| Neurons form complex networks connected by tiny synapses. Imaging neural networks in intact, scattering tissue at the level of single synapses (∼1 mum) requires high-resolution microscopy. This niche is occupied by two-photon excitation laser scanning microscopy (TPLSM). However, building a TPLSM is challenging because it requires custom hardware and software for data acquisition and analysis.; This thesis details the principle, design, and implementation of a TPLSM with dual excitation sources using a simple optical layout without custom electronics using ScanImage for data acquisition. I further describe the creation and distribution of ScanImage, the open source software application for operating laser-scanning microscopes.; Aside from imaging structures in the brain, fluorescence measurements can be coupled to functional probes, most prominently Ca 2+ indicators, to reveal neural activity. Recently, genetically encoded calcium indicators (GECIs) have become available. GECIs promise to revolutionize cellular neuroscience, but little is known about the dynamic properties of these indicators in neurons.; I determine methods to study the properties of synthetic and genetically encoded calcium indicators in vitro and in situ. I characterize the utility of genetically encoded calcium indicators for monitoring neural activity and calcium concentration ([Ca2+]) in situ. I then compare and contrast GECIs and synthetic calcium indicators and propose a mechanism for GECI fluorescence changes upon calcium binding. |
