Saiedeh Saghafi

Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.

3D-ultramicroscopy utilizing aspheric optics.

Using a combination of achromatic aspheric optical elements and achromatic cylindrical lenses, we developed an improved laser light sheet generator for two-side illumination ultramicroscopy. This light sheet generator has a much longer Rayleigh range, a more uniform spatial intensity distribution along x -, y - and z-axis, and reduced aberrations than the standard system consisting of a slit aperture and a single cylindrical lens, which is commonly used in light sheet microscopy. As there is no truncation of the beam by a slit aperture in our design the laser energy is used more efficiently. Applying this light sheet generator to ultramicroscopy of chemically cleared biological samples, such as Drosophila, dissected mouse hippocampi, and entire mouse brains, we achieved a markedly improved resolution of fine details.

Image enhancement in ultramicroscopy by improved laser light sheets.

In the majority of implementations of light sheet microscopy, such as ultramicroscopy, the laser beam illuminating the specimen is truncated by a slit aperture before it is focused to a light sheet by a single cylindrical lens. A light sheet generated in this way can be made very thin near to the focal point, but unfortunately its Rayleigh range is severely limited. This problem can be partially solved by using a smaller slit aperture. However, this also causes a major loss in power, a severe broadening of the beam waist, and thus a significant loss of resolution along the detection axis. We developed improved light-sheet-generation optics, which provide longer Raleigh ranges, whilst retaining beam waists comparable to our standard system with one cylindrical lens. Using the modified system we achieved a marked improvement in the resolution of ultramicroscopy reconstructions of representative biological specimens.

Ultramicroscopy: development and outlook

Abstract. 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.

Immunostaining, Dehydration, and Clearing of Mouse Embryos for Ultramicroscopy

This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethanol and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates

Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.

Dehydration and Clearing of Whole Mouse Brains and Dissected Hippocampi for Ultramicroscopy

This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, mouse brains and hippocampi are carefully dissected and dehydrated, and then cleared in a solution of benzyl benzoate and benzyl alcohol.

P 227. Ultramicroscopy (UM) in neurobiology

UM is a prime example for an interdisciplinary research field invention. It enables researchers to obtain high-resolution 3D-reconstructions of organ systems in different animal models. Knowledge about the interconnections of the neuronal network to the vascular system is often essential neurobiological research. Ultramicroscopy (UM) is a light sheet based fluorescence microscopy. This novel bioimaging technique allows the 3D-visualization of cm-sized biological specimens with μm-resolution ( [Dodt et al., 2007] , [Jahrling, 2011] ). Artifacts of conventional microscopy such as distortion of the tissue are avoided. It is due to optical sectioning instead of mechanical sclicing ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). In UM, a specimen is illuminated by a thin sheet of laser light, formed by one or more cylindrical lenses ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). To generate a light sheet distribution in the standard UM, we basically employ a single cylindrical lens placed in front of a variable rectangular slit aperture ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). Lectinis are proteins that bind to sugar complexes, which are attached to proteins and lipids. We employed an approach using fluorescent conjugated lectins during the transcardial perfusion of mice to contrast the endothelium building up the vascular system ( [Jahrling, 2011] , [Jahrling et al., 2009] ). Biological samples are prepared chemically to become as transparent as possible ( Jahrling, 2011 ). In this study the architecture of the blood vessel system of whole organs of animal models is visualized ( [Jahrling, 2011] , [Jahrling et al., 2009] ). By combining light sheet based UM with lectin-labelling, 3D reconstructions of vascular structures of the mouse spinal cord can be generated. Alteration in optics, morphological analysis of neurobiological disorders, multiple labeling, improving histology of clearing procedure, the analysis of brain tumors are subjects of interests are subjects of interests in our future work.

Dehydration and Clearing of Adult Drosophila for Ultramicroscopy

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Based on the modal analysis method, we developed a model that describes the output beam of a diode-pumped solid state (DPSS) laser emitting a multimode beam. Measuring the output beam profile in the near field and at the constructed far field the individual modes, their respective contributions, and their optical parameters are determined. Using this information, the beam is optically reshaped into a quasi-Gaussian beam by the interference and superposition of the various modes. This process is controlled by a mode modulator unit that includes different meso-aspheric elements and a soft-aperture. The converted beam is guided into a second optical unit comprising achromatic-aspheric elements to produce a thin light sheet for ultramicroscopy. We found that this light sheet is markedly thinner and exhibits less side shoulders compared with a light sheet directly generated from the output of a DPSS multimode laser.