Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

Image analysis in light sheet fluorescence microscopy images of transgenic zebrafish vascular development

The zebrafish has become an established model to study vascular development and disease in vivo. However, despite it now being possible to acquire high-resolution data with state-of-the-art fluorescence microscopy, such as lightsheet microscopy, most data interpretation in pre-clinical neurovascular research relies on visual subjective judgement, rather than objective quantification. Therefore, we describe the development of an image analysis workflow towards the quantification and description of zebrafish neurovascular development. In this paper we focus on data acquisition by lightsheet fluorescence microscopy, data properties, image pre-processing, and vasculature segmentation, and propose future work to derive quantifications of zebrafish neurovasculature development

New live screening of plant-nematode interactions in the rhizosphere

Abstract Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Three-dimensional tracking of small spheres in focused laser beams : influence of the detection angular aperture

Back-focal-plane interferometry is a method capable of determining the three-dimensional position of a particle with high precision ( 0.7 should be used only for dielectric spheres with diameters approximately equal to the laser wavelength. Harmonic optical traps can be calibrated by measurement of the thermal motion of the sphere. We performed Brownian dynamics simulations and subsequent thermal noise analyses to prove that the wrong sin(alpha) incorrectly suggests an increased and nonharmonic axial trapping potential

Resolution in optical microscopy

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