![]() Here is a collection of external links about Compensation.Īll of the pages linked above, also link back here for your convenience. When you are ready to proceed to a more complete overview of the new compensation work flow, please see this document.Īlso, consider the Compensation Tutorial. One other document you will likely find useful is:Īpplying/Renaming – the matrix, a small overview. The Matrix editor – used to view/adjust matrix numbers with a preview of the changes.The Compensation window – used to define a compensation matrix from single stain controls.The Compensation group – in the workspace and tied to compensation Preferences.Single stained controls used to compensate in FlowJo will need to be scaled correctly ahead of time.įor a more complete overview of the theory and principles of compensation, please check out this page.įor version 10, there are three main components to the user interface for Compensation: Single stain controls must be run to determine the amount of secondary fluorescence contributed to each channel. Therefore, in multi-color flow cytometry, each channel will detect the primary fluorescence, but also secondary fluorescence arising from other overlapping fluorophores. However, the fluorophores used in flow cytometry do not adhere to the exact range of emission detected by the instrument. The detectors, or channels, in the instrument are designed to detect a very specific range of emissions. It works like a page layout program, but the graphs are live and will be updated to reflect any change in the data or gating. It is designed to create data display layouts which can contain one or more graphical objects, such as graphs, text and lines. This way you can see the difference before/after compensation.Compensation has undergone a major redesign for FlowJo Version 10!Ĭompensation in flow cytometry is the process of correcting for fluorescence spillover emissions. The Layout Editor is a powerful tool in the creation of both analysis and publication graphics. Checking the “Overlay Uncompensated” checkbox will paint a second layer on each plot that is the uncompensated parameters.Note the heatmap from the matrix is retained here as background of each plot. This view will show current sample’s fluorescent parameters in a grid of either histograms (ALLBY) or NxN bivariate plots.You can use the heatmap to quickly identify the highest/lowest spills of your matrix, which can be useful to track down compensation problems. The color coding is a simple heat map – blues are negative spills, and the increasingly darker shades of yellow are applied to higher values.Modifier keys can be used to accelerate the change – hold down, or, or for finer/coarser changes. You can click inside a field and use up/down arrows to change the numbers. The numbers correspond to the spill values between two parameters.You can uncheck some parameters and then use the “show all” checkbox on top to hide unchecked parameters. ![]() ![]() The checkboxes on the left control which parameters are being used in the display portion (section 3 below).The provides the capability to apply the current selected matrix to a sample or group by dragging and dropping.The Edit button will duplicate the selected matrix and allow you to edit the compensation spillover values.This can make or break an experimental analysis that spans multiple runs, we recommend using a strict naming convention for your matrices to aid in identifying which data set they belong to. The name field tells you which matrix is currently being edited.Since this window is complex, let’s break it up into 3 pieces and discuss them each below: The matrix editor window can be accessed in two ways:ġ) by double-clicking the compensation badge from the workspace window (any compensated sample has this badge next to the sample in the left hand column down the workspace.)Ģ) by clicking the “View Matrix…” button in the main Compensation window. Starting with version 10 of FlowJo, there is a new interface for viewing compensation matrices – the Matrix Editor. ![]()
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