Dunn Kaitlin J, Elias Tresa M, Brown Edward B, Berger Andrew J
University of Rochester, The Institute of Optics, Rochester, NY 14627, USA.
University of Rochester, Dept. of Biomedical Engineering, Rochester, NY 14627, USA.
Biomed Opt Express. 2022 Jul 19;13(8):4236-4246. doi: 10.1364/BOE.461874. eCollection 2022 Aug 1.
Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.
角分辨光散射已被证明是一种早期检测细胞器大小细微变化的方法，因为它对散射体大小和折射率对比度敏感。然而，对于浸没在折射率接近1.33的介质中的细胞，细胞本身就像一个较大的散射体，并贡献其自身的角度特征。这种全细胞散射高度依赖于细胞的形状和大小，很难与所需的细胞器散射信号区分开来。这降低了从角散射中提取细胞器大小信息的准确性。为了减轻这种影响，我们通过将其与水溶性、生物相容性、高折射率液体混合来操纵浸没介质的折射率。这种方法通过最小化细胞质与改性介质之间的折射率对比度，从物理上减少了全细胞散射的量。我们在附着在盖玻片上的活细胞上演示了这种技术，使用傅里叶变换光散射从复杂场图像计算角散射。我们表明，细胞与介质的折射率对比度在高达20度的角度上贡献了显著的散射，并且折射率匹配将这种低角度散射降低了高达4.5倍。这一结果表明了折射率匹配在改善单细胞中细胞器大小分布估计方面的潜力。
