Phasor-based hyperspectral snapshot microscopy allows fast imaging of live, three-dimensional tissues for biomedical applications.

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the rule of linear combinations of the phasor could spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells over time. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging and fit-free analysis based on linear combinations broadly available to researchers and the public.

Non plasmonic semiconductor quantum SERS probe as a pathway for in vitro cancer detection.

Surface-enhanced Raman scattering (SERS)-based cancer diagnostics is an important analytical tool in early detection of cancer. Current work in SERS focuses on plasmonic nanomaterials that suffer from coagulation, selectivity, and adverse biocompatibility when used in vitro, limiting this research to stand-alone biomolecule sensing. Here we introduce a label-free, biocompatible, ZnO-based, 3D semiconductor quantum probe as a pathway for in vitro diagnosis of cancer. By reducing size of the probes to quantum scale, we observed a unique phenomenon of exponential increase in the SERS enhancement up to ~106 at nanomolar concentration. The quantum probes are decorated on a nano-dendrite platform functionalized for cell adhesion, proliferation, and label-free application. The quantum probes demonstrate discrimination of cancerous and non-cancerous cells along with biomolecular sensing of DNA, RNA, proteins and lipids in vitro. The limit of detection is up to a single-cell-level detection.

Chemical imaging of live fibroblasts by SERS effective nanofilm.

Reliable and strong surface enhanced Raman scattering (SERS) signatures of intracellular compartments in live NIH3T3 fibroblasts are collected in real time by means of SERS active thin nanofilm (30 nm) on colloidal silica (1.5 µm). Nanofilm is composed of preformed silver nanoparticles in the matrix of polyacrylic acid, protecting against heating (37 °C) in water, or culture medium or phosphate buffered saline aqueous solution. The SERS enhancement factors (EFs) of the order 10(8) allow single biomolecule detection in the native environment of a single live cell. Primary and secondary SERS hot spots of nanofilm are responsible for such high EFs. A slow SERS EF intensity decay occurs over a broader distance of micron silica with nanofilm, not achievable in a common core-shell model (silver nanoparticle coated with a thin silica layer). Extensive local field EFs and SERS EFs are mainly delivered by prolate silver nanoparticles ("rugby-like" shape). This is achieved if an incident field is polarized along the z-axis and the direction of incident polarization and main axis (z) are perpendicular to each other, not observable in water or on gold.

Composite polymer systems with control of local substrate elasticity and their effect on cytoskeletal and morphological characteristics of adherent cells.

At the interface between extracellular substrates and biological materials, substrate elasticity strongly influences cell morphology and function. The associated biological ramifications comprise a diversity of critical responses including apoptosis, differentiation, and motility, which can affect medical devices such as stents. The interactions of the extracellular environment with the substrate are also affected by local properties wherein cells sense and respond to different physical inputs. To investigate the effects of having localized elasticity control of substrate microenvironments on cell response, we have developed a method to control material interface interactions with cells by dictating local substrate elasticity. This system is created by generating a composite material system with alternating, linear regions of polymers that have distinct stiffness characteristics. This approach was used to examine cytoskeletal and morphological changes in NIH 3T3 fibroblasts with emphasis on both local and global properties, noting that cells sense and respond to distinct material elasticities. Isolated cells sense and respond to these local differences in substrate elasticity by extending processes along the interface. Also, cells grown on softer elastic regions at higher densities (in contact with each other) have a higher projected area than isolated cells. Furthermore, when using chemical agents such as cytochalasin-D to disrupt the actin cytoskeleton, there is a significant increase in projected area for cells cultured on softer elastic regions This method has the potential to promote understanding of biomaterial-affected responses in a diversity of areas including morphogenesis, mechanotransduction, stents, and stem cell differentiation.

Potential of 'flat' fibre evanescent wave spectroscopy to discriminate between normal and malignant cells in vitro.

The present study focuses on evaluating the potential of flattened AgClBr fibre-optic evanescent wave spectroscopy (FTIR-FEWS) technique for detection and identification of cancer cells in vitro using cell culture as a model system. The FTIR-FEWS results are compared to those from FTIR-microspectroscopy (FTIR-MSP) method extensively used to identify spectral properties of intact cells. Ten different samples of control and malignant cells were measured in parallel by the above two methods. Our results show a significant similarity between the results obtained by the two methodologies. The absorbance level of Amide I/Amide II, phosphates and carbohydrates were significantly altered in malignant compared to the normal cells using both systems. Thus, common biomarkers such as Amide I/Amide II, phosphate and carbohydrate levels can be derived to discern between normal and cancer cells. However, marked differences are also noted between the two methodologies in the protein bands due to CH3 bending vibration (1480-1350 cm(-1)). The spectral differences may be attributed to the variation in the penetration depth of the two methodologies. The use of flattened fibre rather than the standard cylindrical fibre has several practical advantages and is considered as an important step towards in vivo measurements in real time, such as that of skin nevi and melanoma using special designs of fibre-optic-based sensors.

ALK-mediated Na+/H+ exchanger-dependent intracellular alkalinization: does it matter for oncogenesis?

In this study we investigated the relevance of the oncogenic protein NPM-ALK in regulating cellular pH (pHi) through the modulation of Na+/H+ exchanger 1 (NHE1)-activity and the consequences of pHi pharmacological manipulation in cells expressing NPM-ALK.

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase.

Myosin light chain kinases (MLCK) phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase (S-MLCK) to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase (L-MLCK) has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules (2Ig) in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased "spike"-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.

Synthesis and characterisation of highly emissive and kinetically stable lanthanide complexes suitable for usage "in cellulo".

The synthesis and photophysical characterisation are reported of a series of cationic, neutral and anionic europium and terbium complexes based on structurally related, nonadentate ligands based on the cyclen macrocycle. Each complex incorporates a tetraazatriphenylene moiety and overall absolute emission quantum yields are in the range 15-40% in aerated aqueous media. Dynamic quenching of the lanthanide excited state occurs with electron-rich donors, e.g. iodide, ascorbate and urate, and a mechanistic interpretation is put forward involving an electron transfer process. The cationic lanthanide complexes are taken up by NlH/3T3 cells and tend to localise inside the cell nucleus.

Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis.

Cytoglobin/stellate cell activation-associated protein (Cygb/STAP) consists of a new class of hexacoordinate globin superfamily, which was recently discovered by a proteome analysis on the rat hepatic stellate cells. Unlike haemoglobin, myoglobin, and neuroglobin, Cygb/STAP is ubiquitously expressed in several organs, although its detailed localization has not been clarified. Immunohistochemistry and immunoelectron microscopy revealed that Cygb/STAP is uniquely localized in fibroblast-like cells in splanchnic organs, namely the vitamin A-storing cell lineage, but neither in epithelial cells, endothelial cells, muscle cells, blood cells, macrophages, nor dermal fibroblasts. The expression of Cygb/STAP was upregulated in fibrotic lesions of the pancreas and kidney in which activated fibroblast-like cells or myofibroblasts are known to increase in number. In cultured hepatic stellate cells, Cygb/STAP expression was augmented by the stimulation with sera, platelet-derived growth factor-BB, and transforming growth factor-beta 1. Overexpression of Cygb/STAP in NIH 3T3 cells induced the cells to lessen migratory activities and increase the expression of collagen alpha1(I) mRNA. These results indicate that Cygb/STAP is a tissue globin uniquely localized in splanchnic fibroblastic cell lineage and may play a role in fibrotic organ disorder.