Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy

Abstract

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.

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

“…S8). These numbers are very close to the order values obtained in similar systems using fluorescence anisotropy (14,21). The standard deviation of j is limited by the measurement noise, which confirms the remarkable homogeneity of molecular orientational order in such single-lipid vesicles.…”

“…Lipid orientational behavior is also governed by the membrane molecular content, which drives lipid packing (14). To explore the contribution of molecular orientational order to lipid organization when local polarity changes, we induced cholesterol depletion in the COS-7 cell membrane using cholesterol oxidase, which specifically converts cholesterol into cholestenone, and methyl-b-cyclodextrin (MBCD), which removes cholesterol from the membrane.…”

“…Since only one of these two parameters is accessible, a measure of molecular orientational order requires working with samples of controlled geometry, where the averaged orientation can be known a priori. This explains the wide range of studies in membranes of round shapes such as red blood cells (13), giant unilamellar vesicles (14), cell blebs (15), or round cells (16). Another way to adapt anisotropy or linear dichroism to oriented structures is to address only particular regions of the sample where the molecules lie on average along or perpendicular to the incident polarization direction, such as horizontal or vertical parts of a membrane in the cell contour image (11,12,17).…”

<title>Measurements of plasma membrane architecture during hypoxia using multiple fluorescent spectroscopic techniques</title>

“…DFPM Measurements:Digitized fluorescence polarization microscopy measurements have been previously performed during hypoxic injury in hepatocytes(9). The details regarding instrument design and data interpretation have been previously published(2,7). Fluorescence polarization ratios (FjJFI) were computed on a pixel-by-pixel basis from digitized image pairs after background subtraction and image alignment.…”

Quantitative imaging of molecular order in lipid membranes using two-photon fluorescence polarimetry

“…In lipid membranes f(q, f) is expected to be a q-dependent cylindrical symmetry function, which can be restricted to the order parameters hP J ¼ 2, 4 6 i defined as hP J i ¼ R f ðqÞP J ðcos qÞsin qdq (with P J (cos q) the J-order Legendre polynomials) (2,4,31).…”