Fig 1.
Workflow of vesicle annotation using 3D ART VeSElecT.
First, the automated registration macro is used, which scales the tomogram via user input of the pixel size and applies various filters in the preprocessing step. Afterwards the foreground is separated, the user semi-automatically selects an area of interest, and the macro applies the watershed algorithm for vesicle segmentation and registration. Second, an optional manual proof-reading step can be applied here, if necessary. Finally, the automatic measurement macro is used to extract results using certain characteristics. All manual steps are colored in yellow, semi-automated steps are in turquoise, automated steps are in blue.
Fig 2.
Analysis of embryonic zebrafish NMJ using 3D ART VeSElecT in comparison to manual analysis using IMOD.
We show in Fig 2 A) the original tomogram of 4dpf zebrafish NMJ, in Fig 2 B) the manual reconstruction is included in the tomogram of A), in B') the 3D reconstruction of the manual annotation (vesicles are colored in light blue) is shown. This is compared to Fig 2 C) which shows the semi-automated vesicle recognition overlaid with the original tomogram, and C') which shows the vesicle pool of the semi-automated annotation as 3D reconstruction (vesicles are in arbitrary colors). In D) boxplots show the results of the comparison of 4dpf and 8dpf zebrafish embryos using manual annotation (left) and semi-automated annotation (right). The box of the box plots shows the mid-50% of data. The line in the box represents the median of all data. Whiskers end at lowest value within 1.5 interquartile range (IQR) of the lower quartile and at the highest value within 1.5 IQR of the upper quartile. Data that is not included in between both whiskers are plotted as outliers with a dot.
Fig 3.
Measurement of inner and outer synaptic vesicle diameter.
In order to get an approximate value of the discrepancies between vesicle diameters of manual and automated measurement we applied the Fiji measurement tool. Fig 3 (A) shows the inner diameter of a vesicle that was annotated by 3D ART VeSElecT, (B) shows the outer diameter. Fig 3 (C) gives the results of the discrepancy of inner and outer diameter of all measured vesicles shown as a histogram (number of measurements = 80).
Table 1.
Tabular schedule showing required time for semi-automated reconstruction.
Table 2.
Error rate of zebrafish tomograms.
Fig 4.
Comparison of N2 and unc-59(e261) C. elegans tomograms using 3D ART VeSElecT.
Fig 4 shows steps of vesicle annotation using 3D ART VeSElecT on N2 (A-A”) and unc-59(e261) (B-B”) C. elegans tomograms. In (A, B) the initial tomogram is illustrated, (A’, B’) show the composite stack that includes the original tomogram plus the annotation of the vesicle pool (vesicles are shown in arbitrary colors). The three dimensional model of the semi-automated annotation (automated registration plus manual proof-reading) of the vesicle pool is shown in (A”, B”). In (C) the semi-automated vesicle pool annotation of (A’) was transferred to IMOD where further structures of the NMJ were complemented. Vesicles are shown in yellow, cell membranes are green, dense projections are blue, endoplasmatic reticulum is white and microtubules are in shades of purple. In (D) synaptic vesicle sizes of N2 and unc-59(e261) were compared. Figure (E) illustrates the comparison of vesicle pool density by determining the three closest vesicles (N2 is illustrated in dark blue, unc-59(e261) is illustrated in light blue). In (F) the approach for determination of the vesicle pool density is illustrated. It is shown that for each vesicle the distance of its center to the center of its three closest neighbors is calculated. (scale bars = 100 nm).
Fig 5.
On the right, upper side a dot plot is shown, representing zebrafish (turquoise) as well as C. elegans (dark red) error rates.
Each dot represents precision and recall of one tomogram (magnified detail plot on the lower right side). Tomograms that are shown as examples on the left side of the figure are represented as a special sign (e.g. triangle or square) instead of a round dot in the plot. On the left side, example 1 shows vesicles of a high quality tomogram, with good results for recall (low number of false negatives) and precision (low number of false positives). In comparison to one tomogram with worse precision (example 2) and two tomograms (example 3 and 4) with a higher rate of false negative vesicles, i.e. lower recall. Furthermore, examples 5 and 6 show vesicles of one tomogram with best precision and recall rate (example 5) in comparison to one tomogram with very low precision. Scale bars: 20 nm.