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FCMPASS Software Download

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FCMPASS Requirements:

  1. Computer [16GB RAM, quad-core processor]
    Note: Mac users on managed devices may experience error on startup due to the software not having read/write permissions by default. Use following the protocol to enable read/write permissions:
  2. Flow cytometer with conventional (perpendicular) side scatter collection optics
    This software's automated light scatter calibration function does not currently cater for non-conventional collection optics e.g. Apogee instruments, though custom collection angles can be inputted for manual light scatter calibration.
  3. 6+ NIST-traceable size standards (if performing light scatter calibration)
    Non-fluorescent, traceable size standards are required for reliable light scatter calibration with the software. This is due to the automated scatter calibration calculating the system collection angle based on specific optical properties of the beads which include diameter and refractive index. Particles with fluorescent properties such as FluoSpheres or MegaMix beads, which lack exact diameter and refractive index assignments, do not produce robust and reliable models for the derivation of collection angle and therefore less suitable as reference materials for reliable diameter calibration.
  4. Fluorescent reference particles (if performing fluorescence calibration)
    The software can be used with any beads that have assigned reference values e.g. MESF or ERF beads.

Software Background

The FCMPASS software has been developed to aid the interpretation and reproducibility single extracellular vesicle flow cytometry. The version 3 release of FCMPASS allows researchers to create databases for each of their cytometers aiding their ability to track cytometer performance and catalogue experiment calibration. FCMPASS is designed to calibrate both fluorescence and light scatter parameters and write these to .fcs files to allow sharing of raw data in standard units. The version 3 release of FCMPASS allows integrates the MIFlowCyt-EV reporting framework as an output and auto-completes the relevant calibration fields to save researchers time.

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

Reference Material Cataloguing - a range of reference materials are used between experiments and over time. To save time whilst maintaining complete records in the outputted calibration reports, all light scatter and fluorescence reference materials are catalogued in the software. This allows allows users of the software to not have to repeatedly input reference values such as diameters, CVs, or MESF values or useful metadata such as manufacturer, catalogue numbers or lot numbers.

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 auto-adjustment - When cataloguing the reference materials for light scatter calibration in the software users are able to pick a composition of polystyrene, silica, or other. Polystyrene and silica refractive indicies are automatically updated based on the light scatter wavelength the user chooses to model using published dispersion data for polystrene and silica, respectively. All other refractive indices are also updated with respect to the dispersion properties of water to maintain their scattering ratios. These can however been updated manually if more accurate data exists.

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.

MIFlowCyt-EV output - upon calibration of the files a spreadsheet is outputted that contains a MIFlowCyt-EV framework with the fields related to calibration auto-completed. To further aid the transparency and reproducibility of the calibrations all parameters and metadata used for fluorescence and light scattering calibration are also included in separate sheets.

Software Protocols

A list of protocols for the current release of FCMPASS can also be found here. These are as published in: Welsh J A, Jones J C, Small Particle Fluorescence and Light Scatter Calibration Using FCMPASS Software, Current Protocols in Cytometry, 94, e79. doi: 10.1002/cpcy.79 [Website]

The protocols outlined and maintained here pertain to the lastest release of the FCMPASS

Release Notes

Release Notes:
  • Update: Quanteon fcs files now supported.
  • Update: MESF calibration accuracy improved.
  • Update: Failed .fcs writes are now logged in ErrorLog.txt for debugging.
Release Notes:
  • Update: FACSAria fcs files now supported.
  • Update: extracting threshold settings from fcs file made more reliable across instrument platforms and acquisition software versions.
Release Notes:
  • New: Software looks for updates on start-up.
  • Bug fix: output report no longer results in error when writing multiple fluorescence calibrations with varying reference bead array sizes.
  • Update: removal of redunatant preferences from v2.
  • Update: database has improved accuracy with scattering cross section units.
  • New: bead catalogue can now append reference bead sets and reference bead information.
  • Bug fix: bead catalogue no longer has errors when adding multiple overlapping bead sets.
Release Notes:
  • Bug fix: updates to handle experiments with multiple thresholds per channel
Release Notes:
  • Bug fix: written fcs files now work with FCSExpress
Release Notes:
  • Update: Calibration figures are exported into .png files rather than .ps
Release Notes:
  • Bug fix: unable to remove bead set from catalogue
Release Notes:
  • Bug fix: unable to add beads to catalogue on some computers
Release Notes:
  • New: Software user interface update enabling cytometer database and dataset tracking
  • New: Fluorescence and light scatter calibration bead cataloguing
  • New: Fluorescence calibration allows for F:P ratio alterations
  • New: Light scatter calibration allows for multiple customisable core-shell and homogeneous sphere compositions
  • New: Light scatter calibration autoatically adjusts for refractive index across models when wavelength is altered
  • New: Light scatter calibration is ~10x faster to calculate with storage being ~5x smaller
  • New: fcs file outputs now retain cytometer metadata from the original files
  • New: Output folder contains spreadsheet with MIFlowCyt-EV report and figures for all calibrations reference in the report
Release Notes:
  • Fixed bug with writing scatter-cross section to .fcs file
  • Fixed bug creating multiple timestamp folders if export took longer than 1 minute
Release Notes:
  • Fixed bug where multiple scattering cross-section conversion parameters were listed if calculated multiple times
  • New: error message appears if criteria not fulfilled when conducting fluorescence
  • New: plot diameter and scattering axis can be resized without recalculating
Release Notes:
  • Fixed bug saving scatter bead datasets
  • Fixed bug saving MESF bead datasets
  • Fixed bug stopping writing to some .fcs files
  • Fixed bug with RI conversions
  • Fixed bug indexing custom angles
Release Notes:
  • New: Updated user interface and loading screens
  • New: more efficient database storage and search system
  • New: Creation of error log file in database folder
  • New: Light scatter modelling up to 800 nm illumination wavelengths
  • Fixed error stopping softwate update search
  • Fixed error in the database storage v2.12
  • Increased start-up speed
  • New: Added user preferences for default input settings
  • New: Added control over a number of graph properties
  • New: Ability to turn parallel computing off (issue for some users)
  • New: Added further metadata for data tracking and reporting
  • New: Added ability to transpose scatter data
  • New: Added help tab in menu bar for reporting bugs and linking how-to guides
  • New: Scatter-Diameter plot does not plot threshold when set to 0
  • New: Exported .fcs file data folders are unique stopping overwriting
  • Fixed spelling error in scatter diameter plot legend
  • New: Adds ability to plot datapoints without using the data to approximate collection angle
  • New: Adds ability to plot predicted light scatter curves without needing reference bead data input
  • New: Adds ability to remove predicted vesicle light scatter range from scat-diam plot.
  • New: Adds ability to customize vesicle channel intercept diameter
  • New: Adds ability to remove EV curves entirely
  • Predicted vs. Acquired plot changed to 'Fold-Difference' by default due to better accuracy.
  • New: Adds coloured reference background to the 'Fold-Difference' plot indicating whether beads fit well or not.
  • Fixed bug stopping the deletion of light scatter bead datasets
Release Notes:
  • Fixed bug causing crash if directory window was cancelled upon startup
  • Fixed bug in setting minimum y-axis limits for scatter-diameter curve
  • New: Added predicted vs. acquired scatter ratio plot
  • New: Added ability to manually update bead directory
Release Notes:
  • New: No limit to input values (previously capped at 10)
  • New: Database integration avoids re-calculation of the same data twice
  • New: fcs file integration
  • New: Diameter determination from fcs
  • New: RI determination from fcs
  • New: SSC to scatter-cross section conversion
  • New: Write parameters to .fcs file format
  • New: Refractive index-diameter plot output
  • New: Predicted vs. Acquired scatter regression output
  • New: Integration of MESF regression