Kinetic analysis of the intracellular processing of sirnas by confocal microscopy.

Here, we describe a method for tracking intracellular processing of siRNA-containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles (sNPs) in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes, and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.