Mapping the Local Organization of Cell Membranes Using Excitation-Polarization-Resolved Confocal Fluorescence Microscopy

Abstract

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

Thioflavine-T and Congo Red Reveal the Polymorphism of Insulin Amyloid Fibrils When Probed by Polarization-Resolved Fluorescence Microscopy

“…The state of polarization for the excitation beam was controlled by rotating sequentially a half waveplate (WPH05M-488, Thorlabs) by means of a step motor in the path of the linearly polarized laser beam. Polarization distortions induced by the dichroic mirror were partially compensated using a wave plate and residual distortions were measured and taken into account in the theoretical computation used for data analysis 26 . Data were recorded by incrementing polarization angles α by steps of 2° between 0 and 178°.…”

“…Data processing includes selection of the region of interest by the user, systematic rejection of data points for which the photon counts is too low (typically below 300 photon counts), and systematic rejection of data points that were affected by sample drift or photobleaching 26 .…”

High-speed polarization-resolved coherent Raman scattering imaging

“…This decrease is essentially due to the lower lipid content measured, a MLV containing typically 50-100 bilayers in the focal volume [16]. It also reflects a lower degree of lipid order due to the presence of different lipid types, proteins, and local membrane folding [31][32][33][34]; normalized S 2n values are indeed about five times less than those observed in MLVs. The lower S 2 signal obtained in red blood cells inherently leads to a higher sensitivity to polarization distortions, in particular, the even slight di-attenuation that was measured between the s and p components of the incident pump polarization (see Supplement 1, Figs.…”

“…Note that this effect, associated to conditions of extreme low signal to noise, is not visible in the MLV measurements described above, which resemble more general conditions used for CARS/SRS imaging. Different strategies can be used to overcome bias in parameter retrieval under such conditions, by either correcting for polarization distortions in the optical setup using compensation optics or by post-processing correction [34].…”

Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

“…Consequently, PR microscopy has been proposed as a unique technique to study molecular structures [1]. PR microscopy has been studied for different light-sample interaction mechanisms, including one- [2][3][4], two- [5,6] and threephoton absorption fluorescence [7], second harmonic generation (SHG) [8][9][10][11][12], sum-frequency generation [13] third harmonic generation [14], or four-wave mixing [15]. PR microscopy has also been combined with fluorescence lifetime imaging [16] and super-resolution techniques, providing an ultimate way to optically image molecular organization and structure [17,18].…”