Basic building units and properties of a fluorescence single plane illumination microscope

The critical issue of all fluorescence microscopes is the efficient use of the fluorophores, i.e., to detect as many photons from the excited fluorophores as possible, as well as to excite only the fluorophores that are in focus. This issue is addressed in EMBL's implementation of a light sheet based microscope [single plane illumination microscope (SPIM)], which illuminates only the fluorophores in the focal plane of the detection objective lens. The light sheet is a beam that is collimated in one and focused in the other direction. Since no fluorophores are excited outside the detectors' focal plane, the method also provides intrinsic optical sectioning. The total number of observable time points can be improved by several orders of magnitude when compared to a confocal fluorescence microscope. The actual improvement factor depends on the number of planes acquired and required to achieve a certain signal to noise ratio. A SPIM consists of five basic units, which address (1) light detection, (2) illumination of the specimen, (3) generation of an appropriate beam of light, (4) translation and rotation of the specimen, and finally (5) control of different mechanical and electronic parts, data collection, and postprocessing of the data. We first describe the basic building units of EMBL's SPIM and its most relevant properties. We then cover the basic principles underlying this instrument and its unique properties such as the efficient usage of the fluorophores, the reduced photo toxic effects, the true optical sectioning capability, and the excellent axial resolution. We also discuss how an isotropic resolution can be achieved. The optical setup, the control hardware, and the control scheme are explained in detail. We also describe some less obvious refinements of the basic setup that result in an improved performance. The properties of the instrument are demonstrated by images of biological samples that were imaged with one of EMBL's SPIMs.