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.

Publisher Correction: Whole-Brain Vasculature Reconstruction at the Single Capillary Level.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

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.

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.

A device for continuous monitoring of true central fixation based on foveal birefringence.

A device for continuous monitoring of central fixation utilizes birefringence, the property of the Henle fibers surrounding the human fovea, to change the polarization state of light. A circular scan of retinal birefringence, where the scanning circle encompasses the fovea, allows identification of true central fixation-an assessment much needed in various applications in ophthalmology, psychology, and psychiatry. The device allows continuous monitoring for central fixation over an extended period of time in the presence of fixation targets and distracting stimuli, which may be helpful in detecting attention deficit hyperactivity disorder, autism spectrum disorders, and other disorders characterized by changes in the subject's ability to maintain fixation. A proof-of-concept has been obtained in a small study of ADHD patients and normal control subjects.

A novel haploscopic viewing apparatus with a three-axis eye tracker.

PURPOSE: To validate the accuracy and precision of a novel haploscope for use in investigating the mechanisms underlying normal eye control as well as the mechanisms of cyclovertical strabismus in pediatric and adult subjects. The accuracy and precision represent the device's ability to reproducibly measure true eye positions. METHODS: A novel haploscope was developed that allows the measurement of eye positions and movements under specified conditions of vergence, head tilting, and cover testing. Equipped with video oculography, the haploscope can aid in the objective assessment of binocular and adaptive mechanisms that maintain oculomotor alignment. The device's accuracy and precision were assessed using a model eye with 3 axes of rotation. The device was then used to measure ocular torsion during the well-documented phenomenon of ocular counter-roll with head tilt. The eye movements of 6 normal subjects were measured as each subject fixated binocularly on the center of radially symmetric targets during head tilting. RESULTS: The device yielded Pearson correlations with the model eye of R = 1.0, with residual error (re), a measure of accuracy, about all 3 ocular axes peaking at re = 19 +/- 5 arcmin. For human subjects, average positional error was re = 21 +/- 9 arcmin. Ocular counter-roll averaged 5.7 +/- 0.9 degrees for left and right eyes. CONCLUSIONS: These results validate the accuracy and precision of this novel haploscope. They support its use in future investigations of the mechanisms of oculomotor control and alignment.