{"doi":"10.1002/cyto.a.24243","title":"Quantification of Light Scattering Detection Efficiency and Background in Flow Cytometry","abstract":"Knowledge of the sensitivity is essential for data interpretation and comparison between flow cytometers, especially when particles with signals close to the detection limit are studied, such as bacteria, extracellular vesicles, viruses, or other nanoparticles. For fluorescence, multiple methods have been developed to quantify sensitivity in terms of the detection efficiency Q and background light signal B (1-6). All methods are based on the fact that light generates photoelectrons at the detector. Q is defined as the number of statistical photoelectrons generated at the detector per fluorochrome molecule passing through the illumination beam (4). B is the background light signal expressed in terms of the equivalent number of fluorochromes (4). The effect of different values of Q and B on the sensitivity of a flow cytometer has been described previously (4, 7). In short, a higher Q and a lower B increases the ability to resolve a dim population from the background noise. Because scattered light also generates photoelectrons at the detector, it is theoretically possible to express the sensitivity of a light scatter detector in terms of Q and B as well. Currently, light scatter sensitivity is often expressed as the smallest detectable polystyrene (PS) bead, which thereby only specifies the detection threshold and provides no information about the ability to resolve dim populations (8). Expression of light scatter sensitivity in terms of Q and B would provide a more complete description of light scatter sensitivity. However, since flow cytometers provide data in arbitrary units (a.u.), a standardized unit is required to compare Q and B between different flow cytometers. In fluorescence, Q and B are commonly expressed in terms of molecules of equivalent soluble fluorophore (MESF), with Q in photoelectrons/MESF and B in MESF. A standardized unit that can be used to express and compare Q and B for light scatter was, hitherto, lacking. Recently, we explained how to use the scatter cross section (σs) in nm2 as a standardized unit for scatter (9), which opened up the possibility of quantifying light scatter sensitivity in terms of Q and B. Here, we explore the feasibility of deriving Q and B to quantify light scatter sensitivity, using σs in nm2 as the standardized unit. The theory behind deriving Q and B for light scatter is similar to that for fluorescence (1-5). Detected signals are assumed to be linear with the light scattering power impinging the detector and dynode noise of the photomultiplier tube (PMT) is ignored. The theory below is derived in analogy to the derivation for fluorescence detectors as published by Chase and Hoffman (3). Because SDint., SDill., and scale linearly with illumination power, CVmeas. corr. bright is independent of illumination power. By varying the illumination power, we can thus study the stochastic process of scattered light at low illumination powers and determine the term SDint.2 + SDill.2 at relatively high illumination powers. By combining Eqs. (12) and (13), Factors affecting Q are Iill (and thus the illumination power and illumination spot size), the acquisition time, the collection angle, the quantum efficiency of the detector, and the transmission efficiency of lenses and spectral filters (4). Factors affecting B include particles in the buffer, particles in the sheath and light scattering of optical components such as the flow cell wall. A bead mixture containing nonfluorescent NIST-traceable PS bead populations with mean diameters of 100, 125, 147, 203, 296, 400, 600, 799, and 994 nm (all 3000 Series Nanosphere Size Standards; Thermo Fisher Scientific, Waltham, MA), and two green fluorescent bead populations of 140 and 380 nm, respectively (G140, G400; Thermo Fisher Scientific) was prepared in distilled water. The concentration of each bead population in the mixture was ~107/ml. The side scatter (SSC) signal of the bead mixture was measured at illumination powers ranging from 20 mW to 200 m","journal":"Cytometry Part A","year":2020,"id":81505,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":14,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9572,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":229529,"name":"Frank A. W. Coumans","orcid":null,"position":1,"is_corresponding":false},{"id":215734,"name":"Joshua A Welsh","orcid":"0000-0002-1097-9756","position":2,"is_corresponding":false},{"id":215736,"name":"Rienk Nieuwland","orcid":"0000-0002-5671-3400","position":3,"is_corresponding":false},{"id":421650,"name":"Ton G. van Leeuwen","orcid":"0000-0002-5642-1133","position":4,"is_corresponding":false},{"id":226787,"name":"Edwin van der Pol","orcid":"0000-0002-9497-8426","position":5,"is_corresponding":false},{"id":226801,"name":"Leonie de Rond","orcid":"0000-0003-3007-1849","position":0,"is_corresponding":true}],"reference_count":16,"raw_metadata":null,"created_at":"2026-07-18T21:52:58.921847Z","pmid":"33085220","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}