Procedures for volume analysis and spatial statistics of 3D point sets in IgorPro. A detailed walkthrough of the analysis procedure is available here. This README contains a brief overview.
Two types of analysis are possible
Microtubules are first segmented in Amira. Amira mesh files are converted to TIFFs and skeletons using a FIJI macro am2skel.ijm. This process .am files to first threshold them and produce categorised 1 px thick MT trajectories in 2D (one for each z-slice). These are called skeletons. The thresholded TIFFs are used for Volume Analysis and the skeletons are used for Spatial Statistics.
###Setup
Place all procedures in User Procedures directory which can be found in:
Mac: /Users/<user>/Documents/WaveMetrics
Windows: C:\Users\<user>\Documents\WaveMetrics
Load VFMasterproc.ipf into a new experiment and then one of the following options:
- Volume Finder
- Spatial Analysis
- All analysis
- Export to R
From the Macros menu.
Caution: Igor 7 only. Most of the code will not compile or run in Igor 6.3 and below.
###Volume analysis This workflow measures the density of microtubules in a stack of TIFFs. Igor will compute the volume of microtubules as a density of the volume in which they are contained. This was written for analysis of segmented data from SBF-SEM (3View).
To do this call VolumeFinder(). Now, point Igor at the directory containing the TIFFs.
You can scale the output to real world values using ScaleIt(xnm,ynm,znm). Using the correct voxel size. This will scale the point volumes and hull volumes to µm^3.
###Spatial Statistics
Skeletons are processed by Igor FindingVectorsFromSkeleton.ipf to form 2D vectors which can then be used for spatial statistical analysis. Vectors are found by a linear fit to xy coordinates.
Igor will produce a report which shows three spatial statistics:
- A comparison of all MT vectors with the spindle axis (defined by two xyz coords at the start of the procedure). This is colour coded (with a key) to show variance in angle from the spindle axis. Histograms summarise this information (4 histograms: 1 for each pole, 1 for all angles, 1 for all angles, abs() values).
- Two further histograms compare MTs that are longer than 60 nm are within 80 nm of other MTs. This is independent of the spindle axis.
- A comparison between the trajectories of MT segments with ellipsoid tangents calculated from an idealised spindle.
Each cell/movie is analysed as a separate pxp. Use SummaryPXP.ipf to make a summary report of all your data. Compare distributions using the command MakeComparison().
####Extra code
A little tool called checkAxis.ipf was developed to help visualise and correct spindle axis in 3D via gizmo. Current workflow is to: 1. run the load and analysis (using input coordinates for poles); 2. use checkaxis to refine the axis (for all datasets); 3. redo the analysis.
###Analysis using R
The analysis can be reproduced in R. Simply use this R markdown file, following the instructions here. The analysis in R requires the xyz coordinates of the start and end points of all lines generated from the skeletons. These coordinates can be output in csv format using ExportToR.ipf, see the instructions for further details. Alternatively, you may use some other non-Igor workflow to get the coordinates and run the analysis in R.
###Calculation of SNR
An ImageJ macro will extract the mean pixel density in segmented MT regions (per slice) and calculate the SD of a halo around the MTs (excluding MTs themselves), SNR is calculated and output as a csv. Igor code Igor code is available to crunch the output.