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COMPENSATION FLOWJO MANUAL
COMPENSATION FLOWJO SOFTWARE

A fluorochrome is excited, and emits a photon in a range of wavelengths.

Your compensation color must be matched to your experimental color.Īnd a bonus rule – collect enough events.Ĭompensation is required for a flow cytometry experiment because of the physics of fluorescence.

Background fluorescence should be the same for the positive and negative control populations for any given parameter.

Controls need to be at least as bright as any sample will apply the compensation to.

Basic Rules of CompensationĬompensation requires that three rules be followed. Secondly, we need to ensure that contributions from fluorochromes not being displayed don’t affect the distributions of the data. In fact many of the current clustering packages being developed require compensated data before they can be run. All automatic compensation uses this theory as the basis of automatic compensation methodologies.įirst is our reliance on one- and two-dimensional visualization and gating tools. The details of the mathematics on how this information is converted into a compensation matrix are found in Bagwell and Adams (1993) (Ann NY Acad Sci 20:167-184). In addition, the O1 signal also includes the S2 signal times a second factor. This factor covers the laser power, the filters used, the sensitivity of the PMT, etc. The O1 signal is composed of the S1 signal times a factor that represents how well that fluorochrome is measured in the specific detector. The “Observed Signal’ represents the fluorescent signal that is reported in the fcs file. The ‘Original Signal’ represents the fluorescent signal on the cells in the sample tube. Automatic compensation is accurate as long as you follow the three basic rules of compensation (see below).įigure 5: Diagrammatic representation of what is happening when you put a sample on your flow cytometer.

COMPENSATION FLOWJO SOFTWARE

If you run a flow cytometer that does not have an automatic compensation package, it is best to collect your data uncompensated and use a third party software package to perform compensation.

COMPENSATION FLOWJO MANUAL

There is no reason an investigator should be performing manual compensation with the propagation of different tools for the job. As described in that paper, and shown in Figure 5 (Figure 2 from Bagwell and Adams’ paper), there are several ways to do this, but the most accurate method is to use automatic compensation ( Herzenberg et al. It is derived from a paper by Bagwell and Adams (1993) (Ann NY Acad Sci 20:167-184). The more accurate and gold standard for compensation is automatic compensation. If you are using an instrument and still performing manual compensation, please ensure you are using statistical data to generate this value. The slope of the line based on the median measurements of the positive and negative gates is calculated to determine the compensation value. This is the spillover of fluorescein into the phycoerythrin (PE) channel.įigure 4: Compensation of the Fluorescein in the PE detector. However, as you can see, there is a percentage of this signal also in the 585/42 filter (approximately 12%). In Figure 2, below, the fluorescein signal is being measured in the BandPass 530/30 filter. PMTs will generate a photocurrent if any photon strikes them therefore, we use filters to restrict the light going to each PMT. As we’ve discussed previously flow cytometers use photomultiplier tubes (PMT) to convert photons to electrons. The problem comes in when we wish to measure this fluorescein signal. As shown in the Figure 1 for fluorescein, this emission occurs at a maximum of about 519 nm, but stretches out far into the orange spectrum (over 600 nm). To release that energy, the molecule emits a photon of a higher wavelength. In the process of excitation, the fluorescent molecule is excited that is, an electron is moved to a higher energy state. It all starts with fluorescence that is, the emission of a photon of light from a molecule that has absorbed a photon of light of a lower wavelength. Figure generated using the Invitrogen spectral viewer. The maximal emission is ~519 nm however fluorescein can emit a photon out into the yellow and red regions of the spectrum. The emission profile, shown in the filled green, represents the probability a photon of a given wavelength will be emitted. The dashed line shows the wavelengths of light that can excite the fluorescein molecule, up to the maximal excitation of ~490 nm.

Figure 1: Fluorescein excitation and emission spectrum.