In situ determination of orientational distributions in Langmuir monolayers by total internal reflection fluorescence

A new application of the polarized total internal reflection fluorescence (PTIRF) technique to study the orientation distribution of a fluorophore within a Langmuir monolayer in situ on an aqueous subphase is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The excitation by the evanescent field is achieved by launching the beam into a prism that is brought into contact with the monolayer from above. We also show here that a combination of PTIRF of monolayers on water and those freshly deposited onto the prism by horizontal lift in the same experiment provide enough data to determine the dielectric constant of the actual local environment of the fluorophore in the monolayer to eliminate the ambiguity of the orientation determination, arising from uncertainty in the normal component of excitation field. The new technique was applied to several model systems: fatty acid monolayers containing amphiphilic dyes DiI or BODIPY and also a monolayer of a synthetic amphiphilic porphyrin-binding peptide AP0. This technique is more accurate than polarized epifluorescence (PEF) in determining the fluorophore orientation distribution due to the much higher normal component of the excitation, achievable in the evanescent field, and to the lack of surface vibrations caused by capillary waves. Comparison of the new PTIRF approach with PEF shows that the monolayer structure is not disturbed by weak van der Waals attachment to the hydrophobic substrate.