Lung surfactant (LS) is a mixture of lipids and proteins that lines the air-water interface of the alveolar walls. It modulates the surface tension in the lungs which greatly reduces the mechanical work of breathing and also prevents the collapse of the alveoli upon expiration. Although lipids are the major constituent of LS, the hydrophobic surfactant proteins SP-B and SP-C play an integral role in proper adsorption of LS to the air-water interface. Understanding the role of these proteins will allow for better design of exogenous LS therapies for the treatment of respiratory distress syndrome. Confocal microscopy's abilities to optically section a sample and simultaneously image multiple dyes provide an ideal tool to study LS adsorption in vitro. We present here a novel technique for imaging the surfactant system in three dimensions, allowing us to determine axial and lateral concentrations of multiple components. Combining three-dimensional imaging with surface tension measurements allows us to determine whether a particular surfactant mixture can successfully transition from bilayer aggregates in the bulk to a functional monolayer on the interface. SP-B and SP-C are believed to play an integral role in both the transport of aggregates to the interface and the unfolding of surfactant bilayers into a monolayer. Confocal microscopy has allowed us to study the importance of SP-B, SP-C, and lipid composition on the transport and unfolding of LS.