Marie Caroline Müllenbroich

A versatile clearing agent for multi-modal brain imaging

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.

Optical mapping of neuronal activity during seizures in zebrafish

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype.

Whole-Brain Vasculature Reconstruction at the Single Capillary Level

The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.

High-fidelity functional and structural whole-brain imaging with Bessel-beam light-sheet microscopy (Conference Presentation)

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6. Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity. We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.

Optimal staining and clearing protocol for whole mouse brain vasculature imaging with light-sheet microscopy

Light-sheet microscopy enables whole mouse brain imaging in association with clearing methodologies. Here, we present a pipeline for optimal investigation of the vascular component, which offers improved image quality for morphological analysis.