With optical techniques, we refer to a group of methods that use of light to perform measurements on matter. Spatial frequency domain imaging (SFDI) is an optical technique that operates in the spatial frequency domain. The technique involves using sinusoidal patterns of light for illumination, to study the reflectance of the target based on the spatial frequency (ƒ_x) of the patterns. By analysing the frequency-specific response with the aid of light transport models, we are able to determine the intrinsic optical properties of the material, such as the absorption coefficient (μ_a) and reduced scattering coefficient (μ'_s) In biological applications, these optical properties can be correlated to physiological structures and molecules, providing a useful tool for researchers and clinicians alike in understanding the phenomena happening in biological tissue. The objective of this work is to contribute to the development of SFDI, so that the technique can be used as a diagnostic tool to study the process of wound healing in tissue. In paper I we introduce the concept of cross-channels, given by the spectral overlap of the broadband LED light sources and the RGB camera sensors used in the SFDI instrumentation. The purpose of cross-channels is to improve the limited spectral information of RGB devices, allowing to detect a larger number of biological molecules. One of the biggest limitations of SFDI is that it works on the assumption of light diffusing through a homogeneous, thick layer of material. This assumption loses validity when we want to examine biological tissue, which comprises multiple thin layers with different properties. In paper IV we have developed a new method to process SFDI data that we call multi-frequency SFDI. In this new approach, we make use of the different penetration depth of the light patterns depending on their ƒx to obtain depth-sensitive measurements. We also defined a 2-layer model of light scattering that imitates the physiology of a wound, to calculate the partial volume contributions to μ's of the single layers. The 2-layer model is based on analytical formulations of light fluence. We compared the performance of three fluence models, one of which we have derived ourselves as an improvement over an existing formulation. In paper II we were able to test our new multi-frequency SFDI method by participating in an animal study on stem-cells based regenerative therapies. We contributed by performing SFDI measurements on healing wounds, in order to provide an additional evaluation metric that complemented the clinical evaluation and cell histology performed in the study. The analysis of the SFDI data at different ƒx highlighted different processes happening on the surface compared to the deeper tissue. In paper V we further refine the technique introduced in paper IV by developing an inverse solver algorithm to isolate the thickness of the thin layer and the layer-specific μ's. The reconstructed parameters were tested both on thin silicone optical phantoms and ex-vivo burn wounds treated with stem cells.