A hypothalamic dopamine locus for psychostimulant-induced hyperlocomotion in mice.

The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain's clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th/Dat1 cells from its anterior subdivision innervate the LS in mice.

FlyClear: A Tissue-Clearing Technique for High-Resolution Microscopy of Drosophila.

Fluorescently labeled transgenic lines of Drosophila melanogaster are a powerful routine tool in fly laboratories. The possibility to fluorescently visualize individual cell populations or entire tissues and the constantly improving microscopy technologies such as two-photon or light-sheet applications, with deep tissue imaging, hold great potential to address central biological questions at an organismic level. However, strong pigmentation and the opaque nature of the D.

Engineering a better light sheet in an axicon-based system using a flattened Gaussian beam of low order.

Lasers are fundamental tools in research and development. The shape of an incident laser beam directly affects the results, when it propagates through complex structured meso-aspheric optical elements. In conic-based systems utilizing elements such as axicons, the impact of secondary lobes is mostly overlooked, although the intensity distributions at the central spot and the side-lobes directly affect the beam properties.

Visualizing minute details in light-sheet and confocal microscopy data by combining 3D rolling ball filtering and deconvolution.

We developed an open-source deconvolution software that stunningly increases the visibility of minute details, as for example, neurons or nerve fibers in light-sheet microscopy or confocal microscopy data by combining rolling ball background subtraction in three directions with deconvolution using a synthetic or measured point spread function. Via automatic block-wise processing image stacks of virtually unlimited size can be deconvolved even on small computers with 8 or 16 GB RAM. By parallelization and optional GPU-acceleration, the software works with high speed: On a PC equipped with a state-of-the-art NVidia graphic board a three dimensional (3D)-stack of about 1 billion voxels can be deconvolved within 5 to 10 minutes.

3D histopathology of human tumours by fast clearing and ultramicroscopy.

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid.

A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity.

Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers.

Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy.

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice.

Deconvolution of light sheet microscopy recordings.

We developed a deconvolution software for light sheet microscopy that uses a theoretical point spread function, which we derived from a model of image formation in a light sheet microscope. We show that this approach provides excellent blur reduction and enhancement of fine image details for image stacks recorded with low magnification objectives of relatively high NA and high field numbers as e.g.

Early Aβ reduction prevents progression of cerebral amyloid angiopathy.

Objective: Clinical trials targeting β-amyloid peptides (Aβ) for Alzheimer disease (AD) failed for arguable reasons that include selecting the wrong stages of AD pathophysiology or Aβ being the wrong target. Targeting Aβ to prevent cerebral amyloid angiopathy (CAA) has not been rigorously followed, although the causal role of Aβ for CAA and related hemorrhages is undisputed. CAA occurs with normal aging and to various degrees in AD, where its impact and treatment is confounded by the presence of parenchymal Aβ deposition.

High-resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO).

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep-tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)-expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP-expressing samples for more than a year.

High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster.

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.

Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy.

In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.

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.

Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy.

Light-sheet microscopy is an effective technique in neuroscience, developmental biology, and cancer research for visualizing and analyzing cellular networks and whole organs in three dimensions. Because this technique requires specimens to be translucent they commonly have to be cleared before microscopy inspection. Here, we provide 3DISCO based protocols for preparing cleared samples of immuno-stained neural networks, lectin-labeled vascular networks, and Methoxy-X04 labeled beta-amyloid plaques in mice.

Outlook on optimizing ultramicroscopy imaging technique through optical characterization.

Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented.

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.

Ultramicroscopy: development and outlook.

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.

Cerebral β-Amyloidosis in Mice Investigated by Ultramicroscopy.

Alzheimer´s disease (AD) is the most common neurodegenerative disorder. AD neuropathology is characterized by intracellular neurofibrillary tangles and extracellular β-amyloid deposits in the brain. To elucidate the complexity of AD pathogenesis a variety of transgenic mouse models have been generated.

Reduction of photo bleaching and long term archiving of chemically cleared GFP-expressing mouse brains.

Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation.

Infrared video microscopy for visualizing neurons and neuronal excitation in brain slices.

Here we describe methods for imaging neurons and neuronal excitation in brain slices with infrared video microscopy. Patch clamping in conjunction with a gradient-contrast system allows electrical recording from fine neuronal processes. Imaging with an infrared (IR)-darkfield system allows visualization of the intrinsic optical signal (IOS).

Prdm6 is essential for cardiovascular development in vivo.

Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice.

Ultramicroscopy: light-sheet-based microscopy for imaging centimeter-sized objects with micrometer resolution.

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.