Resources for working with FACSDiscover S8 and A8 data in FCS Express

The BD FACSDiscover™ S8 and A8 combine full spectrum flow cytometry with BD CellView™ Image Technology, capturing a reconstructed image alongside spectral fluorescence data in real time. The example layout below highlights how FCS Express's Image Cytometry features come together to make sense of that combined data set- including image display.

FACSDiscover A8 Data Layout

In this example layout:

Image-derived parameters such as Diffusivity and Radial Moment are plotted directly alongside standard fluorescence parameters to characterize the spatial distribution of the acrosomal marker ACRV-1, distinguishing a tightly confined signal from a more diffuse one, and indicating intact vs reacted acrosomes.
Gate statistics track population percentages and allow for the calculation of a custom statistic "% Intact Acrosome" built directly from imaging-derived gates.
Throughout, Data Grids let users pull up and review individual event images side-by-side with other recorded features.
Spectrum plots are displayed to compare the spectral signatures of individual populations.

Download the example layout file here Download the example Powerpoint file here

How to Load A8/S8 Spectral and Imaging Data

FCS Express is now compatible with spectral and imaging data acquired on the BD FACSDiscover A8/S8 instruments. As of version 7.32 or later, FCS Express can load A8/S8 images, display the raw spectral data in spectrum plots, and perform spectral unmixing.

Note A8/S8 images must be converted to the .tiff file format to load in FCS Express.

Please refer to the topics below for working with A8/S8 data in FCS Express.

Instrument Specific Settings for A8/S8

New Instrument Specific Settings have been added with version 7.32.0060 for the BD FACSDiscover A8 and S8 instruments. By default, these settings enable the loading of images, and the automatic creation of a spectral parameter (needed for spectrum plots, and spectral unmixing in FCS Express).

To view the new Instrument Specific Settings, navigate to the Ribbon and select File > Options to open the FCS Express User Options window. Then select Data Loading > FCS File Options > Instrument Specific Settings.

If you do not see the four Instrument Specific Settings for the A8 and S8 as depicted above, or you are unable to load images, a spectrum plot, or perform spectral unmixing in FCS Express with A8/S8 data:

Click on the Restore Default Settings button.

This will reset all Instrument Specific Settings to their default values and add the four Instrument Specific Settings for A8 and S8 instruments.

Note the only difference between the four Instrument Specific Settings are:

The instrument the settings apply to, A8 or S8.
The export option selected when exporting data from BD FACSChorus.

How to Turn Off Loading Image Data

The Instrument Specific Settings for the A8/S8 include a new section, Image Options, to enable FCS Express to find and automatically load images associated with A8/S8 fcs files loaded into the layout.

This option is enabled by default. If you would like FCS Express to not load images into the Layout by default, for example if you encounter slower loading or reduced performance when analyzing data with images, uncheck the Load Images box.

How can I apply the updated Instrument Specific Settings to the data files in my layout?

BD A8/S8 Spectral Options

To support the loading of A8/S8 spectral data into spectrum plots and spectral unmixing in FCS Express, new BD A8/S8 Specific Options have been added to the Instrument Specific Settings.

To enable spectral unmixing and loading of A8/S8 data into spectrum plots, the option to Import full spectrum as individual parameter must be checked. Other BD A8/S8 Specific Options include:

Import the spectra of individual lasers as individual parameters – this allows viewing of detectors from specific laser lines on a spectrum plot (i.e. plot only YG detectors or UV detectors).
Hide individual detector parameters from raw data – hides the raw spectral parameters (detectors) from your data on loading.

Loading A8/S8 Spectral and Imaging Data

For optimal loading of A8/S8 data, ensure all Instrument Specific Settings for the A8/S8 are present in your user options.

To load A8/S8 spectral data with images, note the following requirements:

1. FCS Express is not compatible with the .cvw file format used by BD FACSDiscover instruments to store images. To load A8/S8 image data, the .cvw file must be converted into .tiff image files using the BD CellView Image Extractor available within the BD FACSChorus software.

2. See this FAQ for details about the FCS File format to export from FACSChorus.

3. The folder containing the extracted images must be placed in the same folder as the fcs file (see Matching FCS Data to Images).

Matching FCS Data to Images

For FCS Express to properly link images to events, the fcs file and associated image data must be in the same folder.

In the example below, a single fcs file and the image folder containing extracted .tiff images are present in the same folder, “Folder A”.

FCS Express will match an fcs file to the correct images by using the name of the image folder. The image folder itself must start with the fcs filename without the extension (“sample1”), followed by a “_images_*” suffix where “*” can be anything.

The image folder structure will contain numeric subfolder(s) which contain .tiff images. It is critical to keep the image folder structures intact. Do not alter or rename the individual TIFF files or move them from their generated subfolders.

How to View Images With The Data Grid

FCS Express allows you to view the data acquired for every event in A8/S8 data sets, including images, using the Data Grid.

To view images in the Data Grid:

From the Ribbon, select Insert > General > Data Grid to insert a Data Grid on the layout.

The Data Grid displays each parameter or image channel as a column that can be shown or hidden. Each row represents an event. Like all objects in FCS Express, multiple Data Grids can be present on the layout from the same or different data files to compare results.

The Data Grid has unique formatting properties. To format a Data Grid:

Select the Data Grid, then navigate to the Format tab on the ribbon or…
Right-click on the Data Grid, select Format from the pop-up menu, then choose a category from the dropdown to open in the formatting window.

The following formatting options are particularly useful when displaying images in the Data Grid:

a) Parameters To Display – show all or hide specific parameters or image channels. A8/S8 images will be the last parameters listed, allowing for quick selection of only images in the Data Grid.

b) Data Image Options – change the background color of the image columns and other columns to improve visualization of images.

c) Gates to Display – add a new Gates column to show backgating for each event. Gates will be displayed as colored bars in the Gates column.

d) General – assign unique names to each Data Grid on the layout using the Data window name.

Why are my BD FACSDiscover images not colored? Can I assign colors to my images in FCS Express?

How to Apply Gates to the Data Grid

To apply a gate to the Data Grid:

Drag and drop the desired gate from any plot onto the Data Grid.
Select the Data Grid, then from the ribbon click on the Gating > Current Gate dropdown and select the desired gate.

How to Enlarge Images from the Data Grid

The size of images displayed in the Data Grid can be changed in two ways – within the Data Grid and as a separate image outside of the Data Grid. To change the size of the images displayed in a column of the Data Grid:

Select the column header
Click on the bottom border
Drag down to adjust column height

Individual images within the Data Grid can also be embedded on the layout and enlarged:

Left-click and hold to select the image in the Data Grid.
Drag the image to an empty space in the layout.
Release the mouse button.

The image from the data grid will now be inserted as a new picture object on the layout. By default, the picture will appear larger than the image in the Data Grid. The picture can be further resized by selecting the picture and dragging one of the vertices on the pictures border.

FAQs with Tips

How can I apply the updated Instrument Specific Settings to data files that have already been loaded into my layout?

What is the difference between Recommended, All and Small export options for the BD FACSDiscover A8 and S8?

The BD FACSDiscover A8/S8 provides three options for exporting fcs files - All, Recommended and Small.

When an FCS file is loaded, FCS Express will automatically detect whether the Instrument was A8 or S8 and apply the appropriate Instrument Specific Settings, depending on the type of export (All, Recommended or Small).

Importantly, the Recommended and All export options contain unmixed and raw spectral data. FCS files exported with the Small export only contain unmixed spectral data and therefore cannot be used with spectrum plots or to perform spectral unmixing as these tasks require raw spectral data.

Please consult with the BD FACSChorus software manual for further details on the parameter types (i.e. -A, -H, -W, -TTP) included in each of the fcs file export options above.

How do I export images from the BD FACSDiscover for use in FCS Express?

Why are my BD FACSDiscover images not colored? Can I assign colors to my images in FCS Express?

Can I overlay multiple images from the BD FACSDiscover in FCS Express?

Why do my BD FACSDiscover A8/S8 images look different in FCS Express?

Can I view only my A8/S8 images in the Data Grid, like the Image Wall in BD FACSChorus?

Yes, the Data Grid displays each parameter or image channel as a column that can be shown or hidden via the formatting options for Parameters to Display.

To show or hide specific parameters or image channels, use the options “Only the items checked below” or “All items except those checked below”. A8/S8 images will be the last parameters listed in the dropdown, allowing for quick selection of only images in the Data Grid.

Can I make one image or all images larger in the Data Grid?

How long does it take for single color controls to load into the unmixing wizard?

tags: images, image cytometry, FACSDiscover A8, FACSDiscover S8