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

Tailored sample mounting for light-sheet fluorescence microscopy of clarified specimens by polydimethylsiloxane casting

The combination of biological tissue clearing methods with light-sheet fluorescence microscopy (LSFM) allows acquiring images of specific biological structures of interest at whole organ scale and microscopic resolution. Differently to classical epifluorescence techniques, where the sample is cut into slices, LSFM preserves the whole organ architecture, which is of particular relevance for investigations of long-range neuronal circuits. This imaging modality comes with the need of new protocols for sample mounting. Gel matrix, hooks, tips, glues, and quartz cuvettes have been used to keep whole rodent organs in place during image acquisitions. The last one has the advantage of avoiding sample damage and optical aberrations when using a quartz refractive index (RI) matching solution. However, commercially available quartz cuvettes for such large samples are expensive. We propose the use of polydimethylsiloxane (PDMS) for creating tailor-made cuvettes for sample holding. For validation, we compared PDMS and quartz cuvettes by measuring light transmittance and performing whole mouse-brain imaging with LSFM. Moreover, imaging can be performed using an inexpensive RI matching solution, which further reduces the cost of the imaging process. Worth of note, the RI matching solution used in combination with PDMS leads to a moderate expansion of the sample with respect to its original size, which may represent an advantage when investigating small components, such as neuronal processes. Overall, we found the use of custom-made PDMS cuvettes advantageous in term of cost, image quality, or preservation of sample integrity with respect to other whole-mouse brain mounting strategies adopted for LSFM

High-fidelity imaging in brain-wide structural studies using light-sheet microscopy

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution and, in combination with tissue clearing methods, is often employed to reconstruct the cyto-architecture over the intact mouse brain. Inherently to LSM, however, residual opaque objects, always present to some extent even in extremely well optically cleared samples, cause stripe artifacts, which, in the best case, severely affect image homogeneity and, in the worst case, completely obscure features of interest. Here, demonstrating two example applications in intact optically cleared mouse brains, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data for the brain-wide morphology of neuronal and vascular networks. We found that a third of the imaged volume of the brain was affected by strong striated image intensity inhomogeneity and, furthermore, a significant amount of information content lost with Gaussian illumination was accessible when interrogated with Bessel beams. In conclusion, Bessel beams produce high-fidelity structural data of improved image homogeneity and might significantly relax demands placed on the automated tools to count, trace, or segment fluorescent features of interest

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

Light-sheet microscopy (LSM) is a powerful imaging technique that uses a planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM is a variant of LSM that exploits the 2P absorption effect for sample excitation. The light polarization state plays a significant, and often overlooked, role in 2P absorption processes. The scope of this work is to test whether using different polarization states for excitation light can affect the detected signal levels in 2P LSM imaging of typical biological samples with a spatially unordered dye population. Supported by a theoretical model, we compared the fluorescence signals obtained using different polarization states with various fluorophores (fluorescein, EGFP and GCaMP6s) and different samples (liquid solution and fixed or living zebrafish larvae). In all conditions, in agreement with our theoretical expectations, linear polarization oriented parallel to the detection plane provided the largest signal levels, while perpendicularly-oriented polarization gave low fluorescence signal with the biological samples, but a large signal for the fluorescein solution. Finally, circular polarization generally provided lower signal levels. These results highlight the importance of controlling the light polarization state in 2P LSM of biological samples. Furthermore, this characterization represents a useful guide to choose the best light polarization state when maximization of signal levels is needed, e.g. in high-speed 2P LSM.Comment: 16 pages, 4 figures. Version of the manuscript accepted for publication on Biomedical Optics Expres

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

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).Comment: Replacement: accepted version of the manuscript, to be published in Biomedical Optics Express. 36 pages, 15 figure

Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

A versatile clearing agent for multi-modal brain imaging

Extensive mapping of neuronal connections in the central nervous system requires high-throughput um-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 multimodal 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.Comment: in Scientific Reports 201