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Software Overview:

This software aims to be a useful standardization toolbox for researchers who are analyzing single extraceullar vesicles using 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. 

Please contact joshua.welsh@nih.gov if you encounter any bugs/issues using the software or if there are any additions that would be useful to include in future updates.



Software Features:

  • Light Scatter Calibration
    • Flow cytometer collection angle approximation – 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.
    • 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.
    • Particle refractive index approximation – Using the plotted ‘scatter-refractive index’ curve it is possible to interpolate diameter data with the curves to produce particle refractive index distributions. Using this feature would require calibrating a fluorescence parameter to approximate diameter.
  • 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 589 nm measurement to the scatter illumination wavelength being used, which tends to be 488 nm.

  • Fluorescence calibration – Converting arbitrary flow cytometer units to known reference values, such as molecules of equivalent soluble fluorophore (MESF), is a method of fluorescence standardization. The FCMPASS software includes a tool to perform fluorescence regression and write the data to flow cytometry .fcs files for downstream analysis and reporting.

  • Write calibrated data to .fcs - along with outputting calibration plots the FCMPASS software makes it possible to write all of the calibrated data directly to the .fcs file for downstream analysis and sharing with the aim to increase standardization and transparency in reporting of data.