Optical Interference Microscopy Research Articles

Nanoscopic lipid diffusion in the live cell plasma membrane revealed by ultrafast single-molecule tracking.

Membrane organization and dynamics underlie various molecular interactions required for membrane functions. Although the importance of investigating biological membranes at the nanoscale is well recognized, resolving nanoscopic structures and dynamics is technically challenging. In this work, we employ ultrafast single-particle tracking techniques to measure single phospholipid diffusion in the plasma membrane of live cells. We attach a monovalent gold nanoparticle to the headgroup of biotinylated phospholipid and record its diffusive motion on microsecond resolution and nanometer precision by advanced optical interference microscopy. We find that both saturated and unsaturated phospholipids (biotin-PEG-DSPE and biotin-PEG-DOPE, respectively) undergo anomalous subdiffusion in the length scale below 100 nm. The unsaturated lipid diffuses only slightly more freely than the saturated lipid. Moreover, the nanoscopic lipid diffusion is highly heterogeneous in space and time. With statistical analyses, we find that the apparent dual-mobility subdiffusion can describe measured diffusion. The subdiffusion agrees well with the hop diffusion model that describes a Brownian diffuser moving in periodic and partially permeable barriers created by the cytoskeleton. Inhibition of actin polymerization by the chemical drug CK-666 reduces confinement's effective size and strength. Furthermore, we examine the effect of cholesterol on lipid subdiffusion by manipulating the cholesterol concentration. Cholesterol depletion by methyl-b-cyclodextrin leads to a more restricted lipid diffusion. We attribute the more confined diffusion to the formation of solid-like gel-phase nanodomains that move slowly and act as diffusion obstacles. Lowing the system temperature shows similar effects on lipid diffusion, suggesting that the cholesterol-dependent lipid diffusion connects to the nanoscopic phase separation. Overall, this study provides experimental evidence that lipid diffusion in the cell membrane is heterogeneous at the nanoscale and regulated by the membrane cholesterol and actin cytoskeleton.

Subnanometer accuracy of surface characterization by reflected-light differential interference microscopy

We theorize the surface step characterization by reflected incoherent-light differential interference microscopy with consideration of the optical diffraction effect. With the integration of localization analysis, we develop a quantitative differential interference optical system, by which we demonstrate that the axial resolution of measuring surface height variation is sensitive to the shear distance between the two spatially differentiated beams. We fabricate three nanometer-size steps by photolithography, and successfully characterize their 1D height variations with 0.13 nm axial precision. Our result suggests that the optical differential interference microscopy can be used for real-time characterization of surface structure with a subnanometer accuracy and a large field of view, which is greatly beneficial to the surface characterization of micro/nano-electromechanical systems.

Microsphere assistance in interference microscopy with high numerical aperture objective lenses

Various attempts have been discussed to overcome the lateral resolution limit and thus to enlarge the fields of application of optical interference microscopy. Microsphere-assisted microscopy and interferometry have proven that the imaging of structures well below Abbe’s resolution limit through near-field assistance is possible if microspheres are placed on the measured surface and utilized as near-field assisting imaging elements. The enhancement of the numerical aperture (NA) by the microspheres as well as photonic nanojets was identified to explain the resolution enhancement, but also whispering gallery modes and evanescent waves are assumed to have an influence. Up to now, to the best of our knowledge, there is no complete understanding of the underlying mechanisms and no model enabling to examine ideal imaging parameters. This contribution is intended to clarify how much the lateral resolution of an already highly resolving Linnik interferometer equipped with 100 × NA 0.9 objective lenses can be further improved by microspheres. Our simulation model developed so far is based on rigorous near-field calculations combined with the diffraction-limited illumination and imaging process in an interference microscope. Here, we extend the model with respect to microsphere-assisted interference microscopy providing a rigorous simulation of the scattered electric field directly above the sphere. Simulation and experimental results will be compared in the three-dimensional spatial frequency domain and discussed in context with ray-tracing computations to achieve an in-depth understanding of the underlying mechanism of resolution enhancement by the microsphere.

Molecularly Specific and Functional Live Cell Imaging by Label-Free Interference Microscopy

Optical interference microscopy is a powerful bioimaging technique by measuring the complex light fields associated with the specimen. Nowadays, the state-of-the-art interference microscopy makes it possible to directly visualize very small single biological nanoparticles and unlabeled macromolecules. The stable and indefinite linear scattering signal allows for continuous observation of the sample at a high speed, offering the opportunities to investigate single-molecule biophysics with the unprecedented details. Meanwhile, using interference microscopy to explore complex biological samples, such as a biological cell, emerges as an exciting research field. In this Perspective, we share our views on the impacts of optical interference microscopy on live cell imaging. Strategies for discriminating the scattering signals from different cell organelles and biological macromolecules are presented. In particular, the dynamic optical signal of live cells contains rich temporal information that is useful for enhancing the molecular specificity and functional information in label-free cell imaging. Finally, the challenges in three-dimensional imaging and turbidity suppression are discussed.

Measurement of the large area surface relief by optical interference microscope

It was presented the procedure of stitching surface regions measured by optical interference microscope to get a microrelief of large area. It was shown that usage of that procedure allows to perform measurements which are difficult at the processing of measured surface regions separately. Measurement results and stitching are presented.

Chromatin-specific label-free dynamic imaging by high-speed scattering-based interference optical microscopy

The basic unit of chromatin is nucleosome which is composed of a section of DNA wrapped around a histone octamer. The spacing of nucleosomes defines the degrees of packing of chromatin. The organization of chromatin and its dynamics play crucial roles in harmonizing cell functions. Although highly desired, quantitatively measuring the level of chromatin compaction remains challenging. Previous studies demonstrated an approach, the FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM), to evaluate chromatin compaction.

Research Progress in Optical Interference Microscopy Toward Three-Dimensional Imaging of Biological Samples

Research Progress in Optical Interference Microscopy Toward Three-Dimensional Imaging of Biological Samples

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy.

Chromatin is a DNA-protein complex that is densely packed in the cell nucleus. The nanoscale chromatin compaction plays critical roles in the modulation of cell nuclear processes. However, little is known about the spatiotemporal dynamics of chromatin compaction states because it remains difficult to quantitatively measure the chromatin compaction level in live cells. Here, we demonstrate a strategy, referenced as DYNAMICS imaging, for mapping chromatin organization in live cell nuclei by analyzing the dynamic scattering signal of molecular fluctuations. Highly sensitive optical interference microscopy, coherent brightfield (COBRI) microscopy, is implemented to detect the linear scattering of unlabeled chromatin at a high speed. A theoretical model is established to determine the local chromatin density from the statistical fluctuation of the measured scattering signal. DYNAMICS imaging allows us to reconstruct a speckle-free nucleus map that is highly correlated to the fluorescence chromatin image. Moreover, together with calibration based on nanoparticle colloids, we show that the DYNAMICS signal is sensitive to the chromatin compaction level at the nanoscale. We confirm the effectiveness of DYNAMICS imaging in detecting the condensation and decondensation of chromatin induced by chemical drug treatments. Importantly, the stable scattering signal supports a continuous observation of the chromatin condensation and decondensation processes for more than 1 h. Using this technique, we detect transient and nanoscopic chromatin condensation events occurring on a time scale of a few seconds. Label-free DYNAMICS imaging offers the opportunity to investigate chromatin conformational dynamics and to explore their significance in various gene activities.

Two-dimensional modelling of systematic surface height deviations in optical interference microscopy based on rigorous near field calculation

Optical interference microscopes are widespread in topography and roughness measurement on the micro- and nanoscale. In spite of a wide range of scientific and industrial applications, systematic deviations between the measured and the real surface topography occur in certain situations, e.g. at edges or steep flanks. We present a numerical model considering the properties of the measurement instrument as well as the surface structure to be measured in order to get new insights into the physical dependencies of these deviations. The computation is based on a rigorous simulation of the scattered near field combined with a Fourier optics treatment of the image formation in the measurement instrument. In this study, the near fields are calculated with an open-source finite element method (FEM) software and a commercial finite difference time domain method (FDTD) software. The numerical results are compared with an analytical Kirchhoff approach and measurements. The main intention of this paper is to introduce the modelling and point out possible fields of application. In further studies, this model could be extended to 3D and parameter dependencies of systematic deviations, such as the material of the measurement object and the NA of the measurement instrument, could be investigated.

Study of the Structural and Morphological Properties of HPHT Diamond Substrates

The morphological and structural properties of a series of high-pressure high-temperature (HPHT) single-crystal diamond substrates are comprehensively studied by white-light optical interference microscopy, atomic-force microscopy, and X-ray diffraction analysis. Procedures that provide a means for characterizing the substrate parameters most critical for epitaxial application with the laboratory equipment are described. It is shown that the jewelry-type characterization of diamond substrates is insufficient to assess the possibility of their use for the epitaxial growth of chemical-vapor-deposited (CVD) diamond.

Coherence scanning and phase imaging optical interference microscopy at the lateral resolution limit.

To get physical insight into the 3D transfer characteristics of interference microscopy at high numerical apertures we study reflecting rectangular grating structures. In general, the height obtained from phase information seems to be reduced, whereas height values resulting from coherence scanning sometimes seem to be systematically overestimated. Increasing the numerical aperture of an interference microscope broadens the spectra of the resulting interference signals, thus offering a broad variety of wavelength contributions to be analyzed. If phase analysis of a measured far-field interference wavefront is performed at very short wavelengths the periodical profiles obtained from coherence scanning and phase shifting analysis differ only by the measured amplitude. However, at longer wavelength there is a 180° phase shift of the measured profiles obtained from phase analysis compared to coherence peak analysis. Increasing the evaluation wavelength improves the lateral resolution since the long wavelength contributions are related to electromagnetic waves of high angles of incidence. This behavior is to the best of our knowledge not documented in literature so far. It was first observed experimentally and could be confirmed by simulation results obtained from either Kirchhoff diffraction theory or an extended Richards-Wolf model developed in our group. Compared to original input profiles used for the simulation the profiles obtained from phase evaluation correspond quite well at longer wavelength, whereas the results obtained from coherence peak analysis are typically inverted with respect to height.

Исследование структурных и морфологических свойств HPHT алмазных подложек

AbstractThe morphological and structural properties of a series of high-pressure high-temperature (HPHT) single-crystal diamond substrates are comprehensively studied by white-light optical interference microscopy, atomic-force microscopy, and X-ray diffraction analysis. Procedures that provide a means for characterizing the substrate parameters most critical for epitaxial application with the laboratory equipment are described. It is shown that the jewelry-type characterization of diamond substrates is insufficient to assess the possibility of their use for the epitaxial growth of chemical-vapor-deposited (CVD) diamond.

Effect of different surface treatments on bond strength, surface and microscopic structure of zirconia ceramic

ObjectivesTo evaluate the effect of different surface treatments; plasma treatment, silica coating using plasma technology and sandblasting, on bond strength, surface roughness and microscopic structure of yttria-stabilized tetragonal zirconia polycrystals(Y-TZP) after thermo-cycling. Matrials and methodsOne hundred discs (n = 100) of yttria-stabilized tetragonal zirconia were prepared from (Y-TZP) ceramic blocks using MAD/MAM milling technology, and were divided into four equal groups (n = 25) according to the type of surface treatment. Group (1): control (no surface treatment). Group (2): zirconia discs were sandblasted by alumina particles. Group (3): zirconia discs treated by plasma technology to produce surface roughness. Group (4): zirconia discs coated by silica using plasma technology. Samples of each group were subdivided into four subgroups according to different analytical techniques. Subgroup (A): (n = 10) subjected to testing of bond strength of zirconia discs to adhesive resin cement after thermo-cycling. Subgroup (B): (n = 5), to evaluate the microscopic changes of zirconia discs by scanning electron microscope (SEM). Subgroup(C): (n = 5) to evaluate the crystal structure and phase transformation of YZ ceramic by X-ray diffraction (XRD). Subgroup (D): (n = 5) to measure three dimensional surface roughness of YZ ceramic by optical interference microscope. ResultsStatistical analysis of shear bond strength by ANOVA revealed the presence of no statistically significant difference between group (3) and (4); both showed the statistically significantly highest mean shear bond strength values. Group (2) showed statistically significantly lower mean values followed by group (1). SEM showed that the topographic pattern differed by different surface treatments of samples. XRD revealed that; group (1) showed the statistically significantly highest mean % of zirconium oxide (Tetragonal phase). Group (2) showed the statistically significantly lowest mean % of Zirconium oxide (Tetragonal phase) and highest mean % of Boehmite and Zirconium oxide (Anorthic phase); Group (3) and (4) showed the statistically significantly highest mean % of Zirconium oxide (Monoclinic phase) and low % of zirconium oxide (Tetragonal phase). 3D- optical surface roughness showed that group (3) and (4) had highest mean (Ra) values. Group (2) showed statistically significantly lower mean values. Group (1) showed the statistically significantly lowest mean (Ra) values. Conclusions(1) Surface treatments of Y-TZP ceramic together with MDP primer and silane-coupling agent application improve the bond strength to resin cement. (2) Plasma-Silica coating and plasma-oxygen treatment, both are valuable methods that improve the bond strength of resin cement to Y-TZP ceramic. (3) Silica coating by plasma technology provides durable bond strength and can be a promising alternative pretreatment before silane application to enhance bonding with zirconia ceramic. (4) Tetragonal-monoclinic phase transformation had occurred in Y-TZP samples received both types of plasma treatment.

Assessment of replication fidelity of optical microstructures by hot embossing

This work reports the performance evaluation of low-cost hot embossing processes of fine three-dimensional microstructures, namely (i) cylindrical microlens array, (ii) plano-convex aspheric Fresnel lens, and (iii) pyramidal array structures. All those optical elements were replicated by employing a low-cost hot embossing tool in optical quality acrylic material. Scanning electron microscopy and optical interference microscopy techniques were used to quantitatively measure the obtained structures. In terms of the replication fidelity with respect to the mold counterpart, the resulting optical elements presented at least 96 % of dimensional fidelity at micro- and nanometer scale, including the structures with sharp edges present in the Fresnel lens. In terms of the surface optical quality, resulting root mean square surface roughness of at least one tenth of wavelength was obtained, considering applications in the visible range of spectrum. The results showed that even nanostructures generated by the material removal mechanisms during mold fabrication, such as crystal grain elastic recovery, were well replicated with differences in the range of few of nanometers which is within the vertical resolution of the employed optical interference technique.

Thermal melting and ablation of silicon by femtosecond laser radiation

The space-time dynamics of thermal melting, subsurface cavitation, spallative ablation, and fragmentation ablation of the silicon surface excited by single IR femtosecond laser pulses is studied by timeresolved optical reflection microscopy. This dynamics is revealed by monitoring picosecond and (sub)nanosecond oscillations of probe pulse reflection, which is modulated by picosecond acoustic reverberations in the dynamically growing surface melt subjected to ablation and having another acoustic impedance, and by optical interference between the probe pulse replicas reflected by the spalled layer surface and the layer retained on the target surface. The acoustic reverberation periods change during the growth and ablation of the surface melt film, which makes it possible to quantitatively estimate the contributions of these processes to the thermal dynamics of the material surface. The results on the thermal dynamics of laser excitation are supported by dynamic measurements of the ablation parameters using noncontact ultrasonic diagnostics, scanning electron microscopy, atomic force microscopy, and optical interference microscopy of the modified regions appearing on the silicon surface after ablation.

Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.

Influence of surface roughness and interfacial layer on the infrared spectra of V-CVD grown 3C-SiC/Si (1 0 0) epilayers

We have reported room temperature infrared reflectance and transmission measurements at near normal incidence in the frequency range of 200–6500 cm−1 to characterize 3C-SiC epitaxial layers grown on (1 0 0) Si by CVD in a vertical reactor configuration by using different Si/C ratios and growth time ranging from 2 min to 4 h. The effects of surface roughness and ‘conducting interfacial’ transition layer are meticulously included in the effective medium theory for simulating the infrared spectra to explicate observed damping of interference fringe contrasts with increasing frequencies. In a few epitaxially grown samples we have adopted Bruggeman's two-component model to elucidate atypical divots seen within the reststrahlen band by assuming the coexistence of crystalline and intergranular grains forming heterogeneous 3C-SiC. The estimated values of surface roughness and conducting transition layer are correlated very well with the existing data from optical interference and scanning probe microscopy.

Crystallization Mechanisms in Convective Particle Assembly

Colloidal particles are continuously assembled into crystalline particle coatings using convective fluid flows. Assembly takes place inside a meniscus on a wetting reservoir. The shape of the meniscus defines the profile of the convective flow and the motion of the particles. We use optical interference microscopy, particle image velocimetry, and particle tracking to analyze the particles' trajectory from the liquid reservoir to the film growth front and inside the deposited film as a function of temperature. Our results indicate a transition from assembly at a static film growth front at high deposition temperatures to assembly in a precursor film with high particle mobility at low deposition temperatures. A simple model that compares the convective drag on the particles to the thermal agitation explains this behavior. Convective assembly mechanisms exhibit a pronounced temperature dependency and require a temperature that provides sufficient evaporation. Capillary mechanisms are nearly temperature independent and govern assembly at lower temperatures. The model fits the experimental data with temperature and particle size as variable parameters and allows prediction of the transition temperatures. While the two mechanisms are markedly different, dried particle films from both assembly regimes exhibit hexagonal particle packings. We show that films assembled by convective mechanisms exhibit greater regularity than those assembled by capillary mechanisms.

Characterization of Surface Composition and Microstructure of H21 Steel Implanted Using Masking Ti-Implantation

Ti ions were implanted into H21 steel to improve the surface structure of oxides in H21 steel. The wear characteristics of the implanted steel was measured and compared to the performance of the un-implanted steel by a line-cutting apparatus and an optical interference microscope. The Ti concentration depth profile of the implanted steel was measured by Rutherford backscattering spectroscopy (RBS). The structure and composition of oxides were analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD), respectively. The results showed that the improved wear resistance of the implanted steel was mainly due to the structure and composition changes of the surface oxides after Ti ion implantation.

Frequency to Voltage Converter as a Phase Controller in Phase Shifting Interference Microscopy

A new system for the control of a piezoelectric device is introduced. It works as a phase shifter in an interference optical microscope. This circuit translates controllable constant frequency signals, generated with a standard sound card, to electrical signals with constant voltage through a frequency-to-voltage converter; this full analog conversion allows for producing voltage steps of 16 µv, which is a better resolution than a commercial digital to analog converter working (in the desired voltage range), and hence, results in an arbitrary phase uncertainty under 3%. The calibration method allows for verification of the displacement's steadfastness in the arbitrary phase, and for diagnosing of errors that could alter the experimental interferograms. To check the system calibration, it was experimentally and successfully tested on a silicon surface with rectangular holes of known depth.