Rainbow Bead Catalogue
The catalogue is used to store information about flow cytometers, light scatter reference material, fluorescent reference material, and rainbow beads with the aim to reduce inputting repetitive information and being able to directly write detailed meta-data to output reports for sharing data. The catalogue can be opened from the main start menu by clicking the "Catalogue" button.
Upon loading the catalogue, five tabs are available: Cytometers, Light Scatter, Fluorescence, Rainbow Particles, and MIFlowCyt.
- 'Create Set' button takes the current edit field inputs for rainbow particles and creates a set. Note: 'Number of Populations' must be a whole number.
- 'Delete Set' button deletes the currently selected rainbow bead entry from the table. Note: This will delete any associated cross calibrations which will not be recoverable.
- 'Append Set' button appends the current edit field inputs for rainbow particles to the currently selected rainbow bead entry from the table. Note: If the 'Number of Populations' is changed, all cross calibrations must be deleted as they are no longer consistent with the rainbow particles.
- 'Cross Calibration Display Options' are filters to be applied to the cross calibration table on the right-hand side.
- 'Add Fluorophore' button requires a rainbow bead entry to be selected from the rainbow bead table on the left-hand side. This will open up the 'Cross Calibration GUI' and allow for registering a cross calibration to the selected rainbow bead entry.
- 'Delete Fluorophore' button deletes the currently selected cross calibration from the cross calibration table.
- 'Append Fluorophore' button opens up the 'Cross Calibration GUI' and will overwrite the selected cross calibration bead entry
Automated cross-calibration
Cross-calibrating rainbow beads using MESF beads is an efficient and cost-saving method of calibrating data. FCMPASS supports automated cross-calibration, provided each of the peaks is visibly resolved from one another.
- Select the rainbow peak bead you wish to cross-calibrate from left hand table.
- Select 'Add Fluorophore' (No. 5) in the top right corner and a new user-interface will open.
- Click the 'Select MESF .fcs files' button and navigate to the .fcs containing the MESF bead data you wish to cross-calibrate the rianbow beads with.
- Click the 'Select Rainbow .fcs files' button and navigate to the .fcs containing the rainbow bead data you wish to cross-calibrate the rianbow beads with.
The parameter you are performing cross-calibration on must be acquired at the same detector settings. Only common detector settings between the two .fcs files are listed in the 'Select Calibration Parameter' dropdown.
- Select the FSC and SSC parameter used to gate the bead populations, along with parameter that you wish to cross-calibrate.
- Select the catalogued MESF bead from the 'Select Fluorophore' dropdown menu.
- Select the cytometer that the cross-calibration will be specific for.
- Once complete, click 'Next' to proceed to confirming auto-gating accuracy.
Cross-calibrations are filter specific. FCMPASS ties cross-calibrations to the catalogued cytometer to reduce erroneous downstream use of cross-calibrated data.
- If needed, adjust the singlet gating of MESF beads before clicking 'Next'.
- If needed, adjust the singlet gating of rainbow beads before clicking 'Next'.
- Upon completion of cross-calibration a success or error message will be shown and QC data will be automatically outputted to the folder containing the .fcs files.
- The cross-calibration QC data shows the regression accuracy of the MESF bead populations, along with where the MESF regression line intersects with the rainbow bead populations to designate equivalent reference fluorophore units..