Radiation-induced alterations in multi-layered, in-vitro skin models detected by optical coherence tomography and histological methods.

BACKGROUND: Inflammatory skin reactions and skin alterations are still a potential side effect in radiation therapy (RT), which also need attention for patients' health care. METHOD: In a pre-clinical study we consider alterations in irradiated in-vitro skin models of epidermal and dermal layers. Typical dose regimes in radiation therapy are applied for irradiation. For non-invasive imaging and characterization optical coherence tomography (OCT) is used. Histological staining method is additionally applied for comparison and discussion. RESULTS: Structural features, such as keratinization, modifications in epidermal cell layer thickness and disorder in the layering-as indications for reactions to ionizing radiation and aging-could be observed by means of OCT and confirmed by histology. We were able to recognize known RT induced changes such as hyper-keratosis, acantholysis, and epidermal hyperplasia as well as disruption and/or demarcation of the dermo-epidermal junction. CONCLUSION: The results may pave the way for OCT to be considered as a possible adjunctive tool to detect and monitor early skin inflammation and side effects of radiotherapy, thus supporting patient healthcare in the future.

Time-encoded mid-infrared Fourier-domain optical coherence tomography: erratum.

We present an erratum to our Letter [Opt. Lett.46, 4108 (2021)OPLEDP0146-959210.1364/OL.434855] and make a correction to the labeling of the absorption bands (reversed order for CH vibrations) indicated in Fig. 2(a). This does not change the scientific conclusions of the original Letter.

Time-encoded mid-infrared Fourier-domain optical coherence tomography.

We report on a technically simple approach to achieve high-resolution and high-sensitivity Fourier-domain optical coherence tomography (OCT) imaging in the mid-infrared (mid-IR) range. The proposed OCT system employs an InF3 supercontinuum source. A specially designed dispersive scanning spectrometer based on a single InAsSb point detector is employed for detection. The spectrometer enables structural OCT imaging in the spectral range from 3140 nm to 4190 nm with a characteristic sensitivity of over 80 dB and an axial resolution below 8µm. The capabilities of the system are demonstrated for imaging of porous ceramic samples and transition-stage green parts fabricated using an emerging method of lithography-based ceramic manufacturing. Additionally, we demonstrate the performance and flexibility of the system by OCT imaging using an inexpensive low-power (average power of 16 mW above 3µm wavelength) mid-IR supercontinuum source.

Correlative infrared optical coherence tomography and hyperspectral chemical imaging.

Optical coherence tomography (OCT) is a high-resolution three-dimensional imaging technique that enables nondestructive measurements of surface and subsurface microstructures. Recent developments of OCT operating in the mid-infrared (MIR) range (around 4 µm) lifted fundamental scattering limitations and initiated applied material research in formerly inaccessible fields. The MIR spectral region, however, is also of great interest for spectroscopy and hyperspectral imaging, which allow highly selective and sensitive chemical studies of materials. In this contribution, we introduce an OCT system (dual-band, central wavelengths of 2 µm and 4 µm) combined with MIR spectroscopy that is implemented as a raster scanning chemical imaging modality. The fully integrated and cost-effective optical instrument is based on a single supercontinuum laser source (emission spectrum spanning from 1.1 µm to 4.4 µm). Capabilities of the in situ correlative measurements are experimentally demonstrated by obtaining complex multidimensional material data, comprising morphological and chemical information, from a multilayered composite ceramic-polymer specimen.

Dual-band infrared optical coherence tomography using a single supercontinuum source.

Recent developments and commercial availability of low-noise and bright infrared (IR) supercontinuum sources initiated intensive applied research in the last few years. Covering a significant part of near- and mid-infrared spectral ranges, supercontinuum radiation opened up unique possibilities and alternatives for the well-established imaging technique of optical coherence tomography (OCT). In this contribution, we demonstrate the development, performance, and maturity of a cost-efficient dual-band Fourier-domain IR OCT system (2 µm and 4 µm central wavelengths). The proposed OCT setup is elegantly employing a single supercontinuum source and a pyroelectric linear array. We discuss adapted application-oriented approaches to signal acquisition and post-processing when thermal detectors are applied in interferometers. In the experimental part, the efficiency of the dual-band detection is evaluated. Practical results and direct comparisons of the OCT system operating within the employed sub-bands are exhibited and discussed. Furthermore, we introduce the 2 µm OCT sub-system as an affordable alternative for art diagnosis; therefore, high resolution and sensitive measurements of the painting mock-ups are presented. Finally, potentials of the dual-band detection are demonstrated for lithography-based manufactured industrial ceramics.

Sensitivity-Enhanced Fourier Transform Mid-Infrared Spectroscopy Using a Supercontinuum Laser Source.

Fourier transform infrared (FT-IR) spectrometers have been the dominant technology in the field of mid-infrared (mid-IR) spectroscopy for decades. Supercontinuum laser sources operating in the mid-IR spectral region now offer the potential to enrich the field of FT-IR spectroscopy due to their distinctive properties, such as high-brightness, broadband spectral coverage and enhanced stability. In our contribution, we introduce this advanced light source as a replacement for conventional thermal emitters. Furthermore, an approach to efficient coupling of pulsed mid-IR supercontinuum sources to FT-IR spectrometers is proposed and considered in detail. The experimental part is devoted to pulse-to-pulse energy fluctuations of the applied supercontinuum laser, performance of the system, as well as the noise and long-term stability. Comparative measurements performed with a conventional FT-IR instrument equipped with a thermal emitter illustrate that similar noise levels can be achieved with the supercontinuum-based system. The analytical performance of the supercontinuum-based FT-IR spectrometer was tested for a concentration series of aqueous formaldehyde solutions in a liquid flow cell (500 µm path length) and compared with the conventional FT-IR (130 µm path length). The results show a four-times-enhanced detection limit due to the extended path length enabled by the high brightness of the laser. In conclusion, FT-IR spectrometers equipped with novel broadband mid-IR supercontinuum lasers could outperform traditional systems providing superior performance, e.g., interaction path lengths formerly unattainable, while maintaining low noise levels known from highly stable thermal emitters.

Full-field optical coherence tomography in a balanced detection mode.

We discuss balanced time-domain full-field optical coherence tomography (FF-OCT) realized in a Mach-Zehnder configuration. The balanced detection scheme and spatial phase shifting allow single-shot acquisition and reconstruction in FF-OCT. Combined with a 2D quadrature signal-based demodulation technique applying the Riesz transform, previously illustrated for a dual-shot temporal phase shifting in FF-OCT, we demonstrate the concept for single-shot spatial phase shifting. The monitoring of dynamic processes by time-domain FF-OCT is enabled by this approach. The advantage of single-shot acquisition consists of having no failure due to phase changes over time. However, it demands an accurate registration of both spatially shifted interferograms.

Mid-infrared Fourier-domain optical coherence tomography with a pyroelectric linear array.

Optical technology in the mid-infrared wavelength range is currently a rapidly developing field initiated by the availability of novel high-power and spatially coherent sources. Non-destructive testing techniques based on these sources are very promising for industrial and medical applications. However, there are still many engineering problems due to the technical challenges and high prices of the optical elements suitable for the mid-infrared region. In this paper, we report the development and performances of the first mid-infrared Fourier-domain optical coherence tomography based on a supercontinuum source and low-cost pyroelectric detector. The system is designed to operate in the spectral region around 4 µm. Experimental results are demonstrated for detections of embedded microstructures in ceramic materials and subsurface oil paint layers.

In-Situ Optical Coherence Tomography (OCT) for the Time-Resolved Investigation of Crystallization Processes in Polymers.

By application of optical coherence tomography (OCT), an interferometric noncontact imaging technique, the crystallization of a supercooled poly(propylene) melt in a slit die is monitored. Both the quiescent and the sheared melt are investigated, with a focus on experiments where solidification and flow occur simultaneously. OCT is found to be an excellent tool for that purpose since the resultant structures are strongly scattering, which is a prerequisite for application of that method. The resulting images enable for the first time to directly monitor structure development throughout the whole experiment, including final cooling to room temperature. By rendering the setup polarization-sensitive, information on the birefringence of the pertinent structures is obtained.

Full-field optical coherence microscopy with Riesz transform-based demodulation for dynamic imaging.

We present dynamic full-field optical coherence microscope imaging using a scientific complementary metal oxide semiconductor camera in conjunction with a demodulation scheme based on the Riesz transform and monogenic signals. The potential of our approach is verified by a comparison with conventional phase-stepping as well as with an analytic reconstruction method and finally exemplified for dynamic mechanical testing of a polymer/fiber composite structure.

Dynamic optical studies in materials testing with spectral-domain polarization-sensitive optical coherence tomography.

By combining dynamic mechanical testing with spectral-domain polarization-sensitive optical coherence tomography (SD-PS-OCT) performed at 1550 nm we are able to directly investigate for the first time changes within scattering technical materials during tensile and fracture tests. Spatially and temporally varying polarization patterns, due to defects and material inhomogeneities, were observed within bulk polymer samples and used to finally obtain--with the help of advanced image processing algorithms--quantitative maps of the evolving internal stress distribution. Furthermore, locally increased stress within fiber-reinforced composite materials was identified in situ with SD-PS-OCT to cause depolarizing sites of fiber-matrix debonding prior the onset of complete structural failure.

Flexible contrast for low-coherence interference microscopy by Fourier-plane filtering with a spatial light modulator.

We propose a full-field low-coherence interference (LCI) microscope that can provide different contrast modes using Fourier-plane filtering by means of a spatial light modulator. By altering the phase and spatial frequencies of the backreflected wavefront from the sample arm of the interferometer, we are able to change the contrast in the depth-resolved LCI images. We demonstrate that different types of contrast modes, such as, e.g., spiral phase contrast, can successfully be emulated to provide specific enhancement of internal structures and edges and to reveal complementary details within the samples under investigation.

Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.

Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.

Quantitative phase reconstruction for orthogonal-scanning differential phase-contrast optical coherence tomography.

We present differential phase-contrast optical-coherence tomography (DPC-OCT) with two transversally separated probing beams to sense phase gradients in various directions by employing a rotatable Wollaston prism. In combination with a two-dimensional mathematical-reconstruction algorithm based on a regularized shape from shading method, accurate quantitative phase maps can be determined from a set of two orthogonal en-face DPC-OCT images, as exemplified on various technical samples.

Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly.

We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.

Micropatterning for quantitative analysis of protein-protein interactions in living cells.

We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.

DIC image reconstruction on large cell scans.

The width of the emission spectrum of a common fluorophore allows only for a limited number of spectral distinct fluorescent markers in the visible spectrum, which is also the regime where CCD-cameras are used in microscopy. For imaging of cells or tissues, it is required to obtain an image from which the morphology of the whole cell can be extracted. This is usually achieved by differential interference contrast (DIC) microscopy. These images have a pseudo-3D appearance, easily interpreted by the human brain. In the age of high throughput and high content screening, manual image processing is not an option. Conventional algorithms for image processing often use threshold-based criteria to identify objects of interest. These algorithms fail for DIC images as they have a range from dim to bright with an intermediate intensity equal to the background, so as to produce no clear object boundary. In this article we compare different reconstruction methods for up to 100 MB-large DIC images and implement a new iterative reconstruction method based on the Hilbert Transform that enables identification of cell boundaries with standard threshold algorithms.

Switching the inside and the outside of aggregates of water-soluble block copolymers with double thermoresponsivity.

Water-soluble block copolymers were prepared from the nonionic monomer N-isopropylacrylamide (NIPA) and the zwitterionic monomer 3-[N-(3-methacrylamidopropyl)-N,N-dimethyl]ammoniopropane sulfonate (SPP) by sequential free radical polymerization via the RAFT process. Such block copolymers with two hydrophilic blocks exhibit double thermoresponsive behavior in water: the poly-NIPA block shows a lower critical solution temperature, whereas the poly-SPP block exhibits an upper critical solution temperature. Appropriate design of the block lengths leads to block copolymers which stay in solution in the full temperature range between 0 and 100 degrees C. Both blocks of these polymers dissolve in water at intermediate temperatures, whereas at high temperatures, the poly-NIPA block forms colloidal hydrophobic associates that are kept in solution by the poly-SPP block, and at low temperatures, the poly-SPP block forms colloidal polar aggregates that are kept in solution by the poly-NIPA block. In this way, colloidal aggregates which switch reversibly can be prepared in water, and without any additive, their "inside" to the "outside", and vice versa. The aggregates provide microdomains and surfaces of different character, which can be controlled by a simple thermal stimulus.