Preparation of Legionella pneumophila for Cryogenic Electron Tomography

Abstract

Source: Chetrit, D., et al. Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System. J. Vis. Exp. (2020)

The video describes how to prepare Legionella pneumophila samples for cryo-electron tomography by combining bacterial culture, gold nanoparticle marking, and vitrification techniques. The process preserves native cellular structures and enables high-resolution imaging of membrane-associated secretion system components.

Protocol

1. Detection of sfGFP Mass Density with Cryo-ET

1. Sample preparation, data collection, and reconstruction

1. Grow a heavy patch of Legionella pneumophila expressing a fluorescently tagged Dot/Icm protein on CYE-agar-streptomycin plates for 48 hours at 37 °C. Resuspend the cells in ddH₂O to an OD₆₀₀ of 0.7. Add 5 µL of colloidal gold particles (BSA Tracer, 10 nm) to 20 µL of the cell suspension.

2. Pipette 5 μL of the cell mixture onto a freshly glow-discharged holey carbon grid (R 2/1 on Cu 200 mesh) and let stand for 1 min. Blot with filter paper and freeze in liquid ethane using a gravity-driven plunger apparatus.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 nm colloidal gold particles	Aurion	25486
100x Plan Apo objective (1.4 NA)	Nikon
ACES	Sigma-Aldrich	A9758
CoolSNAP EZ 20 MHz digital monochrome camera	Photometrics
Gene Frame, 1.7x2.8 cm, 125 µL	Fisher Scientific	AB-0578
Holey Carbon grid R 2/1 Cu 200 mesh	Quantifoil	Q225-CR1
Iron(III) nitrate nonahydrate	Sigma-Aldrich	216828
K2 Summit camera for cryo-EM	GATAN
Microscope cover slides 22x22 mm	Fisher Scientific	12-542B
Microscope cover slides 24x50 mm	Fisher Scientific	12-545K
Microscope slides 25x75x1 mm	Globe Scientific	1380
SlideBook 6.0	Intelligent Imaging Innovations
Spectra X light engine	Lumencor