Combined Fluorescence Methods to Determine Synapses in the Light Microscope: Multilabel Confocal Laser Scanning Microscopy

The dimensions of synapses are at or below the resolution limit of classical light microscopy. Under optimal conditions, one can appreciate processes of pre- and postsynaptic neurons that appose each other. Such appositions may be casual only and as such not functional in terms of synaptic communication. As a consequence, until quite recently, electron microscopy was the only means available to determine whether identified neurons synapse with each other. Technological developments, however, have created a middle ground between the strictly separated realms of light and electron microscopy. In this chapter I present a triple-fluorescence approach aimed at identifying the apposition of a presynaptic and a postsynaptic neuron, and simultaneously pinpointing a highly specific synapse-associated marker. This third marker identifies the presence of an active zone, necessary to distinguish casual appositions from functional synapses. Methods involved are neuroanatomical tracing, immunofluorescence, confocal laser scanning, and postacquisition computer processing followed by three-dimensional reconstruction and inspection. In my contribution, I will review the theory and practice involved in triple-labeling confocal fluorescence imaging. I begin by dealing with the dimensions of synapses and the structures involved, and relate the physical limitations of light microscopy to the problem of resolving synaptic structure. I then review the principles of image formation in fluorescence microscopy, and present the conditions that must be fulfilled in order to do sound multilabel confocal laser scanning: fluorochromes, lasers, channels, channel separation, and procedures to recognize and suppress unwanted phenomena such as crosstalk. In order to fully illustrate the points discussed, an actual triple visualization experiment will be described. Finally, I will emphasize several important aspects of “operator awareness”, that is, the mind setting necessary to work with an advanced optoelectronic instrument like a confocal microscope and its sophisticated software. An aware user senses when some part of the complicated chain of processes is not producing what it is supposed to produce. If operator awareness is absent, strange results may be obtained.