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. 2017 Mar 30;7:45647. doi: 10.1038/srep45647

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Copyright © 2017, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(A) Confocal images of flow cell (FC) 1 and 2 formed on the L1 sensor chip surface (XY plane) using 10x magnification objectives. POPC SUV labelled with either Rho-PE (control) or Rho-PE and CF were deposited on FC1 and 2, respectively. A 20 μm white scale bar is included. (B) Individual confocal images of FC1 (top) and 2 (bottom) collected at the respective white square ROI in A using a 20x magnification objective, emphasizing the flow cell boundaries. Red (Rho-PE), green (CF) and merge channels are depicted for both A and B. (C) Z-stack image of the L1 sensor chip covered with POPC SUV labelled with Rho-PE and CF. YZ orthogonal projections were produced in order to evaluate the relative Z-position of the lipid membrane and CF probe. A vertical yellow line is located in the middle of the Z projection (40 μm). Red (Rho-PE), green (CF) and merge channels are depicted. A normalized Z plot profile is also shown locating the region of the lipid deposition. (D–H) FRAP analysis of the circular ROI according to the settings explained in the Supplementary Fig. S3. Rho-PE and CF fluorescent signals were collected from the ROI and normalized to the signal on the non-bleached area. The relative fluorescence recovery () is thus shown to evaluate the lateral diffusion of the Rho-PE fluorescent lipids of the bilayer. Briefly, a time series of 4x Zoom 512 × 512 pixels images using a 20x magnification objective were taken: 10 frames were collected before bleaching of the 20 μm radius circular ROI (yellow) with 100 iterations (approx. 40 sec.) of 100% of 488, 514 and 561 laser intensity. After bleaching, 90–150 images were acquired according to the lipid mixture studied (D – POPC, E - POPC:Chol (2:1), F - POPC:Chol:SM (1:1:1), G - POPC:DPPC (1:1), H - POPC (control)) due to fluorescence recovery/sample burning percentage. Representative confocal images are presented in order of their acquisition time. Error bars correspond to the standard deviation of the mean. Experiments were performed in triplicate.

Inline graphic — (A) Confocal images of flow cell (FC) 1 and 2 formed on the L1 sensor chip surface (XY plane) using 10x magnification objectives. POPC SUV labelled with either Rho-PE (control) or Rho-PE and CF were deposited on FC1 and 2, respectively. A 20 μm white scale bar is included. (B) Individual confocal images of FC1 (top) and 2 (bottom) collected at the respective white square ROI in A using a 20x magnification objective, emphasizing the flow cell boundaries. Red (Rho-PE), green (CF) and merge channels are depicted for both A and B. (C) Z-stack image of the L1 sensor chip covered with POPC SUV labelled with Rho-PE and CF. YZ orthogonal projections were produced in order to evaluate the relative Z-position of the lipid membrane and CF probe. A vertical yellow line is located in the middle of the Z projection (40 μm). Red (Rho-PE), green (CF) and merge channels are depicted. A normalized Z plot profile is also shown locating the region of the lipid deposition. (D–H) FRAP analysis of the circular ROI according to the settings explained in the Supplementary Fig. S3. Rho-PE and CF fluorescent signals were collected from the ROI and normalized to the signal on the non-bleached area. The relative fluorescence recovery () is thus shown to evaluate the lateral diffusion of the Rho-PE fluorescent lipids of the bilayer. Briefly, a time series of 4x Zoom 512 × 512 pixels images using a 20x magnification objective were taken: 10 frames were collected before bleaching of the 20 μm radius circular ROI (yellow) with 100 iterations (approx. 40 sec.) of 100% of 488, 514 and 561 laser intensity. After bleaching, 90–150 images were acquired according to the lipid mixture studied (D – POPC, E - POPC:Chol (2:1), F - POPC:Chol:SM (1:1:1), G - POPC:DPPC (1:1), H - POPC (control)) due to fluorescence recovery/sample burning percentage. Representative confocal images are presented in order of their acquisition time. Error bars correspond to the standard deviation of the mean. Experiments were performed in triplicate.