Welcome
Here you will find everything you need to get started and stay informed — from preparing samples for negative stain and cryo-TEM imaging and operating preparative equipment, to screening samples, collecting data, and managing your results.
We are committed to keeping these protocols accurate and up to date, and we welcome your feedback as we continue to grow and improve this resource. As our team carefully reviews and incorporates updates, we appreciate your patience — and encourage you to check back regularly for the latest information.
Negative Stain Sample Preparation
Negative stain TEM is a method for rapidly assessing the quality of your sample for future EM-level imaging experiments. The sample, which can be a purified macromolecule or complex, virus, bacteria, or other object, is applied to an EM grid and embedded in a thin layer of a heavy metal salt solution (stain). The stain improves specimen contrast so that gross and finer details of the sample may be observed.
Below we have provided a primer on negative stain TEM and our basic facility protocol that has been used for preparing negative stain EM grids of proteins, other macromolecules, viruses, and bacteria for downstream imaging in the L120C 120 kV TEM.
Cryo-EM Sample Preparation
Once you have optimized your sample in the ‘wet-lab’ and examined it by negative stain TEM and/or other means, the next step is to prepare your cryo-EM specimens. Depending on the sample, there are a number of approaches to take for sample freezing (vitrification). Traditionally, most aqueous samples of macromolecules, viruses, small non-adherent cells, and other materials can be vitrified successfully by plunge freezing. Briefly, a few microliters of a sample are applied to an EM grid, blotted with filter paper to produce a thin layer of solution across the EM grid, and then plunge frozen into a cryogen. There are several sample vitrification solutions available in the CEMRC and surrounding laboratories at UW-Madison. The two you will most commonly use for samples prepared by plunge freezing are the TFS Vitrobot and Leica EMGP2.
TFS Vitrobot Mark IV. The Vitrobot is a semi-automated vitrification robot for preparing cryo-EM grids via plunge freezing. The Vitrobot program incorporates a number of user-specified settings that allow for flexibility based upon the requirements of the sample and the user’s needs. Below, we have provided the facility protocol manual for using the Vitrobot and the trainee and trainer checklists we use for signing each user off for independent operation of the Vitrobot. Additional links to information about the Vitrobot and its use are presented in the facility protocol manual.
Microscopes and Microscope Operations
The Cryo-EM Microscope Operations section contains standardized protocols and operational guidance for facility instrumentation, including procedures for microscope startup and shutdown, sample loading, alignments, cryogenic handling, contamination prevention, routine maintenance, and troubleshooting. These documents are intended to support safe, consistent, and efficient operation of the microscope systems by trained users and facility staff. Protocols such as the Talos L120C operation guide provide step-by-step instructions and best practices specific to individual microscope platforms and associated equipment.
Data Processing
To access and manage your imaging data using Globus, please follow the steps outlined in this guide.