The effect of swelling on the interference microscopy of fixed biological material

Measurements with the interference microscope show the optical-path difference relative to the medium of many biological objects after histological processing to be less in water than would be expected on the basis of measurements made in other media. This anomalous optical-path difference is found also with sections of gelatin (autoradiographic stripping film), but not if the gelatin is mounted on the slide in such a way that swelling is constrained to a direction parallel to the optical axis of the microscope. Swelling in water has been demonstrated in a variety of formalin-fixed and de-fatted tissues. It is suggested that the anomalous optical-path differences in water are due to swelling causing a change in the lateral dimensions of the object, and hence loss of material from the optical path. The fact that the anomaly has also been found with scanning and integrating interference microscopes is discussed, and it is suggested that the validity of such methods needs re-examination.     

Measurement of the width of thin,cylindrical, transparent objects by phase contrast light microscopy6

Measurement of the width of a thin, cylindrical, transparent object by phase contrast light microscopy has been frustrated by the absence of an established relationship between the true width of the object and its apparent width in the phase contrast image. We have solved this problem by devising a simple method by which individual glass fibres may be measured using both phase contrast light microscopy and electron microscopy. Using this method we have constructed calibration curves relating the diameter measured by phase contrast microscopy to the real diameter of the fibres. These curves are linear in the range 0.10-2.5 μm real diameter, with slopes close to unity and intercepts of about 0.2 μm. Thus widths of such objects are overestimated. The precise value of the intercept is related to the overall numerical aperture of the optical system. Each calibration curve permits the true width of a cylindrical object to be estimated by phase contrast microscopy with an accuracy of better than ±0.05 μm. We have found that greater precision is obtained by taking measurements of light micrographs subjectively using a microcomparator rather than objectively using a microdensitometer.     

Defocusing microscopy

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.     

光纤石英玻璃基板微V槽阵列的精密磨削

针对脆性石英玻璃的微加工,利用自主研发的金刚石砂轮微尖端修整工艺,研发了光纤阵列石英玻璃微V槽磨削技术。分析了60°的微V槽形状偏差对光纤耦合损耗的影响,然后,研究了砂轮微尖端的误差补偿修整工艺。最后,实验分析了微V槽的磨削精度。理论分析显示:微V槽角度、间距和宽度的偏差分别控制在±0.42°、±1.04μm和±1.2μm以内时,耦合损耗小于0.5dB。实验结果表明:开发的数控磨削工艺可加工高精度的60°微V槽阵列;采用数控轨迹和角度补偿修整后,砂轮微尖端半径可平均达到10.46μm,角度精度为(60±0.22)°;对石英玻璃进行微磨削后,微V槽的角度偏差达到0.4°,尖端半径为10.5μm,宽度偏差为0.3μm,间距偏差为0.5μm,可保证光纤阵列的精密对接。     

光栅干涉仪的运动误差原理

本文应用衍射光栅干涉仪的半波相位差面原理模型,说明光栅摆动误差引起的干涉条纹变化情况,进而分析得出光栅干涉仪运动误差公式。可作为设计和应用此种干涉仪的依据之一。     

A 360° single-axis tilt stage for the high-voltage electron microscope

A new type of specimen stage that permits more than 180° of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180° set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within ±0.20°, with a cumulative error of less than 1.0° over a 180° span. The new stage was tested using puffball spores mounted on a micropipette. A 180° tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160° produced results nearly as good as the full data set. Limiting the range to 140°, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120° and 100°.     

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.     

Analysis of polarized light with two quarter-wave plates

The following method is proposed for the analysis of the elliptically polarized light leaving a transparent, birefringent and dichroic specimen (in which the direction of maximum absorption coincides with either of the principal axes of refractive index), oriented with its optic axis at ± 45° relative to the transmission azimuth of the polarizer. Between the specimen and the rotatable analyser are two λ/4 plates; that nearer the specimen is fixed with its slow axis at ± 90° to the polarizer, while that nearer the analyser is rotatable. If the amplitude ratio of the components of light leaving the specimen is tan Y (Y being less than 45°), and their relative retardation is Δ°, at compensation the azimuth of the rotatable λ/4 plate will be Δ/2 (relative to its initial position orthogonal to the polarizer) and that of the analyser will be (45° - Y), relative to the new azimuth of the rotatable λ/4 plate. Apart from involving minimal calculations, the method is capable of high precision with suitable equipment, since all critical settings can be made with the aid of a Nakamura half-shade plate. Although so far used only in transmission polarization microscopy, the method is quite general and could be applied in any work involving polarized light.     

Measurement of the absorption of concentrated dyes and their use for quantitative imaging of surface topography

We propose a method to image the surface topography of transparent objects. The space between the object and the opposite closely positioned surface (such as a cover glass or a slide) is filled with a strongly absorbing dye. The contrast is generated by recording a transmission image at a wavelength where the dye absorbs. Since the transmitted intensity depends on the depth of the dye layer, it carries information about the relief of the tested surface. With sufficiently concentrated dyes, nanometre unevenness of a surface can be detected. By using less-concentrated solutions, it is possible to image and measure larger objects, such as biological cells. At the present stage, biological applications of the method are only semi-quantitative, but the method still provides detailed information about cell shapes that is not readily obtainable with other imaging techniques.
  Conversion of the image grey scale into the units of vertical distance requires knowledge of the absorption coefficient of the dye. The same method that is used for imaging can be adapted to measure the absorption coefficient of concentrated dyes. The solution to be analyzed is placed between a glass slide and a spherical lens of known radius. The absorption coefficient is determined from attenuation of transmitted intensity as a function of the distance to the centre. At the same time, the interference pattern in the reflected image allows measurement of the refractive index of the dye.     

Phase retrieval in TEM using Fresnel images

As an alternative to sideband holography in an electron microscope, methods for phase recovery by in-line holography utilising Fresnel images of aperiodic objects were tested with computed simulations and experimental data. Phases were recovered by minimising an error functional defined as a measure of the differences between experimental and calculated image intensities. The probability of convergence to local minima of the error function was reduced by increasing the ratio of known to unknown parameters, partly by use of several Fresnel images at different defoci, and also by an incremental relaxation of the phase bandwidth. Iterative methods did not converge reliably to a global minimum, but a conjugate gradient algorithm usually recovered the phases exactly, even for object arrays which included large phase variations. In practice, it was essential to use analytic expressions for the error gradients with respect to the phases, defoci and beam direction. Phase shifts of several radians were measured near the edge of a contaminated aperture. The factors that limit the accuracy and reliability of phase recovery from Fresnel images are discussed.     

Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.     

Simple procedures for the transfer of grid images onto glass coverslips for the rapid relocation of cultured cells

Procedures are described whereby electron microscope-grid images may be transferred onto the surface of glass coverslips. By using coordinate grids, patterns are obtained that are ideal for the rapid relocation of cultured cells. Two basic procedures are presented. In the first, metal is evaporated, in a vacuum unit, onto glass coverslips carrying electron microscope grids to produce an exposed, glass replica of the grid pattern. The grid pattern is then etched in the glass surface with hydrofluoric acid and the metal subsequently dissolved away. In the second method silicone monoxide is evaporated onto coverslips carrying a ‘negative grid’ or a negative grid image, to produce a pattern directly visible in the phase contrast or interference microscope. Modifications for scanning electron microscopy are also described.     

Detection of dichroism with the microscope

Two simple, sensitive, visual methods are described for the detection of dichroism in microscopic objects. 1. The object is oriented at ±45° between crossed polars, and either the polarizer or the analyser is rotated through an equal angle alternately clockwise and anti-clockwise. A dichroic object will brighten as the polar is rotated in one direction and darken as the polar is rotated in the other. If the optic axis of the object lies north-east to south-west and the transmission axes of polarizer and analyser respectively east-west and north-south, a positively dichroic object will darken as the polarizer is rotated anti-clockwise, or the analyser is rotated clockwise. 2. A formally similar method, which avoids the need to rotate either polar, is to orient the object at ± 45° between crossed polars, and insert a Nakamura plate between the polars in an optical plane conjugate with the specimen. Dichroism is indicated by inequality of the apparent brightness of the specimen on either side of the dividing line of the plate. Both methods are more sensitive than visual techniques traditionally used in microscopy. The sign of the dichroism of polished smears of dye particles is discussed. It is suggested that polishing in the dry state orients individual dye molecules, while polishing of a damp smear orients dye molecule aggregates. The relation of dichroism to the colour of stained histological structures, as seen either between crossed polars or with bright-field illumination, is discussed, and the elementary optical theory of dichroism is summarized.     

Measurement-based evaluation of optical pathlength distributions reconstructed from simulated differential interference contrast images

Recently a method was presented for reconstructing optical pathlength distributions (OPDs) from images of weak phase objects obtained by a conventional differential interference contrast (DIC) microscope. A potential application of this technique is the determination of the mass of biological objects: by integrating the optical pathlength over the projected surface of the image of an object, a measure of the dry mass, i.e. the total mass of all solid constituents present in the object, is obtained. To assess the possibilities of DIC microscopy for this application, simulations were performed on computer-generated DIC images of objects of various sizes, shapes and orientation angles. After reconstructing the OPDs from these images, the integrated optical pathlength of each of the test objects was determined, and compared with the expected results. The parameter settings used in the reconstruction algorithm were found to be very important in obtaining a reliable measurement. Using optimal parameter settings, errors in the integrated OPD could be limited to a few per cent for circular objects within the investigated size range. For non-circular objects, however, the orientation angle of the object relative to the lateral shift was found to influence the measured values. Ellipses with their long axes perpendicular to the shift direction had a significantly higher integrated OPD than ellipses orientated parallel to the shift. By adjusting the reconstruction parameters the effect could be limited, but complete elimination of the artefact was not possible within the parameter range investigated.     

A confocal video-rate laser-beam scanning reflected-light microscope with no moving parts

A no-moving-parts, 30 frames/s, laser-beam scanning confocal reflected-light microscope has been developed. In principle, the technique can be extended to fluorescence and transmission light microscopy. Acousto-optic beam deflectors controlled by digital electronics move a laser beam in a 512-line interlaced 8·5 times 8·5-mm raster. The light passes through a beam splitter, enters an inverted microscope through the side camera port, and is imaged at the object by the microscope objective. Reflected light returns through the objective, exits the camera port, is reflected off the beam splitter, and is imaged on to the photocathode of an image dissector tube (IDT). Confocality is provided by raster scanning the IDT aperture coincident with the congruent image of the laser beam incident on the object. Real-time jitter-free reflected light images of a variety of biological objects have been produced. Computer-controlled alignment of the laser scan and IDT is performed in several seconds.     

Advances in phase‐sensitive acoustic microscopy studies of polymer blend films: annealing effects and micro‐elastic characterization of PS/PMMA blends

The unique phase‐sensitive acoustic microscope is used for the structural and mechanical characterization of thin films of polystyrene/polymethylmethacrylate blends. The effect of annealing on blends of polystyrene/polymethylmethacrylate spin coated from different solvents unto a substrate is studied. Varying the solvents according to vapour pressure and spin coating at different speeds (for thickness variation) led to changes in phase domain distributions and overall structural properties before annealing. Annealing in vacuum at 190°C for 48 h resulted in the elimination of solvent effects with all samples reverting to a similar morphology irrespective of common solvent and thickness. The Young's moduli at specific points on the film (E_polystyrene= 3.4 ± 0.3 GPa, E_{polymethylmethacrylate}= 4.2 ± 0.4 GPa) and over a given area (E_{polystyrene/polymethylmethacrylate}= 3.9 ± 0.4 GPa) were determined by combinatory use of the atomic force microscope and phase‐sensitive acoustic microscope. These results demonstrate a minimally invasive method for the quantitative characterization of polymer blend films.     

360°高阶非球面反射式全景镜头设计

为了简化系统配置、提高图像采集及处理效率,实现单一光学系统环视高清全景成像,依据折反射式光学系统的工作原理,设计了高阶非球面反射式360°全景镜头,并对光学结构和系统像质进行优化设计。该相机采用高阶非球面反射镜压缩视场角,将垂直光轴方向俯仰角从-55°到20°的环视目标光引入到系统,接着,在后续光路中利用玻璃透镜组对目标光进行接收,并使其聚焦于相机靶面,获得物体的环形全景图像。通过对系统像质的优化,得到高清的360°环视全景图像,并对光学系统的主要性能指标进行了分析。所设计的360°全景镜头采用1片高阶非球面反射镜和10片玻璃球面镜组成,系统的焦距为0.4mm,光圈数为2.2,俯仰角达到75°,像方全视场在150lp/mm处的光学传递函数值均大于0.3。该360°全景镜头采用单一光学系统成像,解决了传统拼接式全景镜头图像采集与图像处理效率低的问题,同时通过简化系统结构,使该产品符合成本低、可量产的要求。     

A microwave interferometer with increased stability for diagnostics of steady-state plasma

The schematic and results of studying an interferometer for a wavelength of 8 mm with homodyne frequency conversion for measuring the electron density of a steady-state plasma are described. The design of its waveguide system allows the drift of the indicated initial phase to be reduced to a level of ±0.1°/h. The maximum phase shift measured is 360°, the tolerable signal-power decay in plasma is 16 dB, and the maximum phase measurement error is ～5%.