Real time monitoring of penetration through the stratum corneum using confocal laser scanning microscopy is correlated with imaging of skin microstructure using cryo-scanning electron microscopy. The z-series dynamic penetration profiles of fluorescent molecules in the stratum corneum were systematically correlated with cross-sections of skin. Fluorescein salt, fluorescent labeled BSA (~6 nm) and polystyrene latex beads (~30 nm) were used as model penetrants to characterize transport through freestanding stratum corneum. Small molecule fluorescein salt penetrates the stratum corneum within 10 minutes. FITC-BSA is most concentrated in the stratum corneum and detectable in the upper epidermis after 2 hours. Thirty-nanometer latex beads are not transported across the stratum corneum in 2 hours. The penetration of selected molecules or nanoparticles is size-dependent. Freestanding stratum corneum is suitable for penetration studies relevant to vaccine antigen permeation and presentation to underlying dendritic cells of the immune system.