Gandhi Sonali A, Sanders Matthew A, Granneman James G, Kelly Christopher V
Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA, 48201.
Center for Molecular Medicine and Genetics, School of Medicine, Wayne State University, Detroit, MI, USA, 40201.
bioRxiv. 2023 Feb 1:2023.01.30.526256. doi: 10.1101/2023.01.30.526256.
The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures the diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed a novel, economical method to simultaneously monitor diffusion and oligomerization with the use of super-continuum laser and spectral deconvolution from a single detector. Customizable excitation wavelengths were chosen from the wide-band source and spectral fitting of the emitted light revealed the interactions for up to four spectrally overlapping fluorophores simultaneously. This method was applied to perform four-color FCCS, as demonstrated with polystyrene nanoparticles, lipid vesicles, and membrane-bound molecules. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. The fluorescence emission passed through a cleanup filter and a dispersive prism prior to being collected by a sCMOS or EMCCD camera with up to 10 kHz frame rates. The emission intensity versus time of each fluorophore was extracted through a linear least-square fitting of each camera frame and temporally correlated via custom software. Auto- and cross-correlation functions enabled the measurement of the diffusion rates and binding partners. We have measured the induced aggregation of nanobeads and lipid vesicles in solution upon increasing the buffer salinity. Because of the adaptability of investigating four fluorophores simultaneously with a cost-effective method, this technique will have wide application for examining complex homo- and heterooligomerization in model and living systems.
磷脂和膜相关蛋白的扩散与重组对于细胞功能至关重要。荧光互相关光谱法（FCCS）可测量生物系统中纳摩尔浓度下的扩散和分子相互作用。我们开发了一种新颖且经济的方法，利用超连续激光和来自单个探测器的光谱解卷积同时监测扩散和寡聚化。从宽带光源中选择可定制的激发波长，对发射光进行光谱拟合可同时揭示多达四种光谱重叠荧光团的相互作用。该方法应用于进行四色FCCS，如用聚苯乙烯纳米颗粒、脂质体和膜结合分子所证明的那样。从广谱光纤激光器中选择多达四个可单独定制的激发通道，以在衍射极限光斑内激发扩散体。荧光发射在通过清洁滤光片和色散棱镜后，由帧速率高达1 kHz的sCMOS或EMCCD相机收集。通过对每个相机帧进行线性最小二乘拟合提取每个荧光团的发射强度随时间的变化，并通过定制软件进行时间关联。自相关和互相关函数能够测量扩散速率和结合伙伴。我们测量了增加缓冲液盐度时溶液中纳米珠和脂质体的诱导聚集。由于该技术能够以经济有效的方法同时研究四种荧光团，因此在检查模型和活体系统中的复杂同型和异型寡聚化方面将有广泛应用。
