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Materials Required:

  • Analysed Scatter reference beads (spherical beads, known diameter & refractive index)
    • Recommend NIST-traceable beads are used which provide diameter and refractive index - examples.
  • Flow Cytometer and Analysis software

Notes:

  • Bead inputs can be edited by clicking on the variable you would like to change and then editing the input fields below. Once edited click the 'Append' button. 
  • Bead sets can also be stored for use at a later time to save having to input the manufacturer specifications in repeatedly. Click the 'Save Set' button and type a name (use only letters and numbers). Sets will then appear in the dropdown menu when the software is opened.

Step-by-step guide:

  1. Scatter reference bead information input
  2. Model settings
  3. Output settings
  4. Refractive index calculation
  5. Light scatter modeling calculation

Scatter reference bead information input 

  1. Open acquired data from scatter reference beads into commercial flow cytometry data analysis software
  2. Gate around each of the bead populations. 
    1. If using a histogram, ensuring that singlets are gated and unwanted noise is removed can the improve accuracy populations statistics. This will in turn provide better modeling accuracy.
  3. Obtain the median and %CV peak-intensity for each bead population for the SSC parameter.
  4. In the FCMPASS software input the manufacturer (or verified) diameter and diameter %CV for each bead. Input the refractive index of the material at the wavelength that the scatter was measured at. The 'Refractive Index Calculator' can help you convert manufacturer specified refractive index measurements at different illumination wavelengths to the illumination wavelength the scatter intensity was measured at. 
  5. Input the acquired scatter intensity measurements from step 3.

Model settings 

  1. Click the 'Model Settings' tab to make adjustments to the vesicles and medium refractive indices, illumination wavelength, and optical collection variables. 

  2. The illumination wavelength, by default, is set to 488 nm. Alter this according to the wavelength that the scatter-intensity is being measured at. Be sure to account the illumination wavelength with inputted bead refractive indices. 
  3. The vesicle and suspension medium refractive index are by default automatically adjusted to the inputted illumination wavelength. For custom vesicle and suspension refractive indices, click the 'Auto-adjust RIs to Wavelength' dropdown menu and select 'Off'. 
  4. The vesicle membrane thickness by default is set to 10 nm and can be edited accordingly from 1 to 20 nm.
  5. The FCMPASS software is set to approximate the collection half-angle of the instrument by default, and assumes a circular collection geometry. The assumed collection angle can be set to 'Circular', 'Square' or 'Both', when the 'Determine Half-Angle' is set to 'On'.
  6. If 'Determine Half-Angle' is switched 'Off', a dropdown menu will appear allowing a choice of 'Basic' or 'Advanced' modeling methods. The 'Basic' method assumes collection half-angles for square and circular collection geometries collected orthogonal to the illumination beam. By selecting 'Advanced' it is possible to edit the collection angle. This is currently limited to side scatter collection angles and angles can therefore only be selected between 0 and 180 degrees.
  7. A dropdown menu labelled 'RI Interpolation' is also present. By default this is set to 'Off'. This feature is extremely computationally intensive and if selected may take several hours to compute. This feature is designed for those that have parameters calibrated to diameter independently of light scatter. Instead of creating an approximated diameter variable, this feature allows for determining the approximate effective refractive index raw light scatter data. This feature is currently inactive while the methodology is validated.
  8. The 'Output Model Settings' allows you to export all of the model settings used to create your scatter calibration model to a spreadsheet that can be shared with publication. If selected a window will appear asking you to select a location to export the settings to upon completion of the scatter calibration calculation.

Output settings 

  1. Under the 'Output Settings' tab are dropdown menus to select which plots will be created during the scatter calibration calculation. There are also options for changing the axis variables and appearance of the threshold for the scatter channel.

Refractive index calculation 

  1. Under the 'RI Calculator' tab are two refractive index calculations
  2. The 'Reference Particle Refractive Index' calculator allows users to obtain refractive indices for water, polystyrene, and silica at different wavelengths using published dispersion data. The dispersion data for water is the same used in the 'Auto adjust RI to wavelength' setting under the 'Model Settings' tab.
  3. The 'Reference Particle Refractive Index Conversion' calculator allow users to convert manufacturer supplied refractive index data for polystyrene and silica to different wavelengths using the Sellmeier equation for polystyrene and silica based on their published dispersion characteristics. i.e. most manufacturer supply refractive index measurements at 589 nm, but most cytometers use 488 nm or 405 nm to detect scatter. The refractive index for these supplied particles therefore needs to be converted from 589 nm to 488/405 nm.

Light scatter modeling calculation 

  1. Once the variables have been inputted and the appropriate parameters selected for modeling click 'Calculate', under the 'Bead Input' tab.
  2. If a database already exists for the inputted beads being modeled, the calculation will take a short (<2 mins) amount of time. If default selections are changed, or new bead variables are being calculated for the first time, databases will need to be constructed which can take time (~5-10 mins each). Databases will only need to be calculated once, however, increases the calculation speed if performing light scatter modeling again.
  3. Once the calculation has been performed diameter conversions can be written to .fcs files. See steps here.