Every once in a while, Version 8 of FlowJo does a funny thing to some of the parameters when biexponential scaling is enabled. It looks something like...
notice how the X parameter is squished from 0 to 10^4 above.
FlowJo has two modes for calculating biexponential scaling - global (all parameters get the same transform) or individual (each parameter gets it's own scale computed, as in the screenshot above).
The unfortunate aspect of this calculation is that once "individual" scaling is calculated, it cannot be reversed. Even the "global" scaling is a one-time-deal - once "on", it cannot be changed (except in the case form Global to Individual).
What determines whether flowjo uses Individual or Global is the method used to invoke the scaling:
- If you do it via Platforms menu, Compensate, define biexponential.. - it will always be calculated uniquely per channel.
- If you do it in your global preferences (flowjo >> preferences >> workspace tab >> define... button) then all channels are treated identically.
Let's do the global scaling exercise. Bring up the lowjo >> preferences >> workspace tab >> define... panel:
notice in the lower-right, there are three variables - these control the biexponential function.
leave the top two fields as they are, but experiment with Width Basis. With basis is the number of channels squished into linear space around 0. I recommend trying values from -50 to -200 or so.
Here is -500 (WB too high, too squished):
Here is -100 (more like it):
-50: not bad!
-10 (default, too "low to the ground")
This titration or Width Basis value hopefully demonstrates what the variable does. Here the best looking value was -50, but slightly lower value would probably be OK too, just not too close to -10.