Yeh Kevin, Kenkel Seth, Liu Jui-Nung, Bhargava Rohit
Department of Bioengineering, ‡Department of Mechanical Science and Engineering, and §Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Anal Chem. 2015 Jan 6;87(1):485-93. doi: 10.1021/ac5027513. Epub 2014 Dec 22.
Infrared (IR) spectroscopic imaging systems are a powerful tool for visualizing molecular microstructure of a sample without the need for dyes or stains. Table-top Fourier transform infrared (FT-IR) imaging spectrometers, the current established technology, can record broadband spectral data efficiently but requires scanning the entire spectrum with a low throughput source. The advent of high-intensity, broadly tunable quantum cascade lasers (QCL) has now accelerated IR imaging but results in a fundamentally different type of instrument and approach, namely, discrete frequency IR (DF-IR) spectral imaging. While the higher intensity of the source provides a higher signal per channel, the absence of spectral multiplexing also provides new opportunities and challenges. Here, we couple a rapidly tunable QCL with a high performance microscope equipped with a cooled focal plane array (FPA) detector. Our optical system is conceptualized to provide optimal performance based on recent theory and design rules for high-definition (HD) IR imaging. Multiple QCL units are multiplexed together to provide spectral coverage across the fingerprint region (776.9 to 1904.4 cm(-1)) in our DF-IR microscope capable of broad spectral coverage, wide-field detection, and diffraction-limited spectral imaging. We demonstrate that the spectral and spatial fidelity of this system is at least as good as the best FT-IR imaging systems. Our configuration provides a speedup for equivalent spectral signal-to-noise ratio (SNR) compared to the best spectral quality from a high-performance linear array system that has 10-fold larger pixels. Compared to the fastest available HD FT-IR imaging system, we demonstrate scanning of large tissue microarrays (TMA) in 3-orders of magnitude smaller time per essential spectral frequency. These advances offer new opportunities for high throughput IR chemical imaging, especially for the measurement of cells and tissues.
红外（IR）光谱成像系统是一种强大的工具，可用于在无需染料或染色剂的情况下可视化样品的分子微观结构。台式傅里叶变换红外（FT-IR）成像光谱仪是目前已确立的技术，它可以高效记录宽带光谱数据，但需要使用低通量光源扫描整个光谱。高强度、宽可调谐量子级联激光器（QCL）的出现现在加速了红外成像，但导致了一种从根本上不同类型的仪器和方法，即离散频率红外（DF-IR）光谱成像。虽然光源的更高强度提供了每个通道更高的信号，但缺乏光谱复用也带来了新的机遇和挑战。在这里，我们将快速可调谐的QCL与配备冷却焦平面阵列（FPA）探测器的高性能显微镜相结合。我们的光学系统根据最近的高清（HD）红外成像理论和设计规则进行概念设计，以提供最佳性能。多个QCL单元被复用在一起，以在我们的DF-IR显微镜中提供跨越指纹区域（776.9至1904.4 cm⁻¹）的光谱覆盖，该显微镜能够进行宽光谱覆盖、宽场检测和衍射极限光谱成像。我们证明该系统的光谱和空间保真度至少与最好的FT-IR成像系统一样好。与具有大10倍像素的高性能线性阵列系统的最佳光谱质量相比，我们的配置在等效光谱信噪比（SNR）方面提供了加速。与最快的可用高清FT-IR成像系统相比，我们展示了在每个基本光谱频率下以小3个数量级的时间扫描大型组织微阵列（TMA）。这些进展为高通量红外化学成像提供了新机遇，特别是对于细胞和组织的测量。
