Alevra M et al. (2012), Direct measurement of diffusion in olfactory ci...

XB-ART-45661

PLoS One 2012 Jan 01;77:e39628. doi: 10.1371/journal.pone.0039628.

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Direct measurement of diffusion in olfactory cilia using a modified FRAP approach.

Alevra M , Schwartz P , Schild D .

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The diffusion coefficient of fluorescein in detached cilia of Xenopus laevis olfactory receptor neurons was measured using spatially-resolved FRAP, where the dye along half of the ciliary length was photobleached and its spatiotemporal fluorescence redistribution recorded. Fitting a one-dimensional numerical simulation of diffusion and photobleaching for 35 cilia resulted in a mean value of the diffusion coefficient (1.20 ± 0.23) · 10(-10)m(2)/s and thus a reduction by a factor of 3.4 compared to free diffusion in aqueous solution.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 mtor

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	Figure 1. Preparation of olfactory cilia.(A–D) SEM micrographs, (A) Top view onto the nostrils of a Xenopus laevis tadpole, (B) one nostril, at the bottom of which a lawn of sensory clila can be seen (C, D), (E) detached cilia (arrow heads) on a Poly-L-Lysine-coated coverslip, imaged with a 100× objective using DIC.
	Figure 2. FRAP scanning protocol and sample data.(A) Schematic of the three-phase scanning protocol showing the position of the illumination line in pixel coordinates. In the first phase, several full frames are acquired to determine initial fluorescence. Half-frames are acquired in the second phase at high frame rate (488 f/s) for photobleaching in the lower half of the cilium. The third phase records the fluorescence redistribution due to diffusion at a low frame rate (28 f/s). Image acquisition (blue) is delayed in respect to the mirror position signal (green) for mirror response linearity. Time axis is not to scale, number of images reduced for simplicity. (B–H) Sample frames from all FRAP phases show evolution of fluorescence distribution, scale bar 5 , frame times relative to first frame. (B) Initial fluorescence. (C,D) First and last half-frame of the bleaching phase, upper half not imaged and displayed as black. (E) First full frame of the recovery phase shows inhomogeneous fluorescence distribution. (F) Mostly homogeneous distribution after 9 frames in the recovery phase. (G) Last frame of the recovery phase. (H) 2D pixel mask used for maximum projection of 2D intensities onto 1D position on cilium. (I) Projected intensity plots (dots) for selected frames (blue: data from frame B, green: bleaching phase (t = 51 ms), red: E, cyan: F), and corresponding best-fits (solid lines, for full data see Fig. 3B).
	Figure 3. Fit of 1D diffusion model to experimental data.(A) Experimental data, shown as 1D fluorescence distribution over frame numbers for a full experiment, with normalized fluorescence () color-coded according to color map. FRAP phases (see Fig. 2A) indicated above. (B) Corresponding data from the best-fit result of the 1D diffusion model. (C) Residuals between A and B, using a smaller colormap range.
	Figure 4. Distribution of diffusion coefficients.Blue: histogram of best-fit results for diffusion coefficients of fluorescein from 35 cilia, with mean and standard deviation as dark gray line and light gray area, respectively. The diffusion coefficient of fluorescein in aqueous solution at 25°C [14] is shown in red, while values corrected for a range of (23±1)°C are shown in green.

References [+] :

Anholt, A partially purified preparation of isolated chemosensory cilia from the olfactory epithelium of the bullfrog, Rana catesbeiana. 1986, Pubmed