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. 2004 Feb 16;164(4):493–499. doi: 10.1083/jcb.200309082

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Copyright © 2004, The Rockefeller University Press

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Figure 2. — Visualization and quantification of mitochondrial fusion using mito-PAGFP. (A) Primary hippocampal neurons (a–c) and HeLa cells (d–e) were cotransfected with mito-DsRED2 (shown processed in Adobe Photoshop with emboss filter) and mito-PAGFP (green). Mito-PAGFP within some of the mitochondria was activated with 413-nm light followed by time-lapse 3D confocal microscopy. Activated and nonactivated mitochondria (arrowheads) are shown 30 s before (a–e) and after (a'–e') mitochondrial fusion and intramitochondrial exchange of matrix contents (visualized by an increase in the amount of activated mito-PAGFP in nonactivated mitochondria). (B) The region indicated by the red circle in a mito-PAGFP–transfected HeLa cell (preactivation) was photoactivated, followed by time-lapse acquisition of images. Between 45 and 50 s the photoactivated mitochondrion divides (arrowheads), followed at 180 s by redistribution of mito-PAGFP from activated to nonactivated mitochondria (arrows,) indicating mitochondrial fusion. To highlight the fluorescence decrease in activated mitochondria and the increase in nonactivated organelles, images were false colored (right). (C) HeLa (a), Cos-7 cells (b), hippocampal neurons (c), WT MEFs (d), and Mfn1^−/− MEFs (e) were transfected with mito-PAGFP, followed by photoactivation of one to four ROIs per cell (white circles) and time-lapse confocal acquisition of a series of z-sections covering the entire thickness of the cell as described in Materials and methods. Images shown are pseudocolored projections of z-series. (D) Changes in the fluorescence intensities within activated (red) and nonactivated (black) mitochondria of HeLa (a), Cos-7 (b), hippocampal neurons (c), WT MEFs (d), and Mfn1^−/− MEFs (e) were measured over time as described in Materials and methods. Note the small change in fluorescence intensity of activated mito-PAGFP in Mfn1^−/− MEFs even 1 h after photoactivation when redistribution of the matrix content greatly decreases fluorescence intensity of mito-PAGFP in other cell types.

Figure 2. — Visualization and quantification of mitochondrial fusion using mito-PAGFP. (A) Primary hippocampal neurons (a–c) and HeLa cells (d–e) were cotransfected with mito-DsRED2 (shown processed in Adobe Photoshop with emboss filter) and mito-PAGFP (green). Mito-PAGFP within some of the mitochondria was activated with 413-nm light followed by time-lapse 3D confocal microscopy. Activated and nonactivated mitochondria (arrowheads) are shown 30 s before (a–e) and after (a'–e') mitochondrial fusion and intramitochondrial exchange of matrix contents (visualized by an increase in the amount of activated mito-PAGFP in nonactivated mitochondria). (B) The region indicated by the red circle in a mito-PAGFP–transfected HeLa cell (preactivation) was photoactivated, followed by time-lapse acquisition of images. Between 45 and 50 s the photoactivated mitochondrion divides (arrowheads), followed at 180 s by redistribution of mito-PAGFP from activated to nonactivated mitochondria (arrows,) indicating mitochondrial fusion. To highlight the fluorescence decrease in activated mitochondria and the increase in nonactivated organelles, images were false colored (right). (C) HeLa (a), Cos-7 cells (b), hippocampal neurons (c), WT MEFs (d), and Mfn1^−/− MEFs (e) were transfected with mito-PAGFP, followed by photoactivation of one to four ROIs per cell (white circles) and time-lapse confocal acquisition of a series of z-sections covering the entire thickness of the cell as described in Materials and methods. Images shown are pseudocolored projections of z-series. (D) Changes in the fluorescence intensities within activated (red) and nonactivated (black) mitochondria of HeLa (a), Cos-7 (b), hippocampal neurons (c), WT MEFs (d), and Mfn1^−/− MEFs (e) were measured over time as described in Materials and methods. Note the small change in fluorescence intensity of activated mito-PAGFP in Mfn1^−/− MEFs even 1 h after photoactivation when redistribution of the matrix content greatly decreases fluorescence intensity of mito-PAGFP in other cell types.