Gaussian weighted local variance computation process.

<p><b>A</b>) During the process, the source pixel is replaced with a Gaussian weighted variance of all the pixels inside the chosen window size. Each pixel integer value in the source image is multiplied by the corresponding value in the overlying Gaussian kernel, and the variance of all the resulting products is computed. The gray value of the source pixel (P_x,y) is then replaced by this Gaussian weighted local variance. This operation is repeated for each pixel in the original image. <b>B</b>) Source image and <b>C</b>) corresponding output image after application of the algorithm.</p

A) Fluorescence snapshots of AVRM expressing HKI-YFP (upper) or HKII-YFP under control conditions and after different periods of anoxia. Note that the mitochondrial network is very dense and uniformly dispersed, making difficult to define appropriate ROI if we wanted to apply the ratiometric ROI method. B) Corresponding spatial variance images. C) Average changes in spatial variance during anoxia, confirming that HK2, but not HK1, translocates to the cytoplasm during anoxia. Note that the measurement time points on differents cells varied, explaining the different number of measurements for each time point.

<p>A) Fluorescence snapshots of AVRM expressing HKI-YFP (upper) or HKII-YFP under control conditions and after different periods of anoxia. Note that the mitochondrial network is very dense and uniformly dispersed, making difficult to define appropriate ROI if we wanted to apply the ratiometric ROI method. B) Corresponding spatial variance images. C) Average changes in spatial variance during anoxia, confirming that HK2, but not HK1, translocates to the cytoplasm during anoxia. Note that the measurement time points on differents cells varied, explaining the different number of measurements for each time point.</p

The local variance algorithm efficiently detects redistribution between compartments.

<p><b>A</b>) Application of the weighted variance map algorithm to the model illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081988#pone-0081988-g002" target="_blank">figure 2</a>. As seen in the images, the spatial local variance is high in the regions of high fluorescence and decreases over time as the fluorescence is redistributed over the whole image. <b>B</b>) From the time course of the change in variance, the time constant of dissociation can be measured. Note that this time constant (6.93 a.u.) is very close to the time constant used to model the translocation (7 a.u., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081988#pone-0081988-g002" target="_blank">figure 2</a>). <b>C</b>) The plot of the local variance mean obtained by the analysis against the true degree of simulated protein translocation, which reveals that the analysis tends to be more sensitive to small changes in variance when the variance is high, whatever the kernel size used for the analysis. This property is an advantage to detect early translocation.</p

Robustness of the method when simulated mitochondria change position over time without otherwise redistributing their fluorescence.

<p><b>A</b>) Example of a sequence of simulated images recapitulating mitochondrial motility by random assignment of the location of the 2D Gaussians, while the size and width of the 2D Gaussians are kept unchanged over time (mitochondrial motility but no protein translocation). <b>B</b>) The corresponding computed spatial local variance for the images remains almost constant. <b>C</b>) The robustness of the method was tested on images simulating 1,000 different patterns of mitochondrial motility over time. Overall, the spatial local variance measured for all the simulations remains constant.</p

Simulation of hexokinase (HK) translocation from mitochondria to cytoplasm.

<p><b>A</b>) Area of high fluorescence arising from mitochondrial HK-bound was simulated by a series of 2D Gaussians, whose redistribution of the fluorescence simulates the translocation from mitochondria to cytoplasm. <b>B</b>) During this process, the height and the width of each 2D Gaussian are changed so the volume under the 2D Gaussian is kept constant, to simulate a redistribution of the fluorescence over the entire cell. <b>C</b>) Top view of the simulation of the translocation of HK from mitochondria to cytoplasm. A uniform random noise is added to generate the final simulation images.</p

Anoxic preconditioning (APC) prevents HKII dissociation from mitochondria during anoxia in NRVM.

<p><b>A & B</b>) Snapshots of fluoresence in NRVM during anoxia, illustrating rapid translocation of HKII-YFP from mitochondria to cytosol (A), which is prevented if NVRM are exposed to 3 short anoxic preconditioning exposures (1 min each) before the prolonged anoxia episode (B). <b>C & D</b>) Time course of changes in spatial variance (C) versus the ratiometric ROI method (D). For the ratiometric ROI methods, a ROI with a high concentration of mitochondria (1) was compared to a ROI placed into a region with few mitochondria (2) as illustrated in the series of images in A & B. Note that the measurement time points on differents cells varied, explaining the different number of measurements for each time point.</p

Hexokinase 2 translocation in living cells exposed to anoxia/reoxygenation.

<p><b>A</b>) Snapshot of fluorescence in CHO cells were subjected to 15 min chemical anoxia using the oxygen scavenger dithionite (1 mM), followed by 10 min of reoxygenation. <b>B</b>) Corresponding spatial variance images. <b>C & D</b>) Plot of the average spatial variance at the various time points, showing the time course of the dissociation (C) and re-association (D) of HK2 with mitochondria during the anoxia/reoxygenation episode. Note that the measurement time points on differents cells varied, explaining the different number of measurements for each time point.</p

Calcein-based assessment of the mitochondrial permeability transition (MPT) in live adult cardiac myocytes.

<p><b>A</b>) The mitochondrial network in AVRM was pre-loaded with Calcein-AM and then superfused with TMRM to record mitochondrial membrane potential. Upon exposure to 0.1 mM Phenylarsine Oxide (PAO) to induce MTP, calcein fluorescence redistributes within 10 min to the cytoplasm indicating MPT (upper series of images), and TMRM fluorescence concomitantly decreases dramatically indicating mitochondrial depolarization. Under each image, the corresponding spatial variance image is reported. <b>B</b>). Despite the differences in total fluorescence for the calcein and TMRM images, the corresponding spatial variance images predict similar times course of redistribution.</p

Time constant for the changes in FRET ratio evoked by addition of pyruvate, AOA and NaCN.

Phase 2t1 is for the addition of pyruvate, phase 3t1 for the addition of AOA, Phases 4t1 and 5t1 for the addition of NaCN and lactate t1 for the addition of lactate. Mean 33.98207 15.79144 20.53946 213.6842 21.87948. SDEV 13.85233 6.90242 11.35711 81.24961 7.461619. ERR Mean 1.74523 0.71193 1.45413 10.40295 0.940076. (TIF)</p

Data obtained in experiments carried out in HEK cells in which HKI had been overexpressed and which have been incubated for 24hrs in the presence of <i>6</i>-Aminonicotinamide (<i>6</i>-AN).

Data obtained in experiments carried out in HEK cells in which HKI had been overexpressed and which have been incubated for 24hrs in the presence of 6-Aminonicotinamide (6-AN).</p