Chapter

# Optical Microscopy

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## Abstract

The microstructure of a material is related directly to its physical, chemical, and mechanical properties as they are influenced by processing and/or the environment. Among the numerous investigative techniques used to study materials, optical microscopy, with its several diverse variations, is important to the researcher and/or materials engineer for obtaining information concerning the structural state of a material. Information gained using optical microscopy is complementary to other techniques and provides unique information to assess the microstructure of the sample. Though predominantly qualitative, in some cases quantitative measurements are made: some examples are for quantification of grain growth, the coarsening of precipitates during a process, or the evolution of structural domains or defects.

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... ; https://doi.org/10.1101/2021.09.29.462090 doi: bioRxiv preprint saturation to the brighter ones. For instance, the bright cell-bodies in R3 have mostly been saturated in case of (2) and (3), whereas they are well preserved in our case (11). Contradicting (2)(3)(4)(5)(6)(7)(8)(9)(10), our method (11) (11). ...
... For instance, the bright cell-bodies in R3 have mostly been saturated in case of (2) and (3), whereas they are well preserved in our case (11). Contradicting (2)(3)(4)(5)(6)(7)(8)(9)(10), our method (11) (11). ...
... Comparison with a few alternative software-based enhancement techniques. (a) ROIs R1, R2 & R3 (from Figure 2) with scale bar of 15 µm, depicted for (1) INP (unprocessed), (2) multiplicative gain enhancement, (3) minimummaximum range adjustment, (4) histogram equalization (HE), (5) contrast-limited adaptive histogram equalization (CLAHE), (6) unsharp masking (UM), (7) morphological erosion, (8) morphological opening, (9-10) rolling-ball and sliding-paraboloid background subtractions & (11) our proposed method; (b) SNR, SBR & contrast-ratio plots for (1-11), gray, orange & cyan bars denote results for R1, R2 & R3, respectively; (c) intensity profiles along L1-3 plotted for(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). ...
Preprint
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With a limited dynamic range of an imaging system, there are always regions with signal intensities comparable to the noise level, if the signal intensity distribution is close to or even wider than the available dynamic range. Optical brain/neuronal imaging is such a case where weak-intensity ultrafine structures, such as, nerve fibers, dendrites and dendritic spines, often coexist with ultrabright structures, such as, somas. A high fluorescence-protein concentration makes the soma order-of-magnitude brighter than the adjacent ultrafine structures resulting in an ultra-wide dynamic range. A straightforward enhancement of the weak-intensity structures often leads to saturation of the brighter ones, and might further result in amplification of high-frequency background noises. An adaptive illumination strategy to real-time-compress the dynamic range demands a dedicated hardware to operate and owing to electronic limitations, might encounter a poor effective bandwidth especially when each digitized pixel is required to be illumination optimized. Furthermore, such a method is often not immune to noise-amplification while locally enhancing a weak-intensity structure. We report a dedicated-hardware-free method for rapid noise-suppressed wide-dynamic-range compression so as to enhance visibility of such weak-intensity structures in terms of both contrast-ratio and signal-to-noise ratio while minimizing saturation of the brightest ones. With large-FOV aliasing-free two-photon fluorescence neuronal imaging, we validate its effectiveness by retrieving weak-intensity ultrafine structures amidst a strong noisy background. With compute-unified-device-architecture (CUDA)-acceleration, a time-complexity of <3 ms for a 1000x1000-sized 16-bit data-set is secured, enabling a real-time applicability of the same.
... Single nano-and microparticles can be investigated by optical microscopy, but conventional techniques give only little insight into structures smaller than half of the used wavelength due to the diffraction limit [54]. Particle characterization with higher resolution can be achieved by using smaller wavelengths, available in X-ray [55] and electron microscopes [56]. ...
... Comparison to equation (2.5) results in the complex dielectric function of the Drude model 54) or, separated into real and imaginary components, ε = ε 1 + iε 2 with ...
... Optical microscopy is the most frequently used method to obtain magnified images of small samples. It is a versatile and essential technique especially for scientific research [54]. In conventional wide-field microscopy the sample is illuminated homogeneously with light. ...
Thesis
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... The maximum incidence angle θ max is determined by the optics used to focus the laser beam-usually and also in our case a microscope objective lens. One of the specifications of an objective is the so-called numerical aperture (or NA, see references [10], [23]). This is a measure for the solid angle over which the objective lens can gather light. ...
... The microscope can be used in either regular transmissive bright-field mode (shown in the figure and described here), in DIC mode (section 4.1) or in epifluorescence mode (section 4.2). All modes make use of Köhler illumination (described in great detail in reference [10]) with a 100 W mercury arc lamp, to ensure a uniformly illuminated microscope image. ...
... Typically, the separation ranges from 150 to 600 nm; see[10]. ...
... One of the rays travels through the crystal at the same velocity in every direction, this is known as the ordinary ray, the other ray travels with a velocity dependent on the propagation direction within the crystal. This is known as the extraordinary ray (Davidson and Abramowitz 2004). The only exception to this behaviour is where light enters along the optical axis, in this case the light behaves as if it were travelling through an isotropic sample. ...
... As a result of this wave difference the waves interfere with each other either constructively or destructively, this often results in a range of colours being observed. (Taken from Davidson and Abramowitz 2004) ...
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... As conventional dark-field objectives are usually designed only for dry conditions, once that they are used with samples covered with a coverslip, chromatic and spherical aberrations could be introduced. However this effect can be considered negligible for dry objectives with low numerical aperture 39 such as in the current case (NA = 0.4). The instrument is also able to perform microscopy measurements by using a diascopic illuminator composed of a 100 W halogen lamp coupled to a dark-field condenser; microscopy measurements are acquired with an RGB CMOS camera (Nikon DS- Fi 2). ...
Article
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Plasmonic nanoparticles are widely used in multiple scientific and industrial applications. Although many synthesis methods have been reported in the literature throughout the last decade, controlling the size and shape of large populations still remains as a challenge. As size and shape variations have a strong impact in their plasmonic properties, the need to have metrological techniques to accurately characterize their morphological features is peremptory. We present a new optical method referred as Dark-Field Single Particle Spectrophotometry which is able to measure the individual sizes of thousands of particles with nanometric accuracy in just a couple of minutes. Our method also features an easy sample preparation, a straightforward experimental setup inspired on a customized optical microscope, and a measurement protocol simple enough to be carried out by untrained technicians. As a proof of concept, thousands of spherical nanoparticles of different sizes have been measured, and after a direct comparison with metrological gold standard electron microscopy, a discrepancy of 3% has been attested. Although its feasibility has been demonstrated on spherical nanoparticles, the true strengthness of the method is that it can be generalized also to nanoparticles with arbitrary shapes and geometries, thus representing an advantageous alternative to the gold-standard electron microscopy.
... First we will have a brief discussion about the simple optical microscope. A more detailed explanation of this microscope can be found in [46]. ...
... Caso tenha acoplado a si um espectrômetro por dispersão de energia de raios X (EDS), o MEV torna-se ainda mais versátil, podendo também realizar análise química elementar com resolução de até aproximadamente 1 µm na superfície (Goldstein et al., 1992). Baseados nestas duas técnicas, foram desenvolvidos sistemas automatizados de identificação e caracterização mineralógica quantitativa (Petruk, 1988;Sutherland & Gottlieb, 1991 A partir da década de 1980, começaram a se popularizar os microscópios com focalização para o infinito (infinity corrected system) e lentes com menos aberrações esféricas e cromáticas (Davidson & Abramowitz, 1999). Paralelamente, houve um excepcional progresso no desenvolvimento de dispositivos para aquisição de imagens ópticas, notadamente, câmeras coloridas CCD (Pirard, 2004). ...
Thesis
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... The SNR between cell membranes and the background result in different refractive indexes, creating a refractive index gradient. 24 Park et al. 10 noted that the inner and outer cell membranes could be observed through HMIs. The refractive index gradients generated by the two light sources appear slightly different for the boundaries between the outer cell membrane and the background. ...
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... The ray diagram shows the destructive interference between the undeviated and deviated light. As the resultant wave is weaker than undeviated light wave, the specimen appears dark [DM,BOJR51]. ...
Thesis
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... Nevertheless, in the last decade, optical microscopy experimented a large growth. Better optics (infinity corrected tube) and new advanced objective lenses provided images with reduced spherical aberration and free of colour distortions (Davidson and Abramowitz, 1999). There was also exceptional progress in CCD devices used in video cameras (Pirard, 2004). ...
Conference Paper
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... Basically, three facts contributed to this trend: better optics, better digital image acquisition devices (Pirard et al., 1999), and the advent of Digital Microscopy. The progress in microscope optics, mainly due to infinity correct tubes and new advanced objective lenses, provided images with reduced spherical aberration and free of colour distortions (Davidson & Abramowitz, 1999), which are more suitable to image analysis and consequently to quantitative microscopy. ...
Chapter
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... The understanding and utilization of the role played by phase in imaging started an era of new imaging techniques that are both qualitative and quantitative. Techniques such as phase contrast and DIC [148] are generally qualitative in the sense that they do not give quantitative phase information but convert that information into suitable intensity modulation to enhance the contrast of the phase objects. Contrary to that, quantitative phase imaging techniques image the phase map of the phase object as a function of its spatial position. ...
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... Some of these are not important if the same microscope and lens are used throughout. Geometrical distortions are nearly zero in optical microscopes with state of the art objective lenses [17], but can be relevant in SEM's [18]. ...
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... The generation of contrast in most optical microscopy techniques relies on the probing of electronic properties of the samples. These approaches can either employ off-resonance conditions or resonant excitation of electronic transitions (Davidson & Abramowitz, 2002). The first type of techniques mostly probes differences in refractive indices. ...
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This chapter gives a comprehensive overview on in situ characterization techniques, which are useful for the characterization of strain localizations and the temporal evolution of deformation processes. The chapter is divided into four parts regarding (i) general remarks, (ii) in situ imaging techniques, (iii) in situ acoustic emission measurements and (iv) in situ full-field measurement techniques. The part of in situ imaging techniques is divided into (i) optical microscopy and scanning electron microscopy, whereas the part of full-field measurement techniques is separated into digital image correlation (measurement of displacement and/or strain fields) and infrared thermography (measurement of thermal fields). For all in situ techniques, a short historical overview is given together with a summary of the basic knowledge and a review of state-of-the-art research (starting from the year 2000 provided in the appendix).
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Biomedical optical imaging is an important subdivision of optical imaging with the aim of understanding the anatomy and function of life. In principle, biomedical optical imaging systems form an image by manipulating the excitation light and detecting the signals originating from light and tissue interactions. Ever since the invention of the first optical microscope over 1000 years ago, biomedical optical imaging technologies have been steadily evolving to enable faster, deeper, and higher resolution imaging. These technologies have led to a more comprehensive understanding of life at the macro-, micro-, and nanoscales and have improved clinical diagnosis and treatment. This tutorial provides an overview of biomedical optical imaging techniques and their applications. Based on the imaging depth, this tutorial classifies the current optical imaging systems into two regimes: diffraction and diffusion. Within each regime, a few commonly used imaging techniques and their biological imaging applications are discussed. Finally, we provide an outlook of future biomedical optical imaging.
Article
Mobile phone technology has led to implementation of portable and inexpensive microscopes. Light-emitting diode (LED) array microscopes support various multicontrast imaging by flexible illumination patterns of the LED array that can be achieved without changing the optical components of the microscope. Here, we demonstrate a mobile-phone-based LED array microscope to realize multimodal imaging with bright-field, dark-field, differential phase-contrast, and Rheinberg illuminations using as few as 37 LED bulbs. Using this microscope, we obtained high-contrast images of living cells. Furthermore, by changing the color combinations of Rheinberg illumination, we were able to obtain images of living chromatic structures with enhanced or diminished contrast. This technique is expected to be a foundation for high-contrast microscopy used in modern field studies.
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The study of digital rock physics has seen significant advances due to the development of X-ray micro computed tomography scanning devices. One of the advantages of using such a device is that the pore structure of rock can be mapped down to the micrometre level in three dimensions. However, in providing such high-resolution images (low voxel size), the resulting file sizes are necessarily large (of the order of gigabytes). Lower image resolution (high voxel size) produces smaller file sizes (of the order of hundreds of megabytes), but risks losing significant details. This study describes the effect of the image resolution obtained by means of hardware-based and software-based approach. Four samples of porous rock were scanned using a SkyScan 1173 High Energy Micro-CT. We found that acquisition using increased pixel binning of the camera (hardware-based resizing) significantly affects the calculated physical properties of the samples. By contrast, voxel resizing by means of a software-based approach during the reconstruction process yielded less effect on the porosity and specific surface area of the samples. However, the decreasing resolution of the image obtained by both the hardware-based and the software-based approaches affects the permeability significantly. We conclude that simulating fluid flow through the pore space using the Lattice Boltzmann method to calculate the permeability has a significant dependency on the image resolution.
Chapter
The analysis of processes on a cellular and sub-cellular level plays a crucial role in life sciences. Commonly microscopic assays make use of stains and cellular markers in order to enhance image contrast, but in many cases, cell imaging requires the sample to be undisturbed during the imaging process, making staining, dying and fixing impractical. Non-destructive techniques are especially useful in long term imaging or in the study of sensitive cell types, such as stem cells, embryos or nerve cells. Novel advances in computation, imaging and incubator technology have recently made it possible to prolong the imaging time, reduced the cost of storing data and opened a door to the development of new computer aided analytical tools based on microscopic image data. Here we illustrate how Hoffman Modulation Contrast imaging and Confocal Microscopy can be combined with visual computing and present results from determination of cell number, volume, spatial location and blastomere connectivity, using examples from embryos grown for in vitro fertilisation. We give examples of how knowledge of the imaging technique can be used to further improve the computer analysis and also how visually guided tools may aid in the diagnostic interpretation of image data and improve the result. Finally we discuss how the use of microscopic data as a basis for embryo modelling may help in both research and educational purposes. The aim of this chapter is to give an example of how microscopic imaging can be combined with standard computer vision techniques to aid in the interpretation of microscopic data, and demonstrate how visual computing techniques can make an essential difference in terms of scientific output and understanding.
Article
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Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.
Conference Paper
This work proposes a new image fusion method that is well suited for merging angle information and extending the field of depth of Differential Interference Contrast (DIC) microscopic images. Many other extend field of depth methods only regularize their solution in a small neighborhood. Such approaches are not suitable for DIC images where reliable depth estimates are only possible at cell walls, which are sparse in the overall images. Therefore, we developed a novel fusion method that first accurately estimates the depth of the cell layer of interest and in a second step extends the field of depth using this depth estimation. The proposed method only fuses information specifically linked to the epidermal cell layer of Arabidopsis leaves, thus avoiding clutter related to underlying cell layers. In addition, our method fuses images with different shear angles, thus overcoming the lack of contrast in certain directions intrinsic for DIC microscopy. By both improving the focal depth and contrast, the proposed method is a useful step towards fully automated analysis of the cellular content of the leaf's epidermal cell layer.
Article
Introduction to Common Particle Properties Analysis of Particle Size Distribution Measurement of the Physical Properties of Particles Testing of Particle Bed Properties Measurement of Particle Bed Movement in Rotary Drums by High-Speed Camera Conclusions References
Chapter
This chapter emphasizes results from all vibrational techniques, in particular those from vibrational circular dichroism (VCD) measurements. It reviews the basic results for peptides, proteins, nucleic acids, and lipids, and also explores some results that demonstrate how imaginative application of these techniques can reveal information that is not available from any other techniques. Owing to their formidable size, low solubility, and structural complexity, proteins were realized early on to be poor subjects for initial studies using vibrational spectroscopy. A plethora of vibrational spectroscopic data exists for various small linear and cyclic peptides. The chapter reviews the basic principles, including the transition dipole coupling model to explain the conformational sensitivity of IR spectroscopy; furthermore, it contains the most comprehensive compilation of amino acid side group vibrations in peptides and proteins, and a thorough discussion of the amide I and II manifolds.
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Fully realizing that immunohistochemistry is a visual art, we therefore need optical aids to assess the results of immunohistochemical reaction. The major optical instrument available to the immunohistochemist is the light microscope — probably the most well-known and well-used research tool in biology and medicine. Yet many students and researchers are unaware of the full range of features that are available in light microscopes. Severe limitations in exploiting the light microscope can cause considerable confusion in interpretation of immunohistochemical stainings. In this chapter we attempt to address this issue without the unnecessary math that often confuses students and postdoctoral researchers. For beginners we highly recommend the handbook “Light Microscopy, Essential Data” by Rubbi (1994) and the review by Davidson and Abramowitz “Optical microscopy” available on the website: http:// micro. magnet. fsu. edu/ primer/ pdfs/ microscopy. pdf. Explanations of the common words used in microscopy may be found in the “RMS Dictionary of Light Microscopy” (Bradbury et al. 1989) and on the website: http:// www. microscope-microscope. org/ basic/ microscope-glossary. htm.
Article
Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings. Copyright © 2014, American Association for the Advancement of Science.
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La evolución de los conocimientos en Hemorreología en las últimas décadas ha hecho de esta rama de la Ciencia una herramienta indispensable para la evaluación de determinadas situaciones clínicas. La necesidad de condiciones experimentales específicas y la definición de parámetros necesarios al enfoque reológico de la sangre y sus componentes conducen a la concepción de nuevos equipos y técnicas generalmente fundamentados en principios ópticos y mecánicos. Las alteraciones en la morfología y en el comportamiento viscoelástico de la membrana eritrocitaria pueden producir serios disturbios en la microcirculación sanguínea. En particular, es de gran interés el estudio de la relación existente entre la viscoelasticidad de la membrana eritrocitaria y diversas alteraciones morfológicas como las observadasen patologías hematológicas, renales y vasculares así también como en diversos tratamientos in vitro y en el almacenamiento de la sangre o de sus componentes. Entre todas las técnicas utilizadas para el estudio de la morfología celular (Microscopía Convencional, Microscopía de Fluorescencia, etc.), la Microscopía Electrónica de Barridoo MEB (Scanning Electron Microscopy o SEM en inglés) es la que ofrece mayores ventajas debido a que presenta mayor poder de reso-lución y mayor profundidad de campo. Palabras clave: microscopía electrónica de barrido, glóbulos rojos, morfología eritrocitaria.
Article
Saccharomyces cerevisiae и конидий микромицетов Trichoderma asperellum, наномо-дифицированных полиэлектролитными пленками и металлическими наночастицами. Рассмотрены техники оптической микроскопии в проходящем и отраженном свете, скани-рующей и просвечивающей электронной микроскопии и атомно-силовой микроскопии. Ключевые слова: наномодифицированные клетки микромицетов, оптическая мик-роскопия, электронная микроскопия, атомно-силовая микроскопия.
Article
A phenol formaldehyde (PF) adhesive was uniformly tagged with iodine such that it yielded sufficient X-ray computed tomography (XCT) gray-scale contrast for material segmentation in reconstructed wood-composite bondlines. Typically, untagged adhesives are organic and have a similar solid-state density as wood cell-walls, and therefore cannot be segmented quantitatively in XCT data. The iodinated PF development involved analysis and comparison of three trial adhesives containing rubidium, bromine, or iodine. Adhesive tag efficacy was measured in terms of X-ray absorption contrast enhancement and tag uniformity along the adhesive polymers. Cured adhesive density, tag element, and concentration were each found to significantly impact XCT contrast results, which in turn agreed with theoretical X-ray attenuation predictions for each resin. Ion chromatography confirmed the absence of free iodide in the liquid PF prior to bonding, and fluorescence microscopy and energy-dispersive spectroscopy (EDS) showed that iodine tags remained associated with the cured adhesive polymers. XCT and EDS results also demonstrated that when contrast agents are simply mixed into resins, rather than attached to the polymer chains, they are free to migrate independent of the penetrating adhesives during bonding. This then can cause complications with quantitative segmentation and analyses. The iodinated PF yielded consistent and uniform XCT gray-scale contrast; its formulation could be adjusted for other viscosity or molecular weight distribution, which would affect its penetration behavior.
Conference Paper
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Observation of pore structures in thin section using the traditional method of impregnation with blue-dyed epoxy becomes difficult when the pores are smaller than about 1 micrometer and/or linear in shape. These types of pores are common in many gas-bearing tight formations, including coal seams, black shales, and lenticular sandstones. Incident-light fluorescence microscopy can be utilized for observation of small, narrow pore structures in tight rocks. Adaptation of this common medical technique to petrography is accomplished by staining the epoxy with fluorescent Rhodamine-B dye instead of the usual blue material, impregnating under vacuum, and preparing a polished thin section in the normal manner. The thin section is observed using an incident-light microscope equipped for fluorescence, which usually involves only a simple lamp and filter change on an existing, universal-type microscope. Under excitation of green light at a wavelength of 540 nm, the Rhodamine fluoresces a brilliant reddish orange, clearly showing impregnated pore space. A dichromatic interference filter in the light path allows only the reddish orange light to pass through, blacking out the mineral grains such that only the pore structure is visible. The incident-light configuration of the microscope has the advantage of inducing brighter fluorescence at higher magnifications due to the more intense concentration of the exciter beam through higher-power objectives. This technique also permits observation of impregnated pore space narrower than the wave-lengths of visible light. Epoxy fluorescing in pores this small behaves like a point source and makes the pore visible. Fluorescence microscopy has much promise for studying pore structures in tight sedimentary rocks and can also be applied to crystalline rocks and other materials containing narrow, linear, or small pores.
Article
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Research on better methods to digitally represent microscopic specimens has increased over recent decades. Opaque specimens, such as microfossils and metallurgic specimens, are often viewed using reflected light microscopy. Existing 3D surface estimation techniques for reflected light microscopy do not model reflectance, restricting the representation to only one illumination condition and making them an imperfect recreation of the experience of using an actual microscope. This paper introduces a virtual reflected-light microscopy (VRLM) system that estimates both shape and reflectance from a set of specimen images. When coupled with anaglyph creation, the system can depict both depth information and illumination cues under any desired lighting configuration. Digital representations are compact and easily viewed in an online setting. A prototype used to construct VRLM representations is comprised only of a microscope, a digital camera, a motorized stage and software. Such a system automatically acquires VRLM representations of large batches of specimens. VRLM representations are then disseminated in an interactive online environment, which allows users to change the virtual light source direction and type. Experiments demonstrate high quality VRLM representations of 500 microfossils.
Article
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We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1, 1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C(20)) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3 -phosphoc holine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D(T), of the DiI-C(20) was measured by FCS. D(T) values at approximately 25 degrees C ranged from approximately 3 x 10(-8) cm(2)/s in the fluid phase, to approximately 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to approximately 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of D(T) in a region of two-phase coexistence on a single vesicle.
Book
List of symbols. Preface. Efficient use of this book. Crystal identification and optical principles. Equipment and preparation of materials. The immersion method. Isotropic crystal identification. Phase relationships and interference. Anisotropic crystals. Uniaxial crystal optics. Uniaxial interference figures. Identification of uniaxial crystals. Biaxial crystal optics. Biaxial interference figures. Identification of biaxial crystals. Crystallographic relations of biaxial crystals. Optic orientation in stereo. Special orientation methods. Use of the dispersion method. Crystal identification practicum Appendix A: optical properties of common rock-forming minerals. Appendix B: identification of fibrous asbestos References. Index.
Chapter
This chapter is concerned with the uses that are made of polarized light in the study of polymer structure. These uses are determined by the type of polymer under examination, i.e. whether it is semicrystalline or amorphous. Semicrystalline polymers crystallize from the melt, in the absence of significant shear or elongational flow, in the form of a characteristic structural entity called a spherulite. Variation in size, shape and type of spherulite with temperature and flow conditions during crystallization is a powerful diagnostic indicator of the change in the factors throughout a complex moulded product. Hence the study of spherulitic texture in these materials will reveal the structural changes caused by varying production conditions. In fact the study of sefnicrystalline polymers by polarized light methods constitutes a large proportion of all light microscope studies on polymers.
Article
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.
Article
Four formalisms are outlined. Crystal field theory explains the color as well as the fluores- cence in transition-metal-containing minerals such as azurite and ruby. The trap concept, as part of crystal field theory, explains the varying stability of electron and hole color centers with respect to light or heat bleaching, as well as phenomena such as thermoluminescence. The molecular orbital formalism explains the color of charge transfer minerals such as blue sapphire and crocoite involving metals, as well as the nonmetal-involving colors in lazurite, graphite and organically colored minerals. Band theory explains the colors of metallic minerals; the color range black-red-orange- yellow-colorless in minerals such as galena, proustite, greenockite, diamond, as well as the impurity-caused yellow and blue colors in diamond. Lastly, there are the well-known pseudo- chromatic colors explained by physical optics involving dispersion, scattering, interference, and diffraction.
Article
The microscopic image of illuminated objects results from a twofold diffraction, at the object and at the lens-aperture. The theories of Rayleigh and of Abbe differ only as to the order in which they consider these diffractions. The Abbe method is here used to calculate the image of a coarse transparent grating with shallow grooves of arbitrary form (phase grating). In the ideal case the image is invisible. The formulae are successively applied to the following practical methods to make the image visible: the schlierenmethod, where the diffraction spectra of the grating are intercepted on one side, the ordinary oblique dark ground illumination, where the central image is intercepted also, the central dark ground illumination, with intercepts the central image only, and the bright ground observation with illumination by a narrow pencil, where the visibility is caused by out-of-focus observation. It is found that none of these methods can show the real groove form. This is possible by the new method of phase contrast, where a path difference of λ/4 is introduced between the spectra and the central image by passing the last through a slightly thicker or thinner part (phase-strip) of a glass plate.In Part II the new method is treated in another way which applies to objects of arbitrary irregular structure. The general result is that by the phase-contrast method transparent details of the object which differ in thickness or in refractive index appear as differences of intensity in the image. An important increase of sensitivity can further be obtained by the use of an absorbing phase strip. The effect of the diffraction by the phase strip is then considered and practical methods discussed to make the resulting diffraction-halo as faint as possible. Various reasons are found why the strip should preferably be of circular form, with a corresponding annular diafragm in the condenser. Finally the methods of preparing phase strips and of placing and adjusting them in the microscope are discussed.
Book
Metallography: Principles and Practice discusses metallographic techniques and their application to the study of metals, ceramics, and polymers. The book concentrates on techniques relevant to visual and light microscopy. Topics include macrostructure, specimen preparation for light microscopy, microstructure, light microscopy, hardness testing, and quantitative microscopy. An extensive collection of macrographs and micrographs is presented to illustrate the various methods discussed and to provide examples of their application to various materials. Appendices including etchants and specimen preparation procedures are included. For information on the print version, ISBN: 978-0-87170-672-0, follow this link.
The Optical Performance of the Light Microscope (2 vols.)
• H W Zieler
H. W. Zieler, The Optical Performance of the Light Microscope (2 vols.). Microscope Publications, Ltd., Chicago, 1972; vol. 1, 102 pp.; vol. 2, 110 pp.
Contrast Methods in Microscopy: Transmitted Light
• M Abramowitz
M. Abramowitz, Contrast Methods in Microscopy: Transmitted Light. Olympus America, Inc., Melville, New York, 1987, 31 pp.
Microscopy From The Very Beginning
• H G Kapitza
H. G. Kapitza, Microscopy From The Very Beginning. Carl Zeiss, Oberkochen, Germany, 1994, 40 pp.
• G Nomarski
G. Nomarski, J. Phys. Radium 16: 9S-11S (1955).
Photomicrography in Theory and Practice
• C P Shillaber
C. P. Shillaber, Photomicrography in Theory and Practice. John Wiley & Sons, Inc., New York, 1944, 773 pp.
S. W. Paddock (ed.), Methods in Molecular Biology, Vol. 122: Confocal Microscopy, Methods and Protocols. Humana Press, Totowa, N. J., 1999, 446 pp.
Structure of Metals Through Optical Microscopy
• A Tomer
A. Tomer, Structure of Metals Through Optical Microscopy. ASM International, Boulder, CO, 1990, 265 pp.
• B Herman
• Fluorescence Microscopy
B. Herman, Fluorescence Microscopy. 2e, BIOS Scientific Publishers Ltd., Oxford, UK, 1998, 170 pp.
The Practical Use of the Microscope
• G H Needham
G. H. Needham, The Practical Use of the Microscope. Charles C. Thomas, Springfield, IL, 1958, 493 pp.
The Polarizing Microscope. 3e, Vickers Instruments
• A F Hallimond
A. F. Hallimond, The Polarizing Microscope. 3e, Vickers Instruments, York, UK, 1970, 302 pp.
Photography Through The Microscope. 9e
• J G Delly
J. G. Delly, Photography Through The Microscope. 9e, Eastman Kodak Co., Rochester, New York, 1988, 104 pp.
• M Pluta
M. Pluta, Advanced Light Microscopy (3 vols.). Elsevier, New York, 1989; vol. 1, 464 pp.; vol. 2, 494 pp.; vol. 3, 702 pp.
Introduction to Light Microscopy
• B Bracegirdle
S. Bradbury and B. Bracegirdle, Introduction to Light Microscopy. BIOS Scientific Publishers Ltd., Oxford, UK, 1998, 123 pp.
• C J R Sheppard
• D M Shotton
C. J. R. Sheppard and D. M. Shotton, Confocal Laser Scanning Microscopy. BIOS Scientific Publishers Ltd., Oxford, UK, 1997, 106 pp.