Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Appl Spectrosc. 2022 Jan;76(1):105-117. doi: 10.1177/00037028211050961. Epub 2021 Oct 21.
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, average 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, using the spectral distance approach, and 0.22 mg/mL and 0.15 mg/mL using an optimal set of discrete frequencies. As expected, averaging and the use of post-processing techniques such as minimum noise fraction 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）来进行表征和优化。在这里，我们报告了对傅里叶变换红外（FT-IR）和离散频率红外（DFIR）成像光谱仪的 LOD 进行的系统分析。除了对样品和空白数据的传统测量外，我们还提出了一种决策理论的观点，将 LOD 的确定作为在噪声均匀性和相关性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、选定频率的吸光度平均值和总光谱距离-以适应获取离散或连续光谱带宽的仪器。通过改进牛血清白蛋白蛋白微阵列的制造，在很宽的范围内（0.05-10 mg/mL）为每个浓度提供来自约 2.8 nL 溶液的八个均匀斑点，从而验证了该分析。使用每种仪器的典型扫描参数，我们使用光谱距离方法分别估计宽场和线扫描 FT-IR 成像系统的 LOD 为 0.16 mg/mL 和 0.12 mg/mL，使用最佳离散频率集的 LOD 为 0.22 mg/mL 和 0.15 mg/mL。正如预期的那样，平均和使用后处理技术，例如最小噪声分数变换，导致 LOD 低至约 0.075 mg/mL，对应于每个斑点的蛋白质质量约为 112 fg/像素。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，可以通过通常的红外光谱交易规则进行改进。这种用于确定 LOD 的系统分析和方法可以为定量测量成像分析物浓度的置信度提供依据，并为进一步改进红外成像技术提供基础。
