From Bench to Beam: A Complete Correlative Cryo Light Microscopy Workflow

In this on-demand webinar, you will hear from experts at the European Molecular Biology Laboratory (EMBL) who have used cryo-CLEM in their own research. They explore the entire workflow, from sample preparation to cryo-electron microscopy data collection.

The cryo-CLEM workflow involves several key steps:

• Sample preparation includes choosing appropriate grids onto which cells are grown, or a specimen is applied before blotting and vitrification is performed using a plunge freezer.
• Subsequently, the vitrified specimens are imaged using cryo-fluorescence microscopy. To ensure a fast and safe loading, a cryo shuttle is used to transfer the vitrified specimens to the cryo stage of the STELLARIS Cryo confocal microscope. Both the cryo stage and cryo objective for low-temperature imaging allow straightforward imaging. Dedicated software helps obtain overviews and high-resolution confocal images, all while the sample remains under controlled cryogenic conditions. Various imaging modalities are leveraged to characterize samples and optimize the signal-to-noise ratio.
• The fluorescence data are leveraged in subsequent workflow steps. A dedicated sample cartridge is used to safely transfer the samples into the FIB instrument. The fluorescent data from the previous step are used to identify regions of interest for milling. The tilted ion beam image in conjunction with 3DCT software helps with the lamella positioning prior to FIB milling.
• Successful lamella milling is followed by a re-check of fluorescence signal at the milled positions, through imaging under the cryo STELLARIS microscope. This additional confocal information can be utilised for correlation with cryo-TEM data at the end of the workflow.