Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells

European Biophysical Journal

Victor V. Skakun1,Ruchira Engel2,3,Jan Willem Borst2,Vladimir V. Apanasovich1,Antonie J.W.G. Visser2

1Department of Systems Analysis and Computer Simulation, Belarusian State University, Minsk 220050, Belarus

2Laboratory of Biochemistry, and Microspectroscopy Centre, Wageningen University, 6703 HA Wageningen, The Netherlands

3Present address: Department of Immunopathology, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

Supplementary Information

*Corresponding Author: Victor V. Skakun

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Table S1 Results of the global analysis of ACF and three PCDs of fluctuating fluorescence intensity data of free GFP (S65T) in Dictyostelium cytoplasm(> 100% denotes an error with more than 100% deviation)

Meas. No. / 1 / 2 / 3 / 4 / 5 / 6 / 7
a1 / -0.196  (>100%) / -0.147  (>100%) / -0.287  (>100%) / -0.644  0.450 / -0.216  (>100%) / -0.327  (> 100%) / -0.563  (>100%)
a2 / 0.117  (>100%) / 0.188  0.042 / 0.229  0.111 / 0.178  0.036 / 0.179  0.022 / 0.187  0.022 / 0.222  0.04
N / 23.04  0.34 / 15.48  0.31 / 22.67  0.38 / 11.22  0.08 / 14.13  0.53 / 16.35  0.13 / 16.346  0.12
qeff0(cpms)(cpms) / 5940  88 / 5346  105 / 6234  105 / 6291  46 / 5918  220 / 6063  46 / 5545  43
qtrue (cpms) / 16801 / 15121 / 17632 / 17794 / 16739 / 17149 / 15684
Tdiff (μs) / 276  9 / 298  9 / 258  8 / 283  5 / 313  18 / 253  4 / 286  5
D (μm2s-1) / 16.17 / 14.98 / 17.30 / 15.77 / 14.26 / 17.64 / 15.61
Ftrip / 0.125  0.023 / 0.196  0.016 / 0.126  0.014 / 0.227  0.032 / 0.204  0.028 / 0.173  0.033 / 0.251  0.048
τtrip(μs) / 14.28  6.18 / 37.14  6.98 / 32.45  8.88 / 5.47  1.37 / 68.34  13.76 / 6.56  2.11 / 4.09  1.17
Gcorr inf / 1.001 / 1.002 / 1.001 / 1.002 / 1.002 / 1.002 / 1.003
χ2 / 1.02 / 0.755 / 0.863 / 0.683 / 0.981 / 1.04 / 1.01

PCDs were calculated with bin times of 20 s, 50 s and 100 s. ACFswere calculated in quasilogarithmic time scale with minimum bin time of 0.25 s. Structure parameter a was fixed to the value, obtained from the calibration experiment (see Table S2). Standard deviations were calculated at the 67% confidence level by the asymptotic standard-errors method[1]. The true value of the brightness qtrue was calculated as qtrue= qeff0/γ2. Diffusion coefficient D was calculated from Tdiff following the standard procedure.

Table S2 Results of the global analysis of ACF and two PCDs of fluctuating fluorescence intensity data of enhanced GFP in buffer

Meas. No. / 1 / 2 / 3 / Calibration
a1 / -0.479 0.075 / -0.4900.070 / -0.344 0.112 / -0.422 0.122
a2 / 0.1510.006 / 0.156 0.005 / 0.128 0.012 / 0.148 0.007
N / 2.183 0.008 / 2.196 0.006 / 2.171 0.008 / 0.362 0.006
qeff0(cpms) / 761526 / 755623 / 787133 / 11560180
qtrue (cpms) / 21538 / 21371 / 22262 / 32697
Tdiff (μs) / 71.80.5 / 71.00.4 / 71.10.6 / 15.930.49
D (μm2s-1) / 62.16 / 62.86 / 62.76
Ftrip / 0.261 0.024 / 0.307 0.030 / 0.251 0.025 / 0.162 0.124
τtrip(μs) / 2.120.26 / 1.480.18 / 1.980.28 / 0.9890.810
a / 6 / 6 / 6 / 6.02 0.47
Gcorr inf / 1.000 / 1.000 / 1.000 / 1.000
χ2 / 0.943 / 0.919 / 1.39 / 1.116

PCDs were calculated with bin times of 50 s and 200 s. ACFs were calculated in quasilogarithmic time scale with minimum bin time of 0.25 s. Rhodamine 110 in water was used for calibration measurements. Structure parameter a was fixed to the value, obtained from the calibration experiment (see the last column). Standard deviations were calculated at the 67% confidence level by the asymptotic standard-errors method. The true value of the brightness qtrue was calculated as qtrue= qeff0/γ2. Diffusion coefficient D was calculated from Tdiff following the standard procedure.

Fig. S1 Graphical results of global analysis of measurement 1 (see Table S1 for the recovered parameters) of GFP (S65T) in Dictyostelium cytoplasm. The experimental intensity fluctuations are shown in the top panel. The red vertical lines on the intensity graphs show the time interval for which the ACF and PCDs were calculated. Experimental and fitted ACF (left) and PCDs (right) are shown in the lower panels. Residuals are plotted below each curve. Fit quality criterion 2 = 1.02

Fig. S2 Graphical results of global analysis of measurement 3 (see Table 1 for the recovered parameters) of GFP (S65T) in Dictyostelium cytoplasm. See legend to Fig. S1 for details. Fit quality criterion 2 = 0.863

Fig. S3 Graphical results of global analysis of one measurement of enhanced GFP in aqueous buffer. The experimental intensity fluctuations are shown in the top panel. See legend to Fig. S1 for details. Fit quality criterion2 = 0.943

Fig. S4One-dimensional visualization of brightness profile functions for measurements 4 to 8 (see Table S1) of GFP (S65T) in Dictyostelium cytoplasm. The Gaussian profile is shown for comparison. The magnified part of the graph is shown in the inset

Fig. S5Graphical results of FCS (top) and PCH (bottom) analyses of measurement 2 of GFP (S65T) in Dictyostelium cytoplasm in the windowing regime (see Table S3 and Table S1 for the recovered parameters in the windowing and global analysis respectively). Intensity traces and graphical results of the global analysis are presented in Fig 1. Length of the window is 2 s

Table S3 Results of FCS and PCH analyses of measurement 2 of GFP (S65T) in Dictyostelium cytoplasm in the windowing regime (deviations with > 100% error are not shown)

Win. No. / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11
PCH
a1 / -1.570.15 / -0.21 / -1.780.14 / -1.30.16 / -0.63 / -0.04 / -0.479 / -0.08 / -1.15 / -1.520.03 / -1.99
a2 / 0.360.06 / 0.0003 / 0.450.45 / 0.280.02 / 0.364 / 1.94 / 0.387 / 0.006 / 1.97 / 0.34 0.01 / 1.42
N / 11.560.27 / 12.660.52 / 11.78 0.48 / 8.720.23 / 13.720.8 / 13.740.71 / 16.821.06 / 16.230.75 / 16.580.81 / 15.140.73 / 14.700.57
qeff0(cpms) / 6605154 / 6570272 / 4662193 / 6940189 / 5668336 / 5944307 / 5033318 / 5408251 / 5111251 / 5330260 / 5378210
χ2 / 0.453 / 1.363 / 0.752 / 0.617 / 2.242 / 1.878 / 2.130 / 1.350 / 1.355 / 1.376 / 0.831
FCS
N / 13.930.54 / 12.760.2 / 18.825.37 / 41.543.25 / 23.053.27 / 12.580.29 / 16.461.32 / 15.440.36 / 16.590.67 / 18.461.56 / 13.710.32
Tdiff (μs) / 40437 / 29611 / 369124 / 9968 / 714163 / 25813 / 27234 / 32217 / 28622.6 / 393 53 / 22712
Ftrip / 0.260.05 / 0.430.13 / 0.390.16 / 0.760.02 / 0.490.07 / 0.1410.05 / 0.180.03 / 0.2550.04 / 0.2420.04 / 0.3090.05 / 0.4060.08
τtrip(μs) / 19.08.8 / 19.90.9 / 741 / 39436 / 21228 / 13.48.6 / 63.635.6 / 12.04.7 / 26.310.3 / 91.524.3 / 33.91.3
Gcorr inf / 1.013 / 1.005 / 1.01 / 1.026 / 0.999 / 1 / 1 / 0.999 / 1.001 / 1.001 / 1
χ2 / 1.481 / 1.219 / 1.036 / 2.688 / 1.032 / 0.946 / 0.898 / 1.072 / 1.095 / 0.847 / 1.32

Length of the analysis window is 2 s. FCS and PCH analyses were performed independently. Structure parameter a was fixed to the value, obtained from the calibration experiment (see Table S2). In the binning correction term of the PCH model we used estimates, obtained from the global analysis (see Table S1, measurement 2). PCDs were calculated with bin times of 20 s. ACF were calculated in quasilogarithmic time scale with minimum bin time of 0.25s. Standard deviations were calculated at the 67% confidence level by the asymptotic standard-errors method.

Fig. S6 Two-dimensional visualization of brightness profile functions. Graphs display the brightness atZ=0 (one-dimensional visualization rotated by 3600) where Z is the optical axis. The contour graph is plotted below.A)Gaussian profile. B) Polynomial profile for measurement 1 (see Table S1) of GFP (S65T) in Dictyostelium cytoplasm

1

[1]M. L. Johnson and L. M. Faunt: Parameter estimation by least-squares methods. Methods Enzymol., 210, 1-37 (1992)