I'm looking in detail into Flow Cytometry. I'm trying to figure out something called Spillover - here's a neat explanation of what it is -
To combat it, software does something called compensation which Im explaining below by example.
However, when I work out the maths I get a different answer. Im looking at an example on page 3 here.
Basically there are 2 fluorescents - FL1-H and FL2-H with values $3.85$ and $1.49$ respectively (measure of the strength of signal). The example then provides a spillover matrix:
This means that $12$% of FL1-H spilled into FL2-H and $26$% of FL2-H spilled into FL1-H.
The true signal of e.g. FL1-H can be calculated from measured FL1-H signal and compensation matrix as a sum of contributions from all.
So, the spillover matrix is:
\begin{bmatrix}1.0&0.12\\0.26&1.0\end{bmatrix}
To get the compensation, we need to invert this matrix. This gives the result:
\begin{bmatrix}1.0322047894302229&-0.12386457473162674\\-0.26837324525185796&1.0322047894302229\end{bmatrix}
Each contribution being equal to measured FL1-H signal multiplied by appropriate compensation coefficient (element of compensation matrix). So the FL1-H fluorescence detected by cytometer is $3.85$, then the true compensated FL1-H fluorescence is: $$(3.85 * 1.0322047894302229) + (3.85 * −0.26837324525185796)$$
Which gives $2.940751445$. But the answer they arrive to is $3.58$. I tried their other examples and I'm way off each time. Not sure what I'm doing wrong....
$\endgroup$1 Answer
$\begingroup$Silly mistake, it should be
(3.85∗1.0322047894302229)+(1.49∗−0.26837324525185796)That gives correct answer
$\endgroup$
