Quantitative Analyses of FRET Efficiency of Dye Molecules Confined within 2-Dimensional Space

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Kazuaki Furukawa
Materials Science Laboratory

@@Lipid bilayer, the fundamental component of cell membranes, can form at the solid-liquid interface by self-organization. We have developed a new type of microchannel device using this self-spreading characteristic of the lipid bilayer [1]. The validity of the device has been confirmed by observing fluorescence resonance energy transfer (FRET). This report is of the successful application of the device to the quantitative determination of FRET efficiency [2].
@@L--PC (extracted from egg yolk) containing 1 mol % of NBD- (a dye for donor) or Texas Red (TR, a dye for acceptor)-conjugated lipid is prepared. They are self-spread from the each side of the straight line pattern with a 10 mm width. Figure 1A shows the confocal laser scanning microscope images from the moment the lipid bilayers collide (set as t = 0) to 600 s. The observation conditions are NBD: 488 nm excitation and 505-525 nm emission, TR: 543 nm excitation and > 610 nm emission. Figure 1B plots the fluorescence intensities averaged over the widths against the position at each time. The collision position is set as = 0.
@@After the collision, two self-spreading bilayers are unified and form a single bilayer that provides the 2-dimensional field for the NBD- and TR-conjugated lipids to diffuse. The average fluorescence intensities can be expressed by the solution of 1-dimensional diffusion equation [2]. As the initial concentration of dye-conjugated lipids are both 1 mol % and their diffusion constants should be almost equal, the sum of the concentrations of two dye lipids are constant, 1 mol %, at any . By analyzing the data in Fig. 1B using these reasonable assumptions, the quantitative determination of FRET efficiency depending on the donor-to-acceptor ratio becomes possible as shown in Fig. 2. A large number of data follow a unique curve, which supports the reliability of our experiments.

[1] K. Furukawa, et al., Lab Chip 6 (2006) 1001.
[2] K. Furukawa, et al., Langmuir 24 (2008) 921.

Fig. 1. A. Confocal laser scanning microscope images of the self-spreading L--PC containing 1 mol % NBD and Texas Red before and after the collision.
B. Fluorescence intensities averaged over the 10 µm width.
Fig. 2. Donor-to-acceptor ratio dependent FRET efficiency.

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