To observe local variations in temperature, oxygenation and blood perfusion over time, four imaging systems were developed and compared: Two systems consisting of white broadband light source and a CCD camera in combination with a Liquid Crystal Tunable Filter, one in the visual domain, 420-730 nm, and one in the infrared domain, 650-1100 nm. Thirdly, a CCD camera in combination with a software controlled hyper-spectral light source consisting of a panel with 600 LEDs divided in 17 spectral groups in the range from 370 to 880 nm so that specific spectral distributions can be generated at high repetition rate (>1000 Hz) and, fourthly a standard IR thermal camera for comparison. From the acquired images at the selected wavelengths chromophores concentration images of oxy and deoxy hemoglobin can be calculated applying different algorithms. These imaging techniques were applied and compared for various clinical applications: Tumor demarcation, early inflammation, effectiveness of peripheral nerve block anesthesia, and localization of epileptic seizure. The relative changes in oxygenation and temperature could be clearly observed in good correlation with the physiological condition. The algorithms and data collection/processing can be optimized to enable a real-time diagnostic technique.