Multimodal imaging of a liver-on-a-chip model using labelled and label-free optical microscopy techniques.

A liver-on-a-chip model is an advanced complex in vitro model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques. In this study, the potential of both labelled as well as label-free multimodal optical imaging techniques for visualization and characterization of the cellular and sub-cellular features of a liver-on-a-chip model was investigated. (1) Cellular uptake and distribution of Alexa 488 (A488)-labelled non-targeted and targeted antisense oligonucleotides (ASO and ASO-GalNAc) in the liver-on-a-chip model was determined using multiphoton microscopy. (2) Hyperspectral stimulated Raman scattering (SRS) microscopy of the C-H region was used to determine the heterogeneity of chemical composition of circular and cuboidal hepatocytes in the liver-on-a-chip model in a label-free manner. Additionally, the spatial overlap between the intracellular localization of ASO and lipid droplets was explored using simultaneous hyperspectral SRS and fluorescence microscopy. (3) The capability of light sheet fluorescence microscopy (LSFM) for full-depth 3D visualization of sub-cellular distribution of A488-ASO and cellular phenotypes in the liver-on-a-chip model was demonstrated. In summary, multimodal optical microscopy is a promising platform that can be utilized for visualization and quantification of 3D cellular organization, drug distribution and functional changes occurring in liver-on-a-chip models, and can provide valuable insights into liver biology and drug uptake mechanisms by enabling better characterization of these liver models.

Infrared spectra and fragmentation dynamics of isotopologue-selective mixed-ligand complexes.

Isolated mixed-ligand complexes provide tractable model systems in which to study competitive and cooperative binding effects as well as controlled energy flow. Here, we report spectroscopic and isotopologue-selective infrared photofragmentation dynamics of mixed gas-phase Au(12/13CO)n(N2O)m+ complexes. The rich infrared action spectra, which are reproduced well using simulations of calculated lowest energy structures, clarify previous ambiguities in the assignment of vibrational bands, especially accidental coincidence of CO and N2O bands. The fragmentation dynamics exhibit the same unexpected behaviour as reported previously in which, once CO loss channels are energetically accessible, these dominate the fragmentation branching ratios, despite the much lower binding energy of N2O. We have investigated the dynamics computationally by considering anharmonic couplings between a relevant subset of normal modes involving both ligand stretch and intermolecular modes. Discrepancies between correlated and uncorrelated model fit to the ab initio potential energy curves are quantified using a Boltzmann sampled root mean squared deviation providing insight into efficiency of vibrational energy transfer between high frequency ligand stretches and the softer intermolecular modes which break during fragmentation.

A Combined Infrared and Computational Study of Gas-Phase Mixed-Ligand Rhodium Complexes: Rh(CO)n(N2O)m+ (n = 1-5, m = 1-4).

In this study, mixed carbonyl and nitrous oxide complexes with Rh+ were studied by mass-selective infrared photodissociation spectroscopy in a molecular beam. The infrared spectra, recorded in the region of the CO and N2O N═N stretches, were assigned and interpreted with the aid of simulated spectra of low-energy structural isomers. Clear evidence of an inner coordination shell of four ligands is observed. The observed vibrational structure can be understood on the basis of local mode vibrations in the two ligands. However, there is also evidence of multiple low-lying isomers and cooperative binding effects between the two ligands. In particular, σ donation from directly coordinated nitrous oxide ligands drives more classical carbonyl bonding than has been observed in pure carbonyl complexes. The observed fragmentation branching ratios following resonant infrared absorption are explained by simple statistical and energetic arguments, providing a contrast with those of equivalent Au+ complexes.

Closing the Shell: Gas-Phase Solvation of Halides by 1,3-Butadiene.

Gas-phase solvation of halides by 1,3-butadiene has been studied via a combination of photoelectron spectroscopy and density functional theory. Photoelectron spectra for X- ⋯(C4 H6 )n (X=Cl, Br, I where n=1-3, 1-3 and 1-7 respectively) are presented. For all complexes, the calculated structures indicate that butadiene is bound in a bidentate fashion through hydrogen-bonding, with the chloride complex showing the greatest degree of stabilisation of the internal C-C rotation of cis-butadiene. In both Cl- and Br- complexes, the first solvation shell is shown to be at least n = 4 ${n = 4}$ from the vertical detachment energies (VDEs), however for I- , increases in the VDE may suggest a metastable, partially filled, first solvation shell for n = 4 ${n = 4}$ and a complete shell at n = 6 ${n = 6}$ . These results have implications for gas-phase clustering in atmospheric and extraterrestrial environments.

Hydrogen Bonding versus Halogen Bonding: Spectroscopic Investigation of Gas-Phase Complexes Involving Bromide and Chloromethanes.

Hydrogen bonding and halogen bonding are important non-covalent interactions that are known to occur in large molecular systems, such as in proteins and crystal structures. Although these interactions are important on a large scale, studying hydrogen and halogen bonding in small, gas-phase chemical species allows for the binding strengths to be determined and compared at a fundamental level. In this study, anion photoelectron spectra are presented for the gas-phase complexes involving bromide and the four chloromethanes, CH3 Cl, CH2 Cl2 , CHCl3 , and CCl4 . The stabilisation energy and electron binding energy associated with each complex are determined experimentally, and the spectra are rationalised by high-level CCSD(T) calculations to determine the non-covalent interactions binding the complexes. These calculations involve nucleophilic bromide and electrophilic bromine interactions with chloromethanes, where the binding motifs, dissociation energies and vertical detachment energies are compared in terms of hydrogen bonding and halogen bonding.

An Infrared Study of Gas-Phase Metal Nitrosyl Ion-Molecule Complexes.

We present a combined experimental and quantum chemical study of gas-phase group 9 metal nitrosyl complexes, M(NO)n+ (M = Co, Rh, Ir). Experimental infrared photodissociation spectra of mass-selected ion-molecule complexes are presented in the region 1600 cm-1 to 2000 cm-1 which includes the NO stretch. These are interpreted by comparison with the simulated spectra of energetically low-lying structures calculated using density functional theory. A mix of linear and nonlinear ligand binding is observed, often within the same complex, and clear evidence of coordination shell closing is observed at n = 4 for Co(NO)n+ and Ir(NO)n+. Calculations of Rh(NO)n+ complexes suggest additional low-lying five-coordinate structures. In all cases, once a second coordination shell is occupied, new spectral features appear which are assigned to (NO)2 dimer moieties. Further evidence of such motifs comes from differences in the spectra recorded in the dissociation channels corresponding to single and double ligand loss.

Halide-propene complexes: validated DSD-PBEP86-D3BJ calculations and photoelectron spectroscopy.

Anion photoelectron spectroscopy has been used to determine the electron binding energies of the X-⋯C3H6 (X = Cl, Br, I) complexes. To complement the experimental spectra the DSD-PBEP86-D3BJ functional has been employed, following comparison with previously calculated halide/halogen-molecule van der Waals complexes. To validate the functional, comparison between the complex geometries and vertical detachment energies with both experimental and CCSD(T)/CBS data for a suite of halide-molecule complexes is also made. PES spectra determine the electron binding energies as 3.89 eV and 4.00 eV, 3.59 eV and 4.01 eV, and 3.26 eV and 4.20 eV for transitions to perturbed 2P states of the chlorine, bromine and iodine complexes respectively. Two contributing structures resulting in the photoelectron spectrum are those where the halide is coordinated by two hydrogens, each from a terminal carbon in C3H6, and when bifurcating the CC bond. These complexes are distinct from the corresponding halide-ethene complexes and represent potential entry pathways to haloakyl radical formation in atmospheric and extraterrestrial environments.

A tale of two conformers: spectroscopic evidence for halide catalysed formic acid isomerisation.

Halide-formic acid complexes have been studied utilising a combined experimental and theoretical approach. Formic acid exists as two conformers, distinguished by the relative rotation about the C-OH bond. Computational investigation of the formic acid isomerisation reaction between the two conformers has revealed the ability of halide anions to catalyse the formation of, and preferentially stabilise, the higher energy conformer. Anion photoelectron spectroscopy has been used to study the halide-formic acid complexes, with the experimental vertical detachment energies compared with simulated photodetachment energies with respect to halide complexes with both formic acid conformers. The existence of experimental spectral features associated with halide complexes of the higher energy formic acid confomer confirms in situ generation, likely as a result of the halide mediated catalytic formation.

Spectroscopic Study of the Br- +CH3 I→I- +CH3 Br SN 2 Reaction.

Mass spectrometry and anion photoelectron spectroscopy have been used to study the gas-phase S N 2 ${{{\rm S}}_{{\rm N}}2}$ reaction involving B r - ${{{\rm B}{\rm r}}^{-}}$ and C H 3 I ${{{\rm C}{\rm H}}_{3}{\rm I}}$ . The anion photoelectron spectra associated with the reaction intermediates of this S N 2 ${{{\rm S}}_{{\rm N}}2}$ reaction are presented. High-level CCSD(T) calculations have been utilised to investigate the reaction intermediates that may form as a result of the S N 2 ${{{\rm S}}_{{\rm N}}2}$ reaction along various different reaction pathways, including back-side attack and front-side attack. In addition, simulated vertical detachment energies of each reaction intermediate have been calculated to rationalise the photoelectron spectra.

Photoelectron Spectroscopy and High-Level Ab Initio Calculations of the Iodide-Methylperoxy Radical Complex.

Anion photoelectron spectroscopy has been used to investigate the structure and dynamics of CH3OOI- van der Waals complexes. Peaks within the photoelectron spectrum are attributed to photodetachment to the perturbed 2P3/2 state of I···CH3OO (3.46 eV) and the two 2P states of bare iodine. A broad feature at 1.7-2.4 eV is attributed to detachment to the excited singlet states from two O2-···CH3I complexes. This represents the first anion photoelectron spectroscopy of a halide-bound methylperoxy radical species. Complex structures have been optimized using MP2/aug-cc-pVQZ with single-point energies at W1w theory for ground-state complexes and NEVPT2 for photodetachment to excited O2. Interactions are dominated by electrostatics, with the anion species interacting with the methyl pocket of the solvating molecule, suggesting conversion via an SN2 mechanism, and excess energy leading to complex dissociation within the timescale of mass spectrometry. The calculated W1w Gibbs energies suggest that while an electron transfer (ET) pathway to conversion is available, it is comparatively unfavored.

Towards an Understanding of Halide Interactions with the Carbonyl-Containing Molecule CH3 CHO.

The anion photoelectron spectra of Cl- ⋅⋅⋅CD3 CDO, Cl- ⋅⋅⋅(CD3 CDO)2 , Br- ⋅⋅⋅CH3 CHO, and I- ⋅⋅⋅CH3 CHO are presented with electron stabilisation energies of 0.55, 0.93, 0.48, and 0.40 eV, respectively. Optimised geometries of the singly solvated species featured the halide appended to the CH3 CHO molecule in-line with the electropositive portion of the C=O bond and having binding energies between 45 and 52 kJ mol-1 . The doubly solvated Cl- ⋅⋅⋅(CH3 CHO)2 species features asymmetric solvation upon the addition of a second CH3 CHO molecule. Theoretical detachment energies were found to be in excellent agreement with experiment, with comparisons drawn between other halide complexes with simple carbonyl molecules.

Spectroscopic Investigation of Chalcogen Bonding: Halide-Carbon Disulfide Complexes.

A combined experimental and theoretical approach has been used to study intermolecular chalcogen bonding. Specifically, the chalcogen bonding occurring between halide anions and CS2 molecules has been investigated using both anion photoelectron spectroscopy and high-level CCSD(T) calculations. The relative strength of the chalcogen bond has been determined computationally using the complex dissociation energies as well as experimentally using the electron stabilisation energies. The anion complexes featured dissociation energies on the order of 47 kJ/mol to 37 kJ/mol, decreasing with increasing halide size. Additionally, the corresponding neutral complexes have been examined computationally, and show three loosely-bound structural motifs and a molecular radical.

Photoelectron Spectroscopy and Structures of X- ⋠⋠⋠CH2 O (X=F, Cl, Br, I) Complexes.

A combined experimental and theoretical approach has been used to investigate X- ⋅⋅⋅CH2 O (X=F, Cl, Br, I) complexes in the gas phase. Photoelectron spectroscopy, in tandem with time-of-flight mass spectrometry, has been used to determine electron binding energies for the Cl- ⋅⋅⋅CH2 O, Br- ⋅⋅⋅CH2 O, and I- ⋅⋅⋅CH2 O species. Additionally, high-level CCSD(T) calculations found a C2v minimum for these three anion complexes, with predicted electron detachment energies in excellent agreement with the experimental photoelectron spectra. F- ⋅⋅⋅CH2 O was also studied theoretically, with a Cs hydrogen-bonded complex found to be the global minimum. Calculations extended to neutral X⋅⋅⋅CH2 O complexes, with the results of potential interest to atmospheric CH2 O chemistry.

Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids.

The development of in vitro culture systems quantitatively and qualitatively recapitulating normal breast biology is key to the understanding of mammary gland biology. Current three-dimensional mammary culture systems have not demonstrated concurrent proliferation and functional differentiation ex vivo in any system for longer than 2 weeks. Here, we identify conditions including Neuregulin1 and R-spondin 1, allowing maintenance and expansion of mammary organoids for 2.5 months in culture. The organoids comprise distinct basal and luminal compartments complete with functional steroid receptors and stem/progenitor cells able to reconstitute a complete mammary gland in vivo. Alternative conditions are also described that promote enrichment of basal cells organized into multiple layers surrounding a keratinous core, reminiscent of structures observed in MMTV-Wnt1 tumours. These conditions comprise a unique tool that should further understanding of normal mammary gland development, the molecular mechanism of hormone action and signalling events whose deregulation leads to breast tumourigenesis.

Halide-Nitrogen Gas-Phase Clusters: Anion Photoelectron Spectroscopy and High Level ab Initio Calculations.

The gas phase anion photoelectron spectra are presented for the halide-nitrogen clusters X(-)···(N2)n, where X = Br and I and n ≤ 5. Electron binding energies for each cluster in the halide series are determined, with no evidence observed for first solvation shell closure in either series. High level ab initio calculations at the CCSD(T) level of theory are presented for the anion and neutral halogen-nitrogen complexes. For the anion species, two minima are predicted corresponding to a loosely bound C2v "T-shaped" species and to a higher energy covalently bound "triangle" C2v symmetry geometry. For the neutral species, three stationary points were located, two of which display similar form to the anion minima and a third which is linear, i.e., C∞v symmetry. The "T-shaped" geometry is a transition state linking equivalent C∞v symmetry minima. Cluster dissociation energies (D0) were determined, for both anion and neutral global minima at the CCSD(T) complete basis set limit, to be 7.8 kJ mol(-1) and 7.0 kJ mol(-1) and 3.5 kJ mol(-1) and 5.0 kJ mol(-1) for the bromine and iodine species, respectively.

Visualizing Actin Architectures in Cells Incubated with Cell-Penetrating Peptides.

Defining the exact role of the actin cytoskeleton in mediating endocytosis through different pathways is a significant challenge. The general consensus is that actin has an important role in organizing the early stages of endocytosis but there is still much to learn. Actin has also been implicated in cell internalization of cell-penetrating peptides (CPPs). It is suggested that CPP variants such as octaarginine (R8) and the HIV Tat peptide induce actin-dependent plasma membrane perturbation and enter via macropinocytosis. Here, we describe confocal microscopy techniques that allow for high-resolution spatial characterization of the actin cytoskeleton in untreated mammalian cells and those incubated with actin-disrupting agents and CPPs. By performing X-Y-Z projection images through different regions of cells to show basal and apical profiles, we initially highlight how these techniques can be used to reveal major differences in cortical and filamentous actin organization between different cell lines. Using these techniques, we demonstrate that the actin-disrupting agent cytochalasin D rapidly changes this framework at concentrations significantly lower than is normally used. Experiments are also performed to highlight that serum starvation significantly sensitizes cells to the effects of R8 on actin-induced ruffling and lamellapodia formation. The techniques described here can be used to gain a higher level of knowledge of the organization of the actin network in individual model cell systems, how this is perturbed using commonly used actin inhibitors, and how plasma membrane reorganization can be induced by the addition of drug delivery vectors such as CPPs.