Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

The modulation contrast microscope: principles and performance

The modulation contrast microscope produces an image of high contrast and resolution. The image has a three-dimensional appearance wherein a rounded object appears dark on one side, bright on the other with grey in between against a grey background. The performance features are optical sectioning, directionality, high resolution and control of contrast and coherence. A bright field microscope is converted to the modulation contrast microscope by adding the modulator, a special amplitude filter, in the objective. A slit aperture part of which is polarized is placed before the condenser. Below this is a rotatable polarizer. The modulator processes light from opposite gradients oppositely, that is brighter for one and darker for the other; thereby preserving the sign. The diffraction theory has been extended to show that gradient image intensity is the intensity of the zero order and when modified by the modulator creates a high contrast image. The modulation contrast microscope is simple and easy to adjust. It is useful in reflected and transmitted light systems, with plastic and glass vessels as well as in combination with fluorescence systems and polarization techniques. There is virtually no limit to the type of specimen that can be studied.     

An electron-optical lens effect as a possible source of contrast in biological preparations

Heavy-metal stain aggregates on the surface of thin sections of biological material have higher contrast than those embedded within the sections and both have greater contrast than can be accounted for by the amplitude image. Disturbances of the incident illumination by a specimen in both light- and electron-optical systems and their possible contribution to image contrast are considered. The hypothesis is proposed that a lens effect produced by the stain aggregates may account for their contrast in the electron microscope in a similar manner to the contrast of glass beads in the light microscope with a low numerical aperture.     

A double detector system for BSE and SE imaging

The double detector system described here is a simple device suitable for any SEM. It permits efficient imaging of specimen surfaces in either the secondary electron (SE) or backscattered electron (BSE) mode. The BSE detector is an annular single-crystal scintillator made of yttrium aluminium garnet (YAG) and the SE detector has a scintillator of the same material. Both detectors have their own light guides which are connected to a single photomultiplier. The choice of signal is made with a mechanical diaphragm mounted on a flange between the light guide and the photomultiplier. The SE detector may be replaced by a second BSE detector to allow the detection of “low” take-off angle BSEs to provide information which differs from that given by the annular BSE detector which operates to detect BSEs with a “high” take-off angle. In this way it is possible to image either material or topographic contrast with high resolution and to take advantage of the choice of detected electrons.     

Contrast and quantitation in uniform regions of thin sections using transmission electron microscopy

A direct approach to quantitative measurements of uniform regions in thin sections is described. Accelerating voltages around 80 kV and objective aperture angles of about 9·3 mrad will provide conditions where contrast is directly proportional to specimen mass thickness. An extensive treatment of electron scattering in Formvar films for wide ranges of electron microscopic operating conditions is summarized in a simple, empirical equation. The extent to which Formvar results may be generalized to other materials, both embedding media and structures within the thin section, is treated. Using these results, precise measurements of local section thickness and of specimen density and/or dry mass of regions which penetrate the entire section thickness are possible, with the accuracy dependent upon irradiation effects and specimen makeup.     

Improving image quality by zero-loss energy filtering: quantitative assessment by means of image cross-correlation

Fourier ring correlation and root-mean-square contrast of pairs of images, taken under identical conditions, were used as criteria of image quality for comparing unfiltered with zero-loss energy-filtered imaging using a TEM equipped with a post-column energy filter. For three different specimens (amorphous carbon film, macromolecules in light negative stain, virus particles in deep negative stain) the dependence of these quantities on electron dose, specimen thickness and defocus was investigated. A model, based on simple assumptions, was used to describe quantitatively their dependence on electron dose and specimen thickness. It was found that energy filtering is most advantageous for low-dose imaging and small defocus values. The gain due to energy filtering strongly increases with specimen thickness, whereby the dependence is linear for light scattering elements. For thick specimens, the gain by energy filtering is more pronounced in the resolution range between 4 and 2 nm than for lower spatial frequencies.     

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.     

Optimized reflection imaging in laser confocal microscopy and its application to neurobiology: Modificationsa to the biorad MRC-500

Reflection images of biological specimens recorded using laser-scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC-500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium-neon laser was not as readily absorbed as the blue and green lines of an argon-ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.     

Evanescent-wave microscopy: A simple optical configuration

Under suitable conditions, the region of the aqueous phase immediately adjacent to a glass-water interface can be selectively illuminated using the evanescent wave created when total internal reflection occurs at the interface. Objects in the aqueous phase away from the glass become effectively invisible, since the intensity of the evanescent wave decays exponentially with distance from the interface. Previous methods of generating evanescent waves for light microscopy have employed accessory light sources and optical components that directed the illumination on the specimen from one side. The asymmetric illumination creates a surprising orientation dependence of visibility for some specimens. Objects such as microtubules are totally invisible unless they are orientated nearly perpendicular to the direction of illumination. An explanation of this phenomenon is provided in terms of the geometry of diffraction of light by long thin objects. A simple method of achieving evanescent-wave illumination is described and shown to be useful in practice for biological specimens. In contrast to previously described methods, the present arrangement has the advantage of producing circularly symmetric illumination, and of utilizing only standard optical microscope components. The system has been used for imaging specimens both by light scattering and by fluorescence. It has proved useful for following the fragmentation of flagella into isolated microtubules, observing microtubules gliding over dynein adsorbed to a surface, and also for determining the arrangement of vinculin and alpha-actinin in cell-substratum attachment sites and termini of growing myofibrils in cardiac cells.     

Comparative light microscopy,scanning electron microscopy and transmission electron microscopy of selected organic walled microfossils

Two simple techniques are described and illustrated. The first is for the study of one specimen by both light microscopy (LM) and scanning electron microscopy (SEM). The second is for the study of one selected specimen by LM, SEM and in ultrathin section by transmission electron microscopy (TEM). Although these techniques were developed for the comparative study of Precambrian organic walled microfossils (OWMs), they could be used for a wide range of other specimens.     

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.     

Iodine vapor staining for atomic number contrast in backscattered electron and X‐ray imaging

Iodine imparts strong contrast to objects imaged with electrons and X‐rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation‐deposition state‐change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas‐tight container along with a small mass of solid I₂. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin‐embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X‐ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron‐ and photon‐sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.     

Thermal enhancement of AFM phase contrast for imaging diblock copolymer thin film morphology.

A simple and effective means of increasing the morphological detail in AFM phase micrographs of microphase separated block copolymer films is presented. Effective AFM phase imaging of microphase separated systems hinges upon the existence of appropriate contrast mechanisms such as differences in elasticity between the microphase separated domains. For some systems, AFM phase imaging at room temperature results in low contrast images due to a paucity of differential mechanical behavior between the microphase domains, e.g. at room temperature both species are glassy. Through the use of a heating apparatus custom-designed for AFM, an elastic contrast mechanism can be created in some systems by raising the specimen to a temperature between the glass transitions of the constituent polymer species. This serves to preferentially soften one species with respect to the other, thus enhancing the phase contrast mechanism, which results in micrographs with superior detail. This simple technique is demonstrated using films of a series of polystyrene-b-poly(n-alkyl methacrylate) diblock copolymers and both commercial and custom-built heating stages. By choosing appropriate measurement temperatures, AFM phase contrast could be greatly enhanced, or indeed created, when compared to room temperature images of these specimens. For these materials, contrast enhancement required that the sample be heated roughly 20 degrees C above the glass transition of the lower-Tg species.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Automated compensation of light attenuation in confocal microscopy by exact histogram specification

Confocal laser scanning microscopy (CLSM) enables us to capture images representing optical sections on the volume of a specimen. The images acquired from different layers have a different contrast: the images obtained from the deeper layers of the specimen will have a lower contrast with respect to the images obtained from the topmost layers. The main reasons responsible for the effects described above are light absorption and scattering by the atoms and molecules contained in the volume through which the light passes. Also light attenuation can be caused by the inclination of the observed surface. In the case of the surfaces that have a steep inclination, the reflected light will have a different direction than the one of the detector. We propose a technique of digital image processing that can be used to compensate the effects of light attenuation based on histogram operations. We process the image series obtained by CLSM by exact histogram specification and equalization. In this case, a strict ordering among pixels must be induced in order to achieve the exact histogram modeling. The processed images will end up having exactly the specified histogram and not a histogram with a shape that just resembles to the specified one, as in the case of classical histogram specification algorithms. Experimental results and theoretical aspects of the induced ordering are discussed, as well as a comparison between several histogram modeling techniques with respect to the processing of image series obtained by confocal microscopy. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

A low cost image and data acquisition system for use with the Vickers M85 scanning microdensitometer

A simple and inexpensive interface has been constructed between the Vickers M85 microdensitometer and a BBC model B microcomputer. The interface incorporates three sensitivity ranges and enables the production of pseudocolour images of the specimen using the two-dimensional scanning mode of the M85. The operator can select a 160times256 pixel image with eight colours or a 320times256 display using only four colours. Each colour represents a defined range of transmittance which is software controlled. The image histogram can be displayed and the interval between colours redefined so as to enable contrast stretching. Intervals between colours can be either linear or logarithmic and the images thus obtained can be stored on disc or videotape, or a hard copy can be obtained using a screen dump routine. Two-dimensional absorption images can thus be obtained at any single wavelength from 400 to 700 nm at normal magnifications of the light microscope. In addition, the system can be used to acquire, store and process data from one-dimensional scans to obtain quantitative information about variations in optical density within the specimen, so considerably increasing the usefulness of the instrument. Although obviously limited in its capabilities, the system produces images of very high quality and one-dimensional data of high sensitivity. The interface can be constructed for less than £40. A small modification to one of the M85 circuit boards is necessary to obtain maximum resolution.     

Transmission and double-reflection scanning stage confocal microscope

A new confocal scanning-stage transmit-ted-light and reflected-light laser microscope is described. In addition to allowing confocal transmitted-light imaging, this microscope has the unique feature of imaging both the top and bottom of a specimen in reflected light, with the two images in perfect registration. Only one detector is used; different imaging modes can be selected by simple rotation of the polarizers. Several confocal images are presented, chosen to illustrate the unique features of the instrument.     

Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimens

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity.