has been essential for bringing especially liposomal drugs into the clinic. [3][4][5] Initial evidence supported that PEGylation reduced opsonization of the liposomal surfaces by creating a steric barrier. [6] However, the complexity of this concept is apparent from studies showing that complement C3 and antibodies efficiently bind PEGylated liposomes, [7,8] hamper the performance of liposomal systems, [9] and in the most extreme cases induce hypersensitivity reactions [10,11] or accelerated clearance upon repeated injections. [12,13] Thus, the current ambiguity regarding the immunological requirements for stealth properties [14] -reduced opsonization [15] or increased dysopsonization [16] -represents a bottleneck for overcoming the widely recognized translational gap in nanomedicine. [17][18][19] Traditionally, anti-liposome antibodies are studied by ELISA. [20][21][22][23] ELISAs are however claimed to suffer from nonspecific binding and -since individual PEG-moieties are simply adsorbed to a flat surface -does not reflect binding to authentic liposomal surfaces with respect to PEG density and topology, lipid mixture, surface curvature, as well as membrane-charge and -fluidity. [24][25][26] Alternatively, liposome opsonization has been studied using bulk biochemical assays, where the protocol includes crude centrifugation steps and/or extensive washing procedures, before opsonization is inferred from indirect detection methods. [7,24,25,27] The validity of such separationbased approaches has been questioned due to concerns about contamination of non-bound proteins in the nanoparticle isolates. [28][29][30][31] In addition, such bulk assays report only average ensemble values, thereby intrinsically assuming all liposomes in the sample to be identical. [32,33] However, it is increasingly recognized that opsonization might be heterogeneous between nanoparticles and that this variation could have great functional importance, [1,31,33,34] prompting the need for new and more sophisticated assays. [24,25,35] Investigating opsonization heterogeneity however means studying individual liposomes in complex biological environments, which is a formidable task due to their nanoscale size. Imaging methodologies capable of directly detecting protein adsorption to individual particles, have only Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibo...