VenturiOne – Overlays in Flow Cytometry Data Analysis


Our rapid data analysis platform to process, interpret, and report on your FCS data files. Find out how to process multiple files, use our compensation wizard, autogating and more on VenturiOne.



Hi, my name is Jo Crofts welcome to the Applied Cytometry flow cytometry software VenturiOne. We’ve recorded some tutorials to help you get started using the software.

To get the most out of the software the tutorials are designed to be used alongside the getting started guide and the operation manual. You will find these at the website below.

Overlays Tutorial

When using VenturiOne you can create both single parameter and dual parameter overlay plots, created these plots will display in the overlays tab. In the overlays tab there are two areas, the area of the overlays themselves and the galleries area which shows you
the individual plots which make up the overlay.

To create an overlay plot you can go to the plots tab and send any individual plots from any sample within your group or work list to a selected overlay plot. From within the plots tab you can select single or dual parameter plots to centre the overlay.

So, using this manual method I can go to any sample in the list and then select the plots that I want to go to the overlay. So, here for example if I want to add the FMC7 plot I click on it to highlight it, then right click and then choose the send selected plot to overlay plot.

As I currently don’t have any overlay plots I will create a new overlay plot with that sample in it. I can then go to the second sample in my playlist highlight and right click on the FMC7 plot and send the selected plot to the overlay and now I can either choose to send this to a new overlay or send to existing overlay, overlay one.

In this case I’m going to add it to overlay one and then I can go to the third sample in my playlist and do exactly the same thing. if i now go back to the first sample, if I want to create an overlay for example that shows the CD79b’s I can again right click on that plot and now I will choose to send that plot to a new overlay plot.

This will be an overlay plot containing the CD79b’s so go to the second sample, right click on the 79b plot and select send to overlay 2. If I want to create an overlay of dual parameter plots, I can do this in the same way.

Right click on the plots then send to overlay plots and in this case I can only send to a new plot because the first two overlays were only single parameter overlay plots. Then I can go to the second sample and send that to overlay three.

To look at the overlay plots click on the overlays tab of the workspace. So, here we see the three overlay plots that we’ve created and the individual plots that make up each of the overlays.

Here in the galleries area looking at the individual plots, I can change the formatting of the plots by going to the overlay format tab on the ribbon. On this tab I can change things like the background colour, the data colour, and I can also fill the histogram with a particular colour.

Any change that is actually made to the gallery will be reflected in the overlay plot. So, now I can draw this one filled as well. The gallery has some tools within it that will help you visualize the overlays, particularly if you are interested in looking at a lot of overlay plots against each other.

You can for example, set one histogram as the control and then that histogram will then show itself on all the other individual plots in the overlay in the gallery. So, if you want to compare individual plots against a control that’s a very useful feature.

You can also choose to see the control in front or the control behind depending on the kind of samples that you’re looking at. In this case the control in front is obviously the better option.

I can send selected plots to the report for printing and I can also move selected plots between overlays, so if I do manage to send a plot to the wrong overlay I can come to the gallery and move that plot to the correct overlay.

When you click on the overlay plot in the overlays area and choose the format tab on the ribbon you have a 3D mode that offsets the individual plots. This can be done for both single and dual parameter plots.

You can also change the rotation of these plots in order to see the overlays more clearly. For histogram plots there is also a smoothing algorithm if you want to smooth any of the plots.

If you want to change the line style, you can do that on the gallery plot so you can have dots or dashes and you can also change the thickness of a line as well. If you want to remove certain elements from the plot for printing or publishing you can turn those elements off and then they won’t appear on the plot, it is quite useful to show the plot though.

With the overlay plots, you can also show a legend. By default the legend will be the plot title, the plot title is automatically set to be the dollar file keyword.

From within the FCS keyword header you can change the legend by double clicking on the plot title and selecting another keyword from within the file or by typing the label that you want to apply.

You can of course customize each of the plot titles and in that way create an appropriate legend. To remove individual plots from the overlay, highlight the plot in the gallery and press the delete button on your keyboard.

To delete an entire overlay, you can either delete the individual gallery plots or highlight the overlay plot and delete that. If you have a lot of samples or want to create a lot of overlays, rather than using the manual method to add the overlay plots you can use the overlay wizard.

The overlay wizard helps you automatically create overlay plots, so head to the overlay tab of the ribbon and select the overlay wizard. If you’re in playlist mode you can add plots from any sample within the playlist, if you are in group mode you can add any plot from within the samples in the individual group.

So the current mode is displayed on the first page of the wizard, click next and here you can choose which plots you want to add. On this page we are looking at what plots are available in the currently highlighted sample and I can select which plots I would like to use from all samples.

So I am selecting three of the single parameter plots and then I will select next. On this page I can choose how I want to display and arrange the plots that I’ve selected.

I can choose to display all plots from the files together, so in this example we will have all three plots from all the samples in the playlist shown on one overlay plot. The second option is to display the plots from the same file together, so here we create an overlay plot
for each file displaying the three plots that I have selected in each and finally and the most used option is to display the same plot from all files together.

So here we will create an overlay plot for each of the parameters that we have selected, so we will have all of the plots from the individual files for that parameter. We can adjust the number of plots that appears in each overlay.

So once you start getting over five or six overlays in a plot it can be very difficult to tell the plot apart. So if you have a large number of samples here we can set a number and that will automatically create a new overlay plot once that number has been reached.

Then at the bottom of the page we have a summary of how many plots we are going to create. So for this sample I have seven files and three plots that I have selected on the previous screen for each sample.

So I’m going to get 21 histogram plots in total and this will be spread over three overlay plots and select next. I can choose to enter a title and I can also choose the plot title.

If I want I can show the legend as well as being able to set the legend to what I want it to be. Select finish and the system will now go through and pick up the individual plots from each sample and add those to the overlay as I’ve selected.

So here you see our three overlay plots and on the right hand side we see the gallery of those plots. If the labels of all the parameters are the same you will see the label appear, if the labels on the x-axis are all different then no label will appear, this can be changed afterwards.

You can send selected overlay plots to a report by highlighting the overlay plot right clicking on the plot and then selecting the send selected plots to report then the overlay plots will be added to the report. Alternatively, you can add individual gallery plots by again highlighting and then right clicking and choosing the send selector plot to report.

If you want to compare samples statistically, you can add regions to a plot. So I can, for example, add a linear region and that linear region will appear for all samples within that overlay plot and the results will be displayed accordingly.

If you want to change the statistic that is displayed you can go to the statistics tab of the ribbon and choose which statistic you want to display. The selected plots will then appear on the report and that is the end of the overlays tutorial.


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