Optical detection in microscopic domains. 3. Confocal analysis of fluorescent amphiphilic molecules.

This series of papers demonstrates the feasibility of novel optical methodologies for microchemistry and analysis in aqueous samples of nano-, pico-, and femtoliter in volume. Not a conventional glass cuvette, but water-immiscible organic liquid, is used as the container for microscopic sample droplets in this approach. In part 1, absorption spectra of excellent quality were obtained and used for analysis from samples as small as a few tens of a micrometer in diameter. In part 2, an inert fluorescence marker as an internal standard was employed for indirectly detecting absorbing but nonfluorescent reagents in microsamples, employing inner filter effects. In this part 3, a third modality, confocal fluorescence microscopy, is added to the techniques being examined. A clearly visible emission ring emanating from an amphiphilic molecule, doxorubicin, at the sample boundary is demonstrated for the first time in "optically sliced" microdroplets. Relative intensity of this ring with respect to sample bulk can be used to study adsorption phenomena at liquid-liquid interfaces with proper calibrations for bulk and boundary. Quantitative separation of these two domains, a precondition to such calibrations, is also discussed.