Simplified equation to extract diffusion coefficients from confocal FRAP data

Abstract

Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane-bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability.

Figure 1. Representative images of the subcellular distribution of the proteins and lipids studied

COS7 cells transfected or exogenously labeled with Alexa conjugated cholera toxin B-subunit (Alexa-CTxB), the model GPI-anchored protein (YFP-GL-GPI), Flotillin-1-RFP (Flot-1-RFP), a fluorescent lipid analog (DiIC₁₆), or EGFP. Scale bar = 10 μm.

Figure 2. Diffusion of Alexa-CTxB on live vs. fixed cells

(A) Representative images of Alexa-CTxB on the plasma membrane during a FRAP experiment on either live or fixed cells for r_n=1.1μm. Scale bar = 1 μm. (B) Postbleach profiles of Alexa-CTxB on the plasma membranes of live (□, n=12) and fixed (○, n=5) cells. (C) FRAP data of Alexa-CTxB on the plasma membranes of live (□, n=12) and fixed (○, n=5) cells. Since the bleach ROI is slightly off center in our system as seen in the images of (A) at t=0, a correction was made to align the center of the postbleach profile to determine r_e.

Figure 3. Confocal FRAP data of Alexa-CTxB, YFP-GL-GPI, Flot-1-RFP, DiIC₁₆, and EGFP

(A) Representative pre- and post-bleach images. Dashed circles represent user-defined bleaching spots with diameter 2.2 μm (i.e. r_n= 1.1 μm). To speed data acquisition for the FRAP analysis, we cropped the imaging window to make it slightly larger than the bleaching spot size. Scale bar, 1 μm. (B) Averaged postbleach profiles for N=12~14 cells along the diagonals of postbleach images (□) and the best fitting Gaussian (Eq. 2, —). Dotted vertical lines show x=±r_n (user defined bleach ROI). Dash-dot horizontal lines are 14% of bleaching depth from the top. (C) Representative FRAP recovery curves (□) and the best fit to Eq. 3 (—). Insets show full FRAP curves for Alexa-CTxB and YFP-GL-GPI. In (B) and (C), y- axes are normalized fluorescence intensities, i.e. F_i = 1. Since the bleach ROI is slightly off center in our system, a correction was made to determine r_e as described in the Materials and Methods.

Figure 4. Comparison of diffusion coefficients determined by various schemes

Comparison of diffusion coefficients determined by FRAP data fitting (D_Fitting, Eq. 3), versus the D_Confocal equation (Eq. 5), or the Soumpasis equation using either r_n (D_rn) or r_e (D_re) in log scale. D’s were found from averaged FRAP curves (N≥12 cells per experiment) for three or more separate experiments (n≥3). Error bars represent standard errors. Dashed boxes show D’s reported in the literature (Table 3). *, p<0.05 compared to D_Fitting, Student’s t-test.

Figure 5. Comparison of diffusion coefficients for EGFP in the cytosol obtained by confocal FRAP under different experimental conditions as calculated using the Soumpasis equation or D_Confocal equation

Diffusion coefficients were determined by FRAP data fitting (D_Fitting, Eq. 3), by the D_Confocal equation (Eq. 5), by the Soumpasis equation using r_n (D_rn), and by the Soumpasis equation using r_e (D_re) for individual confocal FRAP curves (N≥6). Dashed box indicates the range of EGFP’s diffusion coefficients in the cytosol reported in the literature. r_e was measured from an averaged postbleach profile (n=1,N=10 cells) and D’s were obtained from individual FRAP data (n=1, N=8,10,10, and 8 cells). *, p<0.05 compared to D_Fitting, Student’s t-test.

Figure 6. Comparison of values obtained from averaged FRAP data vs. mean of values from individual FRAP data

Comparison of Mean±SE of (A) r_e, (B) τ_1/2, (C) D, and (D) M_f determined using averaged FRAP data from more than three independent experiements with 10 cells (n≥3, N=10) or means from 10 individual FRAP data in a single experiment (N=10). Error bars represent standard errors. p>0.05 Cross comparison, Student’s t-test.

Figure 7. Workflow of applying the D_Confocal equation

Figure 8. Determination of r_e from a postbleach profile