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A high-speed fluorescence microscope operating at a 490 Hz frame rate was used to image two different membrane proteins- the high-affinity IgE receptor FcɛRI, a transmembrane protein, and an outer-leaflet GPI-anchored protein. The IgE receptor was imaged via IgE labeled with Janelia Fluor 646 and the GPI-anchored protein was imaged using a GPI-GFP fusion protein and an ATTO 647 N labeled anti-GFP nanobody. Data was collected for both proteins in untreated cells and cells that had actin stabilized by phalloidin. This dataset can be used for development and testing of single-particle tracking methods on experimental data and to explore the hypothesis that the actin cytoskeleton may affect the movement of membrane proteins.