This has the effect of blocking the laser power, resulting in a low illumination efficiency of the order of 3%.

However, the use of a cylindrical lens for light sheet formation requires a slit in front of the lens to form the illumination profile. The same year, Huisken and Stainier found another solution to the shadow artifact problem by rotating the cylindrical lens around the sample and combining different views of the sample. For large samples like mouse brains, the thickness was set between 25 and 50 μ m, whereas for smaller samples, the thickness could be as low as 6 μ m. The width and thickness of the sheet varied depending on the size of the sample. They used their microscope in combination with benzyl alcohol/ benzyl benzoate (BABB) clearing to detect unique GFP-tagged neurons in excised mouse hippocampi. 16 They used a cylindrical lens to form the sheet in the sample and, to reduce the shadow artifacts created by the single-sided illumination, they added another illumination arm on the opposite side. published in 2007 another version of a light sheet microscope. With a thickness between 3 and 10 μ m, the light sheet had an extended 60 - μ m FOV, that enabled three-dimensional reconstructions of samples up to a cubic millimeter. With this system, they imaged the heartbeat and green fluorescent protein (GFP)-labeled ganglion cells of live medaka fish embryos as well as the embryogenesis of a Drosophila melanogaster embryo over a 17-hr period. described a system they called selective plane illumination microscopy, which also uses a cylindrical lens to illuminate the sample perpendicularly to the axis of the imaging objective. With this system, they succeeded to obtain a wide FOV of 750 μ m with, for the time, an impressive axial resolution of 26.1 μ m. This microscope consisted of a collimated laser beam illuminating the sample through a cylindrical lens. In 1993, Voie and his colleagues used a light sheet microscope to study the internal structures of the cochlea of gerbils, 15 which had been cleared with a five-part solution of methyl salicylate and of three parts of benzyl benzoate. 13 In 1964, McLachlan observed bismuth dendrites by forming a line of light at the focal plane of an imaging lens, through a slit, and scanning the sample to create a virtual light sheet and to form an image. The first description of a light sheet microscope dates back to 1902, when Siedentopf and Zsigmondy observed gold particles using sunlight projected through a slit. After describing historical developments in light sheet microscopy, we will describe a light sheet system that overcomes the limitations of previous designs and achieves a very large FOV with cellular resolution. Indeed, to meet these requirements, the sample must be illuminated with a long and thin beam, which is incompatible with the use of diffractive beams, such as Gaussian beams. In general, the thickness of the sheet is much smaller than the depth of field of the detection objective, resulting in an axial resolution dependent on the light sheet thickness.Īlthough there has been tremendous technological advances in recent years, few light sheet systems are able to capture information from very large fields of view (FOV) of several millimeters while featuring a cellular resolution. The illumination arm forms a sheet of light in the sample, whereas the detection arm collects the emitted signal and forms the image of the illuminated plane on the camera. The configuration of this type of microscope consists of two perpendicular arms, one for the illumination and one for the detection.

1 – 12 The main feature of a light sheet microscope is the selective illumination of a thin plane in the sample.

In the last two decades, interest in light sheet microscopy has remarkably increased in neuroscience due to its rapid and high-resolution imaging capabilities of complex biological samples.

1.35mm manuscript practice sheets