Ellipsometry of thin films of biological objects under conditions of total internal reflection

An analysis of the ellipsometric parameters of the reflected light from the prism test material air system is carried out when circularly polarized light is incident on it under the conditions of the onset of the phenomenon of total internal reflection. At the onset of total internal reflection, the ellipsometry parameter shows high variability with the angle of incidence, in contrast to the parameter r0. It is shown that TIR occurs when the angle of incidence is not equal to the critical angle of the adjacent media for two different materials, these angles differ from each other. In the case of a film, the TIR phenomenon occurs at an angle equal to the critical angle at the prism-air interface and does not depend on the film material. The results obtained show the high efficiency of using the ellipsometric method together with circularly polarized incident radiation for diagnostics of thin films made of biological material.

Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

Thin-Film Frustrated Total Internal Reflection Filter with Plasmonic Nanoparticle Inclusions in the Layers

The influence of plasmonic nanoparticles embedded in the central and side layers of the frustrated total internal reflection filter on the resonant transmission of light is analyzed. It is shown that the frequency dispersion causes the splitting of the filter bandwidth and the angular splitting of the incident beam into several output beams.