We report that single (or selective) plane illumination microscopy (SPIM), combined with a new deconvolution algorithm, provides a three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy in large samples. We demonstrate this by imaging large living multicellular specimens obtained in a three-dimensional cell culture. The ability to rapidly image large samples at high resolution with minimal photodamage provides new opportunities especially for the study of subcellular processes in large living specimens.

A non-blind, shift-invariant image processing technique that fuses multi-view three-dimensional image data sets into a single, high quality three-dimensional image is presented. It is effective for 1) improving the resolution and isotropy in images of transparent specimens, and 2) improving the uniformity of the image quality of partially opaque samples. This is demonstrated with fluorescent samples such as Drosophila melanogaster and Medaka embryos and pollen grains imaged by Selective Plane Illumination Microscopy (SPIM). The application of the algorithm to SPIM data yields high-resolution images of organ structure and gene expression, in some cases at a sub-cellular level, throughout specimens ranging from several microns up to a millimeter in size.

Light-sheet-based fluorescence microscopy (LSFM) is a fluorescence technique that combines optical sectioning, the key capability of confocal and two-photon fluorescence microscopes with multiple-view imaging, which is used in optical tomography. In contrast to conventional wide-field and confocal fluorescence microscopes, a light sheet illuminates only the focal plane of the detection objective lens from the side. Excitation is, thus, restricted to the fluorophores in the volume near the focal plane. This provides optical sectioning and allows the use of regular cameras in the detection process. Compared to confocal fluorescence microscopy, LSFM reduces photo bleaching and photo toxicity by up to three orders of magnitude. In LSFM, the specimen is embedded in a transparent block of hydrogel and positioned relative to the stationary light sheet using precise motorized translation and rotation stages. This feature is used to image any plane in a specimen. Additionally, multiple views obtained along different angles can be combined into a single data set with an improved resolution. LSFMs are very well suited for imaging large live specimens over long periods of time. However, they also perform well with very small specimens such as single yeast cells. This perspective introduces the principles of LSFM, explains the challenges of specimen preparation, and introduces the basics of a microscopy that takes advantage of multiple views.

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opomediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

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.