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