Confocal Scanning Optical Microscope Research Articles

Appositional enamel growth in molars of South African fossil hominids

Enamel is formed incrementally by the secretory activity of ameloblast cells. Variable stages of secretion result in the formation of structures known as cross striations along enamel prisms, for which experimental data demonstrate a correspondence with daily periods of secretion. Patterns of variation in this daily growth are important to understanding mechanisms of tooth formation and the development of enamel thickness. Transmitted light microscopy (TLM) of histological ground sections and scanning electron microscopy (SEM) of bulk specimens or their surface replicas are the usual methods for investigating cross striations. However, these methods pose some constraints on the study of these features in Plio-Pleistocene hominid enamel, the specimens of which may only rarely be sectioned for TLM or examined on only their most superficial surfaces for SEM. The recent development of portable confocal scanning optical microscopy (PCSOM) resolves some of the restrictions on fractured enamel surfaces, allowing the visualization of cross striations by direct examination. This technology has been applied here to the study of Australopithecus africanus and Paranthropus robustus hominid molars from the Plio-Pleistocene of South Africa. We hypothesize that these taxa have increased enamel appositional rates compared with modern humans, because despite having thicker enamelled molars (particularly P. robustus), the enamel crowns of these fossil taxa take an equivalent or reduced amount of time to form. Cross striations were measured in cuspal, lateral and cervical regions of the enamel crowns, and, within each region, the inner, middle and outer zones. Values obtained for A. africanus outer zones of the enamel crown are, in general, lower than those for P. robustus, indicating faster forming enamel in the latter, while both taxa show higher rates of enamel growth than modern humans and the African great apes. This demonstrates a relatively high degree of variability in the mechanisms underlying the development of enamel across taxa.

Phase transitions and domain structures in strained pseudocubic (100)SrTiO3thin films

The mutual interactions between a structural transition and a ferroelectric transition are analyzed for different strain states in a pseudocubic (100) $\mathrm{Sr}\mathrm{Ti}{\mathrm{O}}_{3}$ film by examining the equilibrium solutions of the total free energy as a function of polarization, strain and structural order parameter. The range of possible ferroelectric transition temperatures and the possible ferroelectric states of a strained $\mathrm{Sr}\mathrm{Ti}{\mathrm{O}}_{3}$ film are determined with respect to the variation in the reported properties of bulk $\mathrm{Sr}\mathrm{Ti}{\mathrm{O}}_{3}$ single crystals. The ferroelectric and structural domain morphologies at a biaxial tensile strain ${e}_{0}=0.94%$ were predicted using phase-field simulations. It is shown that variations in the reported values of bulk properties and in the Landau energy coefficients from different literature sources lead not only to a wide range of possible transition temperatures at a given strain, but also to different ferroelectric states (e.g., polarization along the pseudocubic ⟨110⟩ vs ⟨100⟩ directions) thus different domain structures under a biaxial tensile strain. Both optical second harmonic generation and confocal scanning optical microscopy measurements demonstrate that the domain states in $\mathrm{Sr}\mathrm{Ti}{\mathrm{O}}_{3}$ films strained at ${e}_{0}=0.94%$ and 1.16% exhibit polar directions along the pseudocubic ⟨110⟩ directions within the pseudocubic (001) plane of the film.

Coherent confocal microscope with a phase-only filter in its extended source

The phase information of an extended source is reconstructed by use of a two-zone (annular) phase-only filter in a coherent confocal scanning optical microscope. The dependence of its resolution on its source size is investigated theoretically by its three-dimensional optical transfer function (3D OTF). The results show that the resolution is improved, even though the source size is enlarged.

Localized microwave resonances in strained SrTiO3 thin films

Local frequency-dependent polar dynamics of strained SrTiO3 films grown on DyScO3 are investigated using time-resolved confocal scanning optical microscopy. Spectroscopic information is obtained with <1μm spatial resolution over the frequency range 2–4GHz. Most of the DyScO3 film is found to be spatially homogeneous, in contrast to relaxed films. A strong correlation between spatial and spectral homogeneity is revealed. In addition, resonant structures are discovered that are localized both in space and in frequency.

A simple reflection-type two-dimensional refractive index profile measurement technique for optical waveguides

We report a new configuration of a reflection-type confocal scanning optical microscope system for measuring the refractive index profile of an optical waveguide. Several improvements on the earlier design are proposed; a light emitting diode at 650nm wavelength instead of a laser diode or He–Ne laser is used as a light source for better index precision, and a simple longitudinal linear scanning and a curve fitting techniques are adapted instead of a servo control for maintaining an optical confocal arrangement. We have obtained spatial resolution of 800nm and an index precision of 2×10−4. To verify the system’s capability, the refractive index profiles of a conventional multimode fiber and a home-made four-mode fiber were examined with our proposed measurement method.

Reflection-type Optical Waveguide Index Profiling Technique

We report a new configuration of a reflection-type confocal scanning optical microscope system for measuring the refractive index profile of an optical waveguide. Several improvements on the earlier design are proposed; a light emitting diode (LED) at 650 nm wavelength instead of a laser diode (LD) or He-Ne laser is used as a light source for better index precision, and a simple longitudinal linear scanning and curve fitting techniques are adapted instead of a servo control for maintaining an optical confocal arrangement. We have obtained spatial resolution of 700 nm and an index precision of $2\times10^{-4}$ . To verify the system's capability, the refractive index profiles of a conventional multimode fiber and a home-made four-mode fiber were examined with our proposed measurement method.

Three-dimensional polarization imaging of (Ba,Sr)TiO3:MgO composites

The dielectric tuning and loss of (Ba,Sr)TiO3:MgO bulk composites depend strongly on the connectivity and interaction among the two phases. To investigate this relationship, the polar structure and dynamics of these composites is mapped as a function of space and time using a pair of three-dimensional probes: second-harmonic confocal scanning optical microscopy (SH-CSOM), which maps ferroelectric polarization in three dimensions, and time-resolved scanning optical microscopy (TR-CSOM), which maps polarization dynamics along two spatial dimensions and one time dimension. SH-CSOM measurements reveal a high degree of homogeneity within the (Ba,Sr)TiO3 regions, while TR-CSOM measurements indicate that topologically connected regions respond with a spatially uniform phase.

A modified blister test to study the adhesion of thin coatings based on local helium ion implantation

A modified blister test has been developed based on helium ion implantation into selected areas of the metal substrate prior to the coating deposition. After a post-deposition thermal annealing, blisters are formed by agglomeration of the implanted gas at the ceramic–metal interface. This method can be used to control the pressure in the blister which eventually may lead to delamination at the periphery of the blister. A microsieve with a regular array of circular holes is used during the implantation to assure the initial blister size. Two different microsieves were employed in this work, with pore diameters of 1.5 and 4.5 μm, respectively. The distance between the centres of neighbour pores is twice the pore diameter. Scanning Electron Microscopy (SEM) and Confocal Scanning Optical Microscopy (CSOM) observations allowed the determination of the blistering parameters such as the radius, the height and the blister volume. From the gas content and these parameters, the work of adhesion or energy release rate can be obtained. In this work, we present the first results of this blister test applied to W–C:H films and multilayers of Ti and Al deposited by Physical Vapour Deposition on polycrystalline copper substrates. The copper substrates were implanted with 34 keV He + ions up to fluences of 3 and 5×10 16 cm −2 before the deposition of the coatings and annealed afterwards in vacuum at temperatures from 773 to 1073 K for 30 min. Delamination of the Ti/Al multilayer coatings was already detected after annealing at 873 K with an energy release rate estimated to be 0.5 J m −2 at a typical helium pressure of 10 7 Pa. No delamination but only helium swelling was observed for W–C:H coatings annealed at 1073 K. Results of experiments on uncoated copper samples are also shown in order to explain the mechanism of helium bubble growth and helium release that causes the creation of the blisters.

(Ba, Sr)TiO 3 thin films grown by pulsed laser deposition with low dielectric loss at microwave frequencies

We report a method for producing BST films with consistently high figures of merit for tunable microwave applications. (Ba1-x,Srx)TiO3 (x=0.4, target doped with 1% W) thin films have been deposited using pulsed laser deposition onto (100)MgO substrates. Films were deposited at low partial pressures of oxygen (50 mTorr) at a substrate temperature of 730 °C. An analysis of the X-ray diffraction data indicates that the film has a nearly cubic structure, with the overall lattice parameter enlarged relative to the bulk material due to the presence of oxygen vacancies. A post-deposition anneal of the film in flowing oxygen (1000 °C for 6 h) resulted in a decrease in the lattice parameter while remaining nearly cubic. An analysis of the microwave dielectric properties (1–20 GHz) showed that the annealed film exhibited about 10% tunability for an applied bias field of 67 kV/cm with a dielectric Q(1/tanδ)>600. Investigation of the films by time-resolved confocal scanning optical microscopy (CSOM) has revealed that there is an out-of-plane polarization at zero applied field (EDC=0). The results show that the paraelectric response is relatively insensitive to applied field, while the ferroelectric response is correlated with the growth of in-plane nanodomains. We find these results to be consistent with a large number of studies that show that strain-relief is of paramount importance if ferroelectric films are to be developed as microwave circuit components.

Photoelectronic transport imaging of individual semiconducting carbon nanotubes

Photoconductivity in individual semiconducting single-wall carbon nanotubes was investigated using a confocal scanning optical microscope. The magnitude of the photocurrent was found to increase linearly with the laser intensity, and to be maximum for parallel orientation between the light polarization and the tube axis. Larger currents were obtained upon illuminating the tubes at 514.5 nm in comparison to those at 647.1 nm, consistent with the semiconducting tubes having a resonant absorption energy at the former wavelength. Moreover, the determination of the photoresponse as a function of position along single nanotubes has proven to be a useful tool to monitor local electronic structure effects.

Factors that influence confocal apertureless near-field scanning optical microscopy

We demonstrate several influences of a silicon probe of an atomic-force microscope on the fluorescence of single CdSe/ZnS quantum dots on a glass cover slip, measured in a confocal apertureless scanning near-field optical microscope (ANSOM): (a) probe-induced fluorescence quenching with the polarization of excitation orthogonal to probe axes; (b) changes in fluorescence enhancement as the quantum yield of a quantum dot fluctuates (the fluorescence enhancement is much larger in a low quantum yield state); (c) the probe-quantum dot optical interaction depends on very local and sometimes non-uniform optical properties of the probe; some fluorescence ANSOM images of quantum dots are asymmetrical and shifted with respect to topographical images; and (d) probe pressure, not the optical near-field, bleaches quantum dot fluorescence during vertical probe scans, or when the microscope operates in dynamic-force mode and the probe tip taps the sample. The fluorescence measurements have been done using a confocal ANSOM with 10–12% fluorescence collection efficiency under total internal reflection conditions of excitation.

Study of the Ferroelectric Domain Structure and Phase Transitions by Confocal Scanning Optical Microscopy

Confocal scanning optical microscopy (CSOM) has been shown to be a promising method to investigate different properties of ferroelectric domain structures. It combines diffraction-limited spatial resolution with non-invasiveness and sensitivity of optical techniques. We have obtained optical and electrooptic images in ac modulation mode using model ferroelectrics systems, including both single crystals and thin films. The origin of CSOM contrast, its dependence on mutual orientation of the electric field, ferroelectric polarization, and light polarization vector have been established. The direction of ferroelectric polarization in a given point can be determined accurately, within a few degrees. The phase transition in solid solution (Ba,Sr)TiO 3 thin films with different compositions has been studied. It has been shown that paraelectric and ferroelectric areas coexist on both sides of dielectric permittivity maximum. The phase transition was observed to be locally sharp, contrary to wide diffused peak in macroscopic characteristic. Local ferroelectricity was also observed in pure SrTiO 3 thin films, a quantum paraelectric in bulk form.

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy.

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 microm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 degrees C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

Local ferroelectricity in SrTiO3 thin films.

The temperature-dependent polarization of SrTiO3 thin films is investigated using confocal scanning optical microscopy. A homogeneous out-of-plane and an inhomogeneous in-plane ferroelectric phase are identified from images of the linear electro-optic response. Both hysteretic and nonhysteretic behavior are observed under a dc bias field. Unlike classical transitions in bulk ferroelectrics, local ferroelectricity is observed at temperatures far above the dielectric permittivity maximum. The results demonstrate the utility of local probe experiments in understanding inhomogeneous ferroelectrics.

Behavior of the point-spread function in photon-limited confocal microscopy.

We study the behavior of the point-spread function (PSF) of the confocal scanning optical microscope (CSOM) when the available optical energy density from the sample plane is low (<7.5 microJoule/micrometers2). The PSF profile is analyzed under three photon-limited imaging conditions: (1) reflection-type CSOM with a weak source and a perfectly reflecting sample, (2) reflection-type CSOM with a strong illumination source and a weak sample, and (3) fluorescence CSOM with a weak fluorescent sample. Linfoot's image quality criteria of fidelity, structural content, and correlation quality are used to assess the reproducibility of the PSF profile as a function of the photon number. Low photon numbers yield a PSF profile that is difficult to maintain from one location in the sample plane to another. The optical sectioning capability of the CSOM was found to deteriorate more quickly against light power reduction than its transverse resolving power. The signal-to-noise ratio of the scanned CSOM image improves exponentially with the photon number from the sample plane. The noise that is generated by an unstable PSF has an average amplitude that decreases exponentially with the photon number and is significant only at low photon numbers. The CSOM image quality deteriorates because of spurious high-frequency components, degradation in the PSF dynamic range, and varying resolving power.

High-resolution thermoreflectance microscopy

ABSTRACTWe present very high-resolution thermal microscopy using the technique of thermoreflectance, a non-contact measurement of the temperature in and around active semiconductor devices. By measuring the local change in reflectivity and comparing to the optical index versus temperature for the interface materials, thermoreflectance can determine the local temperature distribution. Thermoreflectance allows us to work at wavelengths much smaller than those used in typical blackbody imaging, and thus the spatial resolution is significantly improved over that of traditional thermal microscopy. In our experimental setup, we have a confocal scanning optical microscope with a tunable laser, where reflected light is detected by a silicon photodiode in a heterodyne scheme. The sample consists of a 600 nm wide poly-silicon wire embedded in silicon dioxide on top of a silicon substrate. Varying the amount and temporal shape of the current through the poly-silicon wire, we generate a controlled thermal profile to test the imaging capability. Our preliminary results indicate sub-micron thermal resolution.

Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen

The influence of oxygen on the photophysical properties of individual DiI18 molecules has been investigated by means of both wide field and confocal scanning optical fluorescence microscopy. Excited close to saturation intensity, single-molecule fluorescence detected in wide field by a charge-coupled device camera showed a dramatic increase at exposure to air compared to the fluorescence when the sample was protected from oxygen by nitrogen flush. The change of the triplet lifetime of individual dye molecules due to oxygen quenching was measured in real time by means of time resolved single photon counting. In the presence of oxygen, the triplet state lifetime decreases from several tens of milliseconds down to fractions of a millisecond, whereas no changes of the intersystem crossing quantum yield and the fluorescence lifetime are observed. The triplet lifetimes in the presence and absence of oxygen, respectively, are anti-correlated indicative of heterogeneity of the polymer surrounding the dye molecules.

Nanopolar reorientation in ferroelectric thin films

The influence of varying oxygen pressure P(O2) during the growth of Ba0.4Sr0.6TiO3 thin films is investigated using dielectric and local optical probes. A transition from in-plane to out-of-plane ferroelectricity is observed with increasing P(O2). Signatures of in-plane and out-of-plane ferroelectricity are identified using dielectric response and time-resolved confocal scanning optical microscopy (TRCSOM). At the crossover pressure between in-plane and out-of-plane polarization (Pc=85 mTorr), TRCSOM measurements reveal a soft, highly dispersive out-of-plane polarization that reorients in plane under modest applied electric fields. At higher deposition pressures, the out-of-plane polarization is hardened and is less dispersive at microwave frequencies, and the dielectric tuning is suppressed. Nanopolar reorientation is believed to be responsible for the marked increase in dielectric tuning at P(O2)=Pc.

Direct observation of local ferroelectric phase transitions in BaxSr1−xTiO3 thin films

The temperature dependence of the local ferroelectric polarization of BaxSr1−xTiO3 thin films is measured with submicrometer resolution using confocal scanning optical microscopy. The local ferroelectric hysteresis loop changes shape and disappears as the temperature is increased over a narrow range of temperatures (ΔT∼10 K). The local Curie temperature TC(x,y) varies significantly with position, and the distribution of TC is consistent with the broad temperature dependence of the dielectric permittivity measured through conventional methods. These results directly confirm the hypothesis that the ferroelectric phase transition in these materials is inhomogeneously broadened.

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis.

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.