, 2010).

For in vivo applications, LEDs can be used to fill an optical fiber which is tethered to a behaving animal, but such applications are limited by the highly divergent beam pattern from LEDs with coupling efficiencies of ∼1%; still, with high-power LEDs, this fraction of total power is sufficient to attain the required power density output (Gradinaru et al., 2007 and Petreanu et al., 2007). Possible uses of LEDs include both direct implantation of small LEDs in or on tissue (with heating concerns requiring careful control as noted above), or permanently mounted to optical fiber waveguides carried on the subject (Iwai et al., 2011). Traditional broadband incandescent microscopy light sources, such as arc lamp-based epifluorescence find more illuminators, can be used in optogenetic

experiments with appropriate narrowband spectral filters and the introduction of a shutter to the illumination beam path. Dedicated light sources with built-in high-speed shutters and filter selection are also available (e.g., the Sutter Instruments DG-4; Boyden et al., 2005) and offer pulse durations of as little as 1 ms with pulse repetition rates of up to 500 Hz. Unlike some lasers and LEDs, which offer graded modulation of intensity, shutter-based systems are limited to on/off gating of light pulses; neutral density filters can be used to produce stepped illumination. One significant advantage of the use of filtered broadband light over LEDs or lasers is the ability to select arbitrary illumination wavelengths and spectral linewidth using bandpass filters. Even more flexible Pfizer Licensed Compound Library price are monochromators, which output commanded wavelengths via positioning of a diffraction grating. In light-accessible experimental preparations such as cultured neurons, brain slices, cortical surface, or nematodes, light is typically delivered through a microscope illumination path, passing through the objective and illuminating a

spot within the field of view. Apertures in the illumination path can be used to restrict this spot to a smaller portion of the field. In order to measure the light power density achieved by a given setup, a power meter can be placed Megestrol Acetate below the objective; the total power is measured and divided by the area of the illumination spot (Aravanis et al., 2007). For experiments requiring illumination at multiple sites, or at sites away from the imaged area, an optical fiber-coupled light source (see below) can be mounted on a micromanipulator and used to illuminate the tissue, with light power density similarly calculated from total power and spot size. Laser beams can be coupled into the microscope light path and optically expanded to fill the field of view, and moving optical elements—such as galvanometer-driven mirrors (Rickgauer and Tank, 2009 and Losonczy et al., 2010), digital micromirrors (Farah et al., 2007 and Arrenberg et al., 2010), or diffractive optical elements (Watson et al.