In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, selective plane illumination microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single-photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (∼10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly.
Highly flexible fibers enable simultaneous electrical neural recording, optical stimulation and drug delivery in freely moving mice. Light sheet microscopy promises to revolutionize developmental biology by enabling live in toto imaging of entire embryos with minimal phototoxicity. We present detailed instructions for building a compact and customizable Selective Plane Illumination Microscopy (SPIM) system. The integrated OpenSPIM hardware and software platform is shared with the scientific community through a public website, thereby making light sheet microscopy accessible for widespread use and optimization to various applications.