SIM of KSHV clusters and their detection in PEL cells. (A) The distribution of size and intensities of miniKLacO clusters in SLK cells measured with SIM and CAPS confirms that, in general, they cannot be separated into smaller units. The structures of clusters in one cell (A) with up to sixfold differences in their intensities are homogeneous as resolved by SIM (a–c). A depth-encoded volume projection shows that the largest cluster (d), which has 48-fold higher intensity, is inhomogeneous. See also Video 2. (B) Numbers of KSHV and EBV genomes were measured by FISH and quantitative PCR in two PEL cell lines, BC-1 and BC-2, in the same cell populations and showed clustering of KSHV. Conventional FISH does not distinguish signals derived from one genome versus a cluster of genomes, whereas quantitative PCR measures the mean total number of viral genomes per cell in a population of cells. The number of viral genomes was quantified by detecting the copies of viral genes, LANA1 for KSHV and BALF5 for EBV, with linearized plasmid DNAs as standards and normalized to the copies of cellular Rhodopsin. These measurements (four experiments, two using BC-1 and two using BC-2) show that PEL cells have 30% of their KSHV genomes as clusters; as expected, EBV genomes do not cluster.