When facing an unknown object our first reaction usually is to step back and take a closer look. Surely, visual inspection is one of the oldest tools used by man to understand the nature of his surroundings, and there is a certain charm in the simplicity and intuitiveness of examining something by sight. Much can be deduced by examining the structure and layout of an object. What is its shape, size and consistency? How does it move and change over time? And in the case of living objects: how does it behave and react? It is therefore not surprising that optical tools have been pivotal to unraveling the mysteries of the brain and the nervous system throughout the history of man. The first documented description and discussion of the nervous system was written in 1700 BC by an ancient Egyptian surgeon, and was based on his experiences and insights gained from dissecting human bodies.1 At around 400 BC the renowned Greek philosopher Hippocrates, today considered to be the ’father of medicine’, postulated that the brain is the seat of intelligence and that it is involved with sensations.1 Slowly, more details of the brain were discovered as individual surgeons and natural philosophers studied the appearance and consistency of the brain. Yet lacking any advanced optical tools they were confined to examining macroscopic structures visible to the naked eye or possibly through a single lens.I
IThe oldest discovered lens is 3.000 years old and originates from ancient Assyria. Lenses and their function were known in ancient Egypt, Greece and Rome.
1.1.1. Early development of microscopy and labeling techniques
This all changed dramatically with the turn of the 16thcentury and the invention of the compound light microscope. The power to resolve structures on the millimeter and micrometer scale led to the discovery of the cell (in 1665 by Robert Hooke), allowed the formulation of the cell theory (”All living matter consists of cells, which are the basic building blocks of life”as formulated by Theodor Schwann and Matthias Jakob Schleiden in 1839, and ”all existing cells are formed from pre-existing cells”
as phrased in 1855 by Rudolf Virchow), and enabled the microscopic examination of cellular structures such the nucleus or nerve fibers (in 1717 by Antoni van Leeuwenhoek, who is often called the ”first microbiologist”). Yet the improved spatial resolution of the available optical tools did not change the fact that the brain was a structure with low inherent contrast, making it difficult to discriminate specific details. Without additional methods to provide higher contrast the available imaging techniques were limited in their capacity to visualize interesting structures.
Further complications stemming from the difficulty of sustaining organic tissue needed to be overcome by developing fixation methods (using alcohol or later formaldehyde) for preserving organic structures. These fixation methods were complemented by techniques for cutting and preparing thin slices of fixed organic tissue. Many novel cell staining techniques were developed in the 19th and 20th century, which greatly aided the progression of the neurosciences. TheNissl staining that was developed at the end of the 19th century helped visualize cell bodies (and the endoplasmatic reticulum), which enabled the study of the cytoarchitecture of the brain: the known segments of the brain could again be divided into subdivisions.
Themyelin-sheath stainings and the subsequentsilver impregnation techniques that were developed in the 1950s allowed scientists to follow axons and fiber tracts through the brain and thereby to unravel the organization of the nervous system.
Another milestone was the development of Camillo Golgi’s staining method (la reazione nera). Golgi’s method only stained a small, apparently random subset of cells, but these were stained completely: axons, dendrites and all. Such isolated cells offered very high contrast that could be examined in great detail. This staining method was adopted and later perfected by Santiago Ramòn y Cajal, allowing him to conduct paradigm-shifting research of the central nervous system. He
1.1. A brief history of optical methods in neurosciences
was the first to describe dendritic spines, which are small protrusions located on neuronal dendrites and which were dismissed by his contemporary Camillo Golgi as being staining artifacts. Cajal also produced detailed functional circuit diagrams of the hippocampus, which led him to propose his theory of dynamic polarization (”Neuronal signals are received by dendrites and sent by axons”).
1.1.2. Fluorescence microscopy
The emergence of fluorescent labeling and microscopy methods in the early 1900s laid the path for the study of living brain cells and their dynamics. By introducing fluorescent dyes into cells and tracking the movement of the dye, axonal transport phenomena were observed, enabling the axonal pathways to be mapped much more accurately.II Fluorescent microscopy really kicked off with the invention of antibody labeling techniques in 1941 and the subsequent recognition in 1974 that these antibodies could not only be used for visualizing immune responses, but could target and label all sorts of proteins such as the ubiquitous actin and tubulin.2 Proteins could be labeled by creating a primary antibody that targeted a specific protein of interest and then adding a secondary antibody fused to a fluorescent dye, which then attached itself to the primary antibody, thereby illuminating the structure of interest. This immunostaining technique, which is still widely used today, facilitated the observation of virtually any protein of interest. By using multiple sets of antibodies, each with a differently colored fluorescent dye, colocalization experiments were possible, in which the locations of multiple sets of proteins could be determined simultaneously and in relation to each other. The observation of electrical activity in live neurons with fluorescent microscopy was possible with the development of calcium-sensitive3and voltage-sensitive dyes4around 1980. By the end of the 20th century, widefield fluorescence microscopy had become one of the dominating techniques in fundamental neuroscience.5
IIConcomitant with fluorescent methods, radioactive staining and tracing methods that were viable for live-cell recordings, at least over short times, were developed and implemented