In-vivo microvasculature visualization using hyperspectral imaging

We describe a new noninvasive microscopic near infrared reflectance hyperspectral imaging method for visualizing, in vivo, spatially distributed contributions of oxyhemoglobin perfusing the microvasculature within dermal tissue. Microscopic images of the dermis are acquired, generating a series of spectroscopic images formatted as a function of wavelength consisting of one spectral and two spatial dimensions; a hyperspectral image data cube. The data thus collected can be considered as a series of spatially resolved spectra. For data collection, images are acquired by a system consisting of a near infrared liquid crystal tunable filter (LCTF) and a Focal plane array detector (FPA) integrated with a microscope. The LCTF is continuously tunable over a useful near infrared spectral range (650-950 nm) with an average full width at half-height bandwidth of 6.78 nm. To provide high quantum efficiency without etaloning we utilized a back-illumination FPA with deep -depletion technology. A 30W halogen light source illuminates a dermal tissue area of approximately 18 mm in diameter. Reflected light from the dermal tissue is first passed through the microscope, the LCTF, and then imaged onto the FPA. The acquired hyperspectral data is deconvoluted using a multivariate least squares approach that requires at least two reference spectra, oxy- and deoxyhemoglobin. The resulting images are gray scale encoded to directly represent the varying spatial distributions of oxyhemoglobin contribution. As a proof of principle example, we examined a clinical model of vascular occlusion and reperfusion.