Digital Single Cell Analysis

3DCellAtlas is a computational approach to perform digital single cell analyses in 3D plant organs.

This approach identifies both cell types and cell position within 3D cellular reconstructions of plant organs, enabling equivalent cells in different images to be identified. Both the geometric properties and the abundance of reporter constructs within individual cells can be quantified and compared across different organs at single cell resolution.

Radially symmetric organs

Many plant organs have highly ordered and regular cellular arrangements enabling cellular annotation and internal indexing to be applied. This is particular true for radially symmetric organs. The publication describing this approach can be found here Montenegro-Johnson et al. 2015, The Plant Cell.

Here, a central bezier curve is placed down the centre of the organ and a mesh covering the surface generated.

Bezier curve shown in white

Surface mesh shown in red

Segmented hypocotyl cells shown in cyan

 

 

This guides the alignment of 3 axes describing the 3 principal directions of cell geometry of these approximately cuboid cells. Each individual cells has 3 axes aligned radially within it.

 

 

 

 

Plotting the radial distance on the x-axis and a selected geometric property on the y-axis, discrete clusters of cells corresponding to cell types emerge. These can be selected using a GUI within the interface in MorphoGraphX.

Cells which a mis-annotated can be identified using cellular connectivity and probability distributions of cell-to-cell interactions. This is illustrated below:

This approach works for any and all radially symmetric plant organs including hypocotyls, roots and mature embryos.

 

 

 

 

 

 

 

 

 

Cell identity is stored within the “parents” field for individual segmented cells in MorphoGraphX.

Cell position is identified using an internal cortical cell referencing system, where the first cortical cell is assigned a position of 1, and cell position increases incrementally along the length of the axis. Cell position onto other cell types is projected outwards radially.

 

 

 

 

 

Reporter abundance can be quantified within individual cells using MorphoGraphX. The total volume of reporter captured within the z-stack is quantified at single cell resolution by measuring the total reporter encapsulated within individual cells. Reporters can be expressed in absolute terms, or as concentrations taking cell volume into consideration.

Both cell geometry and reporter abundance can be quantified at single cell resolution. Equivalent cells between different samples can be identified enabling meta-analysis of diverse reporter constructs representing promoter activity, protein abundance or diverse molecular sensors.