Instrument Configurations on the Aria and LSR-II

The FACS DiVA software that runs the Aria and LSR-II has an instrument configuration panel that controls data collection from specific PMTs. The instrument configuration panel is extremely important for at least two reasons:

• It controls whether a PMT is "active" or "inactive". Failure to make a PMT active will result in irretrievable loss of data.
• It sets the "name" of the parameter for each PMT. This is important for appropriate annotation of data. Although improper or uninformative settings of parameter names will not result in loss of data, it may make it more difficult to interpret the data.

The purpose of this page is to propose a new standard for Parameter Names in the FACS DiVA Configuration Panel.

Setting up the Aria PMT Configuration

On an instrument like the FACS Calibur which does not have interchangeable filters, the meaning of the parameter names FL1, FL2, FL3, and FL4 are always the same. On instruments with changeable filters such as the FACS Aria, FACS Vantage, LSR-II and the MoFlo, such names are usually meaningless, as are the detector names like A, B, C.... Continued use of these names will violate the principle that FCS files should contain within them all of the information necessary to interpret the data without cross-reference to a notebook.

The FACS DiVA software that runs the Aria has an Instrument Configuration panel that looks like this (sorry for the low resolution):

You can store a large number of named Instrument configurations, as seen in the box at the left hand side. For each configuration, you can turn on 1-3 lasers, which are labeled Blue (488 nm), Red (633 nm), and Violet (405 nm). Each row in the three-column table to the right of the figure corresponds to a different parameter that you wish to measure; in the figure, the parameters are given familiar names such as FITC, PE, APC, etc., corresponding to the commonly used fluorophores.

I believe that instead of using the familiar fluorophore names in the parameter field, that it would be much better to label the parameters with the specifications of the filters directly in front of the relevant detector. Since the same filters might be used on more than one laser (e.g. a 780/60 filter for PE-Cy7 and APC-Cy7), it will be necessary to also to use a shorthand indication of the laser used in the parameter name (e.g. B, R, V for blue, red, and violet, respectively). The table below provides what might be a typical set of replacements, where the dichroics are not specified.

A second, more complete alternative would include the dichroic filters that are in front of each of the bandpass filers on each of the detectors in the Aria and LSR-II detectors, resulting in the following table:

Note that the last filter in line on the Red and Blue trigons do not require a long-pass filter; in contrast, a 502LP filter is required in front of the 530/30 PMT (Detector E) on the Blue laser, because the SSC detector is downstream of this fluoresence detector.

The advantage of this approach is that it ensures that each FCS file includes a description of the filters that were used to collect the data. In contrast, if common fluorophore names (e.g. FITC, APC, etc.) are used, this information is not included in the FCS files, and can only be retrieved by reference to a notebook. This violates the first principle of Good Data Annotation:

The goal of FACS data annotation should be to include enough information within the FCS file to allow someone else to make sense of the data without requiring crossreferences to your notebook.

Fluorophore Names: Sneaking them back in

In order not to confuse users (and to include appropriate information), it would be good to sneak the fluorophore names back into the FCS files someplace, but where? A good place for this is in the Labels portion of the Experiment Layout dialog box, depicted in the figure below:

Each of the cells in the Experimental Layout dialog box contains two rows. The first row contains the Parameter name, as determined by the Instrument Configuration panel; in the figure, the old familiar names are used, not the new ones I've suggested above. The second row contains the reagent name, such as CD8 as indicated in the blue-colored cells in the figure above. My suggestion is to append the fluorophore to the reagent name; for example, you should use "CD8-APC-Cy7" instead of CD8 as in the example above, providing a final cell that looks like this:

In my opinion, this will provide you with FCS files that do a much better job of containing all of the information relevant to the experiment.

The Dialectic of Data Annotation

At this point, some of you might be saying: "This is takes too much time. Why can't I just use the Parameter Names that I'm familiar with?" Or, "I know what I'm doing, and isn't it good enough?" Or, "I'll do this when it's really important, but I don't see why I have to bother with it for a simple titration."

My answers to these straw-statements are

1. Scientific data collection requires extensive and accurate record keeping.
2. It's a good idea to develop good data annotation habits.
3. It doesn't really cost that much to do it right.
4. It will increase the likelihood that you'll use the right filters, and you'll achieve a better understanding of what you are doing.