Purpose: To identify, characterize, and discuss the current technological status of in vivo corneal diagnostic imaging and target high-priority future development needs. Methods: In vivo tandem scanning microscopy (non-coherent), scanning slit confocal microscopy (noncoherent), and laser scanning confocal microscopy (coherent) are examined. The current and future roles of multi-photon and higher order harmonic imaging are also discussed. Results and Conclusions: This keynote review demonstrates the current abilities and limitations of three currently used clinical imaging modalities to resolve the cellular and structural layers of the cornea temporally and spatially in three or four dimensions (x, y, z, t), with applications to the study of clinical-pathological processes such as inflammation; infection, wound healing, drug toxicity, organ development, differentiation and effects of genetic diseases. Each of these approaches has strengths and weaknesses. Thus, future technological development is essential to provide exciting new insights into understanding the structure and function of not only the cornea and the other ocular structures, but also other multicellular organs in health and disease. These imaging paradigms are among the most important advances in medical science in the past
three decades.
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.
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