Isosurfaces are a common tool for visualising volumetric voxel data sets such as those produced by confocal microscopy. The algorithms and software tools used to generate isosurfaces for confocal can be applied to super-resolution images after performing a 3D density reconstruction (see Image Reconstruction). This indirect approach, however, has a number of disadvantages. To capture detail in the data sets generally requires the use of a small reconstruction voxel size, resulting in exceptionally large datasets. A 10x10x10 µm super-resolved volume with a 5 nm pixel size would give rise to an 8 gigavoxel (32 GB) reconstructed volume. This represents a major computational challenge, in practice limiting such reconstructions to small ROIs and often smoothed and downsampled data. A second limitation is the need to choose this voxel size in advance. Due to the stochastic nature of localisation microscopy, choosing an appropriate reconstruction voxel size is not a trivial problem - different parts of the image could well have a different optimal voxel size.
In PYMEVisualise we have implemented algorithm which permits isosurfaces to be extracted much more efficiently from point datasets without a conventional image intermediate. Our algorithm initially places points into an octree data structure [meagher1980] (Fig. 6 b). We then cut / truncate the octree at a given minimum number of localisations per octree cell (equivalent to a minimum signal to noise ratio (SNR) - see [baddeley2010]). This has the effect of dividing the volume into cubic cells with a size which adapts to the local point density. Cells will be large in areas with few localisations, and small in areas which are localisation dense. The result is a volumetric data structure that contains the same information as a fully sampled reconstruction but with a lot less elements. We calculate a local density of localisations in each cell and then run the Dual Marching Cubes ([schaefer2005]) algorithm on this with a given density threshold (Fig. 6 c).
The algorithm for isosurface generation is accessible from the menu as Fig. 7 a) allowing parameters of the isosurface generation to be adjusted. The parameters are as follows:. This will construct the octree over which the isosurface is calculated and then display a dialog (
- class DualMarchingCubes
Input – the octree name (do not modify)
NPointsMin – the leaf size (number of localisations) at which we truncate the octree. A higher value increases the SNR at the expense of resolution.
ThresholdDensity – the threshold on density (in localisations/nm^3) at which to construct the isosurface.
Remesh – improves mesh quality by subdividing and merging triangles such that triangles are more regularly sized and the number of connections at each vertex is more consistent. This improves both appearance and the reliability of numerical calculations on the mesh (e.g. curvature and vertex normals). Disable when experimenting with thresholds to improve performance.
Repair – will patch holes in the mesh (usually not needed).
Another method of surface extraction from point data sets is to fit spherical harmonics ([singh2011]. When multiple objects are present in a field of view, these will need to be segmented first.). In contrast to the isosurface method (which simply thresholds on density) spherical harmonic fitting assumes that points lie on a surface. Because it is model based it is much better constrained and can extract accurate surfaces from significantly sparser datasets. It is ideally suited to the extraction of the cell nuclear envelope based on a lamin or NPC staining, but is also applicable to other “blobby” structures which are shell-labelled
A number of operations are possible on meshes generated using either isosurfaces or spherical harmonics. These meshes can
be colored by variables, such as
z, and curvature, as in Fig. 7 d. Meshes can be
exported to STL or PLY format, suitable for importing in other software or 3D-printing. There are also a growing number
of analysis options (e.g. which calculates the signed distance between
localisations and the mesh) which operate on the meshes.
Meagher, “Octree Encoding: A New Technique for the Representation, Manipulation and Display of Arbitrary 3-D Objects by Computer,” Rensselaer Polytech. Inst., no. Technical Report IPL-TR-80-111, 1980.
Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal., vol. 16, no. 1, pp. 64–72, 2010.
Schaefer and J. Warren, “Dual marching cubes: Primal contouring of dual grids,” Comput. Graph. Forum, vol. 24, no. 2, pp. 195–201, 2005.
Singh et al., “Non-parametric Population Analysis of Cellular Phenotypes,” Med Image Comput Comput Assist Interv., vol. 14, no. 2, pp. 343–351, 2011.
Barentine et al., “3D Multicolor Nanoscopy at 10,000 Cells a Day,” bioRxiv, 2019.