Couette flow fluorescence detected linear dichroism for analytes in lipid bilayers

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J-1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut-off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the leakage of photons by the long pass filters used is also discussed.

Automated summary

In this study, a Jasco J-1500 circular dichroism spectropolarimeter was used with a microvolume Couette flow cell and appropriate cut-off filters to measure the excitation fluorescence detected linear dichroism (FDLD) of fluorophores in liposomal membranes. The FDLD spectrum selectively probes the fluorophore(s) and eliminates scattering, providing valuable information on analyte orientations in the membrane. Data for gramicidin, anthracene, and pyrene are presented, and issues with photon leakage from the long pass filters used are discussed.