分子转子作为红细胞硬度的细胞内探针
Molecular Rotors as intracellular probes of Red Blood Cell stiffness
摘要：
红细胞的变形性是控制血液流变学及其在体内循环的基本参数。因此，表征血液样品中细胞的刚性及其异质性是理解闭塞现象的关键点，特别是在健康细胞与病理细胞共存的红细胞疾病的情况下。然而，在小的生物区室中测量细胞内流变学需要特定技术的发展。我们提出了一种基于分子转子的技术-粘度敏感荧光探针-来评估上述关键点。DASPI分子转子已被鉴定为具有与细胞质主要成分血红蛋白的光谱荧光特性脱钩的光谱荧光特性。在模型流体中作为粘度探针的转子验证后，我们通过共聚焦显微镜表明，除了与膜结合外，转子自发地均匀地渗透到红细胞中。在温度硬化的红细胞上进行的实验表明，分子转子可以探测它们的整体刚度。一个简单的模型使我们能够分离的贡献的细胞质的膜，提供了定量的细胞质硬化与温度，符合独立测量的血红蛋白溶液的粘度。我们的实验表明，转子可用于量化红细胞在细胞水平的细胞内流变学，以及在血液样品中的这种刚度的异质性。这项技术为生物医学应用、诊断和疾病监测开辟了新的可能性。
英文摘要：
The deformability of red blood cells is an essential parameter that controls the rheology of blood as well as its circulation in the body. Characterizing the rigidity of the cells and their heterogeneity in a blood sample is thus a key point in the understanding of occlusive phenomena, particularly in the case of erythrocytic diseases in which healthy cells coexist with pathological cells. However, measuring intracellular rheology in small biological compartments requires the development of specific techniques. We propose a technique based on molecular rotors-viscosity-sensitive fluorescent probes-to evaluate the above key point. DASPI molecular rotor has been identified with spectral fluorescence properties decoupled from those of hemoglobin, the main component of the cytosol. After validation of the rotor as a viscosity probe in model fluids, we showed by confocal microscopy that, in addition to binding to the membrane, the rotor penetrates spontaneously and uniformly into red blood cells. Experiments conducted on temperature-stiffened red blood cells show that molecular rotors can probe their overall rigidity. A simple model allowed us to separate the contribution of the cytosol from that of the membrane, providing a quantification of cytosol rigidification with temperature, consistent with independent measurements of the viscosity of hemoglobin solutions. Our experiments demonstrate that the rotor can be used to quantify the intracellular rheology of red blood cells at the cellular level, as well as the heterogeneity of this stiffness in a blood sample. This technique opens up new possibilities for biomedical applications, diagnosis and disease monitoring.
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