Electron microscopy requires samples to be viewed in a high vacuum environment to ensure clear electron paths. For biological samples, this creates a unique challenge, leading to the development of special preparation techniques. Among these, negative staining is the most common and simplest method. The process involves spreading a sample droplet on an EM grid with a carbon support film, then adding a heavy metal stain (typically uranyl acetate). After blotting to create a thin film and air drying, the sample is ready for viewing. The term "negative" comes from how the stain surrounds rather than binds to the sample, creating high-contrast images where biological structures appear light against a dark background. This method's popularity stems from its simplicity and effectiveness in producing detailed images, thanks to the strong electron scattering from the metal stains.
Techniques
Negative Staining Technique
Cryo-Electron Microscopy (Cryo-EM)
Unlike negative staining, cryo-electron microscopy (cryo-EM) allows us to view samples in their natural, hydrated state. This technique involves rapid freezing of the sample in liquid ethane at approximately -188°C, creating a vitrified (glass-like) state. The main advantages are preservation of natural structure without the distortions that can occur during traditional staining and drying, and higher resolution imaging due to the natural contrast between proteins and their surrounding solution. The most critical aspect is achieving the proper ice thickness - too thick and electron scattering becomes problematic, too thin and the sample structure might be damaged. This delicate process is now aided by automated devices like the Vitrobot. Samples are typically prepared on specialized holey carbon films rather than continuous carbon films to reduce background noise and improve image contrast.
Single Particle Analysis (SPA)
Electron microscopy images are 2D projections of 3D objects, requiring multiple views from different angles to reconstruct the complete structure. This reconstruction process, called single particle analysis, collects images of identical particles in random orientations (typically 1,000-10,000+ particles). The process involves sophisticated computer analysis where 2D images are first aligned and classified into groups, then averaged to enhance the signal-to-noise ratio. These processed images are used to determine 3D orientations and computationally reconstruct the complete structure. This reconstruction is iteratively refined until a stable 3D model is achieved, with specialized software packages like EMAN, XMIPP, SPIDER and more recently developed RELION and cryoSPARC enabling near-atomic resolution reconstructions.
Single Particle Analysis with Helical Reconstruction
The single particle analysis approach extends beyond globular proteins through the Iterative Helical Real-Space Reconstruction (IHRSR) method, enabling 3D reconstruction of helically ordered structures like actin filaments, tobacco mosaic viruses, and microtubules. The process starts with a basic reference volume (typically a cylinder) and generates multiple projections at different angles. The angular spacing depends on the object's diameter and expected resolution, following the formula 360°*d/(2πD). These reference projections are then compared with thousands of actual image segments, aligned, and used for 3D reconstruction. The process iteratively refines by finding and imposing the optimal helical symmetry parameters (rotation and axial translation) to generate an increasingly accurate model.