1. Click open data This set of slides shows how to generate 3D renderings in Avisio from Tomographic Reconstructions from Inspect 3D or ETomo

2. Step 2 Select Reconstruction file from Inspect 3D session

3. Step 3 A Left click on Loaded Data in project view to display favorite options B Right Click to display all available options 1.Click here Favorite options are displayed after left click

4. Step 4. A Click Interactive ThresholdingB. The following screen now comes up:

5. 4C Now adjust thresholding so you can see your nanofibers, blue color indicates the selected area Slide threshold so you Can see fibers Change slice number here

6. Step 4, Thresholding final step C: Hit Apply to generate thresholded data: 4C: Hit Apply!

7. The steps in project view have been dragged to show the workflow that we just did on the data! Drag colorbars So you can see all objects of the project Thresholded data is now generated hiding in new green colorbar

8. Step 5: To see the thresholded volume in 3D: A) Get rid of slicing plane by deactivating Interactive Thresholding B) Select new green box with thresholded data A) Press Red button to stop displaying threshold plane B) Left click, then right click

9. Step 5 C) Select Volume Rendering, a new yellow double box comes up (it is already connected with an arrow to the thresholded data) D) Use Hand Tool to rotate data freely around in the viewer window C) New colorbar for Volume rendering D) Hand Tool

10. Generate Surface A)Select Thresholded data B)Select Generate Surface Module C)Hit Apply D)Wait (1-3 minutes) A)Left Click B) New module D) New surface data Still displaying thresholded data

11. Surface Simplification is required because the generated surface is too big A)Select surface data (lowest green colorbar) B)Open simplification editor C)Set number of faces to 1,800,000 (add two zeros) D)Hit Simplify Now E)Wait some minutes A)Left Click Simplification Editor (second from left) Initial number of faces Change number of faces Hit Simplify now

12. View Surface A) Deselect Volume Rendering B) Right click on the surface module and select Surface View B) Deselect (color changes from orange to grey) B) New module surface View

13. Auto Skeleton After step 4 (creation of the thresholded data), skeletonizing can be done. The Auto Skeleton function however, usually does not work the way we want it. Left click, right click, type “skeleton” in the search field and select when it shows up

14. Hit Apply on the green button A red colorbar “Auto Skeleton” will appear, left click to select it

15. Manual Skeleton Several Individual steps need to be performed depending on the dataset: resampling, closing, dilating, eroding. But there is no standard way and some trial and error. After applying those steps/filters/functions, the thinning function must be used and only if it yields a graph/line – like skeleton the Auto Skeleton function will result in the desired skeleton and statistical data can be generated

16. Fill in Steps on Surface rendering. Every time I run a surface rendering in Avizio it crashes. Reduce data set first? Best way to do that? Then how to edit surface plot properties ….

17. Different data types File endingContainsDescriptionTypical size Tilt series.mrc.st Header and tilted imagesOne image per angle, angles and general informationare stored in header 2Mb per image (1024x1024x2byte)  280 Mb for 140 angles reconstruction.recAligned imagesGrid (voxels) containing parallel images (pixels) (such as slice and view) 2 bytes per voxel  400nm thick  1.1 Gb Avizo modified.filtered etc Voxels with intensityGrid generated by Avizo after specific function is applied Same as reconstruction or reduced (e.g. when resampled to certain size) Avizo segmentation.labelVoxels (binary) grouped by objects of interest Voxels assigned to segments Intensity  segment# Same as reconstruction Avizo surface.surfaceMesh of Vertices and facescreated from grid using specific algorithm Ca. 10 Mb, fast computer can handle 100Mb Other surface.ply.stl Mesh of vertices and facesCreated from vertices in space Ca. 10 Mb, fast computer can handle 100Mb Avizo skeleton and other measurements.SptGraph etc Skeleton: Lines and nodes, Charts, histograms, etc Skeleton contains of lines and connection of the lines (nodes) modeling thin features, Measurements are numbers in text format and plotted accordingly Very small < 1Mb

18. Tilt series Volume Rendering Label Field Surface Video Data acqui sition Numbers Reco nstru ction Reconstruction file Inspe ction Segm entati on Surface reconst ruction Simplifi cation Animati on Skeleto nizing Spatial Graph Measur ements