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. 2019 May 3;30(18):2066–2109. doi: 10.1089/ars.2018.7534

FIG. 11.

FIG. 11.

Mitochondrial morphofunctional heterogeneity. (A) Summarizing scheme for simultaneous quantification of mitochondrial free ATP levels, membrane potential (Δψ), and mitochondrial morphology by single-cell fluorescence microscopy. Bold symbols (i.e., B, C, D, E, F, G, N) refer to the corresponding panels to the right. PHSFs (control CT5120) were transfected with a mitochondria-targeted variant of “ATeam” (Cox8-ATeam 1.03 or “MitoATeam”), an ATP-sensing CFP/YFP-based FRET (fluorescence resonance energy transfer) reporter molecule (150, 218). The YFP/CFP emission ratio of ATeam can be used as a measure of free ATP concentration. Subsequently, MitoATeam-expressing cells were stained with TMRM (that accumulates in mitochondria in a Δψ-dependent fashion). Next, the acquired images were processed and quantified to assess ATeam and TMRM signals for individual mitochondria and to allow multiparameter analysis. (B) Background-corrected (COR) image depicting the ATeam YFP signal. (C) Binarized (black-and-white; BIN) version of (B) obtained after image processing (explained in Fig. 13). (D) Version of the YFP-BIN image in which non-mitochondrial objects with an area <10 pixels (noise pixels) were removed by thresholding. (E) Masked (MSK) version of (B) obtained by applying a Boolean AND operation to the YFP-COR (B) and YFP-BIN image (C). (F) Same as (E), but now for the CFP-COR (not shown) and YFP-BIN image (C). (G) Same as (E), but now for the TMRM-COR (not shown) and YFP-BIN image (C). (H) ATeam ratio image obtained by dividing the YFP-MSK image (E) by the CFP-MSK image (F). To allow visualization, the ATeam ratio value for each pixel was multiplied by the number 20. (I) Colorized version of (F). Pixels were colorized according to their ratio value (blue = low; red = high). (J) Same as (I), but now for (G). (K) Magnification of the region in (H) depicting individual mitochondria. (L) Same as (K), but now showing a magnification of (I). (M) Same as (K), but now showing a magnification of (J). (N) 3D scatter plot of the ATeam ratio signal versus mitochondrial TMRM signal versus mitochondrial size (area) of the mitochondrial objects in (M). Each dot represents an individual mitochondrial object. Color images are available online.