Thus, cortical circuits can be examined in vivo with connections well preserved. Common two-photon lasers are tunable from 700 nm to 1000 nm or more and are suitable for the excitation of most commercially available fluorophores. There are promising new approaches to extend the quality and versatility of two-photon microscopy and thereby two-photon calcium imaging. Inspired by imaging work that is performed in astronomy OSI-744 the use of adaptive optics in neurobiology aims at correcting in advance (before the illumination light is entering the optical pathway) for spherical aberrations that may distort the laser pulse
and, therefore, may decrease the efficiency of two-photon imaging. These aberrations become increasingly more relevant with increasing depth (Girkin et al., 2009). The purpose of this correction is to obtain the optimal duration and
shape of the laser pulse at the focal spot (Ji et al., 2010, Rueckel et al., 2006 and Sherman et al., 2002). An interesting approach to increase depth penetration in two-photon microscopy is the use of regenerative laser amplifiers, which yields laser pulses with higher photon density, BI 2536 research buy but at lower repetition rate. Because of the increased photon density, the probability for the two-photon effect is elevated, allowing, for example, the recording of sensory-evoked calcium signals from layer 5 pyramidal neuron somata in vivo (Mittmann et al., 2011). Present limitations of this technique are the lack of wavelength tunability and the decreased speed of imaging. Finally, the development of optical parametric oscillators (OPOs) pushes two-photon microscopy
toward excitation wavelengths in the infrared spectrum (>1080 nm) and enables the efficient excitation of red-shifted fluorophores. As a result, it can increase imaging depth because of the reduced absorption Cell press and scattering at longer wavelengths (Andresen et al., 2009 and Kobat et al., 2009). The speed of calcium imaging can be increased by the use of resonant galvo-scanners (Fan et al., 1999, Nguyen et al., 2001 and Rochefort et al., 2009) or the use of acousto-optic deflectors (AOD) (Chen et al., 2011, Grewe et al., 2010, Iyer et al., 2006, Lechleiter et al., 2002 and Otsu et al., 2008), especially when implementing the random-access imaging mode (Iyer et al., 2006, Kirkby et al., 2010 and Otsu et al., 2008). Alternatively, multibeam confocal excitation also allows high imaging speed, but is restricted to superficial layers of nervous tissue and is so far only used in ex vivo preparations (Crépel et al., 2007). Next, there are increasing efforts for 3D imaging, involving various approaches (Cheng et al., 2011, Göbel and Helmchen, 2007 and Göbel et al., 2007). Even when using two-photon microscopy combined with improved depth penetration, imaging depth is ultimately limited (Andresen et al., 2009 and Theer et al., 2003).