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

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

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

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

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

Fast, Image-based Autofocus System for High-resolution Optical Microscopy of Whole Mouse Brains

We describe an image-based method for single-shot measurement and real-time correction of defocus in an optical microscope. The method is validated both in bright-field, epifluorescence and light-sheet microscopy

Confocal multispot microscope for fast and deep imaging in semicleared tissues

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep

Fast multi-directional DSLM for confocal detection without striping artifacts

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast

A versatile new technique to clear mouse and human brain

Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain