This document provides an overview of biomedical electron microscopy methods and interpretations. It discusses topics such as fixatives, dehydration and embedding techniques, freezing and low-temperature embedding, section staining, microscopy, image recording, and three-dimensional reconstructions. The document contains 20 chapters that describe various preparation methods and provide guidance on optimizing techniques and interpreting artifacts.
Formulation and Stability Challenges for Virus TherapeuticsIntegrity Bio
Case Study from Vaccine Development Forum in 2009. Discussion of challenges of virus formulations vs. protein formulations. Includes a comparison of analytical methods and formulation solutions
This document discusses the anti-browning and antimicrobial effects of sodium chlorite on fresh cut guava. The objective was to study the anti-browning and antimicrobial effects of sodium chlorite concentrations ranging from 0.01-0.1% on fresh cut guava. Parameters such as browning index, microbial contamination, and fruit quality were measured. The results showed that sodium chlorite acted as an anti-browning and antimicrobial agent, maintaining quality of fresh cut guava for 7 days of storage.
Hot Melt Extrusion For Amorphous Formulationsasarode
The document discusses hot melt extrusion for amorphous formulations. It summarizes that about 40% of new APIs have poor solubility and are classified as Class II or IV in the Biopharmaceutical Classification System. Hot melt extrusion is presented as a continuous process that can transform crystalline drugs into amorphous forms through melting and mixing with polymers without using solvents. The document reviews the processing, performance, stability, and analytical characterization of drug-polymer hot melt extrudates using solubility parameters, thermal analysis, rheology, dissolution testing, and other techniques. It finds that hot melt extrusion improves drug dissolution by creating amorphous solid dispersions and that performance depends on drug-polymer interactions and pH conditions.
Alessandro Prastaro has a PhD in organic chemistry from the University of Rome La Sapienza. From 2008 to 2010, he worked as a research fellow focusing on organic chemistry, green chemistry, and nano catalysis. He has authored 13 articles with a total impact factor of 4.5. From 2010 to 2011, he worked as quality control supervisor for Birra Peroni, overseeing microbiology and chemical-physical labs and quality systems. He has experience in HPLC, food/beverage analysis, foam stability, microbiology, PCR, GC, UV/VIS spectroscopy, and more. Currently he works as a quality coordinator, monitoring processes, overseeing out of specification/tolerance issues, and collaborating with
Pore engeneering of ZSM-5 by silylation for the side chain alkylation of toluneDr. VIJAYKUMAR MARAKATTI
A novel silylated ZSM-5 zeolite was synthesized for the methylation of toluene to produce p-xylene. The zeolite was characterized and tested as a catalyst for toluene methylation. The zeolite was modified through silylation to tune its shape selectivity. Testing showed the silylated zeolite with a SiO2/Al2O3 ratio of 187 had high p-xylene selectivity of over 90% for toluene conversion. Process parameters like temperature, pressure and water presence were optimized to improve catalyst stability and selectivity.
Crystal Growth and Characterization of Cobalt Doped Barium Tartrate Crystals ...IJERA Editor
Single crystals of Cobalt doped Barium tartrate crystals were grown by single diffusion technique at room temperature. Effect of Cobalt doping in the Barium tartarate crystals has been studied and reported. The XRD pattern shows that Cobalt barium tartarate crystals are polycrystalline in nature and having orthorhombic structure. SEM pictures infer that crystals were grown by layer deposition. The elemental analysis has been carried out by EDAX. The chemical analysis has been performed by FTIR to denote the functional group of grown crystal. The thermal stability has been studied by the TGA, DTG and DSC.
This test report from SGS Taiwan Ltd presents the results of tests conducted on a sample of size (paint) submitted by Rising Trading Co., Ltd. The tests were conducted to analyze if the sample contained regulated substances like cadmium, lead, mercury, hexavalent chromium, polybrominated biphenyls, and polybrominated diphenyl ethers. The test results showed that the concentrations of all regulated substances tested were below the method detection limits.
Formulation and Stability Challenges for Virus TherapeuticsIntegrity Bio
Case Study from Vaccine Development Forum in 2009. Discussion of challenges of virus formulations vs. protein formulations. Includes a comparison of analytical methods and formulation solutions
This document discusses the anti-browning and antimicrobial effects of sodium chlorite on fresh cut guava. The objective was to study the anti-browning and antimicrobial effects of sodium chlorite concentrations ranging from 0.01-0.1% on fresh cut guava. Parameters such as browning index, microbial contamination, and fruit quality were measured. The results showed that sodium chlorite acted as an anti-browning and antimicrobial agent, maintaining quality of fresh cut guava for 7 days of storage.
Hot Melt Extrusion For Amorphous Formulationsasarode
The document discusses hot melt extrusion for amorphous formulations. It summarizes that about 40% of new APIs have poor solubility and are classified as Class II or IV in the Biopharmaceutical Classification System. Hot melt extrusion is presented as a continuous process that can transform crystalline drugs into amorphous forms through melting and mixing with polymers without using solvents. The document reviews the processing, performance, stability, and analytical characterization of drug-polymer hot melt extrudates using solubility parameters, thermal analysis, rheology, dissolution testing, and other techniques. It finds that hot melt extrusion improves drug dissolution by creating amorphous solid dispersions and that performance depends on drug-polymer interactions and pH conditions.
Alessandro Prastaro has a PhD in organic chemistry from the University of Rome La Sapienza. From 2008 to 2010, he worked as a research fellow focusing on organic chemistry, green chemistry, and nano catalysis. He has authored 13 articles with a total impact factor of 4.5. From 2010 to 2011, he worked as quality control supervisor for Birra Peroni, overseeing microbiology and chemical-physical labs and quality systems. He has experience in HPLC, food/beverage analysis, foam stability, microbiology, PCR, GC, UV/VIS spectroscopy, and more. Currently he works as a quality coordinator, monitoring processes, overseeing out of specification/tolerance issues, and collaborating with
Pore engeneering of ZSM-5 by silylation for the side chain alkylation of toluneDr. VIJAYKUMAR MARAKATTI
A novel silylated ZSM-5 zeolite was synthesized for the methylation of toluene to produce p-xylene. The zeolite was characterized and tested as a catalyst for toluene methylation. The zeolite was modified through silylation to tune its shape selectivity. Testing showed the silylated zeolite with a SiO2/Al2O3 ratio of 187 had high p-xylene selectivity of over 90% for toluene conversion. Process parameters like temperature, pressure and water presence were optimized to improve catalyst stability and selectivity.
Crystal Growth and Characterization of Cobalt Doped Barium Tartrate Crystals ...IJERA Editor
Single crystals of Cobalt doped Barium tartrate crystals were grown by single diffusion technique at room temperature. Effect of Cobalt doping in the Barium tartarate crystals has been studied and reported. The XRD pattern shows that Cobalt barium tartarate crystals are polycrystalline in nature and having orthorhombic structure. SEM pictures infer that crystals were grown by layer deposition. The elemental analysis has been carried out by EDAX. The chemical analysis has been performed by FTIR to denote the functional group of grown crystal. The thermal stability has been studied by the TGA, DTG and DSC.
This test report from SGS Taiwan Ltd presents the results of tests conducted on a sample of size (paint) submitted by Rising Trading Co., Ltd. The tests were conducted to analyze if the sample contained regulated substances like cadmium, lead, mercury, hexavalent chromium, polybrominated biphenyls, and polybrominated diphenyl ethers. The test results showed that the concentrations of all regulated substances tested were below the method detection limits.
NYSAS Solid State Spectroscopy Of Materials (Polymorphism)Mark_Sullivan
This document discusses polymorphism in solid-state materials and spectroscopy techniques for characterizing polymorphs. It provides examples of polymorphic drugs and outlines the importance of identifying and quantifying polymorphs. Techniques like FTIR, Raman, terahertz spectroscopy, solid-state NMR, and vibrational spectroscopy combined with multivariate analysis can be used to distinguish, identify, and quantify polymorphs in materials. Understanding polymorphism is crucial for developing drugs and ensuring consistent quality and performance throughout development and commercialization.
An introduction to analytical atomic spectrometry l. ebdonMohamedGhait
Here are the key points from the historical overview of optical spectroscopy:
- Newton discovered the solar spectrum in 1666, finding it was crossed by dark lines later studied by Fraunhofer.
- Kirchhoff in 1859 showed these colors arose from line spectra of elements, not compounds, corresponding to Fraunhofer lines. This demonstrated atomic emission and absorption.
- Atomic absorption spectroscopy, atomic emission spectroscopy, and later atomic fluorescence spectroscopy became associated with advances in astronomy and atomic physics.
- Atomic emission spectroscopy first re-entered analytical chemistry using arc/spark spectrography, then flame techniques from the 1920s.
- Development of the inductively coupled plasma overcame many problems with flames, arcs and sparks
The document provides an overview of the range of expertise at Swiss Commitment for Bioassays, including physico-chemical properties testing, in vitro toxicology, mutagenicity/genotoxicity testing, ecotoxicology testing, biodegradation/environmental fate testing, and analytical chemistry services. It lists guidelines and descriptions for numerous OECD, EU, UN, CIPAC, and other test methods covering areas such as aquatic and terrestrial ecotoxicity, ready and inherent biodegradability, analytical instrumentation, and more. Swiss Commitment for Bioassays offers a comprehensive set of bioassay and analytical chemistry services to support product development and regulatory requirements.
The presentation shortly shows the research that I did during my Ph.D. at the Eindhoven University of Technology. The aim is the microencapsulation of salt hydrates for their use in heat storage applications. The central question of the research is: "How to use Pickering emulsions efficiently for microencapsulation?"
MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.
BioMed Central Physiology, 9:11, 2009
Brian D Metscher
Department of Theoretical Biology, University of Vienna, Austria
OPHTHALMIC NANOSUSPENSION OF PREDNISOLONE ACETATE Yash Nandwani
The document is a presentation summarizing research done towards generating an ophthalmic nanosuspension of prednisolone acetate using NanoCrySP technology. Key aspects of the work included:
- Selection of prednisolone acetate as the drug candidate which is currently only available as a microsuspension.
- Characterization of prednisolone acetate to understand its solid state properties.
- Development of a spray drying method called NanoCrySP to produce nanocrystals of the drug. Parameters like solvent system and spray drying conditions were optimized.
- Excipients like mannitol, xylitol and sorbitol were evaluated for use in a nanosuspension formulation using thermal
A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT IN FORMULATIONSIJSIT Editor
This document describes the development and validation of a novel RP-HPLC method for the quantification of Icatibant in formulations. A simple isocratic elution method was developed using a C18 column with methanol, acetonitrile and water as the mobile phase. The method was validated according to ICH guidelines and showed good linearity, precision, accuracy and robustness. The developed method was successfully applied to analyze Icatibant tablet formulations and bulk drug samples.
Modern Particle Characterization Techniques Series: Laser DiffractionHORIBA Particle
This part two of the webinar series will introduce participants to basic experimental considerations when choosing laser diffraction for particle size analysis. The presentation will explain what makes laser diffraction a “modern technique.” Both wet and dry case studies will be shown along with brief demonstration videos.
In this webinar, you will learn:
- Method development
- Choosing an appropriate refractive index
- Understanding the analysis results
View recorded webinars:
http://bit.ly/particlewebinars
This document provides a manual for quality analyses of soybean products used in the feed industry. It begins with an introduction describing the increasing global production and use of soybeans and soybean products as a dominant source of protein in animal feed. It then describes the various soybean products and their production processes. These include soybean meals produced via expeller or solvent extraction processes, as well as refined products like soy protein isolates and concentrates. The manual provides definitions and applications of different soybean products for accurate identification and classification. It aims to standardize quality analyses to improve knowledge and application of soybean products.
Experiment 2 transport of materials across cell membranes and plant cell wate...Nadine Uy
The document describes experiments measuring diffusion of plant pigments and osmosis in plant cells. Four plant specimens were tested by placing pieces in water, heated water, vegetable oil, or heated oil to observe diffusion of pigments. Results showed lipidsoluble pigments like carotenoids and flavonoids diffused more quickly through oil. A separate experiment on plasmolysis in onion, apple, and spiderwort cells observed changes when placed in hypotonic and hypertonic solutions, showing effects of osmosis. A third experiment determined the sucrose concentration causing plasmolysis in spiderwort cells to estimate their solute concentration.
This document provides a summary of the book "Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters" by Werner Stumm and James J. Morgan. The book covers topics such as chemical thermodynamics and kinetics, acid-base equilibria, dissolved carbon dioxide systems, atmosphere-water interactions, metal ion speciation, precipitation and dissolution, redox reactions, and the solid-liquid interface in natural waters. It also examines the regulation of chemical composition in natural waters and biogeochemical cycles. The book contains 13 chapters and provides a comprehensive reference for understanding important chemical processes that occur in aquatic environments.
Standard test that used to determine the charged molecules, mainly proteins and nucleic acids.
Widely used in biochemistry, forensics, genetics and molecular biology.
Laemmli system of SDS-PAGE was first introduced in 1970s
DETECTION OF HELMINTHS BY USING RADIOACTIVE.SUMBUL AWAN
The document discusses various molecular techniques used to diagnose helminth parasites, including Southern blotting to detect target DNA using labeled probes, polymerase chain reaction (PCR) to amplify parasite DNA, and radioallergosorbent testing (RAST) for serology-based detection. It provides details on extracting and purifying parasite RNA and DNA, performing Southern blotting and PCR, and the basic principles and steps involved in each technique.
1) The document describes an experimental study that tested CO2 foam injection to improve oil recovery from heterogeneous synthetic cores compared to surfactants.
2) A process for manufacturing heterogeneous cores using quartz, sand, and sieves is discussed. The study represents the first high pressure CO2 foam core flooding experiment at the University of Houston.
3) Preliminary results found CO2 foam had an apparent viscosity of 34.4 cP and improved recovery over surfactants alone, laying the foundation for future high pressure core flooding experiments.
Heavy feedstocks present difficult operational challenges for refiners that can add to safety risks and reduce profitability. Processing heavy crudes safely and profitably can require development of new equipment or major changes in operating conditions.
Innovative new methods, which model heavier feedstock processing more accurately, enable refiners to adapt their processes more easily.
Register now to learn more about this important new technology.
Who should attend: Plant Managers, Process Engineers, Engineering Managers, Operations Managers, Process Design Engineers
View OnDemand at: www.real-time-answers.com/refinery
This document reports on a study of the photophysical and electroluminescent properties of a conjugated-nonconjugated multi-block copolymer. Time-resolved fluorescence experiments and comparisons of solution and solid state fluorescence indicate that in the solid state, emission comes from associated species like ground state dimers or excimers, rather than isolated chromophores. Absorption, fluorescence, FTIR and NMR spectroscopy were used to characterize the materials. Light-emitting diodes were fabricated using the copolymer to study its electroluminescent properties.
This study evaluated nanofiltration membranes as a pretreatment for seawater reverse osmosis systems to prevent scaling. Eleven commercially available nanofiltration membranes were tested using synthetic seawater under various operating conditions. Membrane performance was analyzed by measuring rejections of scale-forming ions like sulfate, carbonate, calcium, and magnesium, as well as membrane productivity. Significant differences were observed between membranes in terms of ion rejection and productivity. While sulfate rejection was high for all membranes, rejection of other ions varied from 10-99% depending on the membrane and operating pressure. Further analysis identified the membranes most suitable for shifting solubility equilibrium and preventing scaling in seawater reverse osmosis systems.
Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Si...smistry_us
1. The document discusses the covalent and electrostatic attachment of yeast cytochrome c to a fused silica surface and how its structure and function are affected.
2. Experiments examined how the protein's absorption spectra changed when covalently or electrostatically attached to the surface versus in solution under varying conditions like pH and alcohol concentration.
3. The goal was to better understand how the attachment method and external factors influence the protein's conformation and ability to perform its electron transport function.
1. The document discusses the covalent and electrostatic attachment of yeast cytochrome c to a fused silica surface and how its structure and function are affected.
2. Experiments examined how the protein's absorption spectra changed when covalently or electrostatically attached to the surface versus in solution under varying conditions like pH and alcohol concentration.
3. The goal was to better understand how the attachment method and external factors influence the protein's conformation and ability to perform its electron transport function.
NYSAS Solid State Spectroscopy Of Materials (Polymorphism)Mark_Sullivan
This document discusses polymorphism in solid-state materials and spectroscopy techniques for characterizing polymorphs. It provides examples of polymorphic drugs and outlines the importance of identifying and quantifying polymorphs. Techniques like FTIR, Raman, terahertz spectroscopy, solid-state NMR, and vibrational spectroscopy combined with multivariate analysis can be used to distinguish, identify, and quantify polymorphs in materials. Understanding polymorphism is crucial for developing drugs and ensuring consistent quality and performance throughout development and commercialization.
An introduction to analytical atomic spectrometry l. ebdonMohamedGhait
Here are the key points from the historical overview of optical spectroscopy:
- Newton discovered the solar spectrum in 1666, finding it was crossed by dark lines later studied by Fraunhofer.
- Kirchhoff in 1859 showed these colors arose from line spectra of elements, not compounds, corresponding to Fraunhofer lines. This demonstrated atomic emission and absorption.
- Atomic absorption spectroscopy, atomic emission spectroscopy, and later atomic fluorescence spectroscopy became associated with advances in astronomy and atomic physics.
- Atomic emission spectroscopy first re-entered analytical chemistry using arc/spark spectrography, then flame techniques from the 1920s.
- Development of the inductively coupled plasma overcame many problems with flames, arcs and sparks
The document provides an overview of the range of expertise at Swiss Commitment for Bioassays, including physico-chemical properties testing, in vitro toxicology, mutagenicity/genotoxicity testing, ecotoxicology testing, biodegradation/environmental fate testing, and analytical chemistry services. It lists guidelines and descriptions for numerous OECD, EU, UN, CIPAC, and other test methods covering areas such as aquatic and terrestrial ecotoxicity, ready and inherent biodegradability, analytical instrumentation, and more. Swiss Commitment for Bioassays offers a comprehensive set of bioassay and analytical chemistry services to support product development and regulatory requirements.
The presentation shortly shows the research that I did during my Ph.D. at the Eindhoven University of Technology. The aim is the microencapsulation of salt hydrates for their use in heat storage applications. The central question of the research is: "How to use Pickering emulsions efficiently for microencapsulation?"
MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.
BioMed Central Physiology, 9:11, 2009
Brian D Metscher
Department of Theoretical Biology, University of Vienna, Austria
OPHTHALMIC NANOSUSPENSION OF PREDNISOLONE ACETATE Yash Nandwani
The document is a presentation summarizing research done towards generating an ophthalmic nanosuspension of prednisolone acetate using NanoCrySP technology. Key aspects of the work included:
- Selection of prednisolone acetate as the drug candidate which is currently only available as a microsuspension.
- Characterization of prednisolone acetate to understand its solid state properties.
- Development of a spray drying method called NanoCrySP to produce nanocrystals of the drug. Parameters like solvent system and spray drying conditions were optimized.
- Excipients like mannitol, xylitol and sorbitol were evaluated for use in a nanosuspension formulation using thermal
A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT IN FORMULATIONSIJSIT Editor
This document describes the development and validation of a novel RP-HPLC method for the quantification of Icatibant in formulations. A simple isocratic elution method was developed using a C18 column with methanol, acetonitrile and water as the mobile phase. The method was validated according to ICH guidelines and showed good linearity, precision, accuracy and robustness. The developed method was successfully applied to analyze Icatibant tablet formulations and bulk drug samples.
Modern Particle Characterization Techniques Series: Laser DiffractionHORIBA Particle
This part two of the webinar series will introduce participants to basic experimental considerations when choosing laser diffraction for particle size analysis. The presentation will explain what makes laser diffraction a “modern technique.” Both wet and dry case studies will be shown along with brief demonstration videos.
In this webinar, you will learn:
- Method development
- Choosing an appropriate refractive index
- Understanding the analysis results
View recorded webinars:
http://bit.ly/particlewebinars
This document provides a manual for quality analyses of soybean products used in the feed industry. It begins with an introduction describing the increasing global production and use of soybeans and soybean products as a dominant source of protein in animal feed. It then describes the various soybean products and their production processes. These include soybean meals produced via expeller or solvent extraction processes, as well as refined products like soy protein isolates and concentrates. The manual provides definitions and applications of different soybean products for accurate identification and classification. It aims to standardize quality analyses to improve knowledge and application of soybean products.
Experiment 2 transport of materials across cell membranes and plant cell wate...Nadine Uy
The document describes experiments measuring diffusion of plant pigments and osmosis in plant cells. Four plant specimens were tested by placing pieces in water, heated water, vegetable oil, or heated oil to observe diffusion of pigments. Results showed lipidsoluble pigments like carotenoids and flavonoids diffused more quickly through oil. A separate experiment on plasmolysis in onion, apple, and spiderwort cells observed changes when placed in hypotonic and hypertonic solutions, showing effects of osmosis. A third experiment determined the sucrose concentration causing plasmolysis in spiderwort cells to estimate their solute concentration.
This document provides a summary of the book "Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters" by Werner Stumm and James J. Morgan. The book covers topics such as chemical thermodynamics and kinetics, acid-base equilibria, dissolved carbon dioxide systems, atmosphere-water interactions, metal ion speciation, precipitation and dissolution, redox reactions, and the solid-liquid interface in natural waters. It also examines the regulation of chemical composition in natural waters and biogeochemical cycles. The book contains 13 chapters and provides a comprehensive reference for understanding important chemical processes that occur in aquatic environments.
Standard test that used to determine the charged molecules, mainly proteins and nucleic acids.
Widely used in biochemistry, forensics, genetics and molecular biology.
Laemmli system of SDS-PAGE was first introduced in 1970s
DETECTION OF HELMINTHS BY USING RADIOACTIVE.SUMBUL AWAN
The document discusses various molecular techniques used to diagnose helminth parasites, including Southern blotting to detect target DNA using labeled probes, polymerase chain reaction (PCR) to amplify parasite DNA, and radioallergosorbent testing (RAST) for serology-based detection. It provides details on extracting and purifying parasite RNA and DNA, performing Southern blotting and PCR, and the basic principles and steps involved in each technique.
1) The document describes an experimental study that tested CO2 foam injection to improve oil recovery from heterogeneous synthetic cores compared to surfactants.
2) A process for manufacturing heterogeneous cores using quartz, sand, and sieves is discussed. The study represents the first high pressure CO2 foam core flooding experiment at the University of Houston.
3) Preliminary results found CO2 foam had an apparent viscosity of 34.4 cP and improved recovery over surfactants alone, laying the foundation for future high pressure core flooding experiments.
Heavy feedstocks present difficult operational challenges for refiners that can add to safety risks and reduce profitability. Processing heavy crudes safely and profitably can require development of new equipment or major changes in operating conditions.
Innovative new methods, which model heavier feedstock processing more accurately, enable refiners to adapt their processes more easily.
Register now to learn more about this important new technology.
Who should attend: Plant Managers, Process Engineers, Engineering Managers, Operations Managers, Process Design Engineers
View OnDemand at: www.real-time-answers.com/refinery
This document reports on a study of the photophysical and electroluminescent properties of a conjugated-nonconjugated multi-block copolymer. Time-resolved fluorescence experiments and comparisons of solution and solid state fluorescence indicate that in the solid state, emission comes from associated species like ground state dimers or excimers, rather than isolated chromophores. Absorption, fluorescence, FTIR and NMR spectroscopy were used to characterize the materials. Light-emitting diodes were fabricated using the copolymer to study its electroluminescent properties.
This study evaluated nanofiltration membranes as a pretreatment for seawater reverse osmosis systems to prevent scaling. Eleven commercially available nanofiltration membranes were tested using synthetic seawater under various operating conditions. Membrane performance was analyzed by measuring rejections of scale-forming ions like sulfate, carbonate, calcium, and magnesium, as well as membrane productivity. Significant differences were observed between membranes in terms of ion rejection and productivity. While sulfate rejection was high for all membranes, rejection of other ions varied from 10-99% depending on the membrane and operating pressure. Further analysis identified the membranes most suitable for shifting solubility equilibrium and preventing scaling in seawater reverse osmosis systems.
Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Si...smistry_us
1. The document discusses the covalent and electrostatic attachment of yeast cytochrome c to a fused silica surface and how its structure and function are affected.
2. Experiments examined how the protein's absorption spectra changed when covalently or electrostatically attached to the surface versus in solution under varying conditions like pH and alcohol concentration.
3. The goal was to better understand how the attachment method and external factors influence the protein's conformation and ability to perform its electron transport function.
1. The document discusses the covalent and electrostatic attachment of yeast cytochrome c to a fused silica surface and how its structure and function are affected.
2. Experiments examined how the protein's absorption spectra changed when covalently or electrostatically attached to the surface versus in solution under varying conditions like pH and alcohol concentration.
3. The goal was to better understand how the attachment method and external factors influence the protein's conformation and ability to perform its electron transport function.
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
The chapter Lifelines of National Economy in Class 10 Geography focuses on the various modes of transportation and communication that play a vital role in the economic development of a country. These lifelines are crucial for the movement of goods, services, and people, thereby connecting different regions and promoting economic activities.
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
This presentation was provided by Racquel Jemison, Ph.D., Christina MacLaughlin, Ph.D., and Paulomi Majumder. Ph.D., all of the American Chemical Society, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
Level 3 NCEA - NZ: A Nation In the Making 1872 - 1900 SML.pptHenry Hollis
The History of NZ 1870-1900.
Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
Confiscations, Kotahitanga, Kingitanga, Parliament, Suffrage, Repudiation, Economic Change, Agriculture, Gold Mining, Timber, Flax, Sheep, Dairying,
A Visual Guide to 1 Samuel | A Tale of Two HeartsSteve Thomason
These slides walk through the story of 1 Samuel. Samuel is the last judge of Israel. The people reject God and want a king. Saul is anointed as the first king, but he is not a good king. David, the shepherd boy is anointed and Saul is envious of him. David shows honor while Saul continues to self destruct.
Chapter wise All Notes of First year Basic Civil Engineering.pptxDenish Jangid
Chapter wise All Notes of First year Basic Civil Engineering
Syllabus
Chapter-1
Introduction to objective, scope and outcome the subject
Chapter 2
Introduction: Scope and Specialization of Civil Engineering, Role of civil Engineer in Society, Impact of infrastructural development on economy of country.
Chapter 3
Surveying: Object Principles & Types of Surveying; Site Plans, Plans & Maps; Scales & Unit of different Measurements.
Linear Measurements: Instruments used. Linear Measurement by Tape, Ranging out Survey Lines and overcoming Obstructions; Measurements on sloping ground; Tape corrections, conventional symbols. Angular Measurements: Instruments used; Introduction to Compass Surveying, Bearings and Longitude & Latitude of a Line, Introduction to total station.
Levelling: Instrument used Object of levelling, Methods of levelling in brief, and Contour maps.
Chapter 4
Buildings: Selection of site for Buildings, Layout of Building Plan, Types of buildings, Plinth area, carpet area, floor space index, Introduction to building byelaws, concept of sun light & ventilation. Components of Buildings & their functions, Basic concept of R.C.C., Introduction to types of foundation
Chapter 5
Transportation: Introduction to Transportation Engineering; Traffic and Road Safety: Types and Characteristics of Various Modes of Transportation; Various Road Traffic Signs, Causes of Accidents and Road Safety Measures.
Chapter 6
Environmental Engineering: Environmental Pollution, Environmental Acts and Regulations, Functional Concepts of Ecology, Basics of Species, Biodiversity, Ecosystem, Hydrological Cycle; Chemical Cycles: Carbon, Nitrogen & Phosphorus; Energy Flow in Ecosystems.
Water Pollution: Water Quality standards, Introduction to Treatment & Disposal of Waste Water. Reuse and Saving of Water, Rain Water Harvesting. Solid Waste Management: Classification of Solid Waste, Collection, Transportation and Disposal of Solid. Recycling of Solid Waste: Energy Recovery, Sanitary Landfill, On-Site Sanitation. Air & Noise Pollution: Primary and Secondary air pollutants, Harmful effects of Air Pollution, Control of Air Pollution. . Noise Pollution Harmful Effects of noise pollution, control of noise pollution, Global warming & Climate Change, Ozone depletion, Greenhouse effect
Text Books:
1. Palancharmy, Basic Civil Engineering, McGraw Hill publishers.
2. Satheesh Gopi, Basic Civil Engineering, Pearson Publishers.
3. Ketki Rangwala Dalal, Essentials of Civil Engineering, Charotar Publishing House.
4. BCP, Surveying volume 1
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
4. B/C M D/CAL
LECTRC N
M/C RC C C py
ILLUSTRATED METHODS
AND INTERPRETATIONS
Arvid B. Maunsbach
Department of Cell Biology
Institute of Anatomy
University of Aarhus
Aarhus, Denmark
Bj0rn A. Afzelius
Department of Ultrastructure Research
The Arrhenius Laboratories
Stockholm University
Stockholm, Sweden
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6. CONTENTS
FOREWORD xi 5. Long Fixation Times 40
PREFACE xiii 6. Formaldehyde-Glutaraldehyde
ACKNOWLEDGMENTS XV Combinations 42
7. Potassium Permanganate, Picric Acid, and
Ruthenium Red 44
CHAPTER 1 8. Lead Salts and Tannic Acid 46
9. Uranyl Acetate Postfixation 48
MICROGRAPH INTERPRETATION 10. Tannic Acid-Uranyl Acetate Variations 50
11. Osmium Tetroxide-Potassium Ferrocyanide 52
1. Classical Preparation Method 2
12. Osmium Tetroxide Artifacts 54
2. Low Temperature Approach 4
13. Glutaraldehyde Artifacts 56
3. A Common Test Specimen 6
4. Detection of Objects 8
5. Identification of Artifacts 10 CHAPTER 3
6. Analysis of Geometry 12
7. Biological Identification 14 FIXATIVE VEHICLE
8. Biological Diversity 16
1. Absence and Presence of Buffer 60
9. Analysis of Dynamics: Endocytosis 18
2. Comparison of Buffers 62
10. Analysis of Dynamics: Synthesis 20
11. Comparison of Methods 22 3. Osmolality of Perfusion Fixatives 64
12. Variations in Magnifications 24 4. Effects of Osmolality on Cell Shape 66
13. Interpretation Difficulties 26 5. Effects of Osmolality on Cell Organelles 68
14. Diagnostic Pathology 28 6. Adjustment of Osmolality with Sucrose 70
7. Colloid Osmotic Pressure: Low
Magnification 72
CHAPTER 2 8. Colloid Osmotic Pressure: High
Magnification 74
FIXATIVES 9. Phosphate Buffer Precipitate 76
1. Osmium Tetroxide and Glutaraldehyde at Low
Magnification 32 CHAPTER 4
2. Osmium Tetroxide and Glutaraldehyde at High
Magnification 34 FIXATIVE APPLICATION
3. Glutaraldehyde Concentration: Perfusion
Fixation 36 1. Perfusion-Fixation versus
4. Glutaraldehyde Concentration: Immersion Immersion-Fixation 80
Fixation 38 2. Perfusion-Fixation with Pressure Control 82
7. vi CONTENTS
3. Fixation by Dripping in Vivo 84 3. Holey Films 146
4. Immersion-Fixation 86 4. Thick and Thin Support Films 148
5. Variability within the Tissue 88 5. Folds in Support Film 150
6. Unsuccessful Perfusion-Fixation 90 6. Defects in Formvar Films 152
7. Superficial Tissue Damage 92 7. Common Contaminants 154
8. Early Postmortal Changes 94 8. Volatile Contamination 156
9. Late Postmortal Changes 96
10. Influence of Biopsy Method 98
11. Microwave Treatment 100 CHAPTER 8
ULTRAMICROTOMY
CHAPTER 5
1. Correlation of Light and Electron
DEHYDRATION AND EMBEDDING Microscopy 160
2. Section Thickness: Low Magnification 162
1. Stepwise versus Direct Dehydration 104 3. Section Thickness: High Magnification 164
2. Prolonged Dehydration in Ethanol 106 4. Section Thickness: Half-Micron Section 166
3. Prolonged Dehydration in Acetone 108 5. Determination of Section Thickness 168
4. Inert Dehydration 110 6. Folds in the Section 170
5. Choice of Intermediate Solvent 112 7. Collection of Sections 172
6. Epon, Araldite, and Vestopal: Unstained 8. Surface Topography of Sections 174
Sections 114
9. Knife Scratches 176
7. Epon, Araldite, and Vestopal: Stained
10. Mottling and Flaking 178
Sections 116
11. Worn Glass Knives 180
8. Different Brands of Epoxy Resins 118
12. Transmitted Vibrations 182
9. Spurr and LR White 120
13. Vibrations and Knife Marks 184
10. Embedding of Isolated Cells 122
14. Selective Chatter 186
15. Compression 188
CHAPTER 6 16. Holes and Deformations 190
17. Contamination during Microtomy 192
FREEZING A N D L O W - 18. Extraction during Sectioning 194
TEMPERATURE EMBEDDING 19. Cryoultramicrotomy: Survey Sections 196
20. Collection of Cryosections 198
1. Plunge Freezing 126
21. Thickness of Cryosections 200
2. Contact Freezing of Unfixed Tissue 128
22. Staining of Cryosections 202
3. Contact Freezing of Fixed Tissue 130
23. Defects in Cryosections 204
4. High-Pressure Freezing 132
5. Freeze-Substitution in Methanol/Uranyl
Acetate 134 CHAPTER 9
6. Freeze-Substitution in Osmium Tetroxide/
Acetone 136 SECTION-STAINING
7. Progressive Lowering of Temperature Embedding
in Lowicryl 138 1. Lead Citrate Staining 208
2. Uranyl Acetate Staining 210
3. Enhanced Section Staining 212
CHAPTER 7 4. Effects of Grid Storage 214
5. Section Exposed to Electron Beam 216
SUPPORT FILMS
6. Effect of Electron Beam 218
1. Surface Topography 142 7. Lead-Staining Granularity 220
2. Stability of Film or Section 144 8. Contamination 222
8. CONTENTS vii
9. Block-Staining Precipitate 224 6. Damage to Negatives 300
10. Removal of Contamination 226 7. Damage to Wet Negatives 302
8. Film/Imaging Plate/Charge-Coupled Device (CCD)
Camera 304
CHAPTER 1 0 9. Enlarged Digital Recordings 306
10. Variation in Electron Dose 308
MICROSCOPY
11. Corrections of CCD Camera 310
1. Resolving Power 230
2. Through-Focus Series: Hole and Latex CHAPTER 1 2
Particle 232
3. Through-Focus Series: Myelin Sheath 234 PHOTOGRAPHIC AND
4. Through-Focus Series: Cells 236 DIGITAL PRINTING
5. Minimum Contrast Focusing 238
6. Wobbler Focusing 240 1. Photographic Paper of Different Grades 314
7. Accelerating Voltages 20-100 kV 242 2. Multigrade Paper 316
8. Accelerating Voltages 80-200 kV 244 3. Exposure and Development 318
9. Unsaturated Electron Beam 246 4. Enlargement of Micrograph Details 320
10. Condenser Apertures 248 5. Objective Lens in Enlarger 322
11. Objective Aperture 250 6. Focusing of Enlarger 324
12. Through-Focus Series: Astigmatism 252 7. Intermediate Diapositive 326
13. Image Distortion 254 8. Errors in Photographic Printing 328
14. Chromatic Aberration 256 9. Retouch 330
15. Mechanical Instability 258 10. Comparison of Printers: Low
16. Specimen Drift versus Astigmatism 260 Magnification 332
17. Focus Drift 262 11. Comparison of Printers: Enlarged Prints 334
18. Electrical Instabilities 264 12. Pixel Size at Printing 336
19. Contamination in the Electron Beam 266
20. Radiation Damage 268
CHAPTER 13
21. Radiation Damage and Contamination 270
22. Low-Dose Exposure 272 NEGATIVE STAINING
23. Spectroscopic Imaging: Thin Film 274
24. Spectroscopic Imaging: Thick Section 276 1. Negative Staining Methods 340
25. Spectroscopic Imaging: Carbon 278 2. Properties of Support Film 342
26. Spectroscopic Imaging: Calcium 280 3. Comparison of Stains 344
27. Spectroscopic Imaging: Contrast Changes 282 4. Thickness of Stain 346
28. Cryoelectron Microscopy: Na, K-ATPase 5. Concentration of Specimen 348
Crystals 284 6. Deformation of Specimen 350
29. Defects in Cryoelectron Micrographs 286 7. Radiation Damage 352
CHAPTER 1 1 CHAPTER 14
IMAGE RECORDING AUTORADIOGRAPHY
1. Exposure Time 290 1. Undeveloped Emulsion 356
2. Over/Underexposure 292 2. Developed Emulsion 358
3. Effects of Development 294 3. Resolution 360
4. Exposure Dose Adjustment 296 4. Quantitation 362
5. Primary Magnification 298 5. Preparatory Defects 364
9. viii CONTENTS
CHAPTER 15 7. Complementary Replicas and Stereo
Images 440
CYTOCHEMISTRY 8. Ice Crystals and Etching 442
9. Quick-Freeze Deep Etching 444
1. Influence of Fixation 368 10. Identification of Transport Molecules 446
2. Preincubation Treatment 370 11. Contamination 448
3. Appearance of Reaction Product 372 12. Plastic Distortion 450
4. Composition of Incubation Medium 374 13. Replica Defects 452
5. Cytochemical Resolution 376
6. Unspecific Staining 378
7. Extraction of Reaction Product 380 CHAPTER 18
SAMPLING AND QUANTITATION
CHAPTER 16
1. Calibration of Magnification 456
IMMUNOCYTOCHEMISTRY 2. Sampling and Object Variability 458
3. Sampling of Pellets: Differential
1. Fixation of Sensitive Antigens 384 Centrifugation 460
2. Fixation of Insensitive Antigens 386 4. Sampling of Pellets: Gradient
3. Comparison of Embedding Media 388 Centrifugation 462
4. Influence of Preincubation Solutions 390 5. Micrograph Montages 464
5. Comparison of Primary Antibodies 392 6. Automated Digital Montages 466
6. Dilution of Primary Antibody 394 7. Resolution of Digital Montages 468
7. Quantitation of Gold Particles 396 8. Measurements on Digital Images 470
8. Controls 398 9. Stereological Grids 472
9. Comparison of Gold Probes 400 10. Cycloid Test System 474
10. Amplification of Gold Particles 402
11. Section Staining 404
CHAPTER 19
12. Resolution 406
13. Background Labeling 408 IMAGE PROCESSING
14. Antigen Retrieval by Etching 410
15. Antigen Retrieval with Sodium Dodecyl 1. Digital Contrast Changes 478
Sulfate 412 2. Processing of Scanned Image 480
16. Double Labeling 414 3. Translational Image Enforcement 482
17. Immunonegative Staining 416 4. Averaging of Macromolecular Assemblies 484
18. Freeze-Fracture Replica Labeling 418 5. Rotational Image Enforcement 486
19. Preembedding Labeling 420 6. Photographic versus Computer Averaging 488
20. Semithin Light Microscopic Sections 422 7. Fourier Correction of Section Chatter 490
8. Removal of Image Defects 492
9. Scientific Fraud: Removal of Objects 494
CHAPTER 17 496
10. Scientific Fraud: Manipulation of Labeling
FREEZE FRACTURING
AND SHADOWING CHAPTER 2 0
1. Shadowing of DNA Molecules 428 THREE-DIMENSIONAL
2. Shadowing of Protein Molecules 430 RECONSTRUCTIONS
3. Freeze-Fractured Membrane Faces 432
4. Thickness of Replica: Low Magnification 434 1. Comparison between Transmission and Scanning
5. Thickness of Replica: High Magnification 436 Electron Microscopy 500
6. Rotary Shadowing 438 2. Serial Sectioning 502
10. CONTENTS ix
3. Large Three-Dimensional Objects 504 4. Support Films 526
4. Tilting of Section Cell Nucleus 506 5. Ultramicrotomy 528
5. Tilting of Section" Nuclear Envelope 508 6. Section Staining 530
6. Helical Structures 510 7. Microscopy and Image Recording 532
7. Computer-Analyzed Helices 512 8. Photographic Work 533
8. A Three-Dimensional Model of 9. Negative Staining 534
Na, K-ATPase 514
10. Autoradiography 535
11. immunolabeling 536
APPENDIX 12. Freeze Fracture 538
PRACTICAL M E T H O D S
AUTHOR BIOGRAPHIES 539
1. Fixation 517 ACKNOWLEDGMENTS FOR REPRODUCTION
2. Dehydration and Embedding 522 OF FIGURES 541
3. Low Temperature Embedding 524 INDEX 545
12. FOREWORD
Professors Arvid Maunsbach and Bj6rn Afzelius are publish early, and often, to compete for dwindling grant
uniquely qualified to author a book on methods of speci- funds have led to a decline in the quality of published
men preparation for biomedical electron microscopy. electron micrographs. Meticulous preparation of tissues
They are not newcomers to this field. They were among is essential to avoid artifacts that may lead to interpreta-
the pioneers in the application of the electron microscope tions of questionable validity. This well-written, abun-
to the study of cells and tissues. In distinguished research dantly illustrated, book describes in detail the procedures
careers spanning some 40 years, they have witnessed the required to obtain images of cells and cell organelles free
development of a broad range of methods of specimen of artifact, and provides guidance for image processing
preparation for transmission electron microscopy, and and interpretation. It will be a rich source of information,
have made important contributions to the improvement and inspiration, for younger investigators struggling to
of some of those methods. The electron micrographs in attain optimal results with traditional methods and for
their classic papers on the kidney, liver, and spermatozoa older investigators desirous of applying some of the newer
have set a high standard of quality that others have sought techniques to their own research.
to attain.
There is a real need for this book. Regrettably, the Don W. Fawcett, M.D.
current preoccupation of biomedical scientists with the Hersey Professor of Anatomy and Cell Biology, Emeritus
newer field of molecular biology, and the pressures to Harvard Medical School
xi
14. PREFACE
Electron microscopy has fundamentally changed the procedures in biological electron microscopy, ranging
knowledge about cell structure and function. It is now from fixation through microtomy and microscopy to
an indispensable tool in many fields of cell biology and photographic procedures. The second part exemplifies
medicine and includes a large variety of preparatory more special preparation techniques, including autoradi-
methods. Each of these has its own sets of information ography, cytochemistry, immunoelectron microscopy,
possibilities along with interpretative difficulties and pit- and computer-assisted image analysis of electron micro-
falls. A profound understanding of the methods is there- graphs. Each chapter begins with an introduction, which
fore required for an in-depth analysis and evaluation of we have kept quite short, but which includes a note on
structure-function relationships. In this volume we illus- the early development in the field. The presentations of
trate the basic preparatory methods for transmission elec- the micrographs start with a short section on the prepara-
tron microscopy in biology and medicine, and we hope tion method. Next to this is a description of what can be
that this monograph will be a guide through the broad observed and finally we have some comments on the
spectrum of methods. results. Details of the preparatory procedures used for
From discussions with our research students we have most of the illustrations are included in the Appendix
realized that the interpretation or "reading" of electron (Practical Methods) or can be found in the literature
micrographs often presents more of a problem than the listed at the end of each chapter. These reference lists
actual preparation and microscopy of the biological speci- include both classical studies and more recent key publi-
mens. We have found that the best way to help students cations.
in critically evaluating their micrographs is to show exam- Most illustrations come from preparations that we
ples of t h e i r ~ a n d of o u r ~ g o o d and failed preparations. have performed specifically for this monograph. Some
These comparisons have then formed the basis of discus- originate from our own published papers (see Acknowl-
sions of the biological significance of various structures edgments for Reproduction of Figures). As experimen-
in electron micrographs. tal material we have mainly used tissues and cell types
This monograph should be useful for investigators that have been extensively studied in our laboratories,
working with transmission electron microscopy in biology particularly kidney tubule cells, hepatocytes, and sper-
and medicine~beginners as well as more experienced matozoa. To a large extent, the content of this volume
researchers. Investigators in other fields of biology may therefore reflects our own hands-on experiences. This
use it as a guide in evaluating and interpreting electron focus on a few types of cells and tissues may appear to
micrographs. Research technicians may find it useful for be a limitation, but will facilitate comparisons of dif-
choosing optimal preparation methods and identifying ferent procedures on the same tissue. Moreover, many
artifacts. methods apply almost equally well to different cell
The first part of this monograph presents the basic types.
XlII
16. ACKNOWLEDGMENTS
Much of the inspiration for the present work can be This monograph would not have been completed with-
traced back to our early years of training in biological out the technical help of several persons, particularly
electron microscopy under the critical scientific guidance Karen Thomsen and Else-Merete LOcke in Aarhus and
of Professor Fritiof S. SjOstrand. Ulla Afzelius in Stockholm, who participated in this ven-
Plans for this monograph were already laid in the 1970s ture with great skill and enthusiasm. We also acknowl-
in connection with a series of postgraduate courses in edge the outstanding photographic work of Albert Meier
biological electron microscopy organized by Jos6 David- and thank him for many valuable suggestions. At previous
Ferreira, then director of the Laboratorio Biologia Celu- stages of this project we also received important help from
lar at the Gulbenkian Institute, Oeiras, Portugal. Over Margrethe Aarup, Gunilla Almesj/5, Christina Bohman,
the following years, we continued to assemble material Poul Boldsen, Marianne Ellegaard, Maj Hasselgren, and
during our postgraduate courses in Denmark and Sweden Inger Kristoffersen. Stig Sundelin in Stockholm and Arne
until we decided to finalize the work. Christensen, Ole Moeskja~r, and the late Ejnar Hansen
We are indebted to the late Sven-Olof Bohman, to in Aarhus carefully maintained microscopes and com-
Hans Hebert and BjCrn Johansen for very useful advice puter equipment.
on various chapters of this book; to Jos6 and Karin David- The task of bringing this volume into readable shape
Ferreira for allowing us to include some micrographs; was performed by Dorrit Ipsen, Jytte Kragelund, and
and to our co-authors over the years for inspiring discus- Bente Kragh. We thank them for very competent secre-
sions. We also thank several colleagues and friends who tarial help and editing and for being enormously patient
generously shared their specimens or antibodies with us, with our constant rewriting of the text.
helped to prepare specimens or micrographs, and/or gave Last but not least we thank the staff at Academic
us their constructive comments: Ulla Afzelius, Peter Press in San Diego for very constructive and stimulating
Agre, Pier Luigi Bellon, Emile L. Boulpaep, Erik Ilsr cooperation: Craig Panner for advice and planning, Lori
Christensen, Gunna Christiansen, Romano Dallai, Carl Asbury for production, Michael Remener for design, and
Christian Danielsen, Jens DCrup, Hans JCrgen Gund- Debby Bicher for artwork.
ersen, Anders H/56g, Kaj Josephsen, Peter Leth This project was financially supported in part by the
J0rgensen, Lars Kihlborg, Salvatore Lanzavecchia, Else- Danish Medical Research Council, the Swedish Natural
Merete LOcke, S0ren Mogensen, Jesper Vuust M011er, Science Research Council, the Danish Biomembrane Re-
S0ren Nielsen, T. Steen Olsen, Peter Ottosen, Kaarina search Center, the Karen Elise Jensen Foundation, the
Pihakaski-Maunsbach, Finn Reinholt, Elisabeth Skriver, Novo Nordisk Foundation, and the Aarhus University
Margaret SOderholm, Karen Thomsen, Pierre Verroust, Research Foundation.
Hans Orskov, and several others, who may not always A.B.M. and B.A.A.
have been aware of how valuable their comments were Aarhus and Stockholm
to us. February 1998
XV
18. MICROGRAPH
INTERPRETATION
1. Classical Preparation Method 8. Biological Diversity
2. Low Temperature Approach 9 Analysis of Dynamics: Endocytosis
3. A Common Test Specimen 10 Analysis of Dynamics: Synthesis
4. Detection of Objects 11 Comparison of Methods
5. Identification of Artifacts 12 Variations in Magnifications
6. Analysis of Geometry 13 Interpretation Difficulties
7. Biological Identification 14 Diagnostic Pathology
Present-day ultrastructure research aims at the under- significance of which may not be readily apparent. Inter-
standing of biological structure-function relationships at pretation requires an understanding of the principles of
cellular and molecular levels using a wide variety of pre- the techniques as well as experience in detecting method-
paratory procedures, such as cytochemistry, immunocyto- ological errors. The electron microscope image is charac-
chemistry, negative staining, and image analysis. These terized by its wealth of information. However, only part
methods all depend on a series of basic procedures that of the information is directly related to the biological
are crucial for the outcome of the analyses, e.g., fixation object itself; other parts depend on various preparatory
procedures, ultramicrotomy, and, not the least, mi- steps and to instrument characteristics. As a consequence,
croscopy. electron micrographs are far less accessible to interpreta-
For optimal results a number of methodological as- tion than they appear to be.
pects have to be considered: What preparation method The process of electron micrograph interpretation can
should be used? Should different methods be applied in intentionally or unintentionally be divided into a series
parallel? In what ways will the preparation procedure of consecutive steps or levels of analysis, for example:
influence the object? Is the object frequent or rare?
9 detection of objects
Which magnification should be used for optimal results?
9 identification of artifacts
What quantitative aspects should be considered? What
9 analysis of geometry
is the appearance of a "golden standard" preparation?
9 biological identification
The first part of this chapter serves to illustrate briefly
9 analysis of dynamics
some of these questions. It starts with illustrations of
kidney and liver cells, which we regard to be almost opti- Examples of these five steps of analysis are given in
mally prepared by present-day standards. Comparisons the following. The late steps of interpretation usually
are then made between entirely different methods. The depend on the early ones. An ability to master the initial
necessity to use the right magnifications for the object sequence of interpretation is required for the analysis of
under study is emphasized and we raise the important the late sequence to be meaningful.
question of whether an abnormal pattern is due to a The validity of the final biological conclusions is invari-
preparatory artifact or represents a pathological change ably correlated to the quality of the information obtained.
of the biological object. Thus, emphasis on high technical standards and method-
The last part of this chapter deals with the fact that ological insight is not l'art pour l'art in electron micros-
electron micrographs represent scientific raw data, the copy but a requirement for the analysis.
19. 2 1, MICROGRAPH INTERPRETATION
1, Classical Preparation Method
FIGURE 1.1 A transmission electron micrograph
showing part of the cytoplasm in a rat liver cell. The
anesthetized animal was perfusion fixed through the ab-
dominal aorta with 1% glutaraldehyde in 0.1 M cacodylate
buffer. Excised blocks of liver tissue were postfixed, first
in the same glutaraldehyde fixative and then in 1% os-
mium tetroxide, dehydrated in ethanol, and embedded
in Epon 812. Ultrathin sections were cut on a diamond
knife and stained with uranyl acetate and lead citrate.
x 36,000.
This micrograph is representative of a hepatocyte prepared by a conventional chemi-
cal fixation and embedding procedure. This micrograph is considered as approaching
the "state-of-the-art" preparation. It is free of obvious artifacts, such as disruption
of membranes, extracted regions of ground substance, or organelles, and shows no
instrumental errors, such as astigmatism or specimen drift, yet there is no way to know
the "true" structural appearance of a liver cell or any other cell in a transmission
electron micrograph. For example, one may question whether the membranes of the
R E R in reality have a slightly wavy conformation, as seen here, or are more planar.
Have the mitochondria retained the same diameters and positions as they had in the
living cell? Are the separate mitochondrial profiles seen in this figure in reality in
continuity outside the plane of the section?
The electron micrograph is to be regarded as representing a snapshot of a moment
in the life of the cell. When interpreting the micrograph the investigator must also take
the dynamics and the movements of the organelles in consideration. Mitochondria may
divide or fuse with other mitochondria, various organelles may be transported along
the microtubules, which themselves may grow or shorten, and secretory vesicles open
at the cell surface.
The ultrastructure of cells and tissues as they appear in conventionally fixed and
resin-embedded specimens has not changed appreciably since the early 1960s, despite
the dramatic improvements of the transmission electron microscope itself, which now
features increased flexibility in handling and extended automation due to computeriza-
tion. Indeed, the major improvement in transmission electron microscopy of noncryo-
preparations has been the result of an expanded repertoire of preparatory methods
such as cytochemistry, autoradiography, and, not the least, immunoelectron microscopy.
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