DeepD3, an open framework for automated quantification of dendritic spines
Fig 4
Out-of-the-box performance of DeepD3 in external datasets and advanced image analyses.
A, Validation of DeepD3’s performance on three independently sourced and annotated datasets: In vivo iGluSnFR data was acquired in behaving mice using two-photon microscopy (dataset A [14]). In vivo Thy1-YFP data was acquired in behaving mice using two-photon microscopy (dataset B [14]). In vitro image stacks of counter-stained Biocytin-filled neurons of human brain tissue were acquired using confocal microscopy (dataset C [15]). Top: maximum intensity projections of example images of all three datasets with ground-truth annotation (red crosses) and DeepD3 3D ROI centroids (blue circles). Scale bar is 50 µm. Bottom: DeepD3 performance (recall) on the same datasets relative to the previously determined human IRR of the DeepD3 benchmark dataset (panels A and B). B, Utilization of DeepD3 for determining preferential localization of a nanobody against PSD-95 [18], tagged with mTurquoise2, to dendritic spines measured as the ratiometric spine-to-dendrite ratio (RSDR; see Online Methods). An RSDR value of >1 indicates preferential localization of the construct to the spine over the dendrite. Top: a maximum intensity projection of the raw data is overlayed with the RSDR of each DeepD3-generated spine ROI (purple to yellow, see color bar on the right). Scale bar is 50 µm. Bottom: box- and beeswarm plots of the RSDR measurements of all analyzed (N = 553) dendritic spines. C, Utilization of DeepD3’s dendritic spine segmentation for extracting calcium fluctuations of single dendritic spines using GCaMP7b. Bottom left: average projection of the analyzed calcium-imaging movie with DeepD3-generated spine ROI outlines in color and assigned numbers. Bottom right: calcium transients (ΔF/F0) of the outlined spines. Top: Pearsons’s correlation coefficient r of calcium transients of DeepD3-generated and manually generated spine ROIs using either raw data (blue) or 9-point moving averaged data (turquoise). Scale bar indicates 10 µm.