Results: 3

1.
Fig. 10.2

Fig. 10.2. From: A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy.

Determination of Ebleaching and Ka from Lyn16-CFP-YFP FRET positive control. (a) CFP, FRET, and YFP images before and after YFP photobleaching were acquired from cells expressing Lyn16-CFP-YFP on planar lipid bilayer and processed as described in Section 3. AOIs from three cells were drawn over cell contact areas from each image before (Pre-Bleach) and after bleaching (Post-Bleach). Note that images are not saturated on the 16 bit gray scale. (b) Procedure to obtain Ka from the CFP, FRET, and YFP images in a Microsoft Excel sheet is shown. Shown are a table containing the values that include background-subtracted YFP, Nsen, CFPafter and Dequenching, calculated from the mean fluorescence intensities of AOIs drawn in (a), and intensity plots for Nsen vs YFP and Dequenching vs CFPafter from the values of the table. FRa and Ebleaching were determined from the slope of the line of Nsen vs YFP and Dequenching vs CFPafter, respectively. Finally Ka was obtained from the equation ().

Hae Won Sohn, et al. Methods Mol Biol. ;591:159-183.
2.
Fig. 10.3

Fig. 10.3. From: A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy.

Membrane-bound-antigen-induced association of Lyn with BCR. Cells were dropped onto planar lipid bilayer with or without antigen and time-lapse images were acquired at 37°C for 15 min. (a) Procedure for image processing to obtain the FRET efficiency (Ea) image from experimental cells expressing both Lyn-CFP and Igα-YFP is shown. Images taken 1 min after cell contact to the antigen-bound membrane were processed and shown are the representative images in each step described in Section 3. β*CFP, γ *YFP, Ka* γ*YFP, N4sen and Ea images were obtained from pixel-by-pixel image calculation according to the appropriate equations. Raw; background-subtracted images, Filtered: Lopass-filtered images, Flatten: flatten-filtered images, Mask: masked images from flattened images, Ea-masked: masked image of Ea with both CFP and YFP masks. (b) CFP, FRET, YFP and Ea images at each indicated time after cell contact in the absence (ICAM-1) or presence of antigen (ICAM-1 + Antigen) on a planar lipid bilayer are shown. Images were processed as in (a) and shown are the filtered CFP, FRET, and YFP images and the masked Ea image. Ea is shown as gray scale is from 0 to 0.7. The fluorescence intensities of CFP, FRET, and YFP images are not comparable.

Hae Won Sohn, et al. Methods Mol Biol. ;591:159-183.
3.
Fig. 10.1

Fig. 10.1. From: A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy.

Determination of correction factors: (a) for CFP bleed-through (β) and (b) for YFP crosstalk (γ) from direct excitation of CFP and YFP by 442 nm laser, respectively. Cells expressing either Lyn-CFP alone (CFP+ cell) or Igα-YFP alone (YFP+ cell) were settled on planar lipid bilayer. Cells were excited sequentially with 442 and 514 nm laser light by TIRF microscope. Fluorescent emission was split through DualView to acquire CFP and FRET images (442 nm excitation) and the YFP image (514 nm excitation). Images were aligned, split to obtain raw CFP, FRET, and YFP images from the control cells, and then background subtracted and a Lo-pass filter applied (Filtered) as described in Section 3. To calculate β, intensity data was obtained from a line drawn over the same relative position in the cell in CFP and FRET filtered image and was plotted as FRET vs CFP (a) for each pixel in the line. β was determined from the slope of the line in the plot. γ (B) was calculated similarly using YFP and FRET filtered images. Similar results are obtained by averaging fluorescence intensities from several control cells.

Hae Won Sohn, et al. Methods Mol Biol. ;591:159-183.

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