Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.