Visible Reflectance Hyperspectral Imaging:  Characterization of a Noninvasive, in Vivo System for Determining Tissue Perfusion

Abstract

We characterize a visible reflectance hyperspectral imaging system for noninvasive, in vivo, quantitative analysis of human tissue in a clinical environment. The subject area is illuminated with a quartz-tungsten-halogen light source, and the reflected light is spectrally discriminated by a liquid crystal tunable filter (LCTF) and imaged onto a silicon charge-coupled device detector. The LCTF is continuously tunable within its useful visible spectral range (525-725 nm) with an average spectral full width at half-height bandwidth of 0.38 nm and an average transmittance of 10.0%. A standard resolution target placed 5.5 ft from the system results in a field of view with a 17-cm diameter and an optimal spatial resolution of 0.45 mm. The measured reflectance spectra are quantified in terms of apparent absorbance and formatted as a hyperspectral image cube. As a clinical example, we examine a model of vascular dysfunction involving both ischemia and reactive hyperemia during tissue reperfusion. In this model, spectral images, based upon oxyhemoglobin and deoxyhemoblobin signals in the 525-645-nm region, are deconvoluted using a multivariate least-squares regression analysis to visualize the spatial distribution of the percentages of oxyhemoglobin and deoxyhemoglobin in specific skin tissue areas.

Advanced Optical Imaging of Endocytosis

“…Spectral imaging has been implemented in a variety of optical modalities for biological applications including visible reflectance (Zuzak, et al, 2002), fluorescence (Michalet, et al, 2003) and vibrational spectroscopies such IR absorption (Levin & Bhargava, 2005), Raman scattering (Christensen & Morris, 1998), and surface-enhanced Raman (SERS) (Sharonov, et al, 1994)), as well as in non-optical methods like mass spectrometry (Fletcher, et al, 2008). In practice, higher degrees of multiplexing, higher accuracy, and lower detection limits are achievable with spectral imaging due to the ability to implement multivariate analysis methods to identify and/or classify spectral signatures even in the presence of high degrees of spectral overlap from other labels and cellular autofluorescence (Mansfield, et al, 2005).…”

Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging

“…A CCD-based spectroscopic imaging technique in the NIR and visible regions, implying high spatial resolution, has been used for brain and skin analysis, including Hb quantification and oxygenation [9], [10], [11] and [12]. These imaging approaches cannot provide the actual optical pathlength.…”

“…A spatial resolution of 0.45 mm has been achieved by applying spectral imaging in the visible region but without quantifying the absolute concentration of heme proteins and oxygenation using a device built for perfusion imaging of hand palms [12]. Other NIR imaging systems based on the application of multiple optodes provide spatial resolution in the range of 4 to 40 mm (see, e.g., Refs.…”

A theoretical‐experimental methodology for assessing the sensitivity of biomedical spectral imaging platforms, assays, and analysis methods

“…However, within the last 2 decades, many new applications of spectral imaging have been described, especially in the biomedical imaging field [8][9][10][11][12]. Microscope-based spectral imaging systems have been in use for over a decade [9,10,[13][14][15][16][17][18], while small in vivo animal imaging has also grown in use [19][20][21][22][23][24][25]. In fact, several commercial systems are now available (spectral microscope systems include, but are not limited to, those marketed by Nikon, Olympus, Zeiss, Lightform, and Cytoviva, which utilize spectral imaging detectors, while a subset of confocal spectral imaging microscopes also allow laser lines to be selected using an acousto-optic tunable filter, or AOTF).…”