Fast Inertia-Free Volumetric Light-Sheet Microscope
Fast noninvasive three-dimensional (3D) imag-ing is crucial for quantitatively studying highly dynamic events ranging from flow cytometry to developmental biology. Light-sheet microscopy has emerged as the tool-of-choice for 3D characterization of rapidly evolving systems. However, to obtain a 3D image, either the sample or parts of the microscope are moved, limiting the acquisition speed. Here, we propose a novel inertia-free light-sheet-based scheme for volumetric imaging at high temporal resolution. Our approach comprises a novel combination of an acousto-optic scanner to produce tailored illumination and an acoustic-optofluidic lens, placed in the detection path to provide extended dept…
Light Sheet Fluorescence Microscopy (LSFM) for Two-Photon Excitation Imaging of Thick Samples.
Over the last decades, fluorescence microscopy techniques have been developed in order to provide a deeper, faster and higher resolution imaging of three-dimensional biological samples. Within this framework, Light Sheet Fluorescence Microscopy (LSFM) became an increasingly useful and popular imaging technique able to answer several biological questions in the field of developmental biology [1]. Thanks to the spatial confinement of the excitation process within a thin sheet in the focal plane, it provides an intrinsic optical sectioning and a reduced phototoxicity. On the other side, Two-Photon Excitation (2PE), thanks to the use of IR wavelengths, has become an invaluable tool to improve i…
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 …
Three-dimensional multiple-particle tracking with nanometric precision over tunable axial ranges
The precise localization of nanometric objects in three dimensions is essential to identify functional diffusion mechanisms in complex systems at the cellular or molecular level. However, most optical methods can achieve high temporal resolution and high localization precision only in two dimensions or over a limited axial (z) range. Here we develop a novel wide-field detection system based on an electrically tunable lens that can track multiple individual nanoscale emitters in three dimensions over a tunable axial range with nanometric localization precision. The optical principle of the technique is based on the simultaneous acquisition of two images with an extended depth of field while …
Microscopy method and apparatus for optical tracking of emitter objects
Microscopy method and apparatus for determining the posi tions of emitter objects in a three - dimensional space that comprises focusing scattered light or fluorescent light emitted by an emitter object, separating the focused beam in a first and a second optical beam, directing the first and the second optical beam through a varifocal lens having an optical axis such that the first optical beam impinges on the lens along the optical axis and the second beam impinges decentralized with respect to the optical axis of the varifocal lens , simultaneously capturing a first image created by the first optical beam and a second image created by the second optical beam , and determining the relativ…
4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)
AbstractIn the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of…
Biocompatibility and biodistribution of functionalized carbon nano-onions (f-CNOs) in a vertebrate model
AbstractFunctionalized carbon nano-onions (f-CNOs) are of great interest as platforms for imaging, diagnostic and therapeutic applications due to their high cellular uptake and low cytotoxicity. To date, the toxicological effects of f-CNOs on vertebrates have not been reported. In this study, the possible biological impact of f-CNOs on zebrafish during development is investigated, evaluating different toxicity end-points such as the survival rate, hatching rate, and heart beat rate. Furthermore, a bio-distribution study of boron dipyrromethene (BODIPY) functionalized CNOs in zebrafish larvae is performed by utilizing inverted selective plane illumination microscopy (iSPIM), due to its intri…
Influence of Nanoparticle Exposure on Nervous System Development in Zebrafish Studied by Means of Light Sheet Fluorescence Microscopy
Zebrafish has a remarkable similarity in the molecular signaling processes, cellular structure, anatomy and physiology to other higher order vertebrates, making it an excellent vertebrate model organism (1). Recently, zebrafish has been used for neurotoxicity screening of numerous nanomaterials with a focus on the developmental effects due to the possibility of in vivo visualization of specific neurons and axon tracts by injecting dyes in live animals as well in fixed ones (2). Here we propose Light Sheet Fluorescence Microscopy (LSFM) (3) (4) to perform neurotoxicity studies, in order to study the nervous system architecture and to image 3D structures in the brain of live larvae during the…
A Novel Fast Volumetric Light Sheet Microscopy
Fast noninvasive three-dimensional (3D) imaging is crucial for the quantitative understanding of highly dynamic biological processes. Over the last decades, several fluorescence microscopy techniques have been developed in order to provide a faster and deeper imaging of thick biological samples [1]. Within this framework, Light Sheet Fluorescence Microscopy (LSFM) has emerged as a powerful imaging tool for 3D imaging of thick samples ranging from single cells to entire animals [2,3].However, to obtain a 3D reconstruction either sample or microscope parts usually need to be moved limiting the acquisition speed and inducing possible interferences in volume recording. To solve this problem, he…