Nanosensitive optical coherence tomography for detecting structural changes in stem cells.

Mesenchymal stromal cells (MSCs) are adult stem cells that have been widely investigated for their potential to regenerate damaged and diseased tissues. Multiple pre-clinical studies and clinical trials have demonstrated a therapeutic response following treatment with MSCs for various pathologies, including cardiovascular, neurological and orthopaedic diseases. The ability to functionally track cells following administration in vivo is pivotal to further elucidating the mechanism of action and safety profile of these cells. Effective monitoring of MSCs and MSC-derived microvesicles requires an imaging modality capable of providing both quantitative and qualitative readouts. Nanosensitive optical coherence tomography (nsOCT) is a recently developed technique that detects nanoscale structural changes within samples. In this study, we demonstrate for the first time, the capability of nsOCT to image MSC pellets following labelling with different concentrations of dual plasmonic gold nanostars. We show that the mean spatial period of MSC pellets increases following the labelling with increasing concentrations of nanostars. Additionally, with the help of extra time points and a more comprehensive analysis, we further improved the understanding of the MSC pellet chondrogenesis model. Despite the limited penetration depth (similar to conventional OCT), the nsOCT is highly sensitive in detecting structural alterations at the nanoscale, which may provide crucial functional information about cell therapies and their modes of action.

Development of controlled nanosphere lithography technology.

This work is devoted to the development of nanosphere lithography (NSL) technology, which is a low-cost and efficient method to form nanostructures for nanoelectronics, as well as optoelectronic, plasmonic and photovoltaic applications. Creating a nanosphere mask by spin-coating is a promising, but not sufficiently studied method, requiring a large experimental base for different sizes of nanospheres. So, in this work, we investigated the influence of the technological parameters of NSL by spin-coating on the substrate coverage area by a monolayer of nanospheres with a diameter of 300 nm. It was found that the coverage area increases with decreasing spin speed and time, isopropyl and propylene glycol content, and with increasing the content of nanospheres in solution. Moreover, the process of controllably reducing the size of nanospheres in inductively coupled oxygen plasma was studied in detail. It was determined that increasing the oxygen flow rate from 9 to 15 sccm does not change the polystyrene etching rate, whereas changing the high-frequency power from 250 to 500 W increases the etching rate and allows us to control the decreasing diameter with high accuracy. Based on the experimental data, the optimal technological parameters of NSL were selected and the nanosphere mask on Si substrate was created with coverage area of 97.8% and process reproducibility of 98.6%. Subsequently reducing the nanosphere diameter lets us obtain nanoneedles of various sizes, which can be used in field emission cathodes. In this work, the reduction of nanosphere size, silicon etching, and removal of polystyrene residues occurred in unified continuous process of plasma etching without sample unloading to atmosphere.

OES diagnostics as a universal technique to control the Si etching structures profile in ICP.

In this work, we demonstrate the high efficiency of optical emission spectroscopy to estimate the etching profile of silicon structures in SF6/C4F8/O2 plasma. The etching profile is evaluated as a ratio of the emission intensity of the oxygen line (778.1 nm) to the fluorine lines (685.8 nm and 703.9 nm). It was found that for the creation of directional structures with line sizes from 13 to 100 µm and aspect ratio from ≈ 0.15 to ≈ 5 the optimal intensities ratio is in the range of 2-6, and for structures from 400 to 4000 µm with aspect ratio from ≈ 0.03 to ≈ 0.37 it is in the range 1.5-2. Moreover, the influence of the process parameters on the etching rate of silicon, the etching rate of aluminum, the inclination angle of the profile wall of the etched window, the selectivity of silicon etching with respect to aluminum, and the influence on the overetching (Bowing effect) of the structure was investigated.

Skin cancer margin detection using nanosensitive optical coherence tomography and a comparative study with confocal microscopy.

Excision biopsy and histology represent the gold standard for morphological investigation of the skin, in particular for cancer diagnostics. Nevertheless, a biopsy may alter the original morphology, usually requires several weeks for results, is non-repeatable on the same site and always requires an iatrogenic trauma. Hence, diagnosis and clinical management of diseases may be substantially improved by new non-invasive imaging techniques. Optical Coherence Tomography (OCT) is a non-invasive depth-resolved optical imaging modality based on low coherence interferometry that enables high-resolution, cross-sectional imaging in biological tissues and it can be used to obtain both structural and functional information. Beyond the resolution limit, it is not possible to detect structural and functional information using conventional OCT. In this paper, we present a recently developed technique, nanosensitive OCT (nsOCT), improved using broadband supercontinuum laser, and demonstrate nanoscale sensitivity to structural changes within ex vivo human skin tissue. The extended spectral bandwidth permitted access to a wider distribution of spatial frequencies and improved the dynamic range of the nsOCT. Firstly, we demonstrate numerical and experimental detection of a few nanometers structural difference using the nsOCT method from single B-scan images of phantoms with sub-micron periodic structures, acting like Bragg gratings, along the depth. Secondly, our study shows that nsOCT can distinguish nanoscale structural changes at the skin cancer margin from the healthy region in en face images at clinically relevant depths. Finally, we compare the nsOCT en face image with a high-resolution confocal microscopy image to confirm the structural differences between the healthy and lesional/cancerous regions, allowing the detection of the skin cancer margin.

Accessing depth-resolved high spatial frequency content from the optical coherence tomography signal.

Optical coherence tomography (OCT) is a rapidly evolving technology with a broad range of applications, including biomedical imaging and diagnosis. Conventional intensity-based OCT provides depth-resolved imaging with a typical resolution and sensitivity to structural alterations of about 5-10 microns. It would be desirable for functional biological imaging to detect smaller features in tissues due to the nature of pathological processes. In this article, we perform the analysis of the spatial frequency content of the OCT signal based on scattering theory. We demonstrate that the OCT signal, even at limited spectral bandwidth, contains information about high spatial frequencies present in the object which relates to the small, sub-wavelength size structures. Experimental single frame imaging of phantoms with well-known sub-micron internal structures confirms the theory. Examples of visualization of the nanoscale structural changes within mesenchymal stem cells (MSC), which are invisible using conventional OCT, are also shown. Presented results provide a theoretical and experimental basis for the extraction of high spatial frequency information to substantially improve the sensitivity of OCT to structural alterations at clinically relevant depths.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression.

SIGNIFICANCE: Assessment of disease using optical coherence tomography is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis, and treatment response monitoring. The early manifestation of disease or precancer is typically associated with subtle alterations in the tissue dielectric and ultrastructural morphology. In addition, biological tissue is known to have ultrastructural multifractality. AIM: Detection and characterization of nanosensitive structural morphology and multifractality in the tissue submicron structure. Quantification of nanosensitive multifractality and its alteration in progression of tumor. APPROACH: We have developed a label free nanosensitive multifractal detrended fluctuation analysis(nsMFDFA) technique in combination with multifractal analysis and nanosensitive optical coherence tomography (nsOCT). The proposed method deployed for extraction and quantification of nanosensitive multifractal parameters in mammary fat pad (MFP). RESULTS: Initially, the nsOCT approach is numerically validated on synthetic submicron axial structures. The nsOCT technique was applied to pathologically characterized MFP of murine breast tissue to extract depth-resolved nanosensitive submicron structures. Subsequently, two-dimensional MFDFA were deployed on submicron structural en face images to extract nanosensitive tissue multifractality. We found that nanosensitive multifractality increases in transition from healthy to tumor. CONCLUSIONS: This method for extraction of nanosensitive tissue multifractality promises to provide a noninvasive diagnostic tool for early disease detection and monitoring treatment response. The novel ability to delineate the dominant submicron scale nanosensitive multifractal properties may also prove useful for characterizing a wide variety of complex scattering media of non-biological origin.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression (Erratum).

An error in the 2nd author's name is corrected.

High-temperature etching of SiC in SF6/O2 inductively coupled plasma.

In this work, we demonstrate an effective way of deep (30 µm depth), highly oriented (90° sidewall angle) structures formation with sub-nanometer surface roughness (Rms = 0.7 nm) in silicon carbide (SiC). These structures were obtained by dry etching in SF6/O2 inductively coupled plasma (ICP) at increased substrate holder temperatures. It was shown that change in the temperature of the substrate holder in the range from 100 to 300 °C leads to a sharp decrease in the root mean square roughness from 153 to 0.7 nm. Along with this, it has been established that the etching rate of SiC also depends on the temperature of the substrate holder and reaches its maximum (1.28 µm/min) at temperatures close to 150 °C. Further temperature increase to 300 °C does not lead to the etching rate rising. The comparison of the results of the thermally stimulated process and the etching with a water-cooled substrate holder (15 °C) is carried out. Plasma optical emission spectroscopy was carried out at different temperatures of the substrate holder.

Nanosensitive optical coherence tomography to assess wound healing within the cornea.

Optical coherence tomography (OCT) is a non-invasive depth resolved optical imaging modality, that enables high resolution, cross-sectional imaging in biological tissues and materials at clinically relevant depths. Though OCT offers high resolution imaging, the best ultra-high-resolution OCT systems are limited to imaging structural changes with a resolution of one micron on a single B-scan within very limited depth. Nanosensitive OCT (nsOCT) is a recently developed technique that is capable of providing enhanced sensitivity of OCT to structural changes. Improving the sensitivity of OCT to detect structural changes at the nanoscale level, to a depth typical for conventional OCT, could potentially improve the diagnostic capability of OCT in medical applications. In this paper, we demonstrate the capability of nsOCT to detect structural changes deep in the rat cornea following superficial corneal injury.

Fast and Controllable Synthesis of Core-Shell Fe3O4-C Nanoparticles by Aerosol CVD.

A method for simple and fast (30-60 s) synthesis of spherical "Fe3O4 core-carbon shell" structures by atmospheric pressure aerosol pyrolysis of benzoic acid in dimethylformamide solutions containing dispersed Fe3O4 nanoparticles is described. It has been experimentally shown that it is possible to control both the size of the core-shell particles and the size of Fe3O4 grains and their amount in the particle core by the variation of benzoic acid concentration in solution and using pre-stabilized by mannitol iron oxide nanoparticles. It has been found that particles with an average size of 250-350 nm are formed at the concentration of benzoic acid in the range 0.5-1 mol/L. At a concentration of about 1 mol/L, preliminary stabilization of iron oxide nanoparticles by mannitol with a size of about 180 nm is performed.

Noninvasive detection of nanoscale structural changes in cornea associated with cross-linking treatment.

Corneal cross-linking (CXL) using ultraviolet-A (UVA) irradiation with a riboflavin photosensitizer has grown from an interesting concept to a practical clinical treatment for corneal ectatic diseases globally, such as keratoconus. To characterize the corneal structural changes, existing methods such as X-ray microscopy, transmission electron microscopy, histology and optical coherence tomography (OCT) have been used. However, these methods have various drawbacks such as invasive detection, the impossibility for in vivo measurement, or limited resolution and sensitivity to structural alterations. Here, we report the application of oversampling nanosensitive OCT for probing the corneal structural alterations. The results indicate that the spatial period increases slightly after 30 minutes riboflavin instillation but decreases significantly after 30 minutes UVA irradiation following the Dresden protocol. The proposed noninvasive method can be implemented using existing OCT systems, without any additional components, for detecting nanoscale changes with the potential to assist diagnostic assessment during CXL treatment, and possibly to be a real-time monitoring tool in clinics.

A Theory of Elastic/Plastic Plane Strain Pure Bending of FGM Sheets at Large Strain.

An efficient analytical/numerical method has been developed and programmed to predict the distribution of residual stresses and springback in plane strain pure bending of functionally graded sheets at large strain, followed by unloading. The solution is facilitated by using a Lagrangian coordinate system. The study is concentrated on a power law through thickness distribution of material properties. However, the general method can be used in conjunction with any other through thickness distributions assuming that plastic yielding initiates at one of the surfaces of the sheet. Effects of material properties on the distribution of residual stresses are investigated.

Characterization of an amplified piezoelectric actuator for multiple reference optical coherence tomography: erratum.

This erratum is submitted to correct information regarding Fig. 8 of Appl. Opt.57, E142 (2018)APOPAI0003-693510.1364/AO.57.00E142.

Feasibility of correlation mapping optical coherence tomography angiographic technique using a 200 kHz vertical-cavity surface-emitting laser source for in vivo microcirculation imaging applications.

Optical coherence tomography (OCT) angiography is a well-established in vivo imaging technique to assess the overall vascular morphology of tissues and is an emerging field of research for the assessment of blood flow dynamics and functional parameters such as oxygen saturation. In this study, we present a modified scanning-based correlation mapping OCT using a 200 kHz high-speed swept-source OCT system operating at 1300 nm and demonstrate its wide field-imaging capability in ocular angiographic studies.

Characterization of an amplified piezoelectric actuator for multiple-reference optical coherence tomography.

The characterization of an amplified piezoelectric actuator (APA) as a new axial scanning method for multiple-reference optical coherence tomography (MR-OCT) is described. MR-OCT is a compact optical imaging device based on a recirculating reference-arm-scanning optical delay using a partial mirror that can enhance the imaging depth range by more than 10 times the reference mirror's scanning amplitude. The scanning amplitude of the used APA was varied between 30 µm and 250 µm, depending on the scanning frequency of between 0.8 kHz and 1.2 kHz. A silver-coated miniature mirror was attached to the APA via ultraviolet-cured optical adhesive, and the light source was a super-luminescent diode with 1310 nm center wavelength and 56 nm bandwidth. The sensitivity was measured with and without the partial mirror in the reference delay line as a function of scan speed, frequency, and range, therefore providing results for MR-OCT and TD-OCT modes. It was found that the APA provides more than twice the mechanical scanning range compared to other opto-mechanic actuators, but results indicate degradation of signal-to-noise ratio and sensitivity at larger imaging depths. In conjunction with MR-OCT, the scan range of maximum 200 µm can be enhanced up to 1-1.5 mm by using a reduced amount of orders of reflections, which could be of interest to increase sensitivity in the future.

Label-free ultra-sensitive visualization of structure below the diffraction resolution limit.

For both fundamental study of biological processes and early diagnosis of diseases, information about nanoscale changes in tissue and cell structure is crucial. Nowadays, almost all currently known nanoscopy methods rely upon the contrast created by fluorescent stains attached to the object or molecule of interest. This causes limitations due to the impact of the label on the object and its environment, as well as its applicability in vivo, particularly in humans. In this paper, a new label-free approach to visualize small structure with nano-sensitivity to structural alterations is introduced. Numerically synthesized profiles of the axial spatial frequencies are used to probe the structure within areas whose size can be beyond the diffraction resolution limit. Thereafter, nanoscale structural alterations within such areas can be visualized and objects, including biological ones, can be investigated with sub-wavelength resolution, in vivo, in their natural environment. Some preliminary results, including numerical simulations and experiments, which demonstrate the nano-sensitivity and super-resolution ability of our approach, are presented.

Simultaneous en-face imaging of multiple layers with multiple reference optical coherence tomography.

A technique based on multiple reference optical coherence tomography (MR-OCT) is proposed for simultaneous imaging at multiple depths. The technique has been validated by imaging a reference sample and a fingerprint in-vivo. The principle of scanning multiple selected layers is shown by imaging a partial fingerprint with 200×200×200 voxels of 3×3×0.5 mm size and obtaining an arbitrary amount of layers merely by digital processing. The spacing among the layers can be adjusted arbitrarily, and the SNR roll-off is shown for three different spacings. At a mirror scan frequency of 1 kHz and an A-line rate of 2 kHz, the acquisition time was 20 s for one volume. The results show the feasibility of the application of layer scanning MR-OCT that uses a partial mirror in the reference arm of the Michelson interferometer. The reduced scan range required for layer scanning allows even higher scan rates that are limited only by the voice coil design and the mass-spring system, e.g., mirror mass, spring constant, and damping.

TOF MS Investigation of Nickel Oxide CVD.

NiO layers were deposited by metal-organic chemical vapor deposition using bis-(ethylcyclopentadienyl) nickel (EtCp)2Ni and oxygen or ozone. As a continuation of kinetic study of NiO MOCVD the gas-phase, transformations of (EtCp)2Ni were studied in the temperature range of 380-830 K. Time of reactions corresponding to the residence time of the gas stream in hot zone of the reactor was about 0.1 s under conditions studied. The interaction of (EtCp)2Ni with oxygen started at 450 K and its conversion rate reached the maximum at 700 K. The interaction of (EtCp)2Ni with ozone started at 400 K and its conversion rate reached the maximum at 600 K. Transformations of the gas phase with the temperature in the reaction zone were studied, the model reaction schemes illustrating (EtCp)2Ni transformations in the reaction systems containing oxygen and ozone have developed. In the reaction system (EtCp)2Ni-O2-Ar the main gas-phase products at 380-500 K were CO, CO2, HCO, C2H5OH, CpCOOH, and CpO. Formation of the C2H2O, C3H4O, and C5H8O was found at 630-830 K. The same gas-phase species, (C4H3O)2Ni and dialdehydes was formed in the reaction system (EtCp)2Ni-O3-O2-Ar. Graphical Abstract ᅟ.

In vivo correlation mapping microscopy.

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions.

Functional imaging for regenerative medicine.

In vivo imaging is a platform technology with the power to put function in its natural structural context. With the drive to translate stem cell therapies into pre-clinical and clinical trials, early selection of the right imaging techniques is paramount to success. There are many instances in regenerative medicine where the biological, biochemical, and biomechanical mechanisms behind the proposed function of stem cell therapies can be elucidated by appropriate imaging. Imaging techniques can be divided according to whether labels are used and as to whether the imaging can be done in vivo. In vivo human imaging places additional restrictions on the imaging tools that can be used. Microscopies and nanoscopies, especially those requiring fluorescent markers, have made an extraordinary impact on discovery at the molecular and cellular level, but due to their very limited ability to focus in the scattering tissues encountered for in vivo applications they are largely confined to superficial imaging applications in research laboratories. Nanoscopy, which has tremendous benefits in resolution, is limited to the near-field (e.g. near-field scanning optical microscope (NSNOM)) or to very high light intensity (e.g. stimulated emission depletion (STED)) or to slow stochastic events (photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)). In all cases, nanoscopy is limited to very superficial applications. Imaging depth may be increased using multiphoton or coherence gating tricks. Scattering dominates the limitation on imaging depth in most tissues and this can be mitigated by the application of optical clearing techniques that can impose mild (e.g. topical application of glycerol) or severe (e.g. CLARITY) changes to the tissue to be imaged. Progression of therapies through to clinical trials requires some thought as to the imaging and sensing modalities that should be used. Smoother progression is facilitated by the use of comparable imaging modalities throughout the discovery and trial phases, giving label-free techniques an advantage wherever they can be used, although this is seldom considered in the early stages. In this paper, we will explore the techniques that have found success in aiding discovery in stem cell therapies and try to predict the likely technologies best suited to translation and future directions.