Phagosome neutralization visualized using FITC-yeast. Amoebae of Dictyostelium discoideum were fed living budding yeast (S. cerevisiae strain TH2-1B) that had been labeled with fluorescein isothiocyanate (FITC, 0.25 mg/ml). The amoebae in phosphate buffer (pH 6.4) were examined 1 ½ hours later using a Zeiss LSM510 laser scanning confocal microscope; frames were collected in a single focal plane at 5-second intervals. The yeast fluoresce brightly in the extracellular medium, but are dim in acidic phagosomes because FITC fluorescence is quenched at acidic pH. At the end of the endocytic pathway, a phagosome is neutralized prior to exocytosis of the indigestible yeast carcass; that process is visualized here. The amoebae are also expressing two other fluorescent markers that are barely visible, mRFP-LimE-delta, which labels actin filaments, and VatM-GFP, a subunit of the V-ATPase. Three hundred frames from the time series were exported as .tif images and saved as a stack in ImageJ; they are also shown as a movie (.avi) at 10fps (available at CIL:7328). For related experiments and further details, see Clarke et al., PLoS ONE 5:e8585 (2010).
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