Gelfand Paul, Smith Randy J, Stavitski Eli, Borchelt David R, Miller Lisa M
†Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States.
‡National Synchrotron Light Source-II, Brookhaven National Laboratory, Upton, New York 11973-5000, United States.
Anal Chem. 2015 Jun 16;87(12):6025-31. doi: 10.1021/acs.analchem.5b00371. Epub 2015 May 28.
Fourier-transform infrared (FTIR) spectroscopic imaging is a widely used method for studying the chemistry of proteins, lipids, and DNA in biological systems without the need for additional tagging or labeling. This technique can be especially powerful for spatially resolved, temporal studies of dynamic changes such as in vivo protein folding in cell culture models. However, FTIR imaging experiments have typically been limited to dry samples as a result of the significant spectral overlap between water and the protein Amide I band centered at 1650 cm(-1). Here, we demonstrate a method to rapidly obtain high quality FTIR spectral images at submicron pixel resolution in vivo over a duration of 18 h and longer through the development and use of a custom-built, demountable, microfluidic-incubator and a FTIR microscope coupled to a focal plane array (FPA) detector and a synchrotron light source. The combined system maximizes ease of use by allowing a user to perform standard cell culture techniques and experimental manipulation outside of the microfluidic-incubator, where assembly can be done just before the start of experimentation. The microfluidic-incubator provides an optimal path length of 6-8 μm and a submillimeter working distance in order to obtain FTIR images with 0.54-0.77 μm pixel resolution. In addition, we demonstrate a novel method for the correction of spectral distortions caused by varying concentrations of water over a subconfluent field of cells. Lastly, we use the microfluidic-incubator and time-lapsed FTIR imaging to determine the misfolding pathway of mutant copper-zinc superoxide dismutase (SOD1), the protein known to be a cause of familial amyotrophic lateral sclerosis (FALS).
傅里叶变换红外（FTIR）光谱成像技术是一种广泛应用于研究生物系统中蛋白质、脂质和DNA化学组成的方法，无需额外的标记或标签。该技术对于动态变化的空间分辨和时间研究，如细胞培养模型中的体内蛋白质折叠，具有强大的功能。然而，由于水与位于1650 cm(-1)的蛋白质酰胺I带之间存在显著的光谱重叠，FTIR成像实验通常仅限于干燥样品。在此，我们展示了一种方法，通过开发和使用定制的、可拆卸的微流控培养箱以及与焦平面阵列（FPA）探测器和同步辐射光源耦合的FTIR显微镜，在18小时及更长时间内以亚微米像素分辨率在体内快速获取高质量的FTIR光谱图像。该组合系统通过允许用户在微流控培养箱外进行标准细胞培养技术和实验操作，最大限度地提高了易用性，微流控培养箱的组装可在实验开始前完成。微流控培养箱提供了6-8μm的最佳光程长度和亚毫米的工作距离，以获得像素分辨率为0.54-0.77μm的FTIR图像。此外，我们展示了一种新方法，用于校正亚汇合细胞场中因水浓度变化引起的光谱畸变。最后，我们使用微流控培养箱和延时FTIR成像来确定突变型铜锌超氧化物歧化酶（SOD1）的错误折叠途径，该蛋白质是家族性肌萎缩侧索硬化症（FALS）的病因之一。
