Breaking the diffraction limit of light sheets allows fast isotropic imaging of large samples by ultramicroscopy (original) (raw)
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Cold Spring Harbor protocols, 2013
Ultramicroscopy (UM) is a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. It was developed for specimens in the size range of ∼1-15 mm, such as whole mouse brains, mouse embryos, mouse organs, and Drosophila melanogaster. In UM, the specimen is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. UM is closely related to a growing family of comparable microscopy approaches based on light sheet illumination developed in recent years. This article presents an overview of light-sheet-based microscopy and describes the underlying physics of light sheet generation. The assembly of an "ultramicroscope" for investigating fixed chemically cleared tissue is described in detail, and the functions of the essential components, such as mechanics, camera, and objectives, are discussed. Finally, practical applications of UM...
Ultramicroscopy (UM) is an interdisciplinary imaging technique allowing three dimensional reconstruction of up to cm-sized organs of animal models with mm resolution. Here, we present a review of technical and theoretical aspects of standard laser light sheet fluorescence microscopy. Fine morphological details in structures of animal models such as Drosophila melanogaster (fruit fly) and GFP-expressing mice, produced by UM are presented. Ultramikroskopie – eine neue bildgebende Technologie auf Basis von " Laser Light-Sheets " , entwickelt in Zusammerarbeit mehrerer Forschungsdisziplinen. Die Ultramikroskopie (UM) ist ein interdisziplinä res Verfahren. Diese Technik ermö glicht dreidimensionale Rekonstruktionen von bis zu cm-großen Organen von Tiermodellen mit einer Auflö sung im Mikrometerbereich. In diesem U ¨ bersichtsartikel werden technische und theoretische Aspekte der Standard-" Laser Light Sheet "-Technik erlä utert. Mittels UM kö nnen morphologische Gewebedetails von Tiermodellen, wie etwa von Drosophila melanogaster (Fruchtfliege) und von GFP-exprimierenden transgenen Mä usen, dargestellt werden.
Nature Communications, 2018
The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope setup utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applying image fusion from orthogonal directions. This methodological integration of novel chemical, optical, and computational techniques allows a major advance in the analysis of global neural circuit organisation.
Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy
Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction.The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed.
Ultramicroscopy: development and outlook
Neurophotonics, 2015
We present an overview of the ultramicroscopy technique we developed. Starting from developments 100 years ago, we designed a light sheet microscope and a chemical clearing to image complete mouse brains. Fluorescence of green fluorescent protein (GFP)-labeled neurons in mouse brains could be preserved with our 3DISCO clearing and high-resolution three-dimensional (3-D) recordings were obtained. Ultramicroscopy was also used to image whole mouse embryos and flies. We improved the optical sectioning of our light sheet microscope by generating longer and thinner light sheets with aspheric optics. To obtain high-resolution images, we corrected available air microscope objectives for clearing solutions with high refractive index. We discuss how eventually super resolution could be realized in light sheet microscopy by applying stimulated emission depletion technology. Also the imaging of brain function by recording of mouse brains expressing cfos-GFP is discussed. Finally, we show the first 3-D recordings of human breast cancer with light sheet microscopy as application in medical diagnostics.
We report on an Adaptive Optics (AO) Light-Sheet Fluorescence Microscope compatible with neuroimaging, based on direct wavefront sensing without the requirement of a guide star. We demonstrate fast AO correction, typically within 500ms, of in-depth aberrations of the live adult Drosophila Melanogaster brain, enabling to double the contrast when imaging with structural or calcium sensors. We quantify the gain in terms of image quality on multiply neuronal structures part of the sleep network in the Drosophila brain, at various depths, and discuss the optimization of key parameters driving AO such as the number of corrected modes and the photon budget. We present a first design of a compact AO add-on that is compatible with integration into most of reported Light-Sheet setups and neuroimaging.
Optics Express, 2010
We present and demonstrate the use of an extreme ultraviolet (EUV) microscope that was developed in-house. Images are acquired using Bragg reflection multilayer optics and a laser-produced plasma light source. The upper-limit spatial resolution of the EUV microscope is 130 nm with a 10 ns exposure time and 250 × 250 µm 2 field of view. Resolution is superior to that of visible microscopes with the same size of field of view, and the exposure time is short enough to observe fine structures in-vivo. Observation of the cerebral cortex of a mouse is demonstrated. OCIS codes: (110.0180) Microscopy; (110.7440) X-ray imaging; (180.7460) X-ray microscopy; (230.4170) Multilayers; (340.7460) X-ray microscopy; (340.7480) X-rays, soft Xrays, extreme ultraviolet Vinogradov, I. A. Artyukov, A. G. Ponomareko, V. V. Kondratenko, M. C. Marconi, J. J. Rocca, and C. S. Menoni, "Single-shot extreme ultraviolet laser imaging of nanostructures with wavelength resolution," Opt. Lett. 33(5), 518-520 (2008). 9. I. A. Artioukov, A. V. Vinogradov, V. E. Asadchikov, Yu. S. Kas'yanov, R. V. Serov, A. I. Fedorenko, V. V.
In a previous paper (McConnell et al., 2016) we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently-stained 12.5-day old mouse embryos. Here we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show sub-cellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal disks are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.