Antti Lignell

Bio: Antti Lignell is an academic researcher from California Institute of Technology. The author has contributed to research in topics: Matrix isolation & Molecule. The author has an hindex of 20, co-authored 42 publications receiving 1307 citations. Previous affiliations of Antti Lignell include University of Helsinki & University of Jyväskylä.

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

Abstract: 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.

HKrF in solid krypton

Abstract: A new krypton-containing compound, HKrF, has been prepared in a low-temperature Kr matrix via VUV photolysis of the HF precursor and posterior thermal mobilization of H and F atoms. All three fundamental vibrations have been observed in the FTIR spectra at ∼1950 cm−1 (H–Kr stretch), ∼650 cm−1 (bending), and ∼415 cm−1 (Kr–F stretch). Two distinct sites of HKrF have been identified. The energy difference between the H–Kr stretching vibrations for the two sites is remarkably large (26 cm−1), indicating a strong influence of the environment. In annealing after the photolysis of the precursor, HKrF is formed in two different stages: at 13–16 K from closely trapped H+F pairs and at T>24 K due to more extensive mobility of H and F atoms in the matrix. HKrF in a less stable site decreases at temperatures above 32 K, the other site being stable up to the sublimation temperature of the matrix. The photodecomposition cross section for HKrF has been measured between 193 and 350 nm and compared with the cross sections...

Interaction of rare-gas-containing molecules with nitrogen: Matrix-isolation and ab initio study of HArF⋯N2, HKrF⋯N2, and HKrCl⋯N2 complexes

Abstract: The complexes of HArF, HKrF, and HKrCl with nitrogen molecules have been studied computationally and experimentally. With the help of computations the experimental data can be interpreted as showing the presence of two complex configurations, one linear and one bent. Vibrational properties of the studied molecules are very sensitive to the intermolecular interactions and complexation induces an exceptionally large blueshift (>100 cm−1 for HKrCl) to the H–Ar and H–Kr stretching frequency, especially for the linear configurations. The interaction energies without zero-point energy correction are between 400 and 800 cm−1. According to the energy decomposition scheme, the electrostatic forces provide the most important interaction in the linear complex configurations. For the bent complexes, electrostatic and dispersion forces are competing as a leading attractive interaction.

On theoretical predictions of noble-gas hydrides.

Abstract: We discuss the present status and reliability of theoretical predictions of noble-gas hydride molecules. It is shown that the single-reference MP2 calculations can produce a rather inaccurate energy diagram for the formation of noble-gas hydrides, and this may mislead the theoretical predictions. We suggest that the computational dissociation energy of the HY precursors should always be compared with the experimental values as a checkpoint for the computational accuracy. The computational inaccuracy probably explains why some compounds that are stable with the single-reference MP2 method (HArC4H, HArC3N, and HArCN) did not appear in matrix-isolation experiments, whereas the corresponding compounds with Kr and Xe are known.

Insertion of noble gas atoms into cyanoacetylene: an ab initio and matrix isolation study.

Abstract: A computational and experimental matrix isolation study of insertion of noble gas atoms into cyanoacetylene (HCCCN) is presented. Twelve novel noble gas insertion compounds are found to be kinetically stable at the MP2 level of theory, including four molecules with argon. The first group of the computationally studied molecules belongs to noble gas hydrides (HNgCCCN and HNgCCNC), and we found their stability for Ng = Ar, Kr, and Xe. The HNgCCCN compounds with Kr and Xe have similar stability to that of previously reported HKrCN and HXeCN. The HArCCCN molecule seems to have a weaker H-Ar bond than in the previously identified HArF molecule. The HNgCCNC molecules are less stable than the HNgCCCN isomers for all noble gas atoms. The second group of the computational insertion compounds, HCCNgCN and HCCNgNC, are of a different type, and they also are kinetically stable for Ng = Ar, Kr, and Xe. Our photolysis and annealing experiments with low-temperature cyanoacetylene/Ng (Ng = Ar, Kr, and Xe) matrixes evidence the formation of two noble gas hydrides for Ng = Kr and Xe, with the strongest IR absorption bands at 1492.1 and 1624.5 cm -1 , and two additional absorption modes for each species are found. The computational spectra of HKrCCCN and HXeCCCN fit most closely the experimental data, which is the basis for our assignment. The obtained species absorb at quite similar frequencies as the known HKrCN and HXeCN molecules, which is in agreement with the theoretical predictions. No strong candidates for an Ar compound are observed in the IR absorption spectra. As an important side product of this work, the data obtained in long-term decay of KrHKr + cations suggest a tentative assignment for the CCCN radical.

Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields

Abstract: : The unpolarized absorption and circular dichroism spectra of the fundamental vibrational transitions of the chiral molecule, 4-methyl-2-oxetanone, are calculated ab initio. Harmonic force fields are obtained using Density Functional Theory (DFT), MP2, and SCF methodologies and a 5S4P2D/3S2P (TZ2P) basis set. DFT calculations use the Local Spin Density Approximation (LSDA), BLYP, and Becke3LYP (B3LYP) density functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with experiment. The MP2 force field yields spectra in slightly worse agreement with experiment than the B3LYP force field. The SCF force field yields spectra in poor agreement with experiment.The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreements with experiment. jg

Three-dimensional intact-tissue sequencing of single-cell transcriptional states.

Abstract: Retrieving high-content gene-expression information while retaining three-dimensional (3D) positional anatomy at cellular resolution has been difficult, limiting integrative understanding of structure and function in complex biological tissues. We developed and applied a technology for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. The capabilities of STARmap were tested by mapping 160 to 1020 genes simultaneously in sections of mouse brain at single-cell resolution with high efficiency, accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined gradient distribution of excitatory-neuron subtypes across cubic millimeter–scale volumes (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that could be identified and described with 3D STARmap.

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Abstract: Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

Abstract: In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging – analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry – analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging – digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.