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

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