Infrared (IR) spectroscopic imaging instruments’ performance can be characterized and optimized by an analysis of their limit of detection (LoD). Here we report a systematic analysis of the LoD for Fourier transform IR (FT-IR) and discrete frequency IR (DFIR) imaging spectrometers. In addition to traditional measurements of sample and blank data, we propose a decision theory perspective to pose the determination of LoD as a binary classification problem under different assumptions of noise uniformity and correlation. We also examine three spectral analysis approaches, namely absorbance at a single frequency, sum of absorbance over selected frequencies and total spectral distance – to suit instruments that acquire discrete or contiguous spectral bandwidths. The analysis is validated by refining the fabrication of a bovine serum albumin protein microarray to provide eight uniform spots from 2.8 nL of solution for each concentration over a wide range (0.05 -10 mg/mL). Using scanning parameters that are typical for each instrument, we estimate a LoD of 0.16 mg/mL and 0.12 mg/mL for widefield and line scanning FT-IR imaging systems, respectively, usingthespectraldistanceapproach,and0.22mg/mLand0.15mg/mL using an optimal set of discrete frequencies. As expected, averaging and the use of post-processing techniques such as minimum noise fraction (MNF) transformation results in LoDs as low as 0.075 mg/mL that correspond to a spotted protein mass of 112 fg/pixel. We emphasize that these measurements were conducted at typical imaging parameters for each instrument and can be improved using the usual trading rules of IR spectroscopy. This systematic analysis and methodology for determining the LoD can allow for quantitative measures of confidence in imaging an analyte’s concentration and a basis for further improving IR imaging technology.

红外 (IR) 光谱成像仪器的性能可以通过分析其检测限 (LoD) 来表征和优化。在这里，我们报告了傅里叶变换红外 (FT-IR) 和离散频率红外 (DFIR) 成像光谱仪 LoD 的系统分析。除了样本和空白数据的传统测量之外，我们提出了一种决策理论视角，将 LoD 的确定作为噪声均匀性和相关性的不同假设下的二元分类问题。我们还研究了三种光谱分析方法，即单个频率的吸光度、选定频率的吸光度之和以及总光谱距离，以适合采集离散或连续光谱带宽的仪器。通过改进牛血清白蛋白微阵列的制作来验证分析，以在宽范围 (0.05 -10 mg/mL) 的每个浓度下从 2.8 nL 溶液中提供 8 个均匀点。使用每种仪器的典型扫描参数，我们使用光谱距离方法估计宽视野和线扫描 FT-IR 成像系统的 LoD 分别为 0.16 mg/mL 和 0.12 mg/mL，以及 0.22 mg/mL 和 0.15 mg/mL使用一组最佳的离散频率。正如预期的那样，平均和使用后处理技术（例如最小噪声分数 (MNF) 转换）可使 LoD 低至 0.075 mg/mL，对应于 112 fg/像素的斑点蛋白质质量。我们强调，这些测量是在每种仪器的典型成像参数下进行的，并且可以使用红外光谱的常用交易规则进行改进。这种用于确定 LoD 的系统分析和方法可以对分析物浓度成像的置信度进行定量测量，并为进一步改进红外成像技术奠定基础。