Abstract
Imaging methods are a powerful tool for diagnostic purposes. In this chapter, the most important light-imaging methods, their advantages, and drawbacks are described. The advantages of radiation-free light-based imaging methods relative to traditional radiation methods, such as X-ray, magnetic resonance, or positron emission imaging, are indicated, and the recent advances to improve probing depth, contrast, and resolution in thick tissues are demonstrated. Some historical aspects and recent improvements in light-imaging methods, such as optical coherence tomography, speckle-imaging, second harmonic generation, or light-sheet microscopies, are presented. Due to the recent combination of optical immersion clearing with light-based imaging methods, several studies have been reported, where high-quality images and 3D reconstruction have been obtained for various tissues, providing an alternative to traditional histology or histopathology methods. The purpose of optical clearing is to reduce light scattering, but tissue clearing is obtained through three mechanisms: tissue dehydration, refractive index matching, and protein dissociation. This last mechanism leads to a reduction in the intensity of protein fluorescence, which can be a disadvantage in fluorescence imaging methods. The selection of certain clearing protocols that minimizes or eliminates protein dissociation has been made by some researchers, and a review of such literature is made in the various sections of this chapter.

Abstract
There are several types of measurements that can be performed with biological tissues during optical clearing treatments. When analyzing these methods, two major modes of study must be provided: ex vivo and in vivo. Measurements made from ex vivo samples are more flexible, allowing, for instance, to measure tissue transmittance or sample thickness kinetics. The results obtained from these measurements do not mimic exactly the in vivo situation. In the case of in vivo tissues, results from measurements are more realistic, but a more restrict number is possible, based only on reflectance or imaging methods. In this chapter, we make a brief description and analysis of the various measurement procedures that can be made during treatments of tissues ex vivo and in vivo and present some studies where important information was collected. The valuable results already obtained or possible to obtain in future from measurements described here will be presented and explained in the following sections. A particular case with great interest not only for biophotonics but also for food industry or organ preservation is the estimation of the diffusion properties of water and agents. Such evaluation of parameters is based only on collimated transmittance and thickness measurements made from ex vivo tissues. We will describe these measurements here and exploit their use in the study of diffusion in Chap. 7.

Abstract
[No abstract available]

Abstract
The optical immersion clearing is an effective method to reduce light scattering in tissues, but to optimize each treatment, it is necessary to understand the mechanisms involved. Since these treatments are intended to be temporary, it is also important to know if the mechanisms involved are reversible. Various studies have been made to evaluate and characterize the mechanisms of optical clearing. In all cases studied, two major mechanisms were observed—the tissue dehydration and the refractive index matching mechanisms. Some particular studies have reported that the agents used in treatments also dissolve proteins and suggested that protein dissolution is also a clearing mechanism. All these mechanisms have been reported as reversible, both on ex vivo or on in vivo studies. We make an analysis on these studies and present a method based on ex vivo collimated transmittance and thickness measurements to characterize the major clearing mechanisms—tissue dehydration and refractive index matching. Although this method can only be made with ex vivo tissues, alternative measurements are suggested for in vivo characterization of the clearing mechanisms. © 2019, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Abstract
After making an overview on the most recent progresses regarding the optical immersion treatment technique, we use this chapter to look to the future and perspectives of the following developments and benefits that can be achieved. The increasing number of publications on OC in the last 30 years, which we present in Sect. 9.1, indicates that this is a promising method to aid in the application of optical techniques in clinical practice for diagnosis or treatment purposes. Since several spectroscopy, fluorescence, or imaging methods have recently been used to test and validate the OC effects in various human and animal tissues, a collection of OCAs and OC protocols have been developed. Section 9.2 shows that to get even better results in tissue OC, the discovery of new agents and establishment of new protocols is a work in progress. Section 9.3 indicates the future perspectives for tissue spectroscopy during OC treatment and that the potential of the refractive index matching mechanism can also be evaluated in the ultraviolet range. Section 9.4 discusses the future perspectives of tissue imaging and OC. The establishment of new and faster OC protocols for tissue imaging is suggested, and indication for the necessary efforts to adapt the light-sheet technique to image in vivo is also made. Finally, Sect. 9.5 presents other applications of tissue OC and suggests the cooperation between research fields to increase knowledge in the use of OCAs and their benefits for each field. © 2019, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Abstract
Light interaction with biological materials depends on the material’s optical properties. From those properties, the absorption and scattering coefficients are the most important, since they quantify how much of a light beam is attenuated when traveling inside a tissue. The scattering coefficient is known to be significantly higher than the absorption coefficient in biological materials, meaning that most of the light is scattered, turning optical methods in clinical practice limited. Such difference between the scattering and absorption coefficients is mainly due to a refractive index mismatch between tissue components and fluids. We explain this concept in the present chapter before introducing the technique that efficiently minimizes this effect in the following chapters. © 2019, The Author(s), under exclusive license to Springer Nature Switzerland AG.