Department of Physics, University of Wisconsin, Milwaukee, Wisconsin 53211, United States.
Anal Chem. 2012 Jul 17;84(14):6173-80. doi: 10.1021/ac301080h. Epub 2012 Jul 9.
Fourier transform infrared (FTIR) microspectroscopy is a powerful technique for label-free chemical imaging that has supplied important chemical information about heterogeneous samples for many problems across a variety of disciplines. State-of-the-art synchrotron based infrared (IR) microspectrometers can yield high-resolution images, but are truly diffraction limited for only a small spectral range. Furthermore, a fundamental trade-off exists between the number of pixels, acquisition time and the signal-to-noise ratio, limiting the applicability of the technique. The recently commissioned infrared synchrotron beamline, infrared environmental imaging (IRENI), overcomes this trade off and delivers 4096-pixel diffraction limited IR images with high signal-to-noise ratio in under a minute. The spatial oversampling for all mid-IR wavelengths makes the IRENI data ideal for spatial image restoration techniques. Here, we measured and fitted wavelength-dependent point-spread-functions (PSFs) at IRENI for a 74× objective between the sample plane and detector. Noise-free wavelength-dependent theoretical PSFs are deconvoluted from images generated from narrow bandwidths (4 cm(-1)) over the entire mid-infrared range (4000-900 cm(-1)). The stack of restored images is used to reconstruct the spectra. Restored images of metallic test samples with features that are 2.5 μm and smaller are clearly improved in comparison to the raw data images for frequencies above 2000 cm(-1). Importantly, these spatial image restoration methods also work for samples with vibrational bands in the recorded mid-IR fingerprint region (900-1800 cm(-1)). Improved signal-to-noise spectra are reconstructed from the restored images as demonstrated for a mixture of spherical polystyrene beads in a polyurethane matrix. Finally, a freshly thawed retina tissue section is used to demonstrate the success of deconvolution achievable with a heterogeneous, irregularly shaped, biologically relevant sample with distinguishing spectroscopic features across the entire mid-IR spectral range.
傅里叶变换红外（FTIR）微光谱学是一种用于非标记化学成像的强大技术，它为跨多个学科的许多问题提供了有关异质样品的重要化学信息。基于同步加速器的最先进的红外（IR）微光谱仪可以生成高分辨率图像，但仅在很小的光谱范围内具有真正的衍射限制。此外，在像素数量、采集时间和信噪比之间存在基本的权衡，限制了该技术的适用性。最近委托的红外同步加速器光束线，红外环境成像（IRENI），克服了这种权衡，并在不到一分钟的时间内提供具有高信噪比的 4096 像素衍射限制 IR 图像。所有中红外波长的空间过采样使得 IRENI 数据非常适合空间图像恢复技术。在这里，我们在样品平面和探测器之间的 74×物镜下测量并拟合了 IRENI 的波长相关点扩散函数（PSF）。从窄带宽（4 cm(-1))生成的图像中解卷积无噪声的波长相关理论 PSF，带宽范围为整个中红外范围（4000-900 cm(-1))。使用堆栈的恢复图像重建光谱。与原始数据图像相比，在频率高于 2000 cm(-1)时，具有 2.5 μm 及以下特征的金属测试样品的恢复图像得到了明显改善。重要的是，这些空间图像恢复方法也适用于记录的中红外指纹区域（900-1800 cm(-1))中的振动带的样品。从恢复图像重建的改进的信噪比光谱，如在聚氨酯基质中的球形聚苯乙烯珠的混合物中所示。最后，使用新鲜解冻的视网膜组织切片来证明对具有整个中红外光谱范围内的区别光谱特征的异质、不规则形状、生物相关样品进行去卷积的成功。
