A key advantage of using fluorescence to study synaptic ultrastructure is that multiple protein species can be labeled and monitored concurrently RAD001 in vitro (Micheva and Smith, 2007), including in live neurons. We expect rapid advances in this arena, with high-content studies of synaptic molecular organization leveraging new labeling strategies and chemical biology methods. There has also been dramatic progress in nonlinear optical microscopy. Today, neuroscientists widely appreciate the phenomenon of two-photon excitation, but two-photon effects were once considered esoteric aspects of optical physics. It was the development of solid-state ultrafast lasers, chiefly the development of titanium sapphire laser technology,

that propelled the two-photon microscope innovated by Webb and Denk to become broadly usable by biologists and to permeate neuroscience. We expect continued improvement of not just the hardware elements that comprise

the two-photon microscope, but also general optical strategies for laser scanning, for scanless approaches to laser illumination, and for other new approaches for imaging faster and deeper into tissue (Kobat et al., 2011, Oron et al., 2005, Quirin et al., 2013 and Schrödel et al., 2013). Beyond the current depths presently attainable by two-photon Osimertinib mouse microscopy, nonlinear optical microscopy modalities relying on multiphoton excitation and long-wavelength, ultrashort-pulsed lasers promise to reveal fundamental features of nervous tissue

(Farrar et al., 2011, Horton et al., 2013, Kobat et al., 2011 and Mahou et al., 2012). Due to reduced light scattering at longer wavelengths, three-photon excitation with an illumination wavelength of 1.7 μm has been demonstrated in proof-of-concept studies to reach into even the hippocampus in a live mouse (Horton et al., 2013). However, much work remains to make this a practical technique for day-to-day experimental studies of nervous system structure. Methisazone For deep studies of nervous system dynamics, further development of red fluorescent sensors of neural activity will be required, which presently lag behind green fluorescent sensors such as the highly successful GCaMP6 Ca2+ sensors. There are also crucial issues of optical aberrations to consider when imaging 1 mm or more into dense brain tissue. Adaptive optical methods may help, offering the possibility of correcting aberrations online, and have already made inroads into neuroscience (particularly for ophthalmic imaging of the retina and visualization of single human photoreceptor cells) (Godara et al., 2010 and Hunter et al., 2010). Adaptive optics have also shown utility for two-photon microscopy by improving the resolution of two-photon imaging deep in tissue (Ji et al., 2012). When combined with long-wavelength laser illumination, such as for three-photon excitation, adaptive optics may become even more important.