Abstract
"Beyond the invisible"-more than the title of a song-this motto has become the quest of all microscopists. From its beginning in the 17th century, optical microscopy has opened new fields in the understanding of nature and it has become one of the biologist's favorite companions. However, despite its high lateral resolution in the focal plane, microscopy has always struggled to resolve fine detail in thick and non-transparent samples. Therefore, traditional wide-field microscopy has been limited to the study of thin tissue slices (a few microns). However, the complexity of life cannot be restricted to a single plane and must be considered in three dimensions (3D). Stereology is the 3D interpretation of two-dimensional sections of materials or tissues. It provides a practical means for extracting quantitative information about a 3D material from measurements made from 2D sections. However, this approach still consists in the serial slicing of a tissue, staining and, the observation and digitalization of every slice before 3D reconstruction by a computer. More than tedious, stereology is also time-consuming and it is the source of error. Therefore, during the past 25 years, substantial efforts have been made to conceive and validate microscopy techniques capable of imaging intact tissue volumes. These include variants of confocal imaging, nonlinear (two-photon) microscopy as well and light-sheet microscopy that have considerably increased the imaging depths achievable. Together with the improvement and widespread utilization of tissue-clearing techniques it has become possible to make an entire organ or even animal transparent before imaging it without any physical sectioning. Four centuries after its invention, microscopy has crossed the third-dimension frontier. In this review we focus on the description of the main families of tissue-clearing methods and three-dimensional microscopy approaches to see how-when they are used together-these techniques open new perspectives for anatomy and anatomopathology, at the subcellular level.