Cryo-EM Platform

MBExC Cryo-EM Platform

Introduction

The cryo-EM platform of MBExC has been established to enable high resolution analysis of molecular architecture of biological samples in near-native state. It houses state-of-the-art instrumentation that enables the complete workflow of in situ cryo-electron tomography (cryo-ET) and also single particle analysis (SPA).

Workflow

Vitrification - Vitrobot Mark IV
Vitrification is the starting point of the whole cryo-EM workflow. The vitrobot is a plunge freezer with a humidity and temperature controlled chamber so that purified protein or cells (on a EM grid) are quickly plunged into liquid cryogren (ethane or ethane/propane mix) to achieve vitrification.

Cryo-light microscopy - Cryo CLEM Thunder Imager
Frozen fluorescently labeled samples can be first analyzed by cryo-light microscopy. The cryo CLEM Thunder Imager is a wide field cryo light microscope. It is a fluorescence microscope designed for working with frozen biolgoical samples. It enables cryo correlative light electron microscopy (cryo CLEM) workflow that allows localization of small subcellular target for subsequent steps in cryo-FIB milling and cryo-TEM imaging.

Cryo FIB milling - Aquilos2
Frozen biological samples often need to be thinned down to around 100-200 nm thickness for hight resolution TEM imaging. The Aquilos2 is a dual beam system equipped with a scanning electron microscope (SEM) and a focused-ion-beam (FIB) optimized for thinning down of frozen biological materials. It also includes an integrated fluorescence light microscope (iFLM) for locating feature of interest using cryo-correlative imaging.

Cryo-TEM - Titan Krios G4
The Titan Krios G4 is a cryo Transmission Electron Microscope (cryo-TEM) operating at 300 kV for high resolution imaging frozen materials at liquid nitrogen temperature. It is equipped with an autoloader for quick exchange up to 12 samples, a Falcon4i direct electron detector with a Selectris energy filter for high resolution imaging. The microscope is optimized for single particle analysis (SPA) and also cryo-electron tomography (cryo-ET).