a–i, Comparison of PSD-95 labelled with monoclonal primary antibodies directly conjugated to Alexa647 dye (1°-A647, red) with the same molecules labelled with primary and secondary antibodies conjugated to Cy3 (1°-2°-Cy3, blue) as represented in c. a, b, Comparison between non-synaptic small groups of localizations arising from isolated primary antibodies and secondary antibodies. Schematic shown in a. Standard deviation of localizations in both groups along different dimensions (n = 32 for A647; n = 36 for Cy3) in b. The two types of localizations groups showed similar variation in all dimensions. d, Local density maps of the same PSD-95 cluster labelled with 1°-A647 (top) and 1°-2°-Cy3 (middle) and overlapped distribution of 1°-A647 and 2°-Cy3 with detected nanoclusters highlighted in darker colours (bottom). Scale bar, 200 nm. e, Autocorrelation of synaptic clusters labelled with 1°-A647 and 1°-2°-Cy3. f, Autocorrelation of isolated small groups of localizations of A647 and Cy3 dyes. g, Comparison of the radius at which the autocorrelation function crossed with the random level (g(r) = 1). There was no difference between PSD-95 clusters with different labelling methods, but the r(0) for isolated localization groups were significantly less than r(0) for PSD-95 clusters. **P < 0.01, t-test between the filled and open bars of the same colour. h, Nanoclusters detected in both channels displayed no difference in number, volume, or the fraction of nanoclusters enriched with localizations from the other channel. i, Protein enrichment of localizations detected in each channels with those in the other channel (n = 32 synapses). These results demonstrate that the nanoclusters we detected in our study were not due to aggregation of multiple secondary antibodies to the primary antibodies. j–r, Cells transfected with knockdown-rescue-PSD-95-GFP were labelled with nanobodies against GFP conjugated at a 1:1 ratio with Atto647 (Nb-At647, red) and primary/secondary antibodies against PSD-95 (1°-2°-Cy3, blue) as depicted in l. j, k, Comparison between non-synaptic small groups of localizations arising from isolated Nb-At647 and 1°-2°-Cy3 (as depicted in j, n = 26 and 28, respectively). k, The nanobodies showed a significant smaller size than antibodies. ***P < 0.001, two-way ANOVA, †P < 0.05, ††P < 0.01, pairwise comparison (Tukey test) between nanobodies and antibodies. m–r, Similar comparison as in d–i between PSD-95 clusters labelled with Nb-At647 and 1°-2°-Cy3 (n = 13 synapses). Scale bar, 200 nm. Overall, these results demonstrated that the nanoclusters we detected in our study were unlikely a result of artefacts of antibody binding and labelling. The difference between the size of the isolated localizations groups and PSD-95 clusters calculated by autocorrelation also argues against the possibility that the nanoclusters we detected were owing to repetitive switching of one or a few fluorophores. **P < 0.01, t-test between the filled and open bars of the same colour. s, An example synapse with nanoclusters highlighted before (upper) and after (lower) removal of localizations resulting from fluorophores lasting for multiple frames. Scale bar, 100 nm. t, Paired autocorrelation function of synaptic clusters with and without multiple-frame molecules. P = 0.77, n = 25 synapses for RIM1/2; P = 0.58, n = 25 synapses for PSD-95, two-way ANOVA with repeated measures. u, The tracking removed 13 ± 8% and 17 ± 9% of the localizations for RIM1/2 and PSD-95, respectively, but had no significant effects on autocorrelation function results, nanocluster numbers, or nanocluster volumes. **P < 0.01; ***P < 0.001; NS, P > 0.05; Wilcoxon signed-rank test. All data were pooled from ≥3 replicas.