Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Identification of microplastics using 4-dimethylamino-4′-nitrostilbene solvatochromic fluorescence

In this work, we introduce the use of 4-dimethylamino-4′-nitrostilbene (DANS) fluorescent dye for applications in the detection and analysis of microplastics, an impendent source of pollution made of synthetic organic polymers with a size varying from less than 5 mm to nanometer scale. The use of this dye revealed itself as a versatile, fast and sensitive tool for readily discriminate microplastics in water environment. The experimental evidences herein presented demonstrate that DANS efficiently absorbs into a variety of polymers constituting microplastics, and its solvatochromic properties lead to a positive shift of the fluorescence emission spectrum according to the polarity of the polymers. Therefore, under UV illumination, microplastics glow a specific emission spectrum from blue to red that allows for a straightforward polymer identification. In addition, we show that DANS staining gives access to different detection and analysis strategies based on fluorescence microscopy, from simple epifluorescence fragments visualization, to confocal microscopy and phasor approach for plastic components quantification

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 Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy

The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology

Power-effective scanning with AODs for 3D optogenic applications

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks

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)

Nano-structured myelin: new nanovesicles for targeted delivery to white matter and microglia, from brain-to-brain

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brainextracted myelin were produced. The MyVes have an average diameter of 100-150 nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases

Laser-Fabricated Fluorescent, Ligand-Free Silicon Nanoparticles: Scale-up, Biosafety, and 3D Live Imaging of Zebrafish under Development

This work rationalizes the scalable synthesis of ultrasmall, ligand-free silicon nanomaterials via liquid-phase pulsed laser ablation process using picosecond pulses at ultraviolet wavelengths. Results showed that the irradiation time drives hydrodynamic NP size. Isolated, monodisperse Si-NPs are obtained at high yield (72%) using post-treatment process. The obtained Si-NPs have an average size of 10 nm (not aggregated) and display photoemission in the green spectral range. We directly characterized the ligand-free Si-NPs in a vertebrate animal (zebrafish) and assessed their toxicity during the development. In vivo assay revealed that Si-NPs are found inside in all the early life stages of embryos and larvae growth, showing that the biosafety of Si-NPs and malformation types are independent of the Si-NP dose. Si-NPs were directly imaged inside developing embryos by spinning disk-imaging technique with optical sectioning capability. We showed that Si-NPs can passively enter inside embryos by the pore canals of chorion, can diffuse in the circulatory system, i.e., blood vessel, and accumulate inside larvae midgut and yolk sac, and in the eye lens, indicating the crossing of the blood barrier