In the example below, a comparison of results from the analysis performed on AAV samples using cryoTEM and nsTEM is shown. Three different samples composed mainly of particles representing three classes: empty (S1), filled (S3) and a mix of sample S1 and S3 (S2), were analyzed. The results obtained from the analysis of the images from cryoTEM versus nsTEM are illustrated below:
The results shown in the graphs demonstrate that the two methods, cryoTEM (left) and nsTEM (right), when used to analyze AAV particles with internal dark pixel intensity measure two different characteristics. The cryoTEM shows the expected linear correlation of the increasing concentration of filled particles. This proves that the particles observed with cryoTEM with internal dark pixel intensity represent particles containing DNA. The particles characterized with nsTEM that show internal staining do not show any correlation with the increasing concentration of DNA but instead exhibit a flat trend. This is mainly caused by the differences in the two techniques. In cryoTEM, the sample is embedded in a thin layer of ice, which allows the observation of the internal composition of the particles. Therefore, in cryoTEM images, if the capsid contains DNA there will be an internal pixel density (dark particle), while if the capsid does not contain DNA the internal density will be very close to the background of the image.
In contrast, when using nsTEM there is an embedding of the sample in a thin layer of stain, which means that if the particles are intact the stain will cover the shell of the particle (bright particles) and no information regarding the DNA content will be possible to conclude since only the shell is observed. IIf the particles are damaged or broken, the stain will penetrate into those particles and a darker internal density in the particle (dark particle) will be seen. The internal staining of the particles using nsTEM is caused by the stain creeping into the particle due to disintegration of the capsids and is thus a good method for comparing particle integrity in different sample preparations but not the content of the capsids in terms of DNA.