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Results: 7

1.
Figure 5

Figure 5. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Pixel-wise rate coefficients of fluorescence loss kinetics of eGFP. Using the parameter maps of the FLIP analysis for the cell in Figure 4, rate coefficients were calculated for every pixel position, as outlined in Additional file. A, the resulting 32-bit image stack was color-coded using a FIRE-LUT, and selected frames of the rate coefficients were plotted (blue and yellow-white indicate low and high rate coefficients, respectively). Areas with accelerating speed of FL turn from blue to yellow-white over time. A’ few regions are highlighted with boxes (numbered ‘1’ to ‘5’) for further analysis. Boxes 1 and 2 were placed in the nucleus, while boxes 3 to 5 were in the cytoplasm (inset shows a zoom for the area containing boxes 3 and 4 close to the right edge of the cell). B, rate coefficients as function of time plotted for box 1 to 4. C, x,t-view of the time evolution of the rate coefficients in box 5. See text for further explanations.

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
2.
Figure 3

Figure 3. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Diffusion-limited FLIP of eGFP in the cytoplasm of McA cells. McArdle RH7777 cells expressing eGFP in the cytoplasm and nucleus were placed on a temperature-controlled stage of a confocal microscope maintained at 35 ± 1°C. A 10 pixel diameter circular region in the cytoplasm (white circle) was repeatedly bleached with full laser power, while the whole field was scanned with 0.5% laser output between the bleach scans. This gave a total frame rate of 1.6 sec (see Methods for further details). A, montage of the time series with every 20th frame shown. B-D, fit of the StrExp function to the data giving a map of the stretching parameter (B), the time constant map (C) and its reciprocal, the rate constant map (D). A FIRE-LUT was used for color-coding, where dark blue and yellow indicate lowest and highest values, respectively. The range of values is given below the images with a color bar without units in B, in sec in C and in sec-1 in D. Bar, 5 μm. E, profile of stretching parameters along the line shown in B (grey line, data; black line moving average to smooth the profile).

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
3.
Figure 7

Figure 7. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Multi-compartment modeling of FLIP data reveals dynamics of eGFP-Q73 in inclusion bodies. A FLIP experiment was performed in CHO cells expressing eGFP-Q73 as described in legend to Figure 6. A, montage of every 20th frame of the data. Inset is a zoom of the small box pointing to an IB; a FIRE LUT is used for visualization purposes. A’, sketch of the multi-compartment (MC) model used for determining binding/dissociation parameters. B, FL kinetics for the IB (blue dots) and for a region in the cytoplasm (green dots) with overlayed fit to the MC model for the IB (compartment 1; dark blue line) and for the cytoplasm (compartment 2; dark green line). C, another FLIP sequence imaged with a total frame rate of 2.4 sec. First frame of the time series is shown in green, while selected subsequent frames are overlayed in red. This color coding visualizes movement of two IB’s at the cell edge (large one up and small one below; arrows). Tracking of the mobile IB’s and measurement of their intensity was performed using the SpotTracker plugin for ImageJ [47]. D, x,y-plot of the trajectories of the IB’s; E, mean square displacement (MSD) calculated from the trajectories; F, step length distribution between subsequent steps; G, fluorescence intensity of eGFP-Q73 in the IB’s (blue dots = large IB; red dots = small IB) and in the cytoplasm (green dots) and fit with the MC model for the IB’s (compartments 1, dark blue and red line, respectively) as well as for the cytoplasm (compartment 2, dark green line).

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
4.
Figure 4

Figure 4. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Barrier-limited FLIP of eGFP shuttling between nucleus and cytoplasm. McArdle RH7777 cells expressing eGFP in the cytoplasm and nucleus were placed on a temperature-controlled stage of a confocal microscope maintained at 35 ± 1°C. A 30 pixel diameter circular region in the cytoplasm (white circle) was repeatedly bleached with full laser power, while the whole field was scanned with 0.5% laser output between the bleach scans such that the total frame rate was 2.6 sec. A montage of every 30th frame of the data (upper panel, ‘data’) or of the reconstruction from a pixel-wise fit of data to the StrExp function (lower panel, ‘fit’). B, amplitude map; C, background map, each with three boxes numbered 1 to 3. D, FL in these three boxes along the stack (colored symbols) + fit to StrExp function (colored lines). E-H, parameter maps produced by PixBleach for: E, the stretching parameter (see Eq. 1; the color code for h = 1, the mono-exponential case, is given in small box); F, χ2-values showing the quality of the regression; G, time constant map; H, number of iterations. All values are color-coded using a FIRE-LUT, and the range is given below the images with a color bar. The time constant is given in seconds; all other values are without units. I, J; histograms of the stretching parameters (I) and the time constants (J) for the cell, shown in A-H. Bar, 5 μm.

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
5.
Figure 6

Figure 6. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Quantitative FLIP and single particle tracking of mobile IB’s in the cytoplasm. A FLIP experiment with a bleach region of 10 pixels in diameter was performed in CHO cells expressing eGFP-Q73 on a temperature-controlled stage of a confocal microscope maintained at 35 ± 1°C. The total frame rate was 1.45 sec. A, montage of every 20th frame of the data. Arrows point to an inclusion body (IB) in the cytoplasm. B-E, pixel-wise fit of the data to a StrExp function with B, amplitude map; C, map of the stretching parameter; D, time constant map; E, χ2-values showing the quality of the regression. All values are color-coded using a FIRE-LUT, and the range is indicated by the respective color bar. The time constant is given in seconds; all other values are without units. F, first frame of the time series is shown in green, while selected subsequent frames are overlayed in red (box in F shows movement of the IB). G, FL of eGFP-Q73 in the IB (red symbols) with fit to the StrExp function (black line). H, x,y-plot of the trajectory of the IB during the FLIP experiment. I, mean square displacement (MSD) calculated from the trajectory and linear fit to the five intital data points (red line). J, K, simulation and tracking of a particle experiencing FL with a rate constant, k = 0.01 sec-1. J, trajectory separated in x- (straight lines) and y-direction (dotted lines) for a particle undergoing bleaching (black lines, simulated trajectory; red lines, tracked trajectory). The tracked trajectory coincided with the simulated trajectory until to 180 sec (or frames). K, intensity of the tracked particle (grey lines) compared to a mono-exponential fit with residual. Bar, 5 μm.

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
6.
Figure 2

Figure 2. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Simulation of heterogenous diffusion and fitting with the StrExp function. A 2-dimensional bleaching experiment was simulated on a disk with a circular bleached area of radius r1=0.5 μm using FeniCS, an automated computational modelling suite (http://www.fenicsproject.org). The bleaching rate is set to k=10 sec-1 and the diffusion coefficient is d1=0.2 μm2/sec and d2=0.8 μm2/sec on the left and right half disk, respectively (A). Fluorescence loss inside and outside the bleached region was fitted at every pixel position with the StrExp function. For this purpose the PixBleach plugin to ImageJ was used [40]. The regression recovers a map of the stretching parameter, h, color-coded between 0.4 to 2.0 (B), the time constant distribution color-coded between 1.0 to 15.0 sec (C) and the χ2-map color-coded between 0 to 50 (E). A FIRE-LUT was used for color-coding, where dark blue and yellow indicate lowest and highest values, respectively. The reconstructed stack exactly resembles the simulated data set, as seen in D (upper row, simulated data (‘sim‘), lower row, regression (‘fit‘)). F, profile of time constants, as estimated from fitting the StrExp function to the simulated FLIP data along the line shown in panel C. G-L, rate coefficients were calculated on a pixel-by-pixel basis according to with self-programmed macros to ImageJ. Rate coefficients are color-coded using a FIRE-LUT in the range from 0.017 (dark blue) to 0.97 (yellow). G, shows montage of all time points; H, first frame of montage with some regions of interest (boxes 1 to 4) used for analysis in panel I-L. Rate coefficient as function of time averaged for box 1 (I), box 2 (J), box 3 (K) and box 4 (L) from the whole sequence.

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.
7.
Figure 1

Figure 1. From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation.

Simulation of homogenous diffusion and fitting with the StrExp function.A, Sketch of the FLIP experiment with the cell attached to a surface and filled with eGFP (green) and the cylindrical laser beam focused in the cell center (yellow). B, Geometry of the analytical model for the reaction diffusion system in Eq. 6 to model the FLIP experiment. The cell is assumed to be a flat cylinder with a radius, r2 = 12 μm. The central bleached region with radius r1 = 3 μm is also cylindrical covering the whole cell height (grey shaded area). C-E, The model was solved analytically and simulated for two positions outside the bleached area at a distance of 5 μm (red dot in B and red symbols in C-E) and 10 μm (blue dot in B and blue symbols in C-E) from the origin, respectively. Simulations were performed with a rate constant for the intended bleaching process of k = 10 sec-1 and diffusion constants of D = 0.1 μm2/sec (C), D = 1 μm2/sec (D) and D = 10 μm2/sec (E). A non-linear regression with the StrExp function (black lines) was performed in SigmaPlot (upper panels) including the residuals of the fit (lower panels). F, G, time courses were simulated for D = 0.1 μm2/sec as a function of distance from the origin and fitted to the StrExp function. Fitted parameters including standard deviation of the fit are plotted for the rate constant (‘kfit’; F) and stretching parameter (‘hfit’; G). H, rate coefficients calculated according to Eq. S4 for the parameters in panels F, G as function of distance from bleach ROI (starting at 4 μm from origin and indicated as ‘r’ on the ordinate in H) over time. The scale bar shows rate coefficients color-coded using a FIRE-LUT

Daniel Wüstner, et al. BMC Bioinformatics. 2012;13:296-296.

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