Machine learning-based diagnostics of capsular invasion in thyroid nodules with wide-field second harmonic generation microscopy.

Papillary thyroid carcinoma (PTC) is one of the most common, well-differentiated carcinomas of the thyroid gland. PTC nodules are often surrounded by a collagen capsule that prevents the spread of cancer cells. However, as the malignant tumor progresses, the integrity of this protective barrier is compromised, and cancer cells invade the surroundings.

Widefield polarization-resolved second harmonic generation imaging of entire thyroid nodule sections for the detection of capsular invasion.

The identification of tumor capsular invasion as a sign of malignancy is currently employed in traditional histopathology routines for thyroid nodules. However, its limitations are associated with the assessment criteria for invasion, which often lead to disagreements among observers. The aim of this paper is to introduce a widefield imaging technique combined with quantitative collagen analysis to identify areas of capsular invasion in thyroid neoplasms.

Programmable high-speed confocal reflectance microscopy enabled by a digital micromirror device.

The digital micromirror device (DMD) has been used to achieve parallel scanning in confocal microscopy significantly increasing acquisition speed. However, for confocal reflectance imaging, such an approach is limited to mostly surface imaging due to strong backreflections coming from the DMD that can dominate the signal recorded on a camera. Here, we report on an optical configuration that uses separate areas of DMD to generate multiple spots and pinholes and thereby prevents backreflections from the DMD from reaching the camera.

The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos.

smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease.

Protein-DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans.

Time-domain full-field optical coherence tomography with digital confocal line scanning.

Full-field optical coherence tomography (FF-OCT) is a camera-based interferometric microscopy technique that can image deep in tissue with high spatial resolution. However, the absence of confocal gating leads to suboptimal imaging depth. Here, we implement digital confocal line scanning in time-domain FF-OCT by exploiting the row-by-row detection feature of a rolling-shutter camera.

Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy.

We demonstrated that wide-field second harmonic generation (SHG) microscopy of lung tissue in combination with quantitative analysis of SHG images is a powerful tool for fast and label-free visualization of the fibrosis pathogenesis in pulmonary arterial hypertension (PAH). Statistical analysis of the SHG images revealed changes of the collagen content and morphology in the lung tissue during the monocrotaline-induced PAH progression in rats. First order statistics disclosed the dependence of the collagen overproduction on time, the second order statistics indicated tightening of collagen fiber network around blood vessels and their spreading into the alveolar region.

Visualizing hypochlorous acid production by human neutrophils with fluorescent graphene quantum dots.

In living organisms, redox reactions play a crucial role in the progression of disorders accompanied by the overproduction of reactive oxygen and reactive chlorine species, such as hydrogen peroxide and hypochlorous acid, respectively. We demonstrate that green fluorescence graphene quantum dots (GQDs) can be employed for revealing the presence of the hypochlorous acid in aqueous solutions and cellular systems. Hypochlorous acid modifies the oxygen-containing groups of the GQD, predominantly opens epoxide ring C-O-C, forms excessive C=O bonds and damages the carbonic core of GQDs.

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos.

Surface-Enhanced Raman Spectroscopy of Organic Molecules and Living Cells with Gold-Plated Black Silicon.

Black silicon (bSi) refers to an etched silicon surface comprising arrays of microcones that effectively suppress reflection from UV to near-infrared (NIR) while simultaneously enhancing the scattering and absorption of light. This makes bSi covered with a nm-thin layer of plasmonic metal, i.e.

Characterization of thymine microcrystals by CARS and SHG microscopy.

Identification of chemically homologous microcrystals in a polycrystal sample is a big challenge and requires developing specific highly sensitive tools. Second harmonic (SHG) and coherent anti-Stokes Raman scattering (CARS) spectroscopy can be used to reveal arrangement of thymine molecules, one of the DNA bases, in microcrystalline sample. Strong dependence of CARS and SHG intensity on the orientation of the linear polarization of the excitation light allows to obtain high resolution images of thymine microcrystals by additionally utilizing the scanning microscopy technique.

Macro-, Micro- and Nano-Roughness of Carbon-Based Interface with the Living Cells: Towards a Versatile Bio-Sensing Platform.

Integration of living cells with nonbiological surfaces (substrates) of sensors, scaffolds, and implants implies severe restrictions on the interface quality and properties, which broadly cover all elements of the interaction between the living and artificial systems (materials, surface modifications, drug-eluting coatings, etc.). Substrate materials must support cellular viability, preserve sterility, and at the same time allow real-time analysis and control of cellular activity.

Recognition of Spatial Distribution of CNT and Graphene in Hybrid Structure by Mapping with Coherent Anti-Stokes Raman Microscopy.

The shape of coherent anti-Stokes Raman scattering (CARS) spectral line depends on the ratio of the vibrational and electronic contributions to the third-order susceptibility of the material. The G-mode (1590 cm) of graphene and carbon nanotubes (CNTs) exhibits opposite features in the CARS spectrum, showing "dip" and "peak," respectively. Here, we consider the CARS spectra of graphene and carbon nanotubes in terms of Fano formalism describing the line shapes of CARS resonances.

Influence of the Carotenoid Composition on the Conformational Dynamics of Photosynthetic Light-Harvesting Complexes.

Nonphotochemical quenching (NPQ) is the major self-regulatory mechanism of green plants, performed on a molecular level to protect them from an overexcitation during the direct sunlight. It is believed that NPQ becomes available due to conformational dynamics of the light-harvesting photosynthetic complexes and involves a direct participation of carotenoids. In this work, we perform a single-molecule microscopy on major light-harvesting complexes (LHCII) from different Arabidopsis thaliana mutants exhibiting various carotenoid composition.

Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV.

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field.

The single-molecule Förster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field.

Restriction enzyme Ecl18kI-induced DNA looping dynamics by single-molecule FRET.

Many type II restriction endonucleases require binding of two copies of a recognition site for efficient DNA cleavage. Simultaneous interaction of the enzyme with two DNA sites results in DNA loop formation. It was demonstrated with the tethered particle motion technique that such looping is a dynamic process where a DNA loop is repeatedly formed and disrupted.

Tetramer opening in LacI-mediated DNA looping.

Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain).

Spectral dynamics of individual bacterial light-harvesting complexes: alternative disorder model.

The bacterial (Rhodopseudomonas acidophila) photosynthetic peripheral light-harvesting complex of type 2 (LH2) exhibits rich fluorescence spectral dynamics at room temperature. The fluorescence spectrum of individual LH2 shifts either to the blue or to the red during the experimental observation time of a few minutes. These spectral changes are often reversible and occur between levels of a distinctly different peak wavelength.

Dynamics of the emission spectrum of a single LH2 complex: interplay of slow and fast nuclear motions.

We have studied the relationship between the realizations of static disorder and the emission spectra observed for a single LH2 complex. We show that the experimentally observed spectral fluctuations reflect realizations of the disorder in the B850 ring associated with different degrees of exciton delocalization and different effective coupling of the excitons to phonon modes. The main spectral features cannot be explained using models with correlated disorder associated with elliptical deformations of the ring.

Conformational relaxation of single bacterial light-harvesting complexes.

We have employed the technique of single-molecule fluorescence microspectroscopy to investigate the spontaneous conformational evolution of individual peripheral LH2 complexes from the purple bacterium Rhodopseudomonas acidophila. Fluorescence microscopy is a sensitive tool, which allows the spectral changes of single complexes to be monitored on a time scale from 0.1 s to many minutes.

Comparative study of spectral flexibilities of bacterial light-harvesting complexes: structural implications.

This work presents a comparative study of the frequencies of spectral jumping of individual light-harvesting complexes of six different types: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum; LH1 of Rhodobacter sphaeroides; and two "domain swap mutants" of LH2 of Rhodobacter sphaeroides: PACLH1 and PACLH2mol, in which the alpha-polypeptide C-terminus is exchanged with the corresponding sequence from LH1 of Rhodobacter sphaeroides or LH2 of Rhodospirillum molischianum, respectively. The quasistable states of fluorescence peak wavelength that were previously observed for the LH2 of Rps. acidophila were confirmed for other species.

Spectral trends in the fluorescence of single bacterial light-harvesting complexes: experiments and modified redfield simulations.

In this work we present and discuss the single-molecule fluorescence spectra of a variety of species of light-harvesting complexes: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum and LH1 of Rhodobacter sphaeroides. The emission spectrum of these complexes varies as a function of time as was described in earlier work. For each type of complex, we observe a pronounced and well-reproducible characteristic relationship between the fluorescence spectral parameters of the peak wavelength, width, and asymmetry.

Fluorescence spectroscopy of conformational changes of single LH2 complexes.

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity.

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050.

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum.