The Trebouxia photobiont freshly isolated from Xanthoria parietina (L.) Th. Fr. was used to develop a live cell chondriome (mitochondrial DNA) labelling method. In the initial phase six candidate dyes were tested and compared for mitochondrial labelling utility as assessed by the signal to noise ratio (SNR) of the mitochondrial signal to the adjacent cellular background in standardized confocal images of 30 labelled cells. DIOC7, JC-1 and MitoTracker orange (MTO) dyes showed some labelling ability. MTO had significantly higher utility than the other dyes. In a second phase, MTO concentration was optimized. The final labelling protocol was a 30 minute incubation with 1 μM of MTO. The resultant labelling was suitable for both widefield and confocal microscopy. Both 2D thresholding and 3D volume construction are demonstrated using the resultant data. The protocol can therefore be utilized for both qualitative research and for quantitative measurement of the chondriome in Trebouxia photobionts. This will facilitate a wide range of mitochondrial investigations in lichenology.
