrees to the right to face the GIS for coating. (d) Observation of the coating status by SEM imaging at 40X magnification after GIS coating. (e) Position for cryo-FIB fabrication. The cryo-holder is tilted to −15 degrees. The objective lens of cryo-STAR rises to the focus position. Each part of the system is indicated and labeled. EB, electron beam. GIS, gas injection system.