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.

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. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.

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 set-up 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.

Beam modes are considered by a high-aperture scalar theory based on the complex source-point method. A different form for the beam mode that avoids nonphysical singularities in intensity is introduced. The amplitude in the focal region is explored for the lowest-order mode. Conditions for oscillation of a high-aperture resonator, such as a microcavity, are derived. The amplitude in the focal region is explored for higher-order modes in systems with cylindrical symmetry. These modes are expressed in terms of Jacobi polynomials.