The flow cytometer then uses these values to correct the overlap in each detector for each colour. The matrix is then inverted and gives the actual compensation values. This is done by measuring the spectral overlap of the different fluorochromes and using the measured values to create a matrix. This correction is called compensation.Ĭompensation is necessary in order to be able to differentiate between populations of cells. The ability to correct this stems from the fact, that the overlap is a linear function, so the measured signal can be averaged and thus corrected. The physical overlap between the different emission spectra of fluorochromes can activate different receptors than the ones intended for the given wavelength. When one cell is marked by two or more fluorochromes, the added brightness of one fluorochrome to the other creates significant background noise and affects the strength of the signal. during a two colour experiment, where mouse splenocytes were stained with fluorescein and rhodamine. The first data compensation was done in 1977 by Michael Loken et al. The compensation can be done through different flow cytometry software such as Flowjo, Flowlogic, Kaluza etc. This creates a signal overlap (spillover) which cannot be removed by the optical system and has to be corrected electronically. The photons emitted by fluorochromes have different energies and wavelengths and as flow cytometers use photomultiplier tubes (PMT) in order to convert the photons into electrons, the detector can register the signal from more than one fluorochrome. A manual adjustment compensation shown below: 2 manual adjustment compensation: i.e.The put your experimental tubes into a separate groupĪccessing FlowJo’s compensation controls.First place your “comp” tubes into a separate group.Using FlowJo to calculate compensation 060502 Emory Vaccine Center FCC / Suzanne Mertens / can be used to calculate compensation for multi-color data acquired on a Calibur Also what I realized that the matrix built-in Kaluza is spectral compensation so the numbers don't work in FLowjo, there is a separate.In cytometry, compensation is a mathematical correction of a signal overlap between the channels of the emission spectra of different fluorochromes. #FLOWJO COMPENSATION MATRIX MANUAL#įlowJo expects to see defined positive and negative populations for each single stain Compensation Matrix File When the 'Compute' button in the compensation matrix dialog window is clicked, FlowJo generates a compensation matrix. The user must go ahead and do proper gating to create single stain histograms with positive and negative populations.Ĭhange the value in the matrix, changes in the observed image, and.FlowJo organizes all of your analyses into a workspace. Generate positive and negative populations from single stain histograms In this example, the FITC single stain has been gated for lymphocytes, then displayed as a FITC histogramĬlick and use the histogram bisector tool to quickly separate positive from negativeĭrag and drop gated populations to define the matrix Drag your gated populations to the “AutoAssign Box” amd FlowJo will sort out which population goes in the right Definitions box.
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