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Additional info for Current Protocols in Cytometry
When solvent staining is possible for watersoluble ﬂuorochromes, the spectral characteristics can differ signiﬁcantly from those of ﬂuorochrome in aqueous solution. Surface staining allows many common ﬂuorochromes— especially those used as tags on ﬂuorescent antibodies—to be used for particle staining. , amino group) is covalently bound to a reactive group on the ﬂuorochrome. MESF (molecules of equivalent soluble ﬂuorochrome): measure of particle ﬂuorescence in which the signal from a ﬂuorescent particle is equal to that from a known number of molecules in solution.
Background (noise, ﬂuorescence, scatter): signal present when no particles are ﬂowing in the sample stream. Background noise is one factor that limits the sensitivity of ﬂuorescence detection (see deﬁnitions of ﬂuorescence sensitivity and light-scatter sensitivity below). Depending on how low are the signals that one is trying to detect in the sample, different factors are dominant contributors to the background. , lasers turned off), detector noise is the background limit. For photomultiplier tubes (PMTs), the detector background noise is called dark current and is due to random emission of electrons from the photocathode.
A very useful set of deﬁnitions that generally cover the ﬁeld of clinical diagnostics is NCCLS publication NRSCL8-A, Terminology and Deﬁnitions for use in NCCLS Documents (NCCLS, 1998a). The deﬁnitions for limits below are from the NCCLS deﬁnitions. Additional deﬁnitions related to quantitative ﬂuorescence cytometry are provided by Henderson et al. (1998). The deﬁnitions below should be considered a reasonable point along the way toward authoritative and broadly accepted and understood terminology.