Table 1: Pseudocode for the determination of model-based parameters and estimation of cell number. Histogram analysis of cluster volume and integrated intensity are used to estimate single cell clusters. Model parameters are determined from the estimated single cells. These parameters are used to estimate the number of cells in a given cluster.

Preprocessing and determination of model cell parameters

Load aligned, next image processed cryo-image stack
Perform morphological reconstruction to remove background
User interactively determines , , and
Perform thresholding of ratio image
Perform hysteresis thresholding
Perform connected component analysis
Display histogram of cluster volume
User selects single cell volume range based on largest peak
Display histogram of integrated intensity
User selects single cell integrated intensity range based on largest peak
For all clusters that are within the user defined integrated intensity and volume range
Fit Gaussian with free parameters , , and to each cluster
Perform an average of estimated model parameters to determine and

Estimate number of cells per cluster

For a given cluster
Estimate number of cells per cluster
Test integer numbers of cells within the range
Perform nonlinear optimization to place the model cells in the 3D positions that best fits the input image
The number of cells that best fits the inputted image is chosen as the estimated number of cells in the cluster