Flow cytometer fluorescence compensation adjustment method.

Fluorescence compensation refers to the process of correcting fluorescent optical signals to leak from detector stoeinal stoythin and digitally detect them.
this process has become important since the emergence of a single-laser two-color analysis.
each fluorescent molecule has its own spectral emission range.
there is a superimposon between these emission spectra, which is evident in some cases.
the following small section for you to introduce the flow cytometer fluorescence compensation adjustment method.
example, the chart below shows the overlay of FITC and PE fluorescence emission spectra.
in a dual detector system, FITC fluorescence can be designed to be separated from PE fluorescence.
detectfluorescent emission light based on its wavelength and distribution, a small portion of which will be selected into the detector, which can be obtained by different filters selecting a specific wavelength range of fluorescence from all the emitted light.
so FITC fluorescence is selected by a 530nm filter and fed into the detector, while PE fluorescence is selected by a 575nm filter.
but some FITC fluorescence appears in the PE detector because its emission light covers the filter range of the two detector filters.
this part of the signal we call fluorescent leakage because it is leaked into the PE detector by FITC fluorescence.
note that it is not possible to design a set of fluorescent filters to collect only FITC or PE signals, as fluorescent overlays are always present when both fluorescent dyes are used at the same time.
so that as long as FITC fluorescence exists, you will get a signal in the 530nm region, and at the same time in the 575nm region will get some other signals.
if PE fluorescence is present at this time, it will also be collected in the 575nm region.
How can we count how many signals come from PE and how many signals are caused by FITC? This calculation process, which we call "fluorescence compensation", is to correct the signal in the PE detector by removing the fluorescence of FITC on the PE emission spectrum.
step-by-step fluorescence compensation If the emission light of one fluorescent dye can be detected by two detectors containing different bands of filters, how many signals are available in the other can be calculated based on the signals of one of the detectors.
this is because the two signals vary proportionally (see figure below).
this constant characteristic means that we are able to accurately calculate the curve area of the orange fluorescent detector based on the curve area in the green fluorescent detector.
the ratio of these two values can be calculated without fluorescence other than FITC.
samples used for this purpose are referred to as "fluorescent compensation adjustment samples" or "fluorescent compensation quality controls" and are used to accurately determine compensation values.
for a typical flow cytometer, the signal that FITC emits light in an orange detector is about 15 percent of the green detector.
so if we subtract 15% of the green detector signal from the orange detector, the calibrated orange fluorescent detector signal will always be zero, no matter how many FITC signals are present.
this time PE fluorescence can be added to the system, orange fluorescent detector after deducting 15% of the green signal will show the real PE fluorescence signal, without having to consider the existence of FITC fluorescence.
this is "one-step" fluorescence compensation;
two-step (double) fluorescence compensation from the fluorescence spectrum below can be seen pefluoroxiating also partially leaked into the FITC detector.
this ratio is known by detecting cells that only PE fluorescence, which is generally around 2%.
this is called PE fluorescence compensation correction.
But if PE fluorescence appears in the FITC detector and we use FITC fluorescence to correct the fluorescent signal in the PE detector, is it possible to correct the fluorescence leakage correctly to get a real fluorescent signal? Reagent, according to some mathematical operations is possible to use this method to precisely adjust compensation.
current flow cytometers are automatically regulating fluorescence compensation, but also be prepared.
if we simultaneously detect four fluorescent FITC, PE, Per CP, APC, make compensation adjustment, need the following samples, no staining samples, FITC, PE, Per CP and APC single-dye positive samples, here need a total of 5 tubes.
in a purely handy age, you need to draw a scatter plot of two pairs of these four colors, such as FITC-PE, FITC-Per CP, FITC-APC, PE-Per CP, PE-APC, Per CP-APC, a total of 6 scatterplots.
then, the unstained samples are first taken and the voltage of each fluorescent channel is adjusted so that the cells are located in the lower left corner of each scatterplot.
run each sample to one side before recording to see if the voltage is set correctly.
for example, FITC and PE will overlap, on the FITC positive tube, although other channels will have some signals, but the FITC channel signal should be the strongest.
if there is a situation where the signal of other channels is stronger than the target channel, the voltage should be further adjusted so that the signal of the destination channel is strongest.
this way, each single-dyed sample is passed over to ensure that the signal of the destination channel is strongest.
current instrument software, you don't need to draw these scatterplots yourself, but this step of regulating the voltage is a must and can be done on a negative-to-care page. After
voltage regulation, the signalof the negative control and positive single-dye sample scored, and the machine automatically calculates the compensation value.
If you're going to calculate it manually, here's the principle: TAKE FITC-PE as an example.
is divided into four quadrants on the scatter plot.
if the ordinate is FITC, the horizontal coordinate is PE.
adjustthed voltage, the negative cells are located in the lower left quadrant.
can now derive the average fluorescence intensity values of the negative cell horizontal coordinates (Pe) and ordinate coordinates (FITC). After
voltage is adjusted, the monochromatic positive sample is on.
, for example, on the plate FITC-positive, then do not change the voltage, but adjust the compensation coefficient, the ultimate goal is to make THE FITC-positive cells are located in the downward quadrant, and the cell cluster of the ordinate (PE) fluorescence strength and the value of the negative cell group is consistent.
two compensations for other colors and so on.
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