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Fig. 4. In vivo laser bleaching experiments show that the S. neurona plastid is a continuous organelle. In vivo laser bleaching experiments were performed to test if a fluorescent marker freely diffuses along the entire length of the tubular plastid. The extent of bleaching predicted for organelles with continuous (A) and discontinuous (B) lumen is depicted schematically. FNR-RFP-expressing S. neurona (C-H) and T. gondii (I-K) parasites were imaged in vivo. Plastids were exposed to a series of short laser pulses at the position indicated by the laser symbol (see Materials and Methods for detail). Cells were imaged before (pre) and after (post) bleaching. (C,F,I,L) Single fluorescence images at the focal plane; (D,G,J,M) merged images of C,F,I and L with the respective DIC image; (E,H,K,N) rendered 3D projection of the entire z-stack. (A quicktime movie of these data is available in supplementary material.) Upon exposing the distal end of the S. neurona plastid to multiple laser pulses the entire organelle is bleached (F-H). (I-K) Experiments to control for the spatial precision of the laser were performed with FNR-RFP expressing T. gondii parasites. (L-N) A single plastid was exposed to the laser resulting in bleaching of only the targeted organelle (white arrowhead) without affecting its nearest neighbors.
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