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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. The implemented variation of the Richardson‐Lucy deconvolution algorithm preserves the photogrammetry of the image data by using flux‐preserving regularization, an approach that to our knowledge has not been applied for deconvolving microscopy data before. [ABSTRACT FROM AUTHOR]

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. Using the new light sheet in combination with our objective equipped with a modulator unit to compensate the refractive index mismatch between air and mediums with indices of 1.45–1.56, we present high resolution images of various biological samples that were chemically cleared using different methods. They demonstrate a marked improvement in quality, contrast and resolution. [ABSTRACT FROM AUTHOR]

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. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

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. melanogaster cuticle hinder the penetration of visible light into internal tissues, thereby limiting the application of fluorescent microscopes to analyses of the outermost surfaces of intact samples. In addition, tissue-induced light scattering and optical aberrations quickly blur the view and, hence, require tissue sectioning for further investigation. We have developed a tissue-clearing and depigmentation approach (FlyClear), which preserves endogenous fluorescent signals and is applicable to various developmental stages ranging from larvae to adult fruit flies (Pende et al. Nature communications 9:4731, 2018). In this chapter, we provide a detailed protocol of the experimental steps involved., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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. 3DISCO utilizes the lipophilic solvents tetrahydrofuran (THF) and dibenzylether (DBE) for dehydration and successive clearing. Crucial steps for obtaining transparent tissues and preserving the fragile endogenous GFP are the transcardial perfusion, as well as the proper implementation of the 3DISCO clearing process using peroxide free chemicals. We further provide a protocol for resin embedding of 3DISCO cleared specimens that allows long term archiving of samples for years with virtually no loss in signal quality.