1 BioTools Inc., Jupiter, FL, USA.
2 Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic.
Appl Spectrosc. 2017 Jun;71(6):1117-1126. doi: 10.1177/0003702817701457. Epub 2017 Apr 24.
We report the first vibrational circular dichroism (VCD) measurement of spatial heterogeneity in a sample using infrared (IR) microsampling. Vibrational circular dichroism spectra are typically measured using a standard IR cell with an IR beam diameter of 10 mm or greater making it impossible to investigate the spatial heterogeneity of a solid film sample. We have constructed a VCD sampling assembly with either 3 mm or 1 mm spatial resolution. An XY-translation stage was used to measure spectra at different spatial locations producing IR and VCD maps of the sample. In addition, a rotating sample stage was employed using a dual photoelastic modulator (PEM) setup to suppress artifacts due to linear birefringence in solid-phase or film samples. Infrared and VCD mapping of an insulin fibril film has been carried out at both 3 and 1 mm spatial resolution, and lysozyme films were mapped at 1 mm resolution. The IR spectra of different spots vary in intensity due primarily to sample thickness. The changes in the VCD intensity across the map largely correlate to corresponding changes in the IR map. Closer inspection of the insulin map revealed changes in the relative intensities of the VCD spectra not present in the parent IR spectra, which indicated differences in the degree of supramolecular chirality of the fibrils in the various spatial regions. For lysozyme films, in addition to different degrees of supramolecular chirality, reversal of the net fibril chirality was observed. The large signal-to-noise ratio observed at 1 mm resolution implies the feasibility of further increasing the spatial resolution by one or two orders of magnitude for protein fibril film samples.
我们报告了首例使用红外（IR）微采样技术对样品中的空间异质性进行振动圆二色性（VCD）测量。振动圆二色性光谱通常使用具有 10mm 或更大 IR 光束直径的标准 IR 池进行测量，这使得不可能研究固体膜样品的空间异质性。我们已经构建了具有 3mm 或 1mm 空间分辨率的 VCD 采样组件。使用 XY 平移台在不同空间位置测量光谱，生成样品的 IR 和 VCD 图谱。此外，使用双电光调制器（PEM）设置的旋转样品台抑制了固态或膜样品中线性双折射引起的伪影。在 3mm 和 1mm 空间分辨率下对胰岛素原纤维膜进行了红外和 VCD 测绘，并且在 1mm 分辨率下对溶菌酶膜进行了测绘。由于样品厚度，不同点的 IR 光谱强度不同。图谱中 VCD 强度的变化与 IR 图谱的对应变化主要相关。对胰岛素图谱的仔细检查表明，图谱中存在 VCD 光谱的相对强度变化，而在原始 IR 光谱中不存在，这表明在各个空间区域中纤维的超分子手性程度存在差异。对于溶菌酶膜，除了超分子手性的不同程度外，还观察到净纤维手性的反转。在 1mm 分辨率下观察到的大信噪比意味着对于蛋白质原纤维膜样品，进一步将空间分辨率提高一个或两个数量级是可行的。
