The integration of Fourier transform–infrared (FT-IR) spectroscopy with microscopy facilitates recording of spatially resolved spectral information, allowing the examination of both the structure and chemical composition of a heterogeneous material. While the first such attempt was over 50 years ago (1), present-day instrumentation largely evolved from the point microscopy detection of interferometric signals that developed in the mid-1980s (2). The successful coupling of interferometry for spectral recording and microscopy for spatial specificity in these systems spurred interest in a variety of fields, including the materials (3), forensic (4), and biomedical arenas (5,6). Point microscopy utilizes an aperture to restrict radiation incident on a sample and permits the recording of spatially localized data. The primary utilities of this form of microscopy lay in acquiring accurate spectra from small samples, in determining the chemical structure and composition of heterogeneous phases at specified points, and in building two-dimensional maps of the chemical composition of samples. Because the data were acquired at a single point, composition maps could only be acquired by rastering the sample. Hence, the approach was termed mapping or point mapping and involved as many interferometer scans as the number of pixels in the map.

The use of focal plane array (FPA) detectors for microscopy (7,8) allowed for the acquisition of large fields of view in a single interferogram acquisition sweep. The multichannel detection enabled by array detectors was similar to the concept of recording images with charge-coupled devices (CCDs) in optical microscopy; hence, the approach was termed imaging. The unique advantages of observing an entire field of view rapidly permitted applications that allowed monitoring of dynamic processes, spatially resolved spectroscopy of large samples or many samples, and enhanced quality due to retention of radiation throughput that was lost in point microscopy systems as a result of diffraction at the aperture. Just as for the previous generation of microspectroscopy instruments, applications rapidly followed in the materials (9) and biomedical fields (10–14). Research activity in this area can be divided into three major categories: instrumentation and sampling methodologies, applications, and data extraction algorithms. In this article, we review key advances and recent developments in the context of biomedical imaging. We do not provide a comprehensive overview but selectively highlight certain features of importance for cancer-related imaging. Last, we focus on one emerging application area, namely tissue histopathology, and provide illustrative examples from our laboratory indicating the integrative nature of protocol development.

Instrumentation, Sampling, and Data-Handling Techniques

Instrumentation

Because imaging is largely based upon new detectors with unique performance characteristics for spectroscopy, efforts in instrumentation have largely focused on the efficient integration of FPA detectors with interferometers. Because of the size, different electronics, and unique noise characteristics of FPAs, an optimization of data acquisition methodology was a primary activity in the initial period of availability of instrumentation. The first rational attempt at understanding performance and optimizing the data acquisition process revealed the unique noise characteristics that limited the first generation of array detectors (15). Briefly, this paper established that the general behavior of FT-IR spectrometers is generally held for imaging spectrometers but the detector may serve to limit the applicability of established practices in IR spectrometry. An explicit optimization of the data acquisition time revealed several strategies for speeding data collection for both the step-scan and rapid-scan modes (16). The first example of rapid-scan FT-IR imaging (17) was conducted using asynchronous sampling, followed by descriptions of synchronously triggered sampling and generalized methodologies (18) that could use any detector at any interferometer modulation frequency using post-acquisition techniques. Advances in detector technology have now allowed for rapid-scan imaging to become routine for large FPA detectors, while innovative new detectors have been developed (first by PerkinElmer, Shelton, Connecticut) that trade off a large multichannel detection advantage of arrays against the speed of smaller detector arrays to provide a very high-performance instrument (19).