Advanced 4D tracking of meiotic spindle dynamic
Gunar Fabig, Müller-Reichert lab, Experimental Center, Medical Faculty Carl Gustav Carus, Technische Universität Dresden (TUD), Germany
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
The migration of interneurons in the developing mouse brain
Tibor Harkany Lab Department of Molecular Neuroscience, Center for Brain Research, Medical University of Vienna - Daniela Calvigioni, Zoltán Máté, János Fuzik, Fatima Girach and colleague
Imaging mouse brains using Light-Sheet Microscopy
arivis Vision4D as prerequisite for stitching and visualizing the mouse brain
- After import, individual z-stacks were aligned and stitched using the Tile Sorter
- Resulting mosaics were visualized in 3D using the 4D viewer
- Movies were generated using the storyboard feature
Handling large data sets to understand interneuron heterogeneity
This study benefitted from arivis Vision4D as the only software with the ability to easily stitch, visualize and share large data sets. Therefore, only the combination of Light-Sheet Microcopy and arivis Vision4D allowed the researchers to obtain 3D information of the entire brain and share this via movies in a very demonstrative way. Based on this, a classical tangential migratory route of CCK interneurons while populating the cerebral cortex was ob-served. Overall, this study identified several characteristics of prenatal CCK interneurons and integrates important information for unravelling the diversity of interneuron functionality
Fig 1: Stitching of individual tiles using the Tile Sorter; shown are 35 individual z-stacks covering a dual labelled mouse brain (CCKBAC/DsRed::GAD67gfp/+) at embryonic day 16.5; GA-BAergic neurons are marked by GFP (green) due to expression of GAD67; CCK expression is marked by DsRed (red). Tile 4 is selected and highlighted by a white box. For aligning, the Tile sorter provides different methods including Grid, Manual and Alignments using advanced algorithms. In this case, the z-stack tiles were sorted using the Grid mode; Pixel overlap was set to 10% as during imaging. Several planes were checked to assure correct alignment. Due to stability during image acquisition no further adjustments as algorith-ms were used. Scale options and transparency settings (see selected Tile 4) help to assure correct alignment.
arivis Big data imaging solutions
rivis AG, headquartered in Munich, Germany, is a market leading software company focused on the life sciences industry. arivis AG provides imaging solutions in multi-dimensional microscopy for datasets of basical-ly unlimited file size based on the in-house developed ImageCore technology. With our desktop software arivis Vision4D, scientists are empowered to work with terabyte sized images fast and efficiently on ordinary workstations and laptops. Additional benefit to usability and performance is the possibility to apply color mapping,rendering methods or quantification intuitively with imme-diate feedback and preview of the corresponding results. This potential can be scaled up with arivis WebView, a server-based image analysis framework that allows to access, display and analyze large image data in a standard web browser. With the world’s first and only virtual reality visualization system for real microscopy images, arivis InViewR allows scientists to gain all- dimensional insights by fully immersing into the data. www.arivis.com/imaging-science
Source Data set specifications
Microscope: Lightsheet Z.1 microscope (Zeiss), ×5 (EC Plan Neoﬂuar 5×/0.16) detection objective, ×5/0.1 illumination optics, PCO edge sCMOS camera; Imaging settings: ×0.7 zoom, laser power of 20v%, exposure times of 200 ms, tile scan overlap 10%; Data size: 100-300 GB; Picture size: up to 48 tiles, each 1200 px x 1200 px; Voxel size: 1,3 µm x 1,3 µm x 3,5 µm; z-depth: up to 1500 planes
arivis Software Package
arivis Vision4D Base Package