Confocal Scanning Optical Microscope Research Articles

Tandem-scanning Microscopy of Slow-speed Enamel Cutting Interactions

The aim of this study was to observe the subsurface microstructure of dental enamel during cutting procedures. Real-time confocal scanning optical microscopy, with a tandem-scanning reflected-light microscope, is suitable for making high-resolution images of enamel prisms in intact teeth under near-normal conditions. Blocks of enamel were held on a micro-positioning stage and advanced toward the tip of a diamond knife. The orientation of the samples was varied to simulate common clinical cutting conditions. Oil immersion objectives were used with the knife rigidly mounted in a specially made carrier. This was attached to the objective below its end-lens. The images were recorded with a sensitive video camera and cassette recorder for analysis of the cutting interactions at a slow playback speed. The presence of decussating enamel prisms was particularly effective at slowing crack propagation when loading was applied in an appropriate direction. Residual subsurface cracking was seen when the blade had finished cutting. This was most pronounced in regions where the enamel prisms were cut transverse to their long axis; this was equivalent to the preparation of the cervical margin of an approximately cavity. Where there is surface adhesion to enamel (e.g., glass-ionomer cements), residual subsurface cracking will produce a weakened substrate for bonding that could have important implications for restoration longevity.

Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells.

Dark-grown corn coleoptiles and parsley hypocotyls and their roots were loaded with acetoxymethyl esterified forms of the Ca2+ indicator fluo-3, and the pH indicator 2',7'-bis (2-carboxyethyl)-5(and-6)-carboxyfluorescein. These tissues were treated with the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin analogue, or abscisic acid (ABA), and the cytosolic pH (pHcyt) and cytosolic Ca2+ ([Ca2+]cyt) changes were monitored by confocal scanning optical microscopy. Over a period of 4 min pHcyt decreased 0.1-0.2 pH unit and [Ca2+]cyt increased from 280 to 380 nM in response to 2,4-D. ABS, on the other hand, induced cytosolic alkalinization of 0.05-0.1 pH unit with a concomitant increase in [Ca2+]cyt from 240 to 320 nM over a 4-min period. Responses similar to these were observed in all the tissues tested. We suggest that pHcyt profoundly influences signaling by[Ca2+]cyt, possibly by regulating Ca2+-protein binding, and that the divergent effects of auxin and ABA on pHcyt underlie their mutual antagonism.

Confocal Scanning Optical Microscopy of Meat Products

ABSTRACTConfocal scanning optical microscopy (CSOM) was used to observe the internal structure of frankfurters and summer sausage. This new method allows optical sectioning of thick specimens and thus avoids the potential problem of smearing encountered when fat globules and bacteria are at the surface of sections.

Three-dimensional reconstruction of the rabbit cornea by confocal scanning optical microscopy and volume rendering

The 3-D reconstruction of the central optical zone of the rabbit cornea in an enucleated eye was accomplished. The confocal laser scanning reflected light microscope was used to obtain 3-microm optical serial sections from which the reconstruction was made by computer graphics methods (volume rendering). This volume reconstruction of a living, semitransparent, 400-microm thick, low contrast optical element in the eye presents a new method to visualize the cornea in three dimensions.

Differential interference contrast imaging on a real time confocal scanning optical microscope

The first uses of Nomarski differential interference contrast imaging with the real time confocal scanning optical microscope are described. The advantage of differential interference contrast imaging in a confocal scanning microscope is that both the height and width of an edge can be measured without ambiguity, even if the edge is taller than half of a wavelength. Imaging modes are described in which (1) average reflectivity changes of the sample have been eliminated but the resulting edge enhanced images are nonlinear in phase, or (2) linear phase images are superimposed on an average reflectivity image of the sample. A third method has been developed which both eliminates the reflectivity image of the sample and generates edge enhanced images which are linear in phase for small phase changes. Applications to box-in-box overlay targets, imaging the passivation layer of an integrated circuit, and the measurement of sidewall slope are described.

Architectural organization in the interphase nucleus of the protozoan Trypanosoma brucei: location of telomeres and mini-chromosomes.

We studied the spatial organization of chromatin in the interphase G1, S and G2 nucleus of the protozoan Trypanosoma brucei, applying in situ hybridization with conventional fluorescence and confocal scanning optical microscopy. The majority of the trypanosome telomere GGGTTA repeats from different chromosomes were found clustered together, either extending in a network through the nuclear interior or localized at the nuclear periphery. The population of one hundred mini-chromosomes was often asymmetrically located: either clustered in a narrow band in close association with the nuclear envelope or distributed into several clusters that segregated into roughly one half of the nucleus. The nuclear organization may undergo modifications during the cell cycle and development. We conclude that non-random spatial positioning of DNA exists in the nucleus of this protozoan. Finding a high level of structural organization in the interphase nucleus of T.brucei is an important first step towards understanding chromosome structure and functioning and its role in the control of gene expression.

Dependence of 3-D optical transfer functions on the pinhole radius in a fluorescent confocal optical microscope

The resolution of a fluorescent confocal scanning optical microscope (CSOM) is superior to that of a conventional fluorescent optical microscope. To attain this superiority, the fluorescent CSOM uses a pinhole in front of the detector. Thus, the resolution of the CSOM is dependent on the pinhole radius. Three-dimensional optical transfer functions are calculated for the various radii to elucidate this dependence. The results show that a CSOM with a radius smaller than ~1 optical unit has a bandwidth comparable with that of an infinitely small radius.

Observation of the rabbit cornea and lens with a new real‐time confocal scanning optical microscope

AbstractA new one‐sided tandem scanning microscope (OTSM) has been utilized to optically section a transparent cornea and ocular lens with submicron depth and transverse resolution. Its high resolution, image quality, and contrast, together with color capability, real‐time operation, and easy alignment, have advantages over previous confocal systems. Planes within the nuclei of surface epithelial cells, the epithelial basement membrane, nerve fibers, the interior of endothelial cells, and the transverse structure of the lens fibers are readily observed. The utility of real‐time microscopic observation of live biological structures provides a new paradigm for cell biology and diagnostic ophthalmology.

A method for evaluating microscope objectives to optimize performance of confocal systems

SUMMARYA method for evaluating the performance of microscope objectives on two types of confocal scanning optical microscope is presented. Of these two confocal microscope types, off‐axis beam‐scanning systems are found to require microscope objectives which have been corrected for flatness of field as well as for spherical aberration and astigmatism in order to obtain maximum axial and laterial resolution. In the case of on‐axis specimen‐scanning microscopes, less highly corrected objective lenses (not corrected for flatness of field) may in practice prove to have superior resolving properties.

Multilevel CD/overlay metrology using a real-time confocal scanning optical microscope

Linearity and short-term repeatability have been studied for critical dimension (CD) and overlay measurements using the Real-Time Confocal Scanning Optical Microscope (RSOM). Short-term repeatability of 15nm (three-sigma) has been demonstrated for submicron CD and overlay. CD linearity was measured in the 0.5μm to 1.3μm region using a top-down SEM for reference. Overlay linearity was measured using electrical overlay structures with known offsets in the range -0.2μm to +0.2μm. Response surface methodology was used to determine the optimum focus for achieving repeatable overlay measurements. The results were used to compare the performance of various overlay structures.

Scanning optical microscopes close in on submicron scale

The principles and advantages of the confocal scanning optical microscope (CSOM) are described, and the history of the concept is given. The CSOM is compared with the scanning electron microscope, scanning acoustic microscope, scanning tunneling microscope, scanning force microscope, and near-field scanning optical microscope. The imaging properties and applications of the CSOM are discussed.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Erratum

Journal of Cell Science Volume 94, Part 2, October 1989, pages 175–206. The citation ‘Reproduced, with permission, from Koch et al. (1987).’ given in the figure legend of Fig. 13 is incorrect. The citation should read ‘Reproduced, with permission, from White, J. G. et al. (1987). Further details of the cells, antigen and labelling conditions used, and other examples of such confocal images may be found in Koch et al. (1987).’

Three-dimensional optical transfer function for the fluorescent scanning optical microscope with a slit

The imaging properties of a fluorescent scanning optical microscope (SOM) with a slit detector are investigated by calculating the 3-D optical transfer function (3-D OTF). The bandwidth of the 3-D OTF in the lateral direction varies with the slit direction. The widest bandwidth corresponds to the confocal SOM with an extremely small pinhole detector. The widest longitudinal bandwidth also corresponds to the longitudinal one for the confocal SOM. The contrast for the microscope with the slit is lower than that for the confocal SOM. When the slit width increases, the imaging properties approach those of the conventional optical microscope.

Application of confocal scanning optical microscopy to the study of fibre-reinforced polymer composites

Application of confocal scanning optical microscopy to the study of fibre-reinforced polymer composites

Depth resolution of the fluorescent confocal scanning optical microscope

The fluorescent confocal scanning optical microscope is capable of 3-D observation, for which depth discrimination is vital. To characterize the depth discrimination capability of this microscope, intensity variations depending on fluorescence wavelength are simulated for fluorescent films being moved in the depth direction. Also, the resolution between two films is calculated for a range of fluorescence wavelengths. Results of experiments using photoresist films are compared to simulations. As a practical application, through-holes opened in a thick photoresist film are observed by slice images in the depth direction.

A new microscope for semiconductor luminescence studies

A confocal scanning optical microscope is integrated with a helium cryostat to provide submicron spatial resolution of a specimen immersed in helium. Using gaseous or superfluid liquid helium, specimen temperature can be controlled from 300 to 2 K. Monochromatic illumination is provided and luminescence is analysed by conventional means. The specimen is scanned mechanically and its position relative to the focal point of the optical system is continously monitored by a capacitance displacement transducer and an optical system that senses light reflected from the specimen surface.

Electro-Optic and Non-Linear Optical Coefficients of (Pb, La)(Zr, Ti)O3, BaTiO3, (Sr, Ba)Nb2O6 and Ba2NaNb5O15 Thin Films

ABSTRACTThe electro-optic properties of sputter-deposited PLZT, BaTiO3, SBN, and BNN films on fused silica substrates have been studied using a confocal scanning optical polarization microscope. The Pockels, Kerr, and higher order electro-optic coefficients and their relations to the non-linear optical coefficients in the films are presented. The materials and physical properties of the films are discussed.

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.

Confocal scanning microscopy: three-dimensional biological imaging

Confocal scanning optical microscopy, arguably the most significant advance in biological light microscopy in this decade, enables one to obtain quantitative non-invasive optical sections through labelled biological specimens, virtually free from out-of-focus blur. A set of these optical sections collected at a series of focal levels through an object constitute a three-dimensional image which may then be processed digitally for display as a computer reconstruction, a stereo pair or an animation sequence.

Confocal scanning optical microscopy and its applications for biological specimens

ABSTRACT Confocal scanning optical microscopy (CSOM) is a new optical microscopic technique, which offers significant advantages over conventional microscopy. In laser scanning optical microscopy (SOM), the specimen is scanned by a diffractionlimited spot of laser light, and light transmitted or reflected by the in-focus illuminated volume element (voxel) of the specimen, or the fluorescence emission excited within it by the incident light, is focused onto a photodetector. As the illuminating spot is scanned over the specimen, the electrical output from this detector is displayed at the appropriate spatial position on a TV monitor, thus building up a two-dimensional image. In the confocal mode, an aperture, usually slightly smaller in diameter than the Airy disc image, is positioned in the image plane in front of the detector, at a position confocal with the in-focus voxel. Light emanating from this in-focus voxel thus passes through the aperture to the detector, while that from any region above or below the focal plane is defocused at the aperture plane and is thus largely prevented from reaching the detector, contributing essentially nothing to the confocal image. It is this ability to reduce out-of-focus blur, and thus permit accurate non-invasive optical sectioning, that makes confocal scanning microscopy so well suited for the imaging and three-dimensional tomography of stained biological specimens. In this review, I explain the principles of scanning optical microscopy and blur-free confocal imaging, discuss the various imaging modes of confocal microscopy, and illustrate some of its early applications.