Both confocal and two-photon microscopy use point illumination, which narrows planar focus. In confocal microscopy, the emitted signal is spatially filtered via a pinhole aperture. The light emitted from a single plane creates see more an image, and a progression of images is captured through the thickness of the tissue, resulting in optical sectioning of the specimen (Wilson, 1989). Thus, confocal microscopy eliminates the need for resectioning thick slices typically employed in electrophysiological recording preparations. Furthermore, confocal microscopy provides high spatial resolution, which is particularly important for 3D reconstructions. The temporal stability of the sample is especially relevant

to neuronal reconstructions. Since fluorescently labeled specimens have a limited viability period, it is often necessary to collect image stacks for later

offline tracing of the arbor structure. In two-photon microscopy, fluorophores are excited by the simultaneous absorption of two photons (Denk et al., 1990). The two photons converge simultaneously only at the focal point, yielding sharper images with less background noise. Such a specific illumination removes the need for spatial filtering. Additionally, since the fluorophores outside the focal point are not excited, the specimen undergoes less photobleaching and photodamage (Denk and Svoboda, 1997). Multiphoton microscopy, an extension of two-photon microscopy, uses more than two photons to

Mannose-binding protein-associated serine protease excite the fluorophores, resulting SB203580 manufacturer in a narrower emission region and even less out-of-focus noise. Since the stimulation energy is split between two (or more) photons, a drawback of two-photon (or multiphoton) microscopy is its lower spatial resolution relative to confocal, due to the longer excitation wavelength. Moreover, the necessity to scan the specimen one point at a time greatly increases the time necessary to capture the same field of view (Lemmens et al., 2010). Even with the higher resolution afforded by confocal and two-photon microscopy, subcellular details such as synaptic contacts remain elusive and, until recently, neuronal ultrastructure remained the purview of electron microscopy. However, the advent of superresolution fluorescence microscopy addresses this limit. Two main approaches exist to enable superresolution: one involves the sequential and stochastic switching on and off of fluorophores; the other uses patterned illumination to modulate fluorophore emission. The former includes stochastic optical reconstruction microscopy (STORM; Rust et al., 2006), and (fluorescence) photo-activated localization microscopy (PALM; Betzig et al., 2006; or FPALM; Hess et al., 2006). In this class of techniques, only a subset of fluorophores is illuminated in each imaging cycle and localized to be imaged and reconstructed, and the process is repeated to capture the full distribution of fluorophores.