Extensive mapping of neuronal connections in the central nervous system requires high-throughput µm-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multi-modal optical techniques. Here, we introduce a versatile brain clearing agent (2,2′-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.

Dynamical processes on networks have generated widespread interest in recent years. The theory of pattern formation in reaction–diffusion systems defined on symmetric networks has often been investigated, due to its applications in a wide range of disciplines. Here we extend the theory to the case of directed networks, which are found in a number of different fields, such as neuroscience, computer networks and traffic systems. Owing to the structure of the network Laplacian, the dispersion relation has both real and imaginary parts, at variance with the case for a symmetric, undirected network. The homogeneous fixed point can become unstable due to the topology of the network, resulting in a new class of instabilities, which cannot be induced on undirected graphs. Results from a linear stability analysis allow the instability region to be analytically traced. Numerical simulations show travelling waves, or quasi-stationary patterns, depending on the characteristics of the underlying graph.

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Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440–670 nm; the two-photon excitation range is 900–1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Cutting tissue into thin slices has been the standard practice in histology since its early days. Indeed, this approach allows solving (although roughly) two problems at once, i.e., absence of optical sectioning in conventional wide-field microscopes and tissue opacity. However, biological structures are inherently three-dimensional (3-D); for this reason, scientists have always tried to extend tissue imaging to thick specimens. On the imaging side, the invention of confocal microscopy by Minsky in 1955,¹ the development of two-photon microscopy in 1990,² and the recent revival of light-sheet microscopy,³ have provided a wide choice of microscopy methods allowing optical sectioning, i.e., 3-D resolution. On the other hand, the effectiveness and usability of these techniques in real macroscopic specimens are hampered, because biological specimens are naturally opaque.