Custom-made multiphoton microscope for long-term imaging of neuronal cultures to explore structural and functional plasticity

We use dissociated cultures of E-18 rat cortical neurons to study how they process the information. To correlate electrophysiological data with corresponding network structure, we observe effects of the stimuli on structural changes in this culture using multiphoton microscopy. To keep our 2D and 3D cultures alive for long-term studies, it is necessary to protect them against photodamage. At the same time, we need a flexible microscope design to accommodate our multielectrode array (MEA) electrophysiological station. We have constructed a custom-designed multiphoton microscope based on design of Tsai et al. The microscope is optimized for two-photon imaging to collect maximum possible fluorescent signal using minimum excitation laser intensity. Special attention is paid to get uniformly illuminated images and the ability to use the entire bandwidth of the pulsed laser (700-1000 nm) with the same set of optical components. Flexibility of the design will allow us to easily change or incorporate other optical components suitable for different experimental needs. This microscope will allow us to do electrophysiology and imaging concurrently while maintaining the optimum temperature and CO2 levels.