How do I work with images from the Thermo Scientific Attune CytPix?
Working with data from Attune CytPix in FCS Express
De Novo Software and Thermo Fisher Scientific have collaborated to allow working with raw data AND images from the Attune CytPix instrument in FCS Express. FCS Express is directly compatible with data exported from Thermo Fisher Scientific Attune Cytometric Software (version 6 and later), via .ACS file and .FCS file formats where .ACS files allow access to the imaging component of the data sets. Both .ACS and .FCS file formats allow working with additional extended parameters defined by the the Attune Cytometry Software based on images.
Your multi parameter flow and imaging data can now be easily exported to the .ACS format and analyzed in FCS Express while expanded upon using all of the tools and features FCS Express has to offer. See the Export Instructions below to learn how easy it is to export your .ACS and .FCS files for use in FCS Express.
The Attune Cytometric Software allows you to export either,
- FCS files for standard numeric only analysis, or
- FCS and Image Data for analysis of standard numeric data linked to individual images.
To Export your data from the Attune Cytometric Software to allow working with raw data and images in the .ACS format:
- Right click on either the experiment (to export ACS for all samples) or a single sample.
- Choose the option FCS and Image Data.
Note the circle with the check-mark next to the samples in the image at right. The icon indicates files have been processed in the software and have generated 26 image-based "extended parameters". Users may choose to export data with or without the extended parameters in the next steps.
- Before naming the file and clicking Save, check or uncheck the Extend FCS File check box to choose to save data with the extended parameters (examples below).
- Name the file, and then choose Save.
CytPix .ACS files are loaded into FCS Express like any other standard .FCS files. Data files may be dragged and dropped onto the layout or dragged and dropped into the data list.
When using the blue plus button (+) to load .ACS files into the data list, ensure Thermo Cytpix ACS Files (*.acs) is selected from the Files of Type drop-down (example below).
Images may be viewed through the Data Grid object in FCS Express. The data grid will display all cell by cell multiparametric numeric results and for .ACS files, it will include a column at the far right for "Brightfield" that will contain all images collected by the CytPix. Depending on your acquisition settings, there may be images for all events, or just some events. The "ImageFlag" parameter indicates a value of 1 for events with images and a value of 0 for events without images.
To insert a Data Grid:
- Click on Insert tab> Data Grid.
- Click on an area in the layout to insert the Data Grid.
Note: In the Data Grid each cell has an ImageFlag and Brightfield column.
The Data Grid may then be gated by dragging and dropping gates onto the Data Grid, or by selecting the data grid and choosing a gate from the Gating tab>Current Gate drop down.
Multiple data grids may be inserted with different gates applied to each data grid to visualize cells of interest for different populations.
Formatting the Data Grid to only show images
To Format the Data Grid to show specific parameters or just images, select the Data Grid>right click and choose Format. In the Format window, select Parameters to Display in the drop down.
The Parameters to Display Options allow users to view:
- All Items
- Only the items checked below
- All items except those checked below.
Choose, Only the items checked below. You may then select individual parameters or just Brightfield. In the example below, Event, FSC-A, SSC-A and Brightfield were chosen.
You will now see only these parameters in your Data Grid.
Using the ImageFlag parameter to define a gate and view only events with images.
The ImageFlag parameter is useful if you would like to only view events that have an associated image. By defining a gate around all events with a "1" in the parameter, the gate may be applied to a Data Grid to visualize only events with images. Gates may be created on the parameter using histograms or scatter plots in FCS Express, in the example below we will outline creating a histogram and using a marker converted to a gate to define events with a "1" in the parameter.
- Create a histogram on the layout with ImageFlag set as the X axis parameter.
- Create Markers on the histogram via the Gating tab>Marker. The population on the right are events with a "1" in the ImageFlag parameter that have associated images.
- Right click on the histogram.
- Choose Convert Marker Gate.
- Name the gate as desired.
- Select a Data Grid.
- From Gating tab>Current Gate drop down choose the gate you defined in step 3 above.
You will now see that Data Grid has image only cells (example below).
If you would like view a larger version of the image, enlarge the row height for any event, or select the image in the Data Grid and drag it onto the layout to insert a static image of that cell.
Defining Specific Loading and Scaling Options for Thermo CytPix Data Sets.
Before loading your data, you can also setup Thermo Cytpix ACS Options via File tab>Options>Data Loading>Thermo Cytpix ACS Options. These options will allow you to set preferences of how you would like to visualize and scale data sets.
FCS Express on Mac
Upgrading FCS Express