Central mitosomes associate with the flagellar axonemes. A Giardia cells were gently lysed by the passage through 33G needle, and the ruptured cells were analyzed by immunofluorescence microscopy. The immunolabeling of β-tubulin (green) and mitosomal marker GL50803_9296 (red) revealed that the intact mastigonts bear attached central mitosomes, while the cytoplasmic membrane and rest of cytoplasm was removed during lysis, scale bar 2 μm. Upper and bottom panels represent two examples of the same situation. B 2D STED superresolution microscopy of the mastigonts with associated mitosomes (primary antibodies as in A), scale bar 0.2 μm (0.1 μm in the insets), 30 nm resolution in XY axes. Upper and bottom panels represent two examples of the same situation. C The central mitosomes imaged in the frontal plane, scale bar 2 μm, and in D the transverse plane by 3D STED superresolution microscopy (primary antibodies as in A). Four optical sections are shown, scale bar 0.4 μm; the resolution was 100 nm in Z-axis and 120 nm in XY plane. E The reconstruction of 3D STED by Imaris illustrates the position of mitosomes (red) among eight axonemes (green), sections shown in D are indicated. C, A, P – caudal, anterolateral and posterolateral flagella, respectively.