High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM.

High-lateral-resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a nonlinear, nonequilibrium sputter rate model for the virions and validate the model on the basis of reconstructing the location of the DNA within individual virions. Our experiments with a Cs+ beam show an unexpectedly high initial sputter rate (∼100 um2·nm·pA-1·s-1) with a rapid decline to an asymptotic rate of 0.7 um2·nm·pA-1·s-1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O- and Ga+ beams, yielding similar results. Based on our Cs+ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage.