4/16/2023 0 Comments Flowjo x compensation![]() ![]() In this post I’d like to demonstrate that poor controls are not always the cause. Why is it difficult to get accurate results from automated tools? The only explanation that I’ve heard from the flow cytometry experts is that suboptimal results are caused by poor quality compensation controls. However, people often struggle to get good results from the automated compensation tools and will turn to manual compensation to fix any errors. When calculating compensation, automated tools are the gold standard. For more tips on spectral unmixing specifically, see this video. It saves you time, and generally provides better results. Learn more about AutoSpill here.Įdit October 21, 2021: It should be noted that the tips for gate placement described in this post can be applied to spectral unmixing as well, just replace the term “compensation” with “unmixing”. In essence, AutoSpill uses iterations of linear regressions to get the best compensation or unmixing matrices, and removes the need for gating the positive and negative fractions of your controls. introduced AutoSpill, which resolved the gating issue raised in this blogpost. PMID 330738.Edit January 19, 2023: The 2021 Nature paper by Carlos P. The Journal of Histochemistry and Cytochemistry. "Two-color immunofluorescence using a fluorescence-activated cell sorter". Annals of the New York Academy of Sciences. "Fluorescence spectral overlap compensation for any number of flow cytometry parameters". "Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats". 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. In cytometry, compensation is a mathematical correction of a signal overlap between the channels of the emission spectra of different fluorochromes. ![]()
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