Topical retinoid treatments stimulate biological activities in the skin. The main physical barrier, which limits the efficacy of transdermal drug delivery, is the stratum corneum. Proretinal nanoparticles (PRN) have already been proven to efficiently deliver retinal into the epidermis. In the present study, two transdermal drug delivery systems, microneedles (MN) and PRN, were combined to directly target the dermis. The microchannels induced by the MN, the PRN localization in the microchannels and the skin closure kinetics were investigated by non-invasive imaging techniques, such as dermoscopy, optical coherence tomography and multiphoton tomography. Additionally, the amount of retinal in the epidermis and dermis after application in three different forms (PRN-Loaded microneedles, PRN suspension or conventional retinal solution) was compared. All imaging techniques confirmed the formation of microchannels in the skin, which were partly still detectable after 24 h. Multiphoton tomography showed the release of PRN from the MN within the microchannels. The recovered retinal concentration in the dermis was significantly higher when applied via PRN-loaded microneedles. We hypothesized that this platform of PRN-loaded microneedles can provide a rapid and efficient administration of retinal in the dermis and could be of benefit in some skin conditions such as atrophic scar or photo-aged skin.