The staring biomedical spectral imagers use spectral filters (e.g., filter wheels containing fixed bandpass filters, AOTF, and LCTF) to acquire a single-band 2-D gray image with full spatial information and form a spectral data cube by scanning in the spectral domain through a number of wavelengths. The most basic implementation of staring spectral imaging is the use of a rotatable disk called a filter wheel carrying a set of discrete bandpass filters. Afromowitz et al. have developed a color filter wheel–based multispectral imaging system to evaluate burn depth on human palms and feet.112 This is the earliest study on the staring biomedical spectral imager and its applications. In recent studies, similar systems have been developed for quantitative analysis of the human tooth and ovary.111,113 The advantage of these filter wheel–based imagers is that they are easy to use and relatively inexpensive. However, they also have some disadvantages, such as slow wavelength switching, narrow spectral range and low resolution, mechanical vibration from moving parts, and image misregistration due to the filter movement. Therefore, some other spectral filters need to be used in order to overcome these limitations. There are mainly two filters widely used in the staring biomedical spectral imager: AOTF and LCTF. AOTF is a rapid wavelength scanning solid-state device that operates as a tunable optical bandpass filter based on light–sound interactions in a crystal. The major function of AOTF is to isolate a single wavelength of light from a broadband source in response to an applied acoustic field. The main advantages of AOTF are good transmission efficiency, fast scan times, large spectral range, and flexible controllability and programmability. AOTF is also attached easily to a microscope, endoscope, and camera lens. Schaeberle and Treado, et al. have developed multispectral and HSI microscopy based on AOTF.139,140 Then, Wachman et al. developed an AOTF microscope to map hemoglobin saturation and oxygen tension in mouse brains.99,100 Since then, more AOTF-based microscopy HSI systems have been proposed and used for different biological analysis, for example, the HSI system,86 the molecular HSI (MHSI) system,96 the HSI microscope,141 etc. Besides microscope, endoscope and camera lens also have been attached to AOTF to detect biological tissues in vivo. For example, some AOTF-based endoscope spectral imaging systems have been developed and used to detect skin tumors, thorax surgeries, and cancerous tissues.5,16,84,85,87,88 Furthermore, some camera lens with standard interface can be coupled to an AOTF adapter directly and used for in vivo imaging.80–82 The advantages of AOTF-based imagers include quiet and vibration-free operation, high switching speed, spectral selectivity, spectral purity, and flexibility. On the other hand, LCTF is a solid-state instrument that utilizes electronically controlled liquid crystal cells to transmit light with a specific wavelength and the elimination of all other wavelengths. The LCTF approach also has been used in coupling with the microscope,65,89–92 fundus microscope,114,117–119 and camera lens56–58,60,62,64,68–70,77 to create spectral image cubes for biological imaging in vivo and in vitro. Although the electronic controller of the LCTF is able to shift the narrow bandpass region throughout the entire wavelength range covered by the filter unit, the wavelength switching speed depends on the relaxation time of the liquid crystal as well as the number of stages in the filter. Typically, it takes tens of milliseconds to switch from one wavelength to another, which is far longer than the response time of the AOTF.17 The commercial AOTF and LCTF products also have been widely used to acquire biomedical spectral images in the visible (VIS) and near-infrared (NIR) spectral regions. For example, some researchers have developed different kinds of systems based on the AOTF component (Brimrose, Sparks, MD),5 the AOTF imaging modules (ChromoDynamics, FL),99,141,142 and the CRi LCTF (Cambridge Research and Instrumentation, Woburn, MA).91,92 In addition, some commercial instruments have also been manufactured based on these components, such as the HSi-300 and HSi-440C HSI systems (Gooch & Housego, FL), the Nuance,™ and Vectra™ multiplexing microscopes (CRi). Compared to the fixed interference filters used in the filter wheels, AOTF and LCTF can be flexibly controlled by a computer. They do not have moving parts and, therefore, do not suffer from the problems associated with the rotating filter wheels, such as speed limitation, mechanical vibration, and image misregistration. However, the AOTF- and LCTF-based imagers are in all cases expensive and have delicate and low throughput.12 Therefore, an interferometer also has been used for spectral imaging as it usually has high optical throughput, high and variable spectral resolution. In general, it is possible for Michelson interferometer and Sagnac interferometer to acquire 2-D spatial and one-dimensional spectral information of objects by applying a Fourier transform spectrometer algorithm and the Van Cittert-Zernike theorem. For example, Inoue et al. discussed the performance of Fourier-transform spectral imaging based on the Michelson interferometer in early 1990s. Potter et al. developed an FTIR system by coupling mid-IR Michelson-type step-scan interferometer to an IR microscope.143 Magotra et al. developed an hyperspectral biomedical imager imaging system using a spatially modulated Fourier transform interferometric device. This device is based on a Sagnac interferometer, which creates an interferogram that contains the desired spectral information.144 Fisher et al. obtained spectral images of a tooth by combining imaging optics, a Sagnac interferometer, and an imaging detector.145 Besides the Michelson and Sagnac interferometers, researchers found that Fizeau interferometer, based on double Fourier interferometry, can also be used for spectral imaging.146 In recent years, various interferometric spectral imaging systems and sensors have been developed and used for biomedical analysis, such as the spectral karyotyping (SKY), dynamic monitoring of biomolecular interactions, and live epithelial cancer cells analysis.20,109,147,148 Besides the interferometer, some other devices such as the rotating direct vision prism,149,150 VIF,78 CVF, LVF,42,151 and the dispersion of optical rotation152 can also be used to form biomedical staring imagers. Although the staring biomedical spectral imagers do not need to scan in the spatial dimension, most of them still need to scan in the spectral dimension. The scanning mechanism decreases their temporal resolution and limits their potential use in some real-time imaging. However, the staring spectral imaging systems are still the most popular ones in current biomedical applications.