FM 1-43 can be used as an endocytosis tracer using photo-oxidation electron microscopy.


<p>(<b>A</b>) Experimental outline: IHCs incubated with FM 1-43 take up the dye (red symbols) in endocytotic organelles, but also in the cytosol, as a result of dye penetration through mechanotransducer channels. The dye in the cytosol attaches itself to the external (cytosolic) leaflets of the organelle membranes. After fixation and incubation with DAB (indicated as the blue background), the cells are illuminated with blue light; the FM molecules bleach (gray symbols) and emit reactive oxygen species that cause DAB precipitation (represented as black symbols). The DAB precipitate diffuses in the cytosol, unless it is trapped within the organelles. Therefore, endocytotic organelles are seen as black objects, while the FM dye in the cytosol only causes a general darkening of the cell volume. (<b>B</b>) In presence of the dye numerous dark organelles appear (some examples are indicated by red arrowheads). (<b>C</b>) In the absence of the dye only mitochondria are visibly labeled. Mitochondria are labeled by photo-oxidation by mechanisms independent of the addition of fluorescent markers, and are indicated by white asterisks. (<b>D</b>) Apart from mitochondria, no labeled organelles were found in cells incubated with FM 1-43 at low temperature (2–4°C), confirming that dye photo-oxidation selectively reports endocytotic events – despite the fact that FM 1-43 penetrates into the IHC cytosol at low temperature. The image is over-exposed, to increase the contrast as much as possible, which should allow the detection of any labeled organelles. Nevertheless, none are present, other than mitochondria. Scale bars, 500 nm.</p

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