Negative stain

Negative staining was originally developed for light microscopy in order to surround and delineate unstained bacteria and other biological materials. In the field of EM, Farrant used negative staining in 1954 to study the structure of ferritin. In 1955 phosphotungstic acid was used to stain negatively tomato bushy stunt virus, and in the following years to demonstrate the axial channel in tobacco mosaic virus. The principle of the negative staining is straightforward. An electron dense stain surrounds the biological specimen and penetrates into the structural crevices to give an image in which the biological specimen appears electron lucent against the dark electron dense background. The stain may also penetrate the interior to reveal internal structure as well. The image is mostly formed by the absorption or deflection of the electrons by the stain, giving opacity to these areas. The contrasting electron dense and electron lucent areas project an exact 2D image on the microscope viewing screen or the photographic media.
We use different negative staining methods to evaluate single small organelles like synaptic vesicles or even large individual proteins (molecular weights upwards of
200 KDa). This method is limited on structures between a few nm and maximal approximately 300nm. If the specimen is smaller, the signal to noise ratio is not sufficient to determine the structure. On the other hand, if the specimen is too large the biological material resulting in dark images without any structural information.

Farrant, J.L., An electron microscopic study of ferritin. Biochem., Biophys. Acta, 13, 569, 1954