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 (σ_s) 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 σ_s in nm² as the standardized unit.

了解灵敏度对于流式细胞仪之间的数据解释和比较至关重要，特别是在研究信号接近检测限的颗粒（例如细菌、细胞外囊泡、病毒或其他纳米颗粒）时。对于荧光，已经开发了多种方法来根据检测效率Q和背景光信号B来量化灵敏度( 1-6 )。所有方法都基于光在检测器处产生光电子的事实。Q定义为穿过照明光束 ( 4 ) 的每个荧光染料分子在检测器处生成的统计光电子数。B是以荧光染料的当量数表示的背景光信号 ( 4 )。前面已经描述了不同的Q和B值对流式细胞仪灵敏度的影响( 4, 7 )。简而言之，较高的Q 值和较低的B 值可以提高从背景噪声中分辨出微弱群体的能力。

由于散射光也会在检测器处产生光电子，因此理论上也可以用Q和B来表示光散射检测器的灵敏度。目前，光散射灵敏度通常表示为最小的可检测聚苯乙烯 (PS) 珠，因此它仅指定检测阈值，并且不提供有关解析暗群体能力的信息 ( 8 )。用Q和B表达光散射灵敏度将提供对光散射灵敏度的更完整的描述。然而，由于流式细胞仪以任意单位 (au) 提供数据，因此需要标准化单位来比较不同流式细胞仪之间的Q和B。在荧光中，Q和B通常以等效可溶性荧光团 (MESF) 分子的形式表示，其中Q表示为光电子/MESF，B表示为 MESF。迄今为止，还缺乏可用于表达和比较光散射的Q和B的标准化单位。最近，我们解释了如何使用nm ²的散射截面 ( σ _s ) 作为散射 ( 9 )的标准化单位，这开辟了根据Q和B量化光散射灵敏度的可能性。在这里，我们探索推导Q和B来量化光散射灵敏度的可行性，使用σ _s in nm ²作为标准化单位。