Infrared (IR) spectroscopic imaging instrumentsâ 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 classiï¬cation 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 â to suit instruments that acquire discrete or contiguous spectral bandwidths. The analysis is validated by reï¬ning 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 wideï¬eld 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 conï¬dence in imaging an analyteâs concentration and a basis for further improving IR imaging technology.
Microimaging FT-IR of Head and Neck Tumours. The case of salivary glands