Three-dimensional Surface Reconstruction from Serial Section
Three-dimensional (3-D) reconstruction is an important technique for clarifying the spatialconfigurations of biological objects including plant organelles. Surface reconstruction is particularly
suitable for exploring the 3-D shape because reconstructed structures can be observed from any
arbitrary angle by rendering 3-D models.
Installation of REANT-light“REANT-light” is 3-D reconstruction software performing an interpolation between parallel planar
contours based on the Euclidean distance transformation.
System requirements・Deban GNU/Linux 4.0 i386: http://www.debian.org/

・

GNU Fortran 95 compiler 4.1

・Python 2.4

・

Python modules: numpy, numarray, psyco, pyvtk, PIL.

・MayaVi (scientific data visualization system)

・

ImageJ (image processing software):http://rsb.info.nih.gov/ij/index.html

All required software except ImageJ is included in Debian GNU/Linux 4.0.
Installation of REANT-light
1. Download http://www.biol.s.u-tokyo.ac.jp/users/hasezawa/kbi/reant-light/reant-light.tar.bz2 .
2. Extract files.
In the terminal (" %" represents a prompt of shell):% tar xvfj reant-light.tar.bz2

Preparation of TIFF stack file
1. Acquire a series of cross-sections as digital images.
Although we mainly utilize confocal microscopy for 3-D reconstruction, any approach can beused for the acquisition of sectional images, such as deconvolution algorithms and successive
physical sections.
* Pixel width (a.k.a. Δx) and pixel height (a.k.a. Δy) must be equal.
* Distances between sections (a.k.a. Δz) must be constant in a single series.
* Calculate and note the aspect ratio by Δz / Δx.
2. Convert the image series to a TIFF stack file.

If the image acquisition software does not handle TIFF stack format, the image-processing software“ImageJ” can be used to combine the multiple image files.
2-1. Load multiple image files by ImageJ menu File – Import – Image sequence... .
2-2. Save as a TIFF stack file by ImageJ menu File – Save as – TIFF.
Registration (optional)
If cross-sectional images are acquired by physical sectioning, the geometrical alignment of eachsection is required. This step is called “registration”. The ImageJ plugin “StackReg”(http://bigwww.epfl.ch/thevenaz/stackreg/) is a useful tool for automatic registration. Use the “RigidBody” mode for transformation.
Segmentation
Prior to reconstruction, the original image must be converted to a bi-level image (black and whiteimage). This step is usually called segmentation. The simplest method for the segmentation offluorescence images is intensity thresholding.
Intensity thresholding using ImageJ
1. Open TIFF stack file by the menu File – Open....
2. Create “Threshold” window by ImageJ menu Image – Adjust – Threshold....
3. Adjust horizontal slide-bars in Threshold window.
4. Convert to bi-level image by Apply button.
5. Save as a TIFF stack file by ImageJ menu File – Save as – TIFF.
NOTE: Do NOT overwrite the original TIFF file !

Surface reconstruction using REANT-light
Assumptions:
Filename of original TIFF stack:foo.tif

Filename of bi-level image:foo-bi.tif

Aspect ratio (Δz / Δx):

1.618

In the terminal,% python gen_vtks.pyc -r 2 -z 1.618 -V foo-bi.tif foo.tif

generates an OUT-pol.vtk file containing the 3-D model.Rendering of 3-D model using MayaVi
With default parameters, REANT-light generates the 3-D model as a Visualization Tool Kit (VTK)
file as described above. A scientific data visualization system called “MayaVi” performs therendering (displaying the 3-D model) of VTK format files in Linux and Microsoft Windows.
1. Start MayaVi.
2. Load VTK file by MayaVi menu File – Open – VTK file.
3. Select “Z-coor-pt” as scalar (the depth of focus is used for the coloring of 3-D objects).
4. Display the 3-D model by MayaVi menu Visualize – Modules – PolyData.
5. Capture and save the rendered image to file by MayaVi menu File – Save Scene to – TIFF
image.
The reconstructed 3-D model is interactively rendered in the right panel of MayaVi. The explorationof 3-D space is performed by a point-inputting device (usually a mouse or track-ball) as follows.
Operation of mouse:
Left-button: Rotate
Mid-button: Shift
Right-button: Zoom in/out