Selective Plane Illumination Microscopy: offers optical sectioning, reduced fluorophore bleaching
- Fig. 1: Schematic setup of a Selective Plane Illumination Microscope. Illumination (left) is perpendicular to the detection path (right). Laser light emanating from a fiber is collimated and focused by a cylindrical lens to a light sheet that traverses an aqueous medium-filled chamber. The sample, embedded in agarose gel, is supported in this chamber from above, and can be moved by a translating and rotating stage. Widefield detection is performed with objective lens, filter wheel, tube lens, and CCD camera.
- Fig. 2: In case of an almost transparent sample, multi-view reconstruction provides a nearly isotropic resolution. A single data set of the autofluorescence from a grain of paper mulberry pollen (top) suffers from poor axial resolution (a single slice parallel to the detection axis is shown). Rotation of the sample and acquisition of multiple data sets with different orientations provides overlapping data. The multi-view reconstruction combines the multiple data sets into a single one (bottom) that incorporates all of the high-resolution information available in the individual views. Objective lens: Zeiss Achroplan 63ξ, NA = 0.9, water dipping.
- Fig. 3: Although SPIM provides deep penetration in strongly scattering samples, a single data set may not cover the whole sample (e.g. single view of a GFP moesin labeled Drosophila melanogaster embryo, top). In this case multi-view recordings provide additional information about parts of the sample that are hidden in the single view data. In the reconstruction process, all information is computationally assembled into a superior data set inheriting the high-resolution information from all views (bottom). The sample was rotated about its anterior-posterior axis. Objective lens: Zeiss Achroplan 10 ξ, NA = 0.3, water dipping. Sample provided by F. Jankovics.
Selective Plane Illumination Microscopy (SPIM) offers optical sectioning, reduced fluorophore bleaching, fast, highly efficient image recording, and high depth penetration, especially when multiple views are combined. SPIM performs especially well in large samples such as fish or fly embryos, which can be observed live for several days. However, the SPIM principles are universal and have been successfully applied using objective lenses with magnifications from 5ξ to 100ξ, thus covering sample sizes from >1 mm to <20 μm with isotropic resolutions from 5 μm to 300 nm.
The stereo microscope is the most commonly used microscope in developmental biology. It provides a stereoscopic image of the sample while keeping it under ideal conditions for sorting and selecting embryos (e.g. in a Petri dish filled with an appropriate medium). A camera can be used to record (non-stereoscopic) images or even movies of the developing embryo for later reference. However, no volumetric quantification can be made since the stereo microscope offers little depth discrimination and no optical sectioning. The same holds true for conventional widefield microscopes, which in addition often suffer from short working distances that are not adequate for penetrating large samples.
Optical sectioning is required for a quantitative analysis of images in 3D. Out-of-focus light is rejected in confocal microscopy  by point excitation with a laser beam in combination with point detection through a pinhole, so that mainly in-focus light is detected. This principle works well in relatively thin samples (up to ca. 100 μm); however, in large and scattering objects the signal decreases since most of the fluorescence light is rejected by the pinhole. The confocal microscope, therefore, suffers from a limited penetration depth, and cannot be applied for in toto studies of many embryos. In such samples, the best resolution is obtained by physically sectioning the sample , which is very laborious, destroys the sample irretrievably, and is not applicable for live studies.
Selective Plane Illumination Microscopy
Selective Plane Illumination Microscopy (SPIM)  combines the advantages of stereo or widefield microscopes and confocal scanning microscopes.
SPIM provides optical sectioning in extended objects such as live embryos imaged in a suitable physiological context. In SPIM the sample is illuminated by a sheet of laser light (Fig. 1). A set of lasers, in combination with dichroic mirrors and an acousto-optical tunable filter (AOTF), is used to provide a variety of laser lines for excitation of different fluorescent dyes. In SPIM, cylindrical optics are used to focus the beam of light in one dimension, thereby creating a plane of light. The sample is embedded in a cylinder of agarose immersed in an aqueous medium. The resulting plane of fluorescence excited in the sample is imaged onto a camera using regular fluorescence microscopy optics: an objective lens, a filter wheel, and a tube lens. The sample is moved along the optical axis of the detection system through the light sheet in a stepwise fashion to build up a 3D data set.
3n SPIM optical sectioning is obtained in a direct and efficient way. By illuminating only the plane of interest no out-of-focus light is created in the first place. For this reason bleaching is reduced compared to confocal or widefield techniques, where the complete sample is illuminated even when observing only a single plane. The sectioning capability is defined by the light sheet thickness (usually 2-6 μm), which is adjusted to be nearly uniform across the field of view. Objective lenses with a long working distance and a relatively low numerical aperture (NA) are used for detection with large samples (a few millimeters). The lateral resolution is defined by the NA of the objective lens, whereas the axial resolution of the system is determined by the light sheet thickness (as long as it is thinner than the axial extent of the detection point spread function). Because of the plane illumination, SPIM does not suffer from scattering as much as the confocal microscope does: scattering of the illumination within the plane of the light sheet does not deteriorate the image at all, although scattering out of the plane can widen the thickness of the light sheet. In either event, unlike in the confocal microscope the scattered light is not rejected in SPIM, and can therefore contribute to the imaging process.
Multi-View Recording and Processing
In addition to translating the sample through the light sheet, SPIM offers an optional recording scheme: after recording a 3D stack, the sample is rotated and additional stacks are recorded with different orientations of the sample. These data sets are then combined in a postprocessing step in the computer (multiview reconstruction) . Depending on the optical properties of the sample, multi-view reconstruction can help to fill in information about obscured regions of the sample and/or improve the resolution. If the sample is fully transparent (e.g. the pollen grain in Fig. 2) the different data sets overlap, and the multi-view reconstruction compensates for the anisotropy in a single view and provides isotropic resolution. In the case of a more or less opaque sample (e.g. the Drosophila embryo in Fig. 3), additional views provide information about regions of the sample that are not visible in the single view. Here the multi-view reconstruction combines the information into a complete image of the sample. In many cases an improvement in both the imaged sample volume and the resolution is obtained.
Optical sectioning in fluorescence microscopy can be obtained by selectively illuminating a single plane in the sample. This has several advantages over widefield and confocal scanning techniques. Areas outside the focal plane are not affected by the excitation light, which reduces fluorophore bleaching and phototoxic effects in general. A highly efficient CCD camera is used to collect the fluorescence with much higher frame rates and an improved dynamic range compared to point-scanning devices. Even in strongly scattering samples a high penetration depth can be achieved. Multi-view reconstruction extends the information content of the data set and improves the axial resolution, making the resolution nearly isotropic. Sub-cellular resolution can be obtained in live samples kept in a biologically relevant environment. Applications include invitro studies, 3D cultured cells, multidimensional imaging of a complete spatio- temporal pattern of gene and protein expression, or tracking of tissues during the development of an intact embryo.
 Pawley, J.B., Handbook of Biological Confocal Microscopy, Plenum Press, 1995
 Weninger, W. J., Mohun, T., Nature Genet. 30, 59 (2002)
 Huisken, J., Swoger, J., Stelzer, E.H.K, Science, 305, 1007 (2004)
 Swoger, J., Huisken, J., Stelzer, E.H.K, Opt. Lett., 28, 1654 (2003)
Photos by May Britt Hansen, EMDC.
Jan Huisken, PhD
Jim Swoger European Molecular Biology Laboratory
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