FRaeppli, a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish

Visualizing cell shapes, interactions and lineages of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated tracking cells in in vivo and mapping neuronal connectivity. Nevertheless, integrating multi-fluorophore information into the context of developing tissues in zebrafish is challenging given their cytoplasmic localization and spectral incompatibility with commonly used fluorescent markers. Here, we developed FRaeppli (Fish-Raeppli) expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. By tailoring hyperspectral protocols for time-efficient acquisition, we demonstrate FRaeppli s suitability for live imaging of complex internal organs, like the liver. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting shared developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and tissue lineages at single-cell resolution in zebrafish.

Erratum: Development of a simultaneous imaging system to measure the optical and gamma ray images of Ir-192 source for high-dose-rate brachytherapy

The gamma camera has a 1-mm-thick cerium-doped yttrium aluminum perovskite (YA1O_3: YAP(Ce)) scintillator plate optically coupled to a position-sensitive photomultiplier (PSPMT), and a 0.1-mm-diameter pinhole collimator was mounted in front of the camera to improve spatial resolution and reduce sensitivity.

A spectral demixing method for high-precision multi-color localization microscopy

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. So far, demixing algorithms give suboptimal results in terms of localization precision and correction of chromatic aberrations. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision & drift correction, compensation of chromatic aberrations, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic aberrations at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.

Enthesis strength, toughness and stiffness: an image-based model comparing tendon insertions with varying bony attachment geometries

Tendons of the body differ dramatically in their function, mechanics and range of motion, but all connect to bone via an enthesis. Effective force transfer at the enthesis enables joint stability and mobility, with strength and stiffness arising from a fibrous architecture. However, how enthesis toughness arises across tendons with diverse loading orientations remains unclear. To study this, we performed simultaneous imaging of the bone and tendon in entheses that represent the range of tendon-to-bone insertions and extended a mathematical model to account for variations in insertion and bone geometry. We tested the hypothesis that toughness, across a range of tendon entheses, could be explained by differences observed in interactions between fibre architecture and bone architecture. In the model, toughness arose from fibre reorientation, recruitment and rupture, mediated by interactions between fibres at the enthesis and the bony ridge abutting it. When applied to tendons sometimes characterized as either energy-storing or positional, the model predicted that entheses of the former prioritize toughness over strength, while those of the latter prioritize consistent stiffness across loading directions. Results provide insight into techniques for surgical repair of tendon-to-bone attachments, and more broadly into mechanisms for the attachment of highly dissimilar materials.