Polarized light imaging of white matter architecture

Polarized light imaging (PLI) is a method to image fiber orientation in gross histological brain sections based on the birefringent properties of the myelin sheaths. The method uses the transmission of polarized light to quantitatively estimate the fiber orientation and inclination angles at every point of the imaged section. Multiple sections can be assembled into a 3D volume, from which the 3D extent of fiber tracts can be extracted. This article describes the physical principles of PLI and describes two major applications of the method: the imaging of white matter orientation of the rat brain and the generation of fiber orientation maps of the human brain in white and gray matter. The strengths and weaknesses of the method are set out.     

Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens

We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two‐dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5‐day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F‐Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume‐of‐interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state‐of‐the‐art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech. 79:455–458, 2016. © 2016 Wiley Periodicals, Inc.     

Determining the fibrillar orientation of bast fibres with polarized light microscopy: the modified Herzog test (red plate test) explained

The identification of bast fibre samples, in particular, bast fibres used in textiles, is an important issue in archaeology, criminology and other scientific fields. One of the characteristic features of bast fibres is their fibrillar orientation, referred to as Z‐ or S twist (or alternatively right‐ and left‐handed fibres). An empirical test for determining the fibrillar orientation using polarized light microscopy has been known in the community for many years. It is referred to as the modified Herzog test or red plate test. The test has the reputation for never producing false results, but also for occasionally not working. However, so far, no proper justification has been provided in the literature that the ‘no false results’ assumption is really correct and it has also not been clear up till now, why the method sometimes does not work. In this paper, we present an analytical model for the modified Herzog test, which explains why the test never gives a false result. We also provide an explanation for why the Herzog test sometimes does not work: According to our model, the Herzog test will not work if none of the three distinct layers in the secondary cell wall is significantly thicker than the others.     

Mapping birefringence in three dimensions using polarized light field microscopy: the case of the juvenile clamshell

We report methods to generate three‐dimensional maps of birefringence, its position and orientation in juvenile shells of the Atlantic hard clamshell (Mercenaria mercenaria). For measuring the retardance and optic axis orientation of curved shell surfaces in three dimensions, we developed enhanced acquisition and processing algorithms and combined results from conventional and light field imaging approaches to reconstruct the three‐dimensional shell shape and its anisotropic optical properties. Our work represents the first successful attempt to generate such maps at a spatial resolution of about 2 μm and angular steps of about 9° in terms of the inclination angles of the optic axis. The maps of clamshell birefringence provide structural insights into the early mineralization during juvenile clamshell development.     

PbWO4的偏振吸收光谱及400nm吸收带的结构起因

用改进的Bridgeman方法生长了PbWO4晶体。分别测量了晶轴垂直和平行于表面的该PbWO4晶体样品的吸收谱,同时测量了晶轴平行于表面的偏振吸收谱。结果显示吸收谱在350nm处有一弱吸收带,在400nm处有一强吸收带,在500nm～700nm处有一宽带。400nm的强带在偏振光吸收谱中不显示出二向色性,从而归因于三价铅离子心(Pb3+)。     

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.     

In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C_s corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.     

Practical considerations in the use of polarized light microscopy in the analysis of the collagen network in articular cartilage

Polarized light microscopy is a traditional method for visualizing the collagen network architecture of articular cartilage. Articular cartilage repair and tissue engineering studies have raised new demands for techniques capable of quantitative characterization of the scar and repair tissues, including properties of the collagen network. Modern polarized light microscopy can be used to measure collagen fibril orientation, parallelism, and birefringence. New commercial instruments are computer controlled and the measurements are easy to perform. However, often the interpretation of results causes difficulties, even errors, because the theoretical aspects of the technique are demanding. The aim of this study was to describe the instrumentation and properties of a modern polarized light microscope, to point out some sources of error in the interpretation of the results, and to recall the theoretical background of the polarized light microscopy.     

Polarized light field microscopy: an analytical method using a microlens array to simultaneously capture both conoscopic and orthoscopic views of birefringent objects

For the comprehensive analysis of anisotropic materials, a new approach, called 'polarized light field microscopy' is introduced. It uses an LC-PolScope to which a microlens array was added at the image plane of the objective lens. The system is patterned after the 'light field microscope' that achieves both lateral and axial resolution in thick specimens in a single camera exposure. In polarized light field microscopy, the microlens array generates a hybrid image consisting of an array of small conoscopic images, each sampling a different object area. Analysis of the conoscopic images reveals the birefringence of each object area as a function of the propagation direction of transmitted light rays. The principles and utility of the instrument that we are calling 'light field LC-PolScope' are demonstrated with images of a thin, polycrystalline calcite film, revealing the azimuth and inclination angle of the optic axis for many crystals simultaneously, including crystals with diameters as small as 2 microm. Compared to traditional conoscopy and related methods, the vastly improved throughput and quantitative analysis afforded by the light field LC-PolScope make it the instrument of choice for measuring 3D birefringence parameters of complex structures.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

The development of biological preparative techniques for light microscopy, 1839–1989

The development of techniques of preparing biological material for microscopical observation is summarized, with much greater emphasis being given to the introduction and establishment of the various techniques, largely in the nineteenth century, than to their perfection and extremely widespread use in the twentieth century. The development of the microtome as an instrument of the greatest importance is summarized and an assessment is made of the impact of preparative techniques on the development of the medical sciences.     

A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy

A simple method is described to embed material in resin, in the form of microscope slides, to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block-holder for ultramicrotome are the specific tools of this method.     

Reconstruction of 3D grain boundaries from rock thin sections,using an advanced polarised‐light microscopy method

Grain boundaries play a significant role in materials by initiating reactions and collecting impurities. Here we present FAGO (Fabric Analyser Grain boundary recOnstruction), a first step towards the automatic determination of three‐dimensional (3D) grain boundary geometry using polarised light. The trace of the grain boundaries from 2D rock thin sections are determined primarily from data acquired using an automatic fabric analyser microscope and the FAME software (fabric analyser‐based microstructure evaluation; Peternell and colleagues and Hammes and Peternell). Based on the Fabric Analyser G50's unique arrangement of nine differently oriented light sources the retardation can be determined independently for each light direction and at each pixel in the field of view. FAGO combines these retardation datasets for each individual pixel together with retardation profiles across grain boundaries, to determine the orientations of the boundaries. The grain boundary traces are then broken up into segments of equal orientation, using the profile‐obtained orientation data. Finally, a 3D grain boundary model is reconstructed. The data processing is almost fully automatic using the MATLAB® environment. Only minor manual inputs are required.     

Unstained and in vivo fluorescently stained bacterial nucleoids and plasmolysis observed by a new specimen preparation method for high-power light microscopy of metabolically active cells

Microscope slides were coated with a layer of gelatin, the thickness of the gelatin increasing linearly along the long axis. The bacterial suspension is applied to the dried gelatin and covered by a coverslip. The medium is absorbed by the gelatin and thus the cells applied against the coverslip. By this method, cultures of concentrations below 10⁸ cells/ml provide statistically relevant numbers for observation without prior concentration steps. It is easier to apply than the existing methods for the observation of bacterial nucleoids by phase contrast imaging. Because the cells are maintained in growing conditions the method is useful for the vital fluorescence DAPI-staining of various bacterial species and for observations of plasmolysis and its reversal at different physiological conditions and extracellular osmolalities. The previously generally assumed view that the plasmolytic changes of the cell morphology are immediate upon the hyperosmotic shock and are rapidly repaired when the cell is able to metabolize actively was confirmed; this is in contrast to some recent claims.