Reflection-mode Microscopy Research Articles

Label-free intraoperative histology of bone tissue via deep-learning-assisted ultraviolet photoacoustic microscopy.

Obtaining frozen sections of bone tissue for intraoperative examination is challenging. To identify the bony edge of resection, orthopaedic oncologists therefore rely on pre-operative X-ray computed tomography or magnetic resonance imaging. However, these techniques do not allow for accurate diagnosis or for intraoperative confirmation of the tumour margins, and in bony sarcomas, they can lead to bone margins up to 10-fold wider (1,000-fold volumetrically) than necessary. Here, we show that real-time three-dimensional contour-scanning of tissue via ultraviolet photoacoustic microscopy in reflection mode can be used to intraoperatively evaluate undecalcified and decalcified thick bone specimens, without the need for tissue sectioning. We validate the technique with gold-standard haematoxylin-and-eosin histology images acquired via a traditional optical microscope, and also show that an unsupervised generative adversarial network can virtually stain the ultraviolet-photoacoustic-microscopy images, allowing pathologists to readily identify cancerous features. Label-free and slide-free histology via ultraviolet photoacoustic microscopy may allow for rapid diagnoses of bone-tissue pathologies and aid the intraoperative determination of tumour margins.

Three-dimensional transfer function of optical microscopes in reflection mode.

Three-dimensional (3D) transfer functions build the basis for a comprehensive characterization of optical imaging systems in the spatial frequency domain. Utilizing the projection-slice theorem, the 2D modulation transfer function of an incoherent imaging system can be derived from a 3D transfer function by integration with respect to the axial spatial frequency. For a diffraction limited microscope with homogeneous incoherent pupil illumination, the modulation transfer function equals the 2D autocorrelation function of a circular disc. However, until now to the best of our knowledge no 3D transfer function has been published, which exactly leads to the 2D modulation transfer function of a diffraction limited microscope in reflection mode. In this article, we derive a formula, which after integration with respect to the axial spatial frequency coordinate perfectly fits to the diffraction limited 2D modulation transfer function. The inverse three-dimensional Fourier transform of the 3D transfer function results in a complex-valued 3D point spread function, from which the depth of field, the lateral resolution and, in addition, the corresponding 3D point spread function of both, a conventional and an interference microscope, can beobtained.

On the growth and mechanical properties of nanostructured cobalt foams by dynamic hydrogen bubble template electrodeposition

The growth mechanism of nanostructured cobalt foams prepared by hydrogen bubble template electrodeposition has been studied. To that end, cobalt foams have been synthesized under different electrodeposition times and studied with various characterization techniques, namely scanning electron microscopy (SEM) and confocal microscopy in reflection mode. In addition, the mechanical properties of the resulting foams have been tested by bending and tensile tests. To study the influence of chemical additives in the growth mechanism and final properties of the foams, agar-agar has been added to the electrolytic bath during foam preparation. Results evidence that the addition of agar has successfully modified the microstructure of the final foams, creating a denser porous structure with smaller pore area and reduced growth rate that translates into better mechanical properties. By studying the evolution of the microstructure under different deposition conditions, a growth model of cobalt metallic foams by dynamic hydrogen bubble template is proposed. This easy and scalable route paves the way to produce tailored foams for numerous applications that include, for instance, energy storage and energy conversion.

Surface Morphology Formation of Edible Holographic Marker on Potato Starch with Gelatin or Agar Thin Coatings.

Edible films and coatings based on biopolymers to protect and extend the shelf life of food and medicine can be functionalized, by applying a holographic marker on the coating surface for marking products or sensing storage conditions. In this work, holographic markers were prepared on the surface of thin biopolymer coatings based on starch, gelatin, agar and also starch/gelatin and starch/agar blends by the nanoimprint method from a film-forming solution. The morphology of the surface of holographic markers using optical microscopy in reflection mode was examined, as well as the reasons for its formation using an analysis of the flow curves of film-forming solutions. It was found that the surface morphology of the marker strongly depends on the composition, consistency index of film-forming solution and miscibility of the components. It was shown that the starch/agar film-forming solution at the ratio of 70/30 wt.% has a low consistency index value of 21.38 Pa·s0.88, compared to 64.56 Pa·s0.67 for pure starch at a drying temperature of 30 °C, and the components are well compatible. Thus, an isotropic morphology of the holographic marker surface was formed and the value of diffraction efficiency of 3% was achieved, compared to 1.5% for the marker made of pure starch. Coatings without holographic markers were analyzed by tensile strength and water contact angle, and their properties are highly dependent on their composition.

The effect of martensite volume fraction on topography of dual phase steels

Digital holographic microscopy in reflection mode provides 3D visualization of metallographic surfaces within an adjustable field of view, a lateral resolution close to diffraction limit, and a sub-wavelength axial accuracy. In this paper, we utilize reflective digital holographic microscopy (rDHM) to evaluate the mean height between ferrite and martensite phases in dual phase steel (DPS) samples. The data extracted from processing of several digital holograms show that the mean height difference between the phases is reduced for the samples of higher martensite volume fraction. rDHM has the potential to be used for the multi-phase samples that are difficult to be analyzed by conventional metallographic methods.

Calcite dissolution rate spectra measured by in situ digital holographic microscopy

Digital holographic microscopy in reflection mode is used to track in situ, real-time nanoscale topography evolution of cleaved (104) calcite surfaces exposed to flowing or static deionized water. The method captures full-field holograms of the surface at frame rates of up to 12.5s−1. Numerical reconstruction provides 3D surface topography with vertical resolution of a few nanometers and enables measurement of time-dependent local dissolution fluxes. A statistical distribution, or spectrum, of dissolution rates is generated by sampling multiple area domains on multiple crystals. The data show, as has been demonstrated by Fischer et al. (2012), that dissolution is most fully described by a rate spectrum, although the modal dissolution rate agrees well with published mean dissolution rates (e.g., 0.1μmolm−2s−1 to 0.3μmolm−2s−1). Rhombohedral etch pits and other morphological features resulting from rapid local dissolution appear at different times and are heterogeneously distributed across the surface and through the depth. This makes the distribution in rates measured on a single crystal dependent both on the sample observation field size and on time, even at nominally constant undersaturation. Statistical analysis of the inherent noise in the DHM measurements indicates that the technique is robust and that it likely can be applied to quantify and interpret rate spectra for the dissolution or growth of other minerals.

Upconversion emission obtained in Yb^3+-Er^3+ doped fluoroindate glasses using silica microspheres as focusing lens

An intensity enhancement of the green upconversion emission from a codoped Er(3+)-Yb(3+) fluoroindate glass has been obtained by coating the glass surface with silica microspheres (3.8 µm diameter). The microspheres focus an incoming beam (λ ≈ 950 nm) on the surface of the fluoroindate glass. The green emission (λ ≈ 545 nm) of the Er(3+) ions located in the microsphere focus was measured with a microscope in reflection mode, being the peak intensity 4.5 times the emission of the bare substrate. The transversal FWHM of the upconversion spot was experimentally determined by deconvolution with the experimental Point Spread Function of the system, obtaining a value of 309 nm. This value is in good agreement with Finite-Difference Time-Domain simulations taking into account the magnification of the image due to the microsphere.

Morphology and molten-state rheology of polylactide and polyhydroxyalkanoate blends

Polylactide (PLA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) blends were prepared at different compositions by melt mixing. First, the rheological properties of each individual component are briefly presented focusing on the most important aspects to be taken into account during blends preparation and investigation. The kinetics of PHBV viscosity decrease due to strong polymer degradation in the molten state was recorded. This helped making a choice of the blending parameters and of the way of performing the rheological frequency sweeps. DSC showed that components are immiscible in the whole range of compositions studied. Blends morphology was studied using high-resolution scanning electron microscope and optical microscopy in reflection mode. Nodular and co-continuous morphologies were observed depending on the composition, and minor phase size was roughly estimated. The rheology of PLA/PHBV blends was investigated in the dynamic mode and correlated with the morphology observed. The results showed an important role of the interfaces between PLA and PHBV and a peculiar behaviour of the viscosity of some mixtures at low frequencies. At medium and high frequencies mixture dynamic viscosity follows the mixing law.

Structural and Dielectric Properties of Glycine Phosphite Thin Films

Thin polycrystalline films of amino acid ferroelectrics glycine phosphite (GPI) have been prepared by evaporation method on NdGaO3(001) substrate with interdigital structure of electrodes deposited on the surface. Optical images of films obtained by means of polarized microscope in reflection mode have shown that the films consist of small single crystal blocks of several tens of microns in size. X-ray diffraction analysis proves that the non-polar c* axis in blocks coincides with the normal to the surface, and the polar axis b is oriented in the film plane. Temperature dependences of capacitance of GPI/NdGaO3 structures measured using interdigital structure of electrodes exhibit strong anomaly at ferroelectric phase transition temperature of glycine phosphite single crystals. Dielectric hysteresis loops in glycine phosphite films were measured by Sawyer-Tower method. The difference in frequency dependence of coercive field and remnant polarization in structures with GPI and previously studied betaine phosphite films is analyzed and attributed to considerable difference in dimensions of blocks in these structures.

反射模式近场光学显微镜的局域磁光克尔效应成像

A method for local magneto-optical Kerr effect imaging based on a home-made scanning near-field optical microscope working in reflection mode is presented. Shear force detection is carried out by using a symmetric piezoelectric bimorph sensor, which provides an easy way not only for probe-surface distance control but also for imaging. Polarization-preserving fiber probes used as a local optical detector are fabricated with a heating-pulling technique and the probes' polarization properties are measured. Shear force topographic and near-field magneto-optical images of magneto-optical disk taken with the proposed method are shown.

Local magneto-optical Kerr effect imaging by scanning near-field optical microscope in reflection-mode

Local magneto-optical Kerr effect imaging by scanning near-field optical microscope in reflection-mode

The use of confocal microscopy to assess surface roughness of two milled CAD–CAM ceramics following two polishing techniques

Objective To assess the effects of two polishing techniques on the surface roughness of a commercial and experimental dental ceramic for use in a chairside CAD–CAM system. Methods A standard onlay was reproduced fifteen times in each of the two materials. Each stub was retained and examined using confocal microscopy in reflection mode. Five random samples of each were polished with Sof-Lex aluminium oxide discs, five polished with diamond impregnated polishing wheels and five left in the as-machined state. Surface roughness parameters were measured for each material group in each condition and statistical analyses carried out to determine the efficacy of the polishing techniques for each material. Results The as-machined surfaces of both materials had a statistically similar surface finish. Polishing with Sof-Lex discs was effective at reducing surface roughness of both materials. Whilst the polishing wheels were effective in reducing surface roughness for the commercial material, there was no statistically significant difference in the surface roughness of the experimental material in the as-machined or wheel polished states. Significance Although Sof-Lex discs were effective in reducing surface roughness of the two materials examined in this study, wheel polishing should not be used to reduce surface roughness of the experimental material. This will need to be taken into consideration in any future clinical studies of this material.

Plaque biofilms: The effect of chemical environment on natural human plaque biofilm architecture

The architecture of microbial biofilms especially the outer regions have an important influence on the interaction between biofilm and local environment particularly on the flux of materials into and out of biofilm compartments and as a consequence, biofilm metabolic behaviour. In the case of dental plaque biofilms, architecture will determine access of nutrients including acidogenic substrates and therapeutic materials to the microbial biomass and to the underlying tooth surface. Manipulation of this architecture may offer a means of altering mass transfer into the whole biofilm and biomass and raises the possibility of improving access of therapeutics. Plaque biofilms formed in vivo on human enamel were subjected to a number of different chemical conditions while under observation by confocal laser scanning microscopy in reflection mode. In this way the outer 50-100 microm or so of the biofilms was examined. Density and distribution of biomass were recorded as degree of reflectance. The amount and density of biofilm biomass increased from the plaque saliva interface towards the interior. Plaque biofilms were robust and little affected by mechanical manipulation, high ionic strength or low pH (2.5). Detergent (SLS), however, often appeared to either remove biomass and/or dramatically reduce its density.

Converging spherical wave propagation in a hemispherical solid lens

The propagation of converging spherical waves through a hemispherical solid lens (HSL) is studied, and the multiple reflections inside the HSL are considered. It is found that the reflectivity and transmissivity have an oscillatory behaviour related to the optical radius of the HSL. When the refractive index of the HSL is smaller than that of the sample, the small mismatch in refractive indices is preferable for transmission-mode microscopy. But when the refractive index of the HSL is larger than that of the sample, the reflectivity increases rapidly as the mismatch in refractive indices and the converging angle of incident light increase, the larger mismatch and converging angle are useful for reflection-mode microscopy. The magnitude of the whole reflectivity and its modulation depth are strongly sensitive to the incident polarization, which is also discussed.

Partial Dewetting of Polyethylene Thin Films on Rough Silicon Dioxide Surfaces

The effect of roughness on the dewetting behavior of polyethylene thin films on silicon dioxide substrates is presented. Smooth and rough silicon dioxide substrates of 0.3 and 3.2-3.9 nm root-mean-square roughness were prepared by thermal oxidation of silicon wafers and plasma-enhanced chemical vapor deposition on silicon wafers, respectively. Polymer thin films of approximately 80 nm thickness were deposited by spin-coating on these substrates. Subsequent dewetting and crystallization of the polyethylene were observed by hot-stage optical microscopy in reflection mode. During heating, the polymer films melt and dewet on both substrates. Further observations after cooling indicate that, whereas complete dewetting occurs on the smooth substrate surface, partial dewetting occurs for the polymer film on the rough surface. The average thickness of the residual film on the rough surface was determined by ellipsometry to be a few nanometers, and the spatial distribution of the polymer in the cavities of the rough surface could be obtained by X-ray reflectometry. The residual film originates from the impregnation of the porous surface by the polymer fluid, leading to the observed partial dewetting behavior. This new type of partial dewetting should have important practical consequences, as most real surfaces exhibit significant roughness.

Magnetic beads as interfacial nanoprobes.

We use paramagnetic beads to probe strongly localized magnetic fields from one-dimensional nanomagnets. Using a polarization microscope in reflection mode, we find that light reflected from beads exhibits intensity fluctuations which may help us understand Brownian motion near interfaces. We estimate the height fluctuations and femtonewton forces acting on the beads.

Second‐harmonic far‐field microscopy of random nanostructured gold surfaces

We use a scanning far-field optical second-harmonic microscope in reflection mode to image a gold film surface covered with randomly distributed scatterers. We recorded simultaneously images of the first (FH) and second harmonic (SH) signal in the wavelength range of 750–830 nm for different densities of scatterers. The SH images showed localized enhancement (∼103 the background) in the form of small (∼0.7μm) round bright spots that exhibited both wavelength and polarization dependences in their brightness. We concluded that the overall behaviour of the observed phenomenon is related to the overlap of FH and SH eigenmodes. (© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Second-harmonic scanning optical microscopy of semiconductor quantum dots

Second-harmonic (SH) optical imaging of self-assembled InAlGaAs quantum dots (QD's) grown on a GaAs(0 0 1) substrate has been accomplished at room temperature by use of respectively a scanning far-field optical microscope in reflection mode and a scanning near-field optical microscope in transmission mode. In both cases the SH signal peaks at a pump wavelength of ∼885 nm in correspondence to the maximum in the photoluminescence spectrum of the QD sample. SH near-field optical images exhibit spatial signal variations on a subwavelength scale that depend on the pump wavelength. We attribute this feature to inhomogeneous broadening of the QD energy states.

Spectroscopic and mechanical evidence of the influence of the substrate on an anaerobic adhesive cure

The catalytic effect of different substrates-steel, aluminium and copper-on the curing reaction of a standard anaerobic adhesive is investigated by infrared microscopy in reflection mode. Thus, kinetics of the cure reaction, studied from the decrease of the carbon-carbon double bonds absorption peak, are quite different depending on the substrate under investigation. Among the studied metals, copper has been found as the most reactive surface. The assumed role of saccharine contained in the anaerobic adhesive which is able to oxidise metals into ions will be highlighted. Finally, an attempt will be made to correlate the kinetic results with the mechanical performances of the adhesive joint measured by a single lap shearing test. Thus, fast curing reaction of the anaerobic adhesive on a surface such as copper leads to poor maximal strain while lower surface reactivity may provide better mechanical performances of the adhesive joints.

Confocal imaging of a gap junction protein associated with the keratocytes of the human cornea.

Keratocyte distribution and transparency make it difficult to investigate the precise microanatomy and intercellular connectivity of these cells throughout the cornea in siru. Earlier siudies were limited by the lack of resolution and the inability to achieve three-dimensional conceptualisation of keratocyte morphology.' More recently, human keratocytes have been visualised in situ using tandem scanning confocal microscopy in reflection mode, but resolution was again limited. Similarly, immunohistochemical staining of elements of the keratocyte cytoskeleton imaged by confocal laser scanning microscopy does not enable the keratocyte to be seen in its entirety.' Recently, our group reported the combined use of a new aldehyde fixable cell viability fluoroprobe, 5-chloromethylfluorescein diacetate (CMFDA), and confocal laser scanning microscopy to produce high-resolution images of the entire cytoplasm of the keratocyte ex uizo.'' Here we report on the co-localisation of an antibody to the gap junction protein connexin 50 on human corneal keratocytes stained with CMFDA. Dual channel confocal laser scanning microscopy, digital image reconstruction and merging techniques were used to produce highresolution images of connexin 50 distribution on the cell bodies and fine cell processes of mid-stroma1 keratocytes, and to highlight the functional potential of gap junction proteins in the maintenance of human corneal physiology.