Signal transduction entails the precise interaction of relevant proteins for transmission of signaling pathways that carry out essential functions. In cancer, dysregulated protrusion growth underlies metastasis and disease progression. Breast cancer cell protrusions express HER2 and HER2 has been linked to protrusion growth, but details of how HER2 signaling is conducted in protrusions remains unclear. Here, I demonstrate a sensitive and quantitative method for analyzing the expression and spatial localization of HER2 and pAKT using quantum dots (QDs). I also employ novel monovalent HER2-QD probes using various imaging modalities (single particle tracking, EM, and super-resolution) to characterize the spatiotemporal features of HER2 signaling in breast cancer cell protrusions.