As an alternative to DLS, the size and shape of LNPs was also analyzed by imaging techniques, i.e., AFM and SEM. In general, AFM and SEM yielded similar results regarding the shape of the particles (Figure and Figure ). LNP-1 particles appeared slightly ellipsoidal compared to the more spherical and uniform particles in LNP-2. LNP-3 and LNP-4 showed the presence of agglomerates; however, AFM and SEM did not match well regarding their visible amount. LNP-3 showed more agglomerates in AFM compared to SEM, while the opposite was true for LNP-4. This highlights the influence of sample preparation on particle analysis with AFM and SEM. In contrast to shape, the measured particle size did not match between AFM and SEM at all. Radii obtained by SEM were 40-100 % smaller (64, 103, 41, and 70 % for LNP-1, LNP-2, LNP-3, and LNP-4, respectively) compared to radii obtained by AFM or offline DLS (Table1). SEM preparation causes dehydration of the particles, relative to ambient drying of AFM or fully hydrated sizes of DLS. Also, tip convolution effects related to the diameter of the AFM probe can increase the size of the particles. However, a major disadvantage of imaging techniques such as AFM or SEM is the limited sampling size that is analyzed, rendering quantitative analysis prone to error. Moreover, LNPs tend to form aggregated clusters during the drying of the LNP dispersion on mica surfaces or SEM stubs. Consequently, the particle composition can be vastly different throughout the analyzed specimen, thus results obtained by AFM or SEM are highly dependent on the spots which are chosen for (quantitative) analysis (Figure ). Increasing the accuracy of these methods requires further tedious work, e.g., analysis of multiple spots and specimens, and frequent change of the AFM probe. Still, very inhomogeneous samples that contain aggregates in their distribution (i.e., LNP-3 and LNP-4) are challenging to analyze with either technique (e.g., AFM, SEM, offline DLS).