Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana

Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae and algal rhizoids). High-resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that light sheet fluorescence microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosol-localized ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterized their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing

Oblique plane microscopy is a method enabling light-sheet fluorescence imaging through a single microscope objective lens by focusing on a tilted plane within the sample. To focus the fluorescence emitted by the oblique plane on a camera, the light is imaged through a pair of remote objective lenses, facing each other at an angle. The aperture mismatch resulting from this configuration limits the effective numerical aperture of the system, reducing image resolution and signal intensity. This manuscript introduces an alternative method to capture the oblique plane on the camera. Instead of relying on angled objective lenses, an electrically tunable lens is employed. This lens adjusts the focal plane of the microscope synchronously with the rolling shutter of a scientific CMOS camera. In this configuration the entire aperture of the objective is effectively employed, increasing the resolution of the system. Moreover, a variety of objective lenses can be employed, enabling the acquisition of wider axial fields of view compared to conventional oblique plane microscopy

Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency

Modeling Lung Carcinoids with Zebrafish Tumor Xenograft

Lung carcinoids are neuroendocrine tumors that comprise well-differentiated typical (TCs) and atypical carcinoids (ACs). Preclinical models are indispensable for cancer drug screening since current therapies for advanced carcinoids are not curative. We aimed to develop a novel in vivo model of lung carcinoids based on the xenograft of lung TC (NCI-H835, UMC-11, and NCI-H727) and AC (NCI-H720) cell lines and patient-derived cell cultures in Tg(fli1a:EGFP)(y1) zebrafish embryos. We exploited this platform to test the anti-tumor activity of sulfatinib. The tumorigenic potential of TC and AC implanted cells was evaluated by the quantification of tumor-induced angiogenesis and tumor cell migration as early as 24 h post-injection (hpi). The characterization of tumor-induced angiogenesis was performed in vivo and in real time, coupling the tumor xenograft with selective plane illumination microscopy on implanted zebrafish embryos. TC-implanted cells displayed a higher pro-angiogenic potential compared to AC cells, which inversely showed a relevant migratory behavior within 48 hpi. Sulfatinib inhibited tumor-induced angiogenesis, without affecting tumor cell spread in both TC and AC implanted embryos. In conclusion, zebrafish embryos implanted with TC and AC cells faithfully recapitulate the tumor behavior of human lung carcinoids and appear to be a promising platform for drug screening

An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics

The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. The toolbox includes newly developed algorithms and interactive tools for image pre-processing and segmentation, estimation of significant single-neuron single-trial signals, mapping event-related neuronal responses, detection of activity-correlated neuronal clusters, exploration of population dynamics, and analysis of clusters' features against surrogate control datasets. The modules are integrated in a modular and versatile processing pipeline, adaptable to different needs. The clustering module is capable of detecting flexible, dynamically activated neuronal assemblies, consistent with the distributed population coding of the brain. We demonstrate the suitability of the toolbox for a variety of calcium imaging datasets. The toolbox open-source code, a step-by-step tutorial and a case study dataset are available at https://github.com/zebrain-lab/Toolbox-Romano-et-al

Imaging of calcium gradient oscillations in plant root hairs by light sheet fluorescence microscopy

Root hairs are a delicate single-cell system whose growth is regulated by a fine mechanism characterised by the presence of a tip-high Ca2+ gradient that shows regular oscillations in growing root hairs. We show a method based on the use of Light sheet fluorescence microscopy (LSFM) which allows the quasi-physiological analysis of Arabidopsis thaliana plant roots hairs with excellent spatial and temporal resolution over a wide field of view. We show how the healthy growing root hairs are linked to precise oscillations and how a disruption of this mechanism can be associated to specific genes

M3D-BIO - Microfluidics-Enabled 3D Printing for Biofabrication

Microfluidics market is the fastest growing research area in the world, and they have shown much promise in biofabrication and 3D bioprinting of tissues and organs. However, microfluidics is conventionally produced using drawn-out and expensive lithographic methods, hindering their wider uptake. To this end, we have established a streamlined pipeline which incorporates simulation, design, fabrication and validation processes to produce versatile microfluidic chip nozzles for a range of applications in biofabrication. The microfluidic devices are produced by combining material extrusion additive manufacturing (MEAM) with innovative design approaches to achieve leak-free and low-surface roughness channels without any need of special tubing. These microfluidic chip nozzles create complex anisotropic fibrous core-shell structures matching blood vessels at resolutions not reported previously. The results of this study show that the novel microfluidics system can be adopted in a wide range of applications from tissue scaffolds, cell culture systems, biochemical sensors and lab-on-a-chips, paving ways for next generation of 3D-printed microfluidics in biofabrication.</p