4. Spot quantification

To quantify spot features (module 4), SpotMAX uses the pixels of a spheroid mask with an expected spot size centred at the spot centre. See the parameter Spot (z, y, x) minimum dimensions (radius) for more details.

These features include several intensity distribution metrics (mean, max, median, quantiles, etc.) from the raw, Gaussian-filtered, and DoG-filtered images including different background correction strategies.

Spots can also be further quantified with a Gaussian peak fitting procedure (SpotFIT). We optimised this procedure for high-density spots where multiple spots are fitted together with a sum of 3D Gaussian functions.

The SpotFIT framework computes additional features, including the total integral of the Gaussian function, the size of the spot, the amplitude, and the background level.

Calculated features are explained in this section Description of the parameters.