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Abstract: Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by IR wavelength making subcellular study challenging. Recently we have reported, with the use of a prototype ZnS transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron– IR microscopy with a 36x objective with numerical aperture (NA) of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron single point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time whilst measurements from the scanning methods produced a smoother image. Synchrotron single point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.

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Abstract: Nanocomposites comprising metal organic frameworks (MOFs) embedded in a polymeric matrix are promising carriers for drug delivery applications. While understanding the chemical and physical transformations of MOFs during the re-lease of confined drug molecules is challenging, this is central to devising better ways for controlled release of therapeutic agents. Herein we demonstrate the efficacy of synchrotron microspectroscopy to track the in situ release of 5-fluorouracil (5-FU) anticancer drug molecules from a drug@MOF/polymer composite (5-FU@HKUST-1/polyurethane). Using experimental time-resolved infrared spectra jointly with newly developed density functional theory calculations, we reveal the detailed dynamics of vibrational motions underpinning the dissociation of 5-FU bound to the framework of HKUST-1 upon water exposure. We discover that HKUST-1 creates hydrophilic channels within the hydrophobic polyurethane matrix hence helping to tune drug release rate. The synergy between a hydrophilic MOF with a hydrophobic polymer can be harnessed to engineer a tunable nanocomposite that alleviates the unwanted burst effect commonly encountered in drug delivery.

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Abstract: Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic sampling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic “bottlenecks” introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN– ligands, linked to the protein via bridging and terminal cysteine sulfur ligands, as well as an electron relay chain of iron sulfur clusters. Infrared spectroscopy is ideal for probing hydrogenases because the CO and CN– ligands are strong IR absorbers, but the suite of IR-based approaches we describe here will be equally valuable in studying substrate- or intermediate-bound states of other metalloenzymes where key mechanistic questions remain open, such as nitrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase. We therefore hope that this Account will encourage future studies which unify information from different techniques across bioinorganic chemistry.

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Abstract: Research on the broadband dielectric response of metal-organic frameworks (MOFs) is an emergent field that could yield exciting device applications, such as smart optoelectronics, terahertz sensors, high-speed telecommunications and microelectronics. Hitherto, a detailed understanding of the physical mechanisms controlling the frequency-dependent dielectric and optical behavior of MOFs is lacking because a large number of studies have focused only on static dielectric constants. Herein we employed high-resolution spectroscopic techniques in combination with periodic ab initio density functional theory (DFT) calculations to establish the different polarization processes for a porous copper-based MOF, termed HKUST-1. We used alternating current measurements to determine its dielectric response between 4 Hz and 1.5 MHz where orientational polarization is predominant, while synchrotron infrared (IR) reflectance was used to probe the far-IR, mid-IR, and near-IR dielectric response across the 1.2 THz to 150 THz range (ca. 40 – 5000 cm-1) where vibrational and optical polarizations are principal contributors to its dielectric permittivity. We demonstrate the role of pressure on the evolution of broadband dielectric response, where THz vibrations reveal distinct blue and red shifts of phonon modes from structural deformation of the copper paddle-wheel and the organic linker, respectively. We also investigated the effect of temperature on dielectric constants in the MHz region pertinent to microelectronics, to study temperature-dependent dielectric losses via dissipation in an alternating electric field. The DFT calculations offer insights into the physical mechanisms responsible for dielectric transitions observed in the experiments and enable us to explain the frequency shifts phenomenon detected under pressure. Together, the experiments and theory have enabled us to glimpse into the complex dielectric response and mechanisms underpinning a prototypical MOF subject to pressure, temperature, and vast frequencies.

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Abstract: The mid‐infrared (IR) spectra of human cystic fibrosis (CF) cells acquired by Fourier transform infrared microspectroscopy (microFTIR) were compared to those of non‐CF cells. Within the 1700‐1480cm‐1 spectral domain of amides, unsupervised explorative principal component analysis identified a few variables reflecting quantitative and qualitative vibrations arising from protein secondary structures and amino acid side chains. Their pattern reflected α‐helix to β‐sheet transitions in bronchial epithelial cells and in immortalized peripheral blood mononuclear cells from patients with R1162X missense or in‐frame F508del mutations in the cystic fibrosis transmembrane regulator gene (Cftr). Similar transitions have been described in IR spectra of cells, tissues and body fluids of patients affected with some neurodegenerative diseases characterized by the accumulation of misfolded protein aggregates. The variables pattern was able to distinguish CF cells from non‐CF cells and was modified by molecular compounds used to rescue the unbalanced folding process of mutated cystic fibrosis transmembrane regulator (CFTR) anion channel. To our knowledge, this is the first experimental evidence of spectroscopic biomarkers of the impaired biogenesis of CFTR by IR microanalysis in the spectra of human CF bronchial epithelial and lymphoblasotid cells.