Live-cell imaging of meiotic events in C. elegans
Tackling challenges in image analysis with arivis Vision4D
“The segment tracking enabled us to analyze the spindle elongation over time very efficiently. So far, it is the only working solution for us to analyze our data to our full satisfaction.”
Analysis pipeline using arivis Vision4D
Based on these advantages, Gunar Fabig created an analysis pipeline within arivis Vision4D to standardize the analysis of spindle elongation:
- After import, a denoising filter was applied.
- Individual meiotic events within the gonad were analyzed separately by creating 3D Regions of interest to export single spindles.
- Within these ROIs, spindle poles marked by GFP were defined in 3D using an intensity threshold segmentation.
- To exclude small, false positive segments, a filtering step based on volume size was applied.
- The segmented spindles were tracked over time by using the arivis Vision4D tracking module including the track editor.
- The analysis report was exported as Excel file including the center of mass coordinates of each segment over time.
Towards a big picture of the meiotic spindle
Based on this fast, quantitative analysis of spindle dynamics in wild-type C. elegans using arivis Vision4D, the Müller-Reichert lab is now able to further complete the big picture of the meiotic spindle and address outstanding questions. Altogether, this analysis will shed light onto the molecular mechanism, which allows the spindle apparatus to accurately segregate paired and unpaired chromosomes. Visit: https://tu-dresden.de/med/mf/cfci/forschung.
Fig. 1: 4D segmentation and 4D tracking of spindle dynamics; (left) 3D representation of male meiosis I in living C. elegans; spindle poles are marked by GFP fused to γ-tubulin, chromosomes are marked by mCherry fused to histone H2B. Segmented spindle poles are shown in green, chromosome are shown in red using the 4D Viewer of arivis Vision4D. Tracks of segmented spindle poles over time are depicted as lines. The left track is selected and can be easily identified using the track editor (right), highlighted in blue. Within the track editor, individual tracks are shown on the left, time frames on the top. Every segment is visualized as a small circle. A series of connected segments that form a track is visualized by a horizontal line connecting the segment circles. Merging or splitting of tracks can be easily done by clicking on segments and dragging them to their desired position.
Source Data set specifications
Microscope: Olympus IX83, invers; Andor IQ 3.2; equipped with Yokowaga CSU-X1, Andor iXON Ultra 897 EMCCD detection camera and Olympus U Plan S Apo 60x / 1.2 W objective; Data size: 1 GB to 3 GB; Time series: stack acquisition every 30 s for 45 to 60 min; Picture size: 512 x 512 px; Voxel size: 0.222 µm x 0.222 µm x 0.33 µm; z-depth: ~60 planes, 19,8 µm;
arivis Software Package
arivis Vision4D: Base Module, Analysis and Tracking Modules