FCM PASS

This software package will be freely available once the related manuscript is published. To be notified of its release click here and send the email.


This software aims to be a useful standardisation toolbox for researchers who are quantifying extraceullar vesicles using vesicle flow cytometry. The software is built around Mie scattering theory, which can be used to calculate the quantity of light scattered by a single spherical particle of known diameter and refractive index. These calculations have been further expanded to predict how much light a detector receives from a spherical particle traversing a laser, taking into account the flow cytometer geometry that is surrounding the particle scattering light. 

This software’s core scripts are based upon those written in MATLAB by Matzler, who converted the work of Bohren and Huffman into code. These scripts were then further edited by Horak and myself.

Features include:

  • Flow cytometer collection angle derivation – This method enables researchers to derive the collection angle of their conventional flow cytometers, simply by acquiring beads of known diameter and refractive index. 
  • Scatter-Diameter plotting – Using a known collection angle, or utilising the above feature, researchers can approximate the scatter parameter sensitivity of their flow cytometer irrespective of refractive index. This method also enables approximation of particle refractive index and/or particle diameter with the correct controls.
  • EV Particle Diameter Approximation – Using the plotted ‘scatter-diameter’ curve it is possible to interpolate raw SSC data with the curves to produce particle diameter distributions.
  • Refractive index calculator – provides researchers with a calculator to convert polystyrene and silica refractive indices to the wavelength of detection for use in models. For example, many bead refractive index measurements are provided at 589 nm. Using this calculator would allow conversion of the 589nm measurement to the scatter illumination wavelength being used, which tends to be 488 nm.
  • MESF calculator – Converting arbitrary flow cytometer units to molecules of equivalent soluble fluorophore (MESF) units is a method of fluorescence standardisation. This calculator produces a regression curve from MESF beads and outputs the FlowJo ‘derive’ equation for converting arbitrary units scale to MESF units by copying and pasting it. This allows for an easier method of performing FCM fluorescence standardisation.