This dissertation focuses on an electron microscopy investigation of the microstructure of SiC layers in TRISO coated particles deposited by chemical vapour deposition under different experimental conditions, which include temperature, concentration of gases and deposition time. The polycrystalline β-SiC was deposited from the decomposition of methyl trichlorosilane MTS in the presence of hydrogen (H2) as carrier gas. Scanning electron microscopy (SEM), using the backscattered electron (BSE) mode, was used to image the microstructure of and defects in the SiC layers of TRISO particles. Electron backscatter diffraction (EBSD) in the SEM was used to determine the SiC grain sizes and distribution thereof in TRISO particles deposited under different conditions. For samples with a poor EBSD indexing rate, transmission Kikuchi diffraction and transmission electron microscopy (TEM) investigations were also carried out. From the results, the effects of growth temperature on the SiC microstructure, specifically on the grain size and shape and the porosity were determined. The effects of cooling or non-cooling of the gas inlet nozzle on the SiC microstructure were also investigated. TEM and scanning TEM (STEM) analyses of the SiC layers in TRISO particles were performed to image the defects and reveal the crystallinity of SiC layers. The microstructure and composition of SiC tubes fabricated by reaction bonding (RB) was also investigated by using electron microscopy and Raman spectroscopy. SEM-BSE imaging of RBSiC samples allowed the identification of impurities and free silicon in the RBSiC. Finally, the penetration of the metallic fission product, palladium, in reaction bonded SiC at a temperature of a 1000ºC is determined. A brief comment on the suitability of RBSiC as candidate for fuel cladding in a PWR is made. A short discussion of the suitability of the characterisation techniques used is included at the end.

A culmination of theory, techniques and devices stemming from a wide variety of sources and disciplines, optical microscopy presents vast possibilities for visualisation of small structures. One of the most fundamental yet significant optical microscopy techniques is Confocal Fluorescence Microscopy (CFM). CFM is studied here by analysing its performance with respect to the two most important metrics - Signal-to-noise ratio and 3D optical resolution. Several authors have commented on the inherent inefficiency of imaging systems such as CFM to utilise the available light when providing resolution beyond the well-known diffraction limit, primarily due to the precise mechanisms that help realise the resolution gain in the first place. In CFM, the detection pinhole is the key mechanism that helps realise up to 1.4 times resolution improvement over conventional wide-field microscopy techniques by trading off SNR. First, an investigation of the inherent SNR-resolution trade-off in a CFM system is studied; the impact of the detection pinhole geometry on the performance of a CFM is examined by means of an effective trade-off curve. Using alternative pinhole geometries in conjunction with new detection schemes, it is next shown how performance gains are realised in both the lateral and axial directions. Examined next is a recently developed detection scheme called subtractive imaging; wherein a special annular pinhole is used to divide the confocal point spread function signal into two detectors. By using fast point detectors in place of CCD arrays, it is shown how using numerical optimisation yields an optimum “differential pinhole” to achieve considerable 3D resolution gains over conventional (circular pinhole based) CFM systems. By examining the trade-off curves it is also shown that the proposed design is able to offer simultaneous and maximum performance gains up to a considerably high SNR in comparison to conventional (circular pinhole) based CFM systems. Lastly, the work will propose the use of a deconvolution technique and an alternative detection scheme to demonstrate substantially higher improvements in the quality of images acquired by a CFM system. Image reconstruction is a tried and tested image post processing strategy to realise super resolution. An image reconstruction technique, based on an expectation maximisation maximum likelihood (EM-ML) algorithm is used in conjunction with array detectors to demonstrate enhanced resolution and noise performance of a CFM system. The point scan method used here renders the algorithm slow with long run times. To mitigate this, structured illumination is used to show how similar resolution gains in the array detector based CFM systems could be realised but in a much shorter time.

No description available.

Because of difficulty experienced with visual observation of bleached, unstained softwood tracheids, a study was initiated to determine the best microscopic system, for observation of this type of material. A comparison was made between bright-field, dark-field, polarising, phase, electron, fluorescence and interference microscopic systems. In addition, the optical characteristics of each type of microscope were studied to determine the reasons for the differences in visual results. The study was expanded to include an evaluation of the interference microscope and its use in measuring refractive index of bleached wood pulp. The interference microscope was found to give visual images superior to those obtained with any of the other systems examined and thus constitutes an important, contribution to the practice of microscopy. Inconclusive results were obtained in the evaluation of the system of measurement of refractive index by means of the interference microscope. / Forestry, Faculty of / Graduate

Evaluation of several methods of fixing chicken skin for scanning electron microscopy (SEM) indicated standard chemical fixation using glutaraldehyde and osmium tetroxide followed by chemical dehydration with 2,2-dimethoxypropane to be the method of choice. SEM revealed that chicken skin has a convoluted surface. Two types of chicken skin, distinguishable on the basis of chemical composition and appearance were observed. Type I has a filamentous surface with 55% moisture and 25% fat, whereas Type II skin has a globular appearance, 52% fat and 33% moisture. The fatty acid profiles of Types I and II skin are the same. Bacteria have greater affinity for Type II than Type I skin. Attachment studies indicated that Salmonella typhimurium quickly attach to the skin surface and cannot be removed easily by washing with water or with water containing a surfactant. / Land and Food Systems, Faculty of / Graduate

Orientador: Americo Bortolazzo Correr / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T03:22:53Z (GMT). No. of bitstreams: 1 Sfalcin_RavanaAngelini_D.pdf: 1513616 bytes, checksum: bd6dc4a4843283522343b56a58ef8ec7 (MD5) Previous issue date: 2015 / Resumo: O objetivo neste trabalho foi avaliar as propriedades físico-químicas de infiltrantes resinosos com adição de partículas bioativas, bem como sua capacidade de penetração e dureza da profundidade em lesões subsuperficiais de esmalte. Uma blenda contendo TEGDMA (75% em peso) e BisEMA (25% em peso) foi manipulada e a partir dela foram incorporados 5 tipos de partículas bioativas (10% em peso): hidroxiapatita (HAp), fosfato de cálcio amorfo (ACP), vidro bioativo policarboxilato de zinco (BAG Zn), vidro bioativo 45S5 (BAG 45S5), cimento de silicato de cálcio modificado por ?-TCP (HCAT-?). Um material comercial foi utilizado (ICON®) como controle. Dez espécimes foram confeccionados para cada grupo de cada teste: rugosidade superficial (Ra) antes e após a escovação; Resistência à flexão por 3 pontos (RF) e módulo de elasticidade (ME); resistência coesiva à tração (RC); dureza Knoop (KHN); densidade de ligação cruzada (DLC); grau de conversão (GC); sorção (S) e solubilidade (SL) em água; e micro-dureza (KHN). Os dados foram submetidos a ANOVA e teste Tukey (?=0.05). A penetração dos infiltrantes resinosos no esmalte humano desmineralizado foi qualitativamente avaliada em Microscopia Confocal de Varredura a Laser (n=5). Os resultados mostraram que os menores valores de rugosidade (antes e após a escovação foram apresentados pelo ACP. Com relação à resistência a flexão e módulo de elasticidade, T+B apresentou o maior valor e ICON® mostrou o menor valor. ICON® também mostrou o menor valor de resistência coesiva à tração; não houve diferença significativa entre os grupos T+B, HAp, ACP, BAG Zn, BAG 45S5 e HCAT-?. Para o teste de dureza Knoop, ICON® obteve o menor valor e BAG Zn mostrou o maior valor. Para densidade de ligação cruzada, ICON® apresentou maior quantidade de ligação cruzada e HAp, menor quantidade de ligação cruzada. ICON® apresentou grau de conversão significantemente menor que os infiltrantes experimentais, que não diferiram entre eles. ICON® apresentou a maior sorção de água e HAp a menor. Não houve diferença significativa entre os demais grupos. Para solubilidade, ICON® apresentou os maiores valores, mas sem diferença de ACP. BAG 45S5 apresentou a menor solubilidade. Com relação a micro-dureza, não houve diferença estatisticamente significante entre as profundidades avaliadas (50 µm, 200 µm, 350 µm e 500 µm). BAG 45S5, BAG Zn e HCAT-? não mostraram diferença estatística entre eles. Entretanto, HCAT-? e BAG Zn foram similares ao ICON® e ACP. O grupo cariado mostrou menor valor quando comparado a todos os grupos testados. A análise em microscopia confocal mostrou que todos os materiais apresentaram boa capacidade de penetração nas lesões iniciais, exceto para FCA. Pôde ser concluído que adição de partículas bioativas em um infiltrante experimental melhorou as propriedades mecânicas e não afetou a capacidade de penetração dos infiltrantes. O infiltrante resinoso contendo fosfato de cálcio amorfo foi o que apresentou o melhor desempenho no teste de rugosidade de superfície antes e após a escovação / Abstract: The aim of this study was to evaluate the physical-chemical properties of the experimental infiltrants with the addition of bioactive particles as well as their capability of penetration and depth Knoop hardness into caries-like lesions. A control blend was made with TEGDMA (75 wt%) and BisEMA (25 wt%). Five bioactive fillers were added in the control blend (10 wt%): Hydroxyapatite (Hap), amorphous calcium phosphate (ACP), Zinc-polycarboxylated bioactive glass (BAG-Zn), bioactive glass 45S5 (BAG 45S5), and ?-TCP modified calcium silicate cements (HCAT-?). An available commercially material was used (ICON®). Ten specimens were comprised by each group for the following tests: Surface roughness (Ra) before and after brushing abrasion; flexural strength (FS) and elastic modulus (E-Modulus); tensile cohesive strength (TCS); Knoop hardness (KHN); softnening ratio (SR); degree of conversion (DC); water sorption (WS) and solubility (SL); and micro-hardness (micro-KHN). Data were subjected to ANOVA and Tukey¿s test (?=0.05). Confocal Scanning Laser Microscopy was used to evaluate qualitatively the penetration capability of resin infiltrants into demineralized human enamel. Results showed that ACP had the lowest Ra before and after brushing abrasion. Regarding to the FS and E-modulus, T+B showed the higher value and ICON® showed the lower value. Also, ICON® showed the lower value of TCS, but there was no significant statistically difference among the groups T+B, HAp, ACP, BAG Zn, BAG 45S5 e HCAT-?. To the KHN, ICON® obtained the lower value and BAG Zn showed the higher value. According to the SR, ICON® showed lower SR and HAp, the higher SR. ICON showed DC significantly lower than experimental resin infiltrants. Regarding to the WS, ICON® presented the highest water sorption and HAp the lowest one. There was no significant statistically difference among the other groups. ICON showed the highest SL results; however, the results were similar to ACP. The lowest SL was found for BAG 45S5. Regarding to the micro-KHN, there was no statistically difference among the analyzed depths (50 µm, 200 µm, 350 µm and 500 µm). BAG 45S5, BAG Zn and HCAT- ? did not show statistical difference among them. However, HCAT- ? and BAG Zn were similar to ICON® and ACP. Carious group showed lower value when compared to all the tested groups. Confocal microscopy analysis showed good capability of penetration into the initial lesions for all materials, except for ACP. It could be concluded that the addition of bioactive particles into an experimental infiltrant improved the mechanical properties and did not affect the capability of penetration into the experimental infiltrants. The resin infiltrant with amorphous calcium phosphate presented the best performance to the roughness surface before and after brushing abrasion / Doutorado / Materiais Dentarios / Doutora em Materiais Dentários

Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the “Receptome”, the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in naïve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi. / Menschen neigen schon immer dazu, vor allem das zu glauben, was sie mit eigenen Augen sehen können, weswegen mikroskopische Methoden seit ihrer Erfindung im 17. Jahrhundert schon immer sehr beliebt waren. Mit der Einführung der hochauflösenden Mikroskopie konnte das Auflösungslimit von ~200 - 250 nm durchbrochen werden, was genauere Einblicke in biologische Proben ermöglichte. Insbesondere die Einzelmolekül-Lokalisations-Mikroskopie Methode dSTORM bietet hierbei die Möglichkeit der quantitativen Bildgebung. Sie nutzt das wiederholte Schalten organischer Farbstoffe in Anwesenheit von Thiolen, was eine Auflösung von bis zu 20 nm möglich macht. Eine weitere kürzlich entwickelte hochauflösende Mikroskopiemethode ist die Expansionsmikroskopie (ExM), in welcher die Probe isotrop vier- bis sogar zwanzigfach vergrößert wird, womit sich auch die Auflösung um diesen Faktor vergrößert. Um dies zu ermöglichen, wird die Probe in ein Hydrogel eingebettet, mittels einer unspezifischen Proteinase homogenisiert und in destilliertem Wasser expandiert. Innerhalb dieser Arbeit wurden beide Methoden genutzt, um sowohl die Verteilung von Plasmamembran Rezeptoren als auch unterschiedliche bakterielle und pilzliche Pathogene zu beleuchten Im ersten Teil dieser Arbeit wurde dSTORM genutzt, um das „Rezeptom“, die Gesamtheit aller Membranrezeptoren, sowohl von Jurkat T-Zellen als auch von primären Patientenzellen zu entschlüsseln. In dieser Arbeit konnten die Rezeptoren CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 und CD105 erfolgreich visualisiert und quantifiziert werden, welche Dichten von 0,8 Cluster pro µm² im Falle von CD20 und 81,4 Cluster pro µm² für den stark exprimierten Rezeptor CD45 in aktivierten primären T-Zellen auf der basalen Membran aufwiesen. Hierbei konnten wir für einen Großteil der Rezeptoren eine homogene Verteilung nachweisen, wohingegen nur wenige andere Rezeptoren Cluster zeigten. Für CD3 konnten sowohl in Jurkat T-Zellen als auch in aktivierten primären Zellen Cluster detektiert werden, was auf deren Aktivierung hinweist, wohingegen CD3 in naiven Zellen homogen verteilt war. Im Weiteren wurde dSTORM im Rahmen von drei klinischen Fragestellungen angewandt, in welche die Rezeptoren CD38, BCMA und CD20 involviert waren, die in Immuntherapien mit monoklonalen Antikörpern oder auch CAR T-Zellen adressiert werden. In den beiden erstgenannten Projekten wurde dSTORM genutzt, um die Erhöhung der Rezeptoren-Expression nach Zugabe verschiedener Medikamente sowohl in der Zelllinie MM1.S als auch in primären Zellen von Patienten mit multiplen Myelomen zu bestimmen. Durch das CD20-Projekt hingegen wurde die überlegene Sensitivität von dSTORM gegenüber der Durchflusszytometrie unter Beweis gestellt. Hier konnte verglichen mit der Durchflusszytometrie eine deutlich höhere CD20-positive Fraktion in Patientenzellen detektiert werden, welche nach Behandlung mit CD20 CAR T-Zellen eliminiert wurde. Hierauf folgte hochauflösende Bildgebung ganzer Zellen sowohl mit LLS-3D dSTORM als auch 10x ExM, um Interaktionen mit der Glasoberfläche ausschließen zu können. Bei 10x ExM wurde eine Signalamplifikation mittels Biotin und Streptavidin ATTO 643 benötigt, wonach sogar einzelne Antikörper, welche gegen den Heterodimer CD11a gerichtet waren, an einem herkömmlichen konfokalen Mikroskop detektiert werden konnten. Obwohl dies aufgrund der Prozesse von Gelierung, Verdau und Expansion während des ExM-Protokolls vermutlich nicht quantitativ ist, konnten sogar mutmaßliche Dimere des Rezeptors CD2 mit 10x ExM-SIM visualisiert werden, welche ähnlich in dSTORM Experimenten auftraten. Im zweiten Teil dieser Arbeit wurde die Expansionsmikroskopie für bakterielle und pilzliche Pathogene eingesetzt. ExM ermöglichte nicht nur eine isotrope vierfache Expansion von Chlamydia trachomatis, sondern auch die Unterscheidung der beiden Entwicklungsformen, der Retikulär- und Elementarkörperchen, aufgrund der Größe der einzelnen Chlamydien. Anschließend wurde ein neues α-NH2-ω-N3-C6-Ceramid eingeführt, was eine effiziente Fixierung und zum ersten Mal die Nutzung von Lipiden in 4x und 10x ExM ermöglichte, was wir Sphingolipid ExM nannten. Diese Verbindung wurde genutzt, um die Ceramid-Aufnahme und den -Einbau in die Zellmembran von Chlamydia trachomatis und Simkania negevensis zu untersuchen. Im Falle von Chlamydia trachomatis wurde die hohe Auflösung von 10x ExM mit SIM kombiniert, was die Visualisierung beider bakterieller Membranen in einem Abstand von ~30 nm ermöglichte. Hiernach wurde ExM bei den drei unterschiedlichen Pilzen Ustilago maydis, Fusarium oxysporum und Aspergillus fumigatus nach enzymatischen Verdau der pilzlichen Zellwand angewandt. Im Falle von Ustilago maydis Sporidien konnte der Verdau sowohl an lebenden Zellen, was in Protoplasten resultierte, als auch an fixierten Zellen verwendet werden, was die Morphologie erhielt. Mittels dieses neuen Protokolls konnten sowohl Immunfärbungen als auch fluoreszierende Proteine der drei genannten Pilze expandiert werden.

Fluorescence imaging is indispensable to biology and neuroscience. The need for large-scale imaging in freely behaving animals has further driven the development in miniaturized microscopes (miniscopes). However, conventional microscopes and miniscopes are inherently constrained by their limited space-bandwidth-product, shallow depth-of-field, and inability to resolve 3D distributed emitters such as neurons. In this thesis, I present a Computational Miniature Mesoscope (CM2) that leverages two computation frameworks to overcome these bottlenecks and enable single-shot 3D imaging across a wide imaging field-of-view (FOV) of 7~8 mm and an extended depth-of-field (DOF) of 0.8~2.5 mm with a high lateral (7 um) and axial resolution (25 um). The CM2 is a novel fluorescence imaging device that achieves large-scale illumination and single-shot 3D imaging on a compact platform. This expanded imaging capability is enabled by computational imaging that jointly designs optics and algorithms. In this thesis, I present two versions of CM2 platforms and two 3D reconstruction algorithms. In addition, pilot studies of in vivo imaging experiments using a wearable CM2 prototype are conducted to demonstrate the CM2 platform's potential applications in large-scale neural imaging. First, I present the CM2 V1 platform and a model-based 3D reconstruction algorithm. The CM2 V1 system has a compact lightweight design that integrates a microlens array (MLA) for 3D imaging and an LED array for excitation on a single compact platform. The model-based 3D deconvolution algorithm is developed to perform volumetric reconstructions from single-shot CM2 measurements, achieving 7 um lateral and 200 um axial resolution across a wide 8 mm FOV and 2.5 mm DOF in clear volumes. This mesoscale 3D imaging capability of CM2 is validated on various fluorescent samples, including resolution target, fibers, and particle phantoms in different geometry. I further quantify the effects of bulk scattering and background fluorescence in phantom experiments. Next, I investigate and improve the CM2 V1 system for both the hardware and the reconstruction algorithm. Specially, the low axial resolution (200 um), insufficient excitation efficiency (24%), and heavy computational cost of the model-based 3D deconvolution hinder CM2 V1's biomedical applications. I present and demonstrate an upgraded CM2 V2 platform augmented with a deep learning-based 3D reconstruction framework, termed CM2Net, to address the above limitations. Specially, the CM2 V2 design features an array of freeform illuminators and hybrid emission filters to achieve 3 times higher excitation efficiency (80%) and 5 times better suppression of background fluorescence, compared to the V1 design. The multi-stage CM2Net combines ideas from view demixing, lightfield refocusing and view synthesis to account for the CM2’s multi-view geometry and achieve reliable 3D reconstruction with high axial resolution. Finally, trained purely on simulated data, I show that the CM2Net can generalize to experimental measurements. A key element of CM2Net's generalizability is a 3D Linear Shift Variant (LSV) model of CM2 that simulates realistic measurements by accurately incorporating field varying aberrations. I experimentally validate the CM2 V2 platform and CM2Net achieve faster, artifact-free 3D reconstructions across a 7 mm wide FOV and 800 um DOF with 25 um axial and 7 um lateral resolution in phantom experiments. Compared to the CM2 V1 with model-based deconvolution, the CM2Net achieves a 10 times better axial resolution at 1400 times faster reconstruction speed without sacrificing the imaging FOV or lateral resolution. The new system design of CM2 V2 with the LSV-embedded CM2Net provides an intriguing solution to large-scale fluorescence imagers with a small form factor. Built from off-the-shelf and 3D printed components, I envision that this low-cost and compact computational imaging system can be adopted in various biomedical and neuroscience labs. The CM2 systems and the developed computational tools can have impact in a wide range of large-scale 3D fluorescence imaging applications.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this study was to demonstrate the clinical application of the television microscope for direct intraoral micromeasurement of cavity margin deterioration. Mesiocclusal alloy restorations were placed in fifty-one maxillary second deciduous molars. A cast gold overlay with two proximal margin observation holes, one hole in the occlusal one-third and one hole in the gingival one-third, was fabricated for each restored tooth. The mesiobuccal proximal margins of the restorations were observed by the television microscope, and the marginal deterioration was electronically measured at intervals of one week, two weeks, four weeks, 12 weeks, 24 weeks, and 36 weeks postoperatively. The average gingival marginal deterioration ranged from 4.9 microns at one week to 37.8 microns at 36 weeks; the average occlusal marginal deterioration ranged from 5. 4 microns at one week to 60.1 microns at 36 weeks. The data indicated that the gingival area of the proximal margin deteriorated at a faster rate during the first 12 weeks postoperatively, and the occlusal area of the gingival margin deteriorated at a faster rate during the last 24 weeks. Greater marginal alloy flash in the gingival area was believed to be responsible for the initial gingival deterioration, and repeated masticatory stresses was believed to be major causative factor for the occlusal deterioration during the last 24 weeks of the study.

When the writer took an introductory course in Microtechnique in 1954-55, he was impressed by the inadequacy of some microtechnical procedures recommended in triple staining, and he felt disappointed and discouraged by the consistently inferior results. He resolved to attempt modifications or to develop a new technique which could be relied upon to achieve anticipated results. This is a report on two years of efforts to develop a simple dependable technique in polychrome staining for histological studies. The writer feels that the results are of importance to students and teachers of biological science and to microtomists. The herein proposed technique enables the observer readily to recognize fine details of microscopic structures, and it presents a more attractive picture than does the common Harris’ hematoxylin counterstained with eosin. This report also includes a list of polychrome procedures, divided into four groups: (1) hematoxylin and combinations, (2) safranin and combinations, (3) acid fuchsin and combinations, and (4) miscellaneous.