Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 106 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.
Keywords: fluorescence microscopy; labeling efficiency; liquid-phase electron microscopy; membrane protein; nanoparticle; single-molecule analysis.