Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy

Abstract

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C(20) or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions.

Figure 1

Schematic (A) and digital photograph (B) of the pTIRFM/AFM instrument. See text for details on the optical components and their arrangement. The numbered circles in panel B correspond to the regions of the optical train also labeled in panel A by numbered circles.

Figure 2

Chemical structures of the molecules used in model lipid bilayer experiments of this study. (A) DOPC. (B) DSPC. (C) Cholesterol. (D) DiI-C₂₀. (E) BODIPY-PC. The location and orientation of the transition dipole moments are indicated by double-ended arrows.

Figure 3

Calibration of the polarized TIRF microscope. (A) A ψ-image stack of a randomly oriented, dilute solution of TRITC dye molecules dissolved in ethanol (532 nm excitation). The image contrast settings are the same for these images allowing the gradual decrease in fluorescence intensity as ψ is increased from 0° to 90° to be visualized. The intensity spatial distribution of these images reflects the nearly Gaussian-shaped profile (TEM₀₀ mode) of the TIRF illumination spot. (B) Calculated order parameter image of the ψ-image stack in panel A. This image is colored according to the color scale in the image histogram shown in panel C. The plotted image histogram is restricted to the 20 × 20 μm region of interest in panel B. The histogram is peaked close to the expected value of 〈P₂〉 = 0 for a random distribution of fluorophore absorption dipoles. (D) The normalized fluorescence intensity of five random pixel positions from the central-most region of the illumination spot in the ψ-image stack in panel A are plotted against the theoretical curve (solid line, Eq. 13; 〈θ_c〉 = 54.7°, 〈P₂〉 = 0, B = 0.27) expected for a random distribution of dye molecules when imaged by the pTIRFM technique. The good agreement between the measured points and the theoretical curve indicates that it is valid to calculate the order parameter on a single pixel level and that the pTIRFM technique can be measured with diffraction-limited spatial resolution.

Figure 4

Representative pTIRFM/AFM image set of a (1:1) DOPC/DSPC/30 mol % Cholesterol/1 mol % BODIPY-PC lipid bilayer at room temperature. (A) The ψ-image stack showing the gradual decrease in image intensity as the evanescent field polarization angle is increased from 0° to 90°. Panels B and C compare the p- and s-polarized images of the 20 × 20 μm region enclosed by the open square in the first frame of panel A. (D) Calculated order parameter image of this same region. The very low order parameter spots in this image (solid) are unfused lipid vesicles that are also visible in the corresponding AFM topography image (E).

Figure 5

Representative pTIRFM/AFM image set of a (1:1) DOPC/DSPC/30 mol % Cholesterol/1 mol % DiI-C₂₀ lipid bilayer at room temperature. (A) The ψ-image stack showing the gradual increase in image intensity as the evanescent field polarization angle is increased from 0° to 90°. Panels B and C compare the p- and s-polarized images of the 20 × 20 μm region enclosed by the open square in the last frame of panel A. (D) Calculated order parameter image of this same region. The bright spots with an order parameter close to zero (open) are unfused lipid vesicles (also visible in B) that were swept away by the raster scanning motion of the AFM tip and subsequently do not appear in the corresponding AFM topography image (E).

Figure 6

Summary data for pTIRFM/AFM bilayer experiments containing various amounts of cholesterol (5, 15, and 30 mol %). (A) (1:1:x) DOPC/DSPC/Cholesterol bilayers labeled with DiI-C₂₀. (B) (1:1:x) DOPC/DSPC/Cholesterol bilayers labeled with BODIPY-PC. (Solid squares) pTIRFM order parameter values associated with high topography membrane regions. (Open squares) pTIRFM order parameter values associated with low topography membrane regions. Each measured point in the graphs is an average of at least 1000 pixels averaged over the appropriate topography regions in a 20 × 20 μm pTIRFM order parameter image. The error bar for each point is determined by the standard deviation of measured pixel values around the average.

Figure 7

pTIRFM/AFM images of a 1:1 DOPC/DSPC/15 mol % cholesterol/1 mol % DiI-C20 lipid bilayer at room temperature. The s-polarized (A) and p-polarized (B) pTIRFM images. (C) Order parameter image. (D) AFM topography image of same field of view in the pTIRFM images. This image shows that the large dark feature in panels A and B is a membrane defect that exposes the mica substrate (∼6 nm height difference). Panels E and F are smaller scanned AFM topography images of the boxed regions in panels D and E, respectively, and they reveal domain substructures that are not resolved optically. The dashed white lines in these AFM images correspond to the plotted height line profiles below the images. The bright spots over the membrane in the p-polarized image in panel B are unfused vesicles that are swept away by the raster-scanning motion of the AFM tip during imaging.