Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

The aim of this study was to investigate if load cycling affects interfacial integrity of glass ionomer cements bonded to sound- or caries-affected dentin. A conventional glass ionomer, Ketac Bond, and a resin-modified glass ionomer (Vitrebond Plus), were applied to dentin. Half of the specimens were load cycled. The interfaces were submitted to dye-assisted confocal microscopy evaluation. The unloaded specimens of sound and carious dentin were deficiently hybridized when Ketac Bond was used. Ketac Bond samples showed an absorption layer and an adhesive layer that were scarcely affected by fluorescein penetration (nanoleakage), in sound dentin. Nevertheless, a higher degree of micropermeability was found in carious dentin. In Ketac Bond specimens, load cycling improves the sealing capability and remineralization at the cement–dentin interface as porosity and nanoleakage was reduced. In contrast, samples treated with Vitrebond Plus exhibited a Rhodamine B-labeled absorption layer with scarce nanoleakage in both sound and carious unloaded dentin. The adhesive layer was affected by dye sorption throughout the porous cement–dentin interface. Samples treated with Vitrebond Plus had significant increases in nanoleakage and cement–dye sorption after load cycling. Within the limitations of an in vitro study, it is expected that conventional glass ionomers will provide major clinical efficacy when applied to carious-affected or sound dentin.

Light microscopy is a powerful tool that allows for many types of samples to be examined in a rapid, easy, and nondestructive manner. Subsequent image analysis, however, is compromised by distortion of signal by instrument optics. Deconvolution of images prior to analysis allows for the recovery of lost information by procedures that utilize either a theoretically or experimentally calculated point spread function (PSF). Using a laser scanning confocal microscope (LSCM), we have imaged whole impact tracks of comet particles captured in silica aerogel, a low density, porous SiO2 solid, by the NASA Stardust mission. In order to understand the dynamical interactions between the particles and the aerogel, precise grain location and track volume measurement are required. We report a method for measuring an experimental PSF suitable for three-dimensional deconvolution of imaged particles in aerogel. Using fluorescent beads manufactured into Stardust flight-grade aerogel, we have applied a deconvolution technique standard in the biological sciences to confocal images of whole Stardust tracks. The incorporation of an experimentally measured PSF allows for better quantitative measurements of the size and location of single grains in aerogel and more accurate measurements of track morphology.

The “LEXT” confocal laser scanning microscope has been used for the three-dimensional (3D) imaging of the surface of specimens, especially in materials science fields, by the penetration of near-infrared (NIR) light without mechanical cutting, deposition, or other specimen pretreatment. Noninvasive investigation of various biological tissues such as human spinal dura mater, rat aorta, and cornea without the dehydration process was successfully carried out with the “LEXT,” in order to access both surface and internal topographic images of the biological structures at a good status of the wet tissue such as in vivo, especially in measuring tissue thickness. The confocal NIR laser microscopy offers the viable means to visualize tissue architecture and its thickness in microdomain to integrate 3D images efficiently. We believe that the “LEXT” has a good application for biological researchers to study biomaterials, and it would be useful as a diagnostic tool in the near future.

A confocal scanning laser holography (CSLH) microscope that uniquely combines the concepts of confocal microscopy with holography has been validated for making nonintrusive, full three-dimensional (3D) intensity and phase measurements of objects from a single viewpoint of observation without loss of object information. The phase measurements have been used to determine the 3D refractive indices of a point source heated silicone oil. The refractive indices are converted to 3D temperature measurements, which are useful for heat transfer studies. An important feature of CSLH is its nonintrusive 3D scanning method, which enables its application to the study of Marangoni convection in microgravity with minimal operational vibrations affecting the motion of fluid in the specimen.

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between “acceptable” and “unacceptable” performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

A radioactive particle embedded in a sediment collected from the Irish Sea was examined with spectrometric methods to understand the potential bioavailability of its constituents. Images of the particle surface were acquired in the backscattering mode by scanning electron microscopy. The elemental composition of the particle surface layer was measured using energy dispersive (SEM-EDX) and wavelength dispersive (SEM-WDX) X-ray spectrometers. The investigation showed that the sample consists of a calcite matrix in which uranium is present in the form of separate inclusions. The diameter of U inclusions was less than 10 μm. Synchrotron radiation based X-ray fluorescence in confocal geometry (confocal μ-XRF) was used to determine the spatial distribution of elements in the particle. Three-dimensional reconstructions of the Ca, Cr, Mn, Fe, Zn, Sr, Ba, Pb, and U distributions were performed. The oxidation state of uranium in the different inclusions was determined by synchrotron radiation based X-ray absorption in confocal geometry (confocal μ-XANES). The isotopic composition of uranium was measured by secondary ion mass spectrometry. The results revealed that uranium was depleted in 235U. Pu and other actinides were not detected.

To describe the wide-field ganglion cells, they were injected intracellularly with Neurobiotin using an in vitro preparation of macaque retina and labeled with streptavidin-Cy3. The retinas were then labeled with antibodies to choline acetyltransferase and other markers to indicate the depth of the dendrites within the inner plexiform layer (IPL) and analyzed by confocal microscopy. There were eight different subtypes of narrowly unistratified cells that ramified in each of the 5 strata, S1–5, including narrow thorny, large sparse, large moderate, large dense, large radiate, narrow wavy, large very sparse, and fine very sparse. There were four types of broadly stratified cells with dendritic trees extending from S4 to S2. One type resembled the parvocellular giant cell and another the broad thorny type described previously in primates. Another broadly stratified cell was called multi-tufted based on its distinctive dendritic branching pattern. The fourth type had been described previously, but not named; we called it broad wavy. There was a bistratified type with its major arbor in S5, the same level as the blue cone bipolar cell; it resembled the large, bistratified cell with blue ON-yellow OFF responses described recently. Two wide-field ganglion cell types were classified as diffuse because they had dendrites throughout the IPL. One had many small branches and was named thorny diffuse. The second was named smooth diffuse because it had straighter dendrites that lacked these processes. Dendrites of the large moderate and multi-tufted cells cofasciculated with ON-starburst cell dendrites and were, therefore, candidates to be ON- and ON–OFF direction-selective ganglion cells, respectively. We concluded that there are at least 15 morphoplogical types of wide-field ganglion cells in macaque retinas.

An integrated optical method for measuring deformation of micro-mechanical systems with better than sub-micron resolutions is detailed. Both a confocal laser scanning microscope and a photon tunneling microscope were integrated into a single microscopy system due to their complimentary capabilities for examining sub-micrometer deformations. A halogen lamp and laser were adopted as the two light sources for the measurements. Since topographic information of samples up to a 15μm by 15μm area can be measured, a three-dimensional displacement field of the sample was extracted by comparing topographies of the same specimen area before and after deformation. The bending and twisting deformation of a micro-mirror driven by the electrostatic force was measured to demonstrate the capability of this newly developed instrument. The experimental data obtained agrees reasonably well with the theoretical results calculated by adopting an analytical solution and a finite element method. The small discrepancy in the result can be traced to the surface roughness effect, which is often non-negligible in micro-systems.

Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses, milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation; this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 g fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.

Our recent observation that microtubules (MTs) are required for completion of division (abscission) led us to analyze MT organization during cytokinesis. Although many studies of MTs in sea urchin eggs have been done, computer-aided analysis of optical sections described herein reveals a new MT assemblage, which we call furrow MTs. This assemblage comprises bundles of MTs that lie in the cleavage furrow. Furrow MTs become apparent when the furrow has progressed approximately one-third of the way through the egg and persist to abscission. Furrow MTs are 8–24-µm long and arc across the base of the cleavage furrow. Acetylated tubulin is localized primarily in the furrow suggesting a distinct MT population. Three-dimensional analysis of optical sections suggests that furrow MTs are spatially distinct from midbody and astral MTs.

We present a novel combined atomic force microscope (AFM) and confocal laser scanning microscope (CLSM). The sample is supported from the side by a boom from a piezo tube scanner, allowing top and bottom access to the sample. The sample is scanned above an inverted microscope objective with a fixed optical path for fluorescent CLSM imaging. An AFM positioned directly above the sample simultaneously measures surface topography. The piezo tube scanner is angled, placing its center of scan curvature directly above the microscope objective. This geometry allows flat scans up to 300 μm on a side. Because the sample is scanned, the AFM and CLSM images are acquired in direct registration.

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.