Department of Health Technology, Technical University of Denmark, Kongens Lyngby, DK-2800, Denmark.
Teamator AB, SE-25023, Helsingborg, Sweden.
Cytometry A. 2019 Aug;95(8):917-924. doi: 10.1002/cyto.a.23797. Epub 2019 May 23.
The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.
近年来，利用高通量流式细胞术对纳米颗粒进行特性分析受到了越来越多的关注。然而，为了充分发挥流式细胞术在纳米级物体特性分析方面的潜力，必须开发适用于尺寸估计的校准品，并提高传统流式细胞仪的灵敏度。基于散射信号，通常使用硅和塑料微球作为流式细胞术的尺寸校准品，以评估细胞外囊泡和人工囊泡（脂质体）的尺寸。然而，一些研究表明，这些微球无法准确地将散射强度与囊泡尺寸相关联。在这项工作中，我们提出了一种新的方法，即基于荧光激活细胞分选（FACS）制备的脂质体尺寸校准品，通过流式细胞术估计单个脂质体的尺寸，我们称之为荧光激活纳米颗粒分选（FANS）。这些校准脂质体具有与被研究的颗粒相同的尺寸、结构和折射率，因此可以作为独特的校准品。首先，制备了一份多分散荧光标记的单层脂质体样品，并通过流式细胞术进行分析。然后，根据荧光强度对多分散脂质体的不同部分进行 FANS 分选。此后，我们采用纳米颗粒跟踪分析（NTA）来评估 FANS 分选的脂质体部分的脂质体尺寸。最后，我们将 FANS 分选的脂质体部分的流式细胞术读出值（侧向散射和荧光强度）与其通过 NTA 获得的相应尺寸相关联。通过这种方法，我们可以将脂质体的荧光强度转换为所有检测到的单个脂质体的纳米级脂质体尺寸。我们结合 FANS 校准品，用流式细胞术对多分散脂质体样品的尺寸分布进行了验证，结果与纳米颗粒尺寸的标准方法（包括 NTA 和冷冻传输电子显微镜）一致。这项工作还强调了使用流式细胞术侧向散射读数来确定小尺寸（30-300nm）人工囊泡尺寸的局限性。 © 2019 国际细胞分析协会。
