Pharmaceutical applications of Confocal Laser Scanning Microscopy D. Cosco 1,2 , S. Bulotta 1 , D. Paolino 1,2 and M. Fresta 1,2,* 1 Department of Health Sciences, University “Magna Græcia”of Catanzaro, Campus Universitario “S. Venuta” - Building of BioSciences, Viale S. Venuta, I-88100 Germaneto - Catanzaro, Italy 2 IRC FSH- Interregional Research Center for Food Safety & Health, University “Magna Græcia”of Catanzaro, Campus Universitario “S. Venuta” - Building of BioSciences, Viale S. Venuta, I-88100 Germaneto - Catanzaro, Italy * Corresponding author: fresta@unicz.it Confocal laser scanning microscopy (CLSM) is a suitable tool used for investigating the physico-chemical features of pharmaceutics and analyzing their performance in numerous in vitro and in vivo models. Even though the applications of this technology are manifold, during the last decade it has been particularly applied in the field of drug delivery. In fact, CLSM allows the recognition of the interaction phenomena that occur between micro-/nano-systems and cells, the investigation of the localization of many colloids inside cells, and the exploration of the permeation/diffusion rates of these systems through biological barriers (i.e. blood brain barrier, gastrointestinal epithelium, pulmonary tissue, skin layers). In detail, CLSM is helpful in distinguishing the cell interaction/permeation profiles of fluorescent micro- and nano- pharmaceutics as a function of their technological properties and in furnishing the evidence of efficient selective targeting through the use of suitable compounds. The cellular localization of nanosystems is another important aspect that has been intensely investigated with the help of this technique in order to study the selective delivery of specific compounds, such as in the case of various genetic materials--pDNA, mirNAs, sirNAs, etc. In this chapter these aspects will be described with the aim of providing a brief report about the most important applications of CLSM in micro- and nano-drug delivery. Keywords: CLSM; drug delivery systems; liposomes; micro- and nanoparticles 1. Introduction The 20 th -century scientific and technological revolution led to sweeping changes in the field of microscopy, producing new tools able to bypass the theoretical resolution limits derived from physical laws. As a result, it is now possible to have information from living organisms at a resolution of 200 nm, the limit imposed by the formula of Ernst Abbe: where Ȝ is the wavelength of the collected light; sinα has to do with the angular aperture of the objective lens; and n is the refractive index that describes light propagation when the lens is immersed in a medium. Among the new instruments resulting from the aforementioned technological revolution, the confocal laser scanning microscope (CLSM) is dependable to the 3 rd spatial dimension of biological systems. Due to its scarce invasiveness, fluorescence microscopy is an essential investigation tool in biomedical research, since the structures observed in the laboratory are very close to the biological situations to which they actually belong. This is because, unlike electron microscopy, the sample preparation required for CLSM is totally biocompatible [1-3]. Conventional fluorescence microscopy is based on wide-field illumination involving the homogenous illumination of the sample. As a consequence, all the fluorescent molecules inside the specimen will be excited and the final image will be produced both from the light of the plane of interest but also the light coming from adjacent planes (out-of-focus). CLSM solves this problem by introducing two new elements resulting from the presence of tiny holes or “pinholes” along the optical pathway of the microscope [4,5]. The first element is that the focal plane of the objective lens is illuminated by a laser crossing an illumination pinhole in front of it; the second is that the collection of light emission is limited by a detection pinhole with a controlled opening in front of the detector (Fig.1). An optical section is created since the detection pinhole filters only the light of the focal plane, blocking light emission from layers above and below it. Both the illumination and detection pinholes have the same focal plane, hence the term “confocal”. In wide-field microscopy, the acquisition of the whole image is immediate because fluorescence is induced simultaneously at all points in the field of interest, while in confocal microscopy image acquisition is performed by sequential, point by point illumination of the specimen, followed by three-dimensional reconstruction. Ȝ d = 2n sinα Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________ 995 © FORMATEX 2014