Confocal microscopy of the in-situ crystalline lens

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.     

The use of high-voltage electron microscopy and semi-thick sections for examination of cyanobacterial thylakoid membrane arrangements

High-voltage electron microscopy (HVEM) of semi-thick sections was evaluated as a technique for studying thylakoid membrane arrangements in cyanobacterial cells. Semi-thick sections (0·25 μm) provided important information that was relatively difficult or impractical to obtain by viewing either randomly or serially cut thin sections. Specifically, the semi-thick sections were better suited for visualizing (i) overall thylakoid arrangements and (ii) interconnections between the thylakoids and the cytoplasmic membrane. By comparison, randomly cut thin sections frequently yielded deceptively incomplete or inconsistent data in regard to these specific features. Tilting of thick sections about two perpendicular axes served to improve the clarity of complex membranous intersections and other cell features.     

Images of arterioles in unfixed tissue obtained by acoustic microscopy

A scanning acoustic microscope operating at 600 MHz was used to observe arterioles in a thin sheet of collagenous connective tissue dissected from the submucosa of the guinea-pig small intestine. The arterioles were clearly defined in images made using transmitted ultrasound, and the acoustic attenuation (α) of the arteriolar wall was estimated to be 120 cm^?1. Images made using reflected ultrasound did not show the arterioles clearly.     

Cutting work in thick section cryomicrotomy

The forces during cryosectioning were measured using miniature strain gauges attached to a load cell fitted to the drive arm of the Porter-Blum MT-2 cryomicrotome. Work was calculated and the data normalized to a standard (1 mm × 1 mm × 0·5 μm) section. Thermal energy generated was also calculated. Five parameters were studied: cutting angle, thickness, temperature, hardness, and block shape. Force patterns could be divided into three major groups thought to represent cutting (Type I), large fracture planes > 10 μm in length (Type II), and small fracture planes < 10 μm in length (Type III). Type I and Type II produced satisfactory sections. Work in cutting ranged from an average of 78·4 μJ to 568·8 μJ. Cutting angle and temperature had the greatest effect on sectioning. Heat generated would be sufficient to cause through-section melting for 0·5 μm thick sections assuming the worst possible case, namely that all heat went into the section without loss. Presence of a Type II pattern (large fracture pattern) is thought to be presumptive evidence against thawing.     

Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index

The effect of refractive-index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.     

Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers

The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR-39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono-energetic neutrons of energy 1–2 MeV, (b) exposed to the alpha radiation from ²²²Rn and its progeny. Both groups were irradiated at normal incidence. Radiation-induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488-nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z-scan mode was taken through each selected etched track at vertical spacings of 0·642 μm. Successive reconstructions of Z-sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha-particle-induced tracks ranges from 7·9 to 11 μm, whereas for protons the same parameter ranges between 2·0 and 3·8 μm for a specific electrochemical etch procedure.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

Acoustic microscopy of ceramic-fibre composites

A variety of ceramic-fibre composites has been studied by acoustic microscopy, at 1·9 GHz with a resolution of 0·8 μm. The materials studied were Nicalon-reinforced borosilicate glass, SiC fibres in a magnesium–aluminosilicate matrix and a calcium-aluminosilicate matrix, and SiC monofilaments in a Ti–6Al–4V matrix. In all the specimens the contrast was dominated by strong excitation of Rayleigh waves in the surface. This gave strong contrast from different phases, and revealed interfaces and cracks by characteristic crack patterns. Quantitative agreement between observed and calculated fringe patterns was found, and values of shear and Young's modulus were measured. In the SiC monofilament specimens, various stages of progressive deterioration as a result of thermal ageing treatments were observed.     

Measurement of probe displacement to the thermal resolution limit in photonic force microscopy using a miniature quadrant photodetector

A photonic force microscope comprises of an optically trapped micro-probe and a position detection system to track the motion of the probe. Signal collection for motion detection is often carried out using the backscattered light off the probe-however, this mode has problems of low S/N due to the small backscattering cross sections of the micro-probes typically used. The position sensors often used in these cases are quadrant photodetectors. To ensure maximum sensitivity of such detectors, it would help if the detector size matched with the detection beam radius after the condenser lens (which for backscattered detection would be the trapping objective itself). To suit this condition, we have used a miniature displacement sensor whose dimensions makes it ideal to work with 1:1 images of micrometer-sized trapped probes in the backscattering detection mode. The detector is based on the quadrant photo-integrated chip in the optical pick-up head of a compact disc player. Using this detector, we measured absolute displacements of an optically trapped 1.1 μm probe with a resolution of ～10 nm for a bandwidth of 10 Hz at 95% significance without any sample or laser stabilization. We characterized our optical trap for different sized probes by measuring the power spectrum for each probe to 1% accuracy, and found that for 1.1 μm diameter probes, the noise in our position measurement matched the thermal resolution limit for averaging times up to 10 ms. We also achieved a linear response range of around 385 nm with cross talk between axes ?4% for 1.1 μm diameter probes. The detector has extremely high bandwidth (few MHz) and low optical power threshold-other factors that can lead to its widespread use in photonic force microscopy.     

Angular measurement of acoustic reflection coefficients by the inversion of V(z, t) data with high frequency time-resolved acoustic microscopy

For inspection of mechanical properties and integrity of critical components such as integrated circuits or composite materials by acoustic methodology, it is imperative to evaluate their acoustic reflection coefficients, which are in close correlation with the elastic properties, thickness, density, and attenuation and interface adhesion of these layered structures. An experimental method based on angular spectrum to evaluate the acoustic coefficient as a function of the incident angle, θ, and frequency, ω, is presented with high frequency time-resolved acoustic microscopy. In order to achieve a high spatial resolution for evaluation of thin plates with thicknesses about one or two wavelengths, a point focusing transducer with a nominal center frequency of 25 MHz is adopted. By measuring the V(z, t) data in pulse mode, the reflection coefficient, R(θ, ω), can be reconstructed from its two-dimensional spectrum. It brings simplicity to experimental setup and measurement procedure since only single translation of the transducer in the vertical direction is competent for incident angle and frequency acquisition. It overcomes the disadvantages of the conventional methods requiring the spectroscopy for frequency scanning and/or ultrasonic goniometer for angular scanning. Two substrates of aluminum and Plexiglas and four stainless plates with various thicknesses of 100 μm, 150 μm, 200 μm, and 250 μm were applied. The acoustic reflection coefficients are consistent with the corresponding theoretical calculations. It opened the way of non-destructive methodology to evaluate the elastic and geometrical properties of very thin multi-layers structures simultaneously.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

The use of a low-viscosity epoxy resin in the preparation of undecalcified bone sections for light microscopy

A method using the Spurr low-viscosity epoxy resin medium for the preparation of Von Kossa impregnated undecalcified bone sections for light microscopy is described. The method gives high quality thin sections (0·6–1·0 μm) of cancellous bone and overcomes some of the difficulties experienced with other plastics. The procedure is relatively simple and is well suited for use in a routine diagnostic laboratory.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Method for the study of the three-dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy

A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 μm inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.     

Measurements of cells in culture by scanning acoustic microscopy

A novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation. Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.     

Glycol methacrylate embedding of bone and cartilage for light microscopic staining

A method is described for embedding bone and cartilage in glycol methacrylate (GMA) for light microscopy. Dehydration-infiltration of the hard tissue is with aqueous GMA solutions minimizing solvent and dehydration artefact, and polymerization is by UV light in the cold to minimize thermal damage. Over fifty stains, enzyme localizations and related histochemical methods for 0·5–3·0 μm thick sections of GMA embedded tissue are listed. The increased resolution plus the localization of cellular and extracellular chemical moieties is now easier and more accurate providing an improved method for the study of the musculo-skeletal system by light microscopic histochemistry.     

Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy

The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol-mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.     

Ponceau 2R staining of proteins and periodic acid bleaching of osmicated subcellular structures on semi-thin sections of tissues processed for electron microscopy: a simplified procedure

An aqueous solution (pH 1·5) of 0·5% Ponceau 2R (C.I. 16150) and 2% periodic acid was used at 318 K to stain proteins (brilliant red) and simultaneously to bleach the osmicated non-proteinaceous cell structures on 1–2 μm thick sections of tissues fixed in glutaraldehyde-osmium tetroxide and embedded in epoxy resins. This H₅IO₆ bleaching-Ponceau 2R staining procedure only stains the proteins so that black-and-white films can be used.