Antti Lignell

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.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. Summary: Single-molecule hybridization chain reaction, combined with tissue clearing, allows the near-quantitative and spatially localized detection of mRNAs in thick tissue samples.

Photochemical activity of Titan's low-altitude condensed haze

Titan, the largest moon of Saturn and similar to Earth in many aspects, has unique orange-yellow colour that comes from its atmospheric haze, whose formation and dynamics are far from well understood. Present models assume that Titans tholin-like haze formation occurs high in atmosphere through gas-phase chemical reactions initiated by high-energy solar radiation. Here we address an important question: Is the lower atmosphere of Titan photochemically active or inert? We demonstrate that indeed tholin-like haze formation could occur on condensed aerosols throughout the atmospheric column of Titan. Detected in Titans atmosphere, dicyanoacetylene (C₄N₂) is used in our laboratory simulations as a model system for other larger unsaturated condensing compounds. We show that C4N2 ices undergo condensed-phase photopolymerization (tholin formation) at wavelengths as long as 355 nm pertinent to solar radiation reaching a large portion of Titans atmosphere, almost close to the surface.

Spatial Genomic Analysis: A Multiplexed Transcriptional Profiling Method that Reveals Subpopulations of Cells Within Intact Tissues

Here, we present Spatial Genomic Analysis (SGA), a quantitative single-cell transcriptional profiling method that takes advantage of single-molecule imaging of individual transcripts for up to a hundred genes. SGA relies on a machine learning-based image analysis pipeline that performs cell segmentation and transcript counting in a robust way. SGA is suitable for various in situ applications and was originally developed to address heterogeneity in the neural crest, which is a transient embryonic stem cell population important for formation of various vertebrate body structures. After being specified as multipotent neural crest stem cells in the dorsal neural tube, they go through an epithelial to mesenchymal transition in order to migrate to different destinations around the body, and gradually turn from stem cells to progenitors prior to final commitment. The molecular details of this process remain largely unknown, and upon their emergence, the neural crest cells have been considered as a single homogeneous population. Technical limitations have restricted the possibility to parse the neural crest cell pool into subgroups according to multiplex gene expression properties. By using SGA, we were able to identify subgroups inside the neural crest niche in the dorsal neural tube. The high sensitivity of the method allows detection of low expression levels and we were able to determine factors not previously shown to be present in neural crest stem cells, such as pluripotency or lineage markers. Finally, SGA analysis also provides prediction of gene relationships within individual cells, and thus has broad utility for powerful transcriptome analyses in original biological contexts.