Cryo electron microscopy is a structural biology technique that images biological molecules in a near native, frozen hydrated state. Samples such as proteins, nucleic acids and large complexes are rapidly vitrified in a thin layer of amorphous ice, preserving their structure without chemical fixation or staining. Imaging is performed at liquid nitrogen temperatures in a transmission electron microscope, using very low electron doses to minimize radiation damage.

In single particle analysis, thousands to millions of images of individual particles in random orientations are recorded. Sophisticated image processing and classification algorithms align these projections and reconstruct a three dimensional density map. Advances in direct electron detectors, microscope stability, automated data collection and computational methods have pushed the achievable resolution to near atomic levels. This “resolution revolution” allows visualization of side chains, ligands and water molecules in many cases, enabling detailed mechanistic insights.

Cryo electron tomography extends the approach to larger, more heterogeneous or contextualized systems, such as organelles, cells or viruses in situ. A series of tilted images is collected from the same region, then combined into a three dimensional volume that reveals native ultrastructure. Subtomogram averaging can improve resolution for repeated motifs.

Modern workflows integrate sample preparation, microscope automation and high performance computing. Remaining challenges include preferred particle orientation, beam induced motion, radiation damage and image processing bottlenecks. Nonetheless, the method has transformed understanding of membrane proteins, large molecular machines and dynamic assemblies that are difficult to crystallize, and it increasingly complements or replaces X ray crystallography and nuclear magnetic resonance in structural biology.