Microtubules are first segmented in Amira. A detailed walkthrough of the analysis procedure is available here.

csv with a list of conditions in each image (encoded with an integer).Procedures for volume analysis and spatial statistics of 3D point sets in IgorPro.

log file from blind_analysis for unblinding the images.

csv with locations of particle xy coords.

To run Stereology Workflow the following files are needed: ijm) so that the person locating particles is blind to the conditions of the experiment.ДЄ segmented version of each EM image is also required (in a directory called Masks). To do this a images are stripped of metadata and the filenames encoded (using blind_analysis. The locations of particles in images are recorded in ImageJ and saved as a csv. The function LoadTIFFFilesForAnalysis() works on a directory of TIFFs, but it requires several waves loaded into the experiment to do this properly. The location of the peaks is then used to calculate the SNR. In the file FTAnalysisSNR.ipf, you will find tools to fit 2D Gaussian functions to FerriTag particles. There are two layouts which can be saved in a variety of formats. Igor will then display several graphs of the analysis. Drag the Cursors from the little box below the graph.Д¬lick continue and the next one will display until you have loaded all models from your directory. Use Cursors A and Cursor B to mark out what you judge to be the start and end of the pit. Igor will load each model and display it to you with a graphical interface. To run the analysis, select Macros > FerriTag Analysis or run IMODModelAnalysis() from the command line. We make a csv and load it in using Igor's Load > Data > Load Delimited Text. This is important and the analysis will not run without it.

PixelSize (numeric) size in nm of a pixel.

FileName (textwave) containing the names of each txt file.

You need to provide this as two waves called: Igor needs scaling information for each image/model to do the analysis. These txt files should be in a directory with no other txt files (other filetypes are OK). Model2point -fl -ob -z example_Model_IMOD example.txt Output models as text files using model2point. To start you need to segment your images in IMOD. These functions (in IMODModelAnalysis.ipf) will load and analyse FerriTagged particles near clathrin-coated pits visualised by EM. To estimate the number of measurements are required, type PowerTest(8,22).

To run the simulation many times (settings are hardcoded), type RunMultiple(n) where n is the number of times you'd like to run the program. Note, that the main program requires a wave of observations called FTMeasWave. Plasma membrane can be generated using PMMaker(). To generate a 3D figure to show the pose of FerriTag and the measurements taken, run FerriTag(16,6.5,70,100) and append posSect and posWave as scatter in a gizmo. To run the simulation, type LookAtPDFs(7,18), for example. Utility functions (called from other functions only) Run the main program many times to test robustness of simulation MakeFTPlot() compares real data with the modelД®stimation of the number of observations required to determine the spatial resolution of the method.LookAtPDFs() visualise probability density functions.Igor procedure file Ferritag.ipf featuring: Stereological analysis of particles in EM images.Signal-to-noise ratio calculation and 2D Gauss fitting of FerriTag particles.Nanoscale mapping FerriTag distributions using IMOD outputs.

Computer simulations and spatial analysis of FerriTag in Igor Pro.

Igor pro 7 gizmo