Fluorescent Dyes and Electron Microscopy Techniques
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Explain how benzimides differ from most fluorescent stains in terms of cell membrane permeability.

Benzimides, such as H-33342 and H-33258, can penetrate the cell membrane without the need for permeabilization, unlike most fluorescent stains that require cells to be fixed or permeabilized before staining.

What is the primary fluorescence emission of propidium iodide when excited by UV or blue light?

Propidium iodide fluoresces red or orange when excited by UV or blue light.

Describe the specificity of Hoechst 33342 and 33258 stains in relation to their interaction with DNA.

Hoechst 33342 and 33258 stains specifically bind to adenine-thymidine (AT) rich regions in DNA, particularly to thymidine.

What considerations must be made when using ethidium bromide for measuring DNA due to its binding properties?

<p>Ethidium bromide binds to both RNA and DNA, so RNA must be removed if only DNA measurement is desired.</p> Signup and view all the answers

Identify the fluorophore commonly used in immunohistochemistry and describe its fluorescence characteristics.

<p>Fluorescein isothiocyanate (FITC) is commonly used in immunohistochemistry and fluoresces green when excited.</p> Signup and view all the answers

What is the primary difference between the lenses used in light microscopy (LM) and electron microscopy (EM)?

<p>LM uses glass or quartz lenses, while EM uses magnetic coils.</p> Signup and view all the answers

Why must specimens be placed in a vacuum for electron microscopy?

<p>Specimens must be in a vacuum to prevent electron scattering by air molecules.</p> Signup and view all the answers

What is the actual limit of resolution typically achieved by electron microscopy compared to the theoretical limit?

<p>The theoretical limit is 0.002 nm, but the actual limit is around 0.1 nm.</p> Signup and view all the answers

What role do glutaraldehyde and osmium tetroxide play in electron microscopy specimen preparation?

<p>Glutaraldehyde acts as a fixative by cross-linking molecules, while osmium tetroxide helps preserve cell membranes.</p> Signup and view all the answers

What is the typical thickness of ultrathin sections prepared for transmission electron microscopy (TEM)?

<p>Ultrathin sections for TEM are usually 50-70 nm thick.</p> Signup and view all the answers

How does the presence of unsaturated fatty acyl chains in cell membranes help organisms survive in cold environments?

<p>Unsaturated fatty acyl chains maintain membrane fluidity at lower temperatures, allowing normal growth and reproduction even as environmental temperatures decrease.</p> Signup and view all the answers

What role does cholesterol play in influencing membrane fluidity at varying temperatures?

<p>Cholesterol helps maintain membrane fluidity by preventing fatty acyl chains from binding at low temperatures, but can reduce fluidity at 37°C if present in excess.</p> Signup and view all the answers

Explain how the phase transition of lipids affects cell membrane functionality.

<p>The phase transition from fluid to gel state restricts membrane movement, which can impair normal cellular functions such as growth and reproduction.</p> Signup and view all the answers

In what way does the interaction of cholesterol’s hydroxyl group with phospholipid head groups affect membrane structure?

<p>The hydroxyl group forms interactions with phospholipid head groups, contributing to the stabilization and fluidity of the membrane.</p> Signup and view all the answers

What are the implications of excess cholesterol in eukaryotic cell membranes at physiological temperatures?

<p>Excess cholesterol can lead to reduced membrane fluidity, potentially disrupting cellular processes and functionality.</p> Signup and view all the answers

What is the role of phospholipid translocators or flippases in the cell membrane?

<p>Phospholipid translocators or flippases transfer selected phospholipids from the cytosolic monolayer to the non-cytosolic side of the membrane.</p> Signup and view all the answers

How does the presence of cis-double bonds affect the fluidity of the lipid bilayer?

<p>Cis-double bonds introduce kinks in the fatty acyl chains, making it more difficult to pack the chains together, thereby increasing membrane fluidity.</p> Signup and view all the answers

Explain how polyunsaturated fatty acids (PUFAs) influence ion permeability in cell membranes.

<p>High PUFA content enhances the permeability of cell membranes particularly to Na+ ions, necessitating increased activity of the Na+/K+ pump.</p> Signup and view all the answers

What symmetrical and asymmetrical properties are present in the lipid bilayer of human red blood cells (RBCs)?

<p>The outer leaflet contains phosphatidylcholine and sphingomyelin, while the inner leaflet contains phosphatidylethanolamine and phosphatidylserine, exhibiting lipid composition asymmetry.</p> Signup and view all the answers

Describe the significance of cholesterol in cell membranes.

<p>Cholesterol enhances ion permeability properties and helps stabilize the membrane structure by fitting between phospholipid molecules.</p> Signup and view all the answers

What is the effect of membrane peroxidation, and how does it relate to fatty acid composition?

<p>Membrane peroxidation occurs due to high PUFA content, leading to increased free radical damage and degradation of cell membranes.</p> Signup and view all the answers

How is membrane fluidity monitored during experiments?

<p>Membrane fluidity can be monitored using techniques such as fluorescence recovery after photobleaching (FRAP) and electron spin resonance (ESP).</p> Signup and view all the answers

What is the main function of membrane proteins in relation to ion composition?

<p>Membrane proteins regulate the ion composition of compartments within the cell, ensuring selective permeability to various ions.</p> Signup and view all the answers

What role does SDS play in SDS-PAGE?

<p>SDS imparts a negative charge to proteins and solubilizes them by disrupting non-covalent interactions.</p> Signup and view all the answers

How does beta-mercaptoethanol contribute to protein analysis in SDS-PAGE?

<p>Beta-mercaptoethanol serves as a reducing agent, breaking disulfide bridges and allowing proteins to unfold.</p> Signup and view all the answers

Why might SDS-PAGE give misleading molecular weight estimates for glycoproteins?

<p>If a protein has a large amount of carbohydrate, its migration on the gel may be affected, leading to inaccurate molecular weight estimates.</p> Signup and view all the answers

What occurs to proteins at their isoelectric point during isoelectric focusing?

<p>At the isoelectric point, proteins have no net charge and stop migrating in the electric field.</p> Signup and view all the answers

Describe the effect of pH on the charge of proteins.

<p>At low pH, proteins typically carry a net positive charge, while at high pH they carry a net negative charge.</p> Signup and view all the answers

What is the purpose of using a molecular weight marker in SDS-PAGE?

<p>A molecular weight marker is used as a reference to estimate the molecular weight of the proteins in the sample.</p> Signup and view all the answers

In the context of PAGE, what results from the separation of proteins in two-dimensional electrophoresis?

<p>Two-dimensional PAGE allows for the separation of proteins based on both isoelectric point and molecular weight.</p> Signup and view all the answers

Explain how polypeptide chain size affects migration speed in SDS-PAGE.

<p>Smaller polypeptide chains migrate faster through the gel compared to larger ones due to less resistance from the gel pores.</p> Signup and view all the answers

What role do watery asteroids play in shaping the chirality of amino acids on Earth?

<p>Watery asteroids may have amplified the abundance of left-handed amino acids, biasing the origin of life towards this form.</p> Signup and view all the answers

How did researchers find evidence of left-handed isovaline in meteorites?

<p>They studied meteorites that had ancient exposure to liquid water, noting a stronger left-handed bias with longer water persistence.</p> Signup and view all the answers

What is the significance of meteoritic compounds for the origin of life on Earth?

<p>Meteorites likely seeded Earth with prebiotic compounds like amino acids while favoring the left-handed version necessary for life.</p> Signup and view all the answers

What is the effect of polarized starlight on amino acids found in asteroids?

<p>Polarized starlight may preferentially destroy right-handed amino acids in asteroids, enhancing the left-handed form.</p> Signup and view all the answers

Describe the complexity of cellular structures mentioned in the context of life.

<p>Cells, whether prokaryotic or eukaryotic, are small yet complex with significant intercellular differences and a complicated molecular composition.</p> Signup and view all the answers

What are the limitations in understanding molecular interactions in cells?

<p>The intricate structural and functional features of biomolecules make it challenging to grasp their interactions, despite advances in omics.</p> Signup and view all the answers

How have giant viruses changed our understanding of viruses?

<p>Giant viruses, found in sludge and exceeding 1 micrometer, challenge traditional notions of virus size and complexity.</p> Signup and view all the answers

What unique characteristic is noted about the cellular basis of life?

<p>The cellular basis of life includes both prokaryotic and eukaryotic cells, and their structures can often be difficult to see and understand.</p> Signup and view all the answers

Flashcards

Asteroid Theory of Life

The theory that life on Earth began with molecules from space, specifically left-handed amino acids carried by asteroids.

L-form Amino Acid

A type of amino acid that exists in two mirror-image forms: left-handed and right-handed. Life on Earth uses predominantly left-handed amino acids.

Water Amplification of L-amino Acids

The phenomenon where the presence of water on asteroids amplified the left-handed amino acid molecules, potentially contributing to the dominance of left-handed amino acids in life on Earth.

Cell

The smallest unit of life. It can be either prokaryotic (simple, without a nucleus) or eukaryotic (complex, with a nucleus).

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Omics

The study of all the molecules in a cell or organism. It involves understanding the functions and interactions of these molecules.

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Virus

A microscopic infectious agent that can only replicate inside the cells of other living organisms. They are much smaller than cells.

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Giant Virus

Viruses that are unusually large in size, even when compared to other viruses. They are much bigger than typical viruses.

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Giant Viruses in Sludge

A type of virus that is known to be much larger than regular viruses. They were discovered in sludge and can be over twice the size of typical viruses.

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Fluorescent nucleic acid stains

Fluorescent dyes used to stain nucleic acids like DNA and RNA. They bind to the bases of these molecules, allowing for visualization and analysis.

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Flow cytometry and fluorescent stains

In the context of cell analysis, these dyes help us understand the amount of DNA a cell has (ploidy) and where it is in the cell cycle (growth and division stages).

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Intercalating fluorescent stains

These dyes insert themselves between the base pairs of double-stranded DNA and RNA, causing them to fluoresce.

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Benzimide dyes (H-33342 & H-33258)

These dyes can pass through the cell membrane without needing the cell to be pre-treated. This makes them useful for quick and easy staining.

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Fluorescein isothiocyanate (FITC)

This dye is commonly used in studies where proteins need to be identified and visualized by binding to specific antibodies. It glows green under specific light conditions.

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3D reconstruction from CSFM images

The process of combining CSFM images captured at different depths to create a three-dimensional view of the specimen.

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Electron Microscopy (EM)

A type of microscope that uses an electron beam instead of light to illuminate and magnify a specimen, revealing detailed structures at the nanoscale.

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Limit of resolution for EM

The smallest distance between two points that can be distinguished as separate entities. In EM, it is typically around 0.1 nm, allowing for the visualization of individual atoms and molecules.

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Glutaraldehyde

A common fixative used in electron microscopy, glutaraldehyde cross-links molecules by forming bonds between them, preserving the specimen's structure.

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Osmium tetroxide

A heavy metal stain often used in electron microscopy, osmium tetroxide reacts with organic compounds, enhancing contrast and highlighting cell membranes.

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Membrane Fluidity

The ability of a membrane to maintain a flexible, fluid state. It's crucial for cell growth and reproduction.

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Cholesterol

A type of lipid that plays a crucial role in regulating membrane fluidity. It can insert itself into the membrane altering its physical properties.

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Fluid to Gel Phase Transition

The process where a membrane transitions from a fluid state to a more rigid, gel-like state. This occurs at specific temperatures depending on the fatty acid composition.

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Unsaturated Fatty Acids (PUFAs)

A type of fatty acid with at least one double bond. They contribute to membrane fluidity, especially at low temperatures.

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How Organisms Adapt to Cold Environments

In cold environments, organisms increase the proportion of unsaturated fatty acids in their membranes to maintain fluidity.

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SDS (Sodium dodecyl sulfate)

A detergent and reducing agent used to solubilize proteins for SDS-PAGE. It disrupts protein structure and imparts a negative charge.

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Beta-mercaptoethanol

A powerful reducing agent that breaks disulfide bonds in proteins, leading to their unfolding.

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SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)

A technique that separates proteins based on their size. Proteins are denatured and given a negative charge. Smaller proteins migrate faster through the gel.

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Isoelectric Point (pI)

The pH at which a protein has no net electrical charge. Each protein has a unique isoelectric point.

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Isoelectric Focusing

A technique used to separate proteins based on their isoelectric points. Proteins migrate through a pH gradient until they reach their pI, where they stop.

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Two-dimensional PAGE

A technique that combines two dimensional separation methods – isoelectric focusing and SDS-PAGE. This gives a more comprehensive and detailed protein separation.

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Selective Permeability

The ability of a cell membrane to allow certain substances to pass through while blocking others. It is essential for maintaining cellular homeostasis and communication.

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Flippase

A specific type of membrane protein that actively moves selected phospholipids from one side of the cell membrane to the other.

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Scramblase

A type of membrane protein that randomly moves phospholipids in both directions across the membrane.

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Cis-double Bonds

Double bonds in fatty acid chains contribute to membrane fluidity by making the chains more difficult to pack together due to the 'kinks' they introduce.

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PUFA and Permeability

Highly unsaturated fatty acids (PUFAs) increase membrane fluidity, but also make the membrane more permeable to ions, especially sodium (Na+).

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Lipid Bilayer Asymmetry

The difference in lipid composition and charge between the two layers of the cell membrane. For example, the outer leaflet of the human RBC membrane is enriched in phosphatidylcholine (PC) and sphingomyelin (SM), while the inner leaflet has more phosphatidylethanolamine (PE) and phosphatidylserine (PS).

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FRAP for Lipid Movement

The process of directly observing the movement of lipids within a membrane using fluorescence recovery after photobleaching (FRAP). This technique highlights the dynamic nature of the membrane.

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Study Notes

Cellular and Biochemical Basis of Life - 19th October 2023

  • Miller-Urey Prebiotic Experiment: In 1959, Stanley Miller and Harold Urey attempted to generate biochemicals under simulated primitive Earth conditions (water, methane, ammonia, hydrogen, electric arc). Within a week, more than 20 amino acids formed.
  • Biochemicals of life: Abundant evidence of major volcanic eruptions 4 billion years ago released various gases into the atmosphere. Subsequent experiments have produced more than 20 different amino acids, more than the 20 that naturally occur.
  • Stereoisomerism: Stereoisomers are molecules with the same atoms but different arrangements. Enantiomers (D and L isomers) are mirror images that cannot be superimposed.
  • Asteroids and L-forms of amino acids: Water on asteroids may have amplified the left-handed amino acid molecules. Meteorites may have provided prebiotic compounds, including amino acids, to Earth.
  • Cellular and Biochemical Basis of Life: Prokaryotic and eukaryotic cells, viruses, viruses, and small but complex structures are all considered part of the cellular basis of life along with molecular composition. Methods of determining structure are increasingly complex with omics contributing to understanding.
  • Viruses: Some giant viruses are relatively large compared with regular viruses and have around 2500 genes
  • Viroids: Plant pathogens composed of short RNA (200-300 nucleobases), mostly circular single-stranded RNA, but with some double-stranded regions.
  • Prions: Misfolded, protease-resistant proteins that can transmit diseases and cause brain damage.
  • Microscopy Techniques There are many different types of microscopy techniques. These include light microscopy, phase contrast, fluorescent microscopy, confocal microscopy, TEM, SEM, STEM, atomic force microscopy, flow cytometry, etc. The resolution of tools is critical to determining findings.

Experimental Methods in Cell Biology

  • Microscopy 1 & 2: Light microscopy (LM), phase contrast, fluorescent microscopy, confocal microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), and atomic force microscopy (AFM) are used to visualize various structures at different scales.
  • Cell Culture Techniques: A variety of techniques are used to isolate cells for study under specific environmental conditions and grow them in culture for further experimentation. Cells are typically isolated and suspended in an appropriate media and environment.
  • Fluorescent Microscopy: Excitation and emission wavelengths, filter sets are specific to specific dyes, filters used to produce a sharp 2D image of the exact plane of focus, beam scanners
  • Experimental Methods: Tissue prep involves fixing, embedding/sectioning, de-waxing, staining, dehydrating, and mounting.

Staining

  • Basic stains: Haematoxylin (basophilic structures, blue). Eosin (acidophilic or basophilic, pink/red).
  • Mordants: Metal salts are used to enhance the staining of haematoxylin.
  • Other stains: Fast green, safranin (Mallory's trichrome), and many others depending on the tissue structure.

Other Techniques

  • FISH (Fluorescent In Situ Hybridization): Used to localise specific DNA sequences on chromosomes
  • Immunocytochemistry: Used to localise molecules using antigen/antibody complex.
  • Pulse-chase Autoradiography: Used to trace the pathway of molecules in cells
  • Cell Culture Techniques: These techniques used for studying processes like DNA replication and transcription and cell cycles.
  • Centrifugation and cell sorting: Used for separating cell compounds/extracts and separating/isolating specific cells from a mixture.
  • SDS-PAGE electrophoresis: Using a detergent (SDS), it separates proteins by molecular weight.
  • Isoelectric focusing: Separates proteins based on isoelectric points (pI), which is the pH at which a protein has no net charge.
  • Chromatography: Separate molecules using stationary and mobile phases based on differences in size, charge, etc. Types include ion-exchange, gel filtration (size exclusion), and affinity chromatography.

Advanced Microscopy:

  • Confocal scanning Fluorsecent Microscopy (CFSM): Fine laser beam scans through several layers, creating three-dimensional images.

Cell Cycle and Apoptosis

  • Cell Cycle: Cell cycle control system, phases (G1, S, G2, and M); cyclin-dependent kinases (Cdks), interphase.
  • Apoptosis: The programmed death pathway of cells.
  • Necrosis: The accidental death of a cell.
  • Apoptotic vs. Necrosis: Both pathways lead to cell death, with apoptosis as a regulated process, and necrosis, as harmful accidental event.
  • Proteins in Apoptosis/Necrosis: Cellular proteins, pathways.

Other

  • General principles of centrifugation: Fixed angle and swinging bucket rotors
  • Molecular Biology of Proteins: Types, characteristics, functional significance. Structure - primary, secondary, tertiary, quaternary.
  • Common Chemistry of Amino Acids: Basic building components, properties (polar, non-polar, charged, etc.)
  • Differences between Various Procedures: The various techniques that use specific steps and instruments.

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Description

This quiz explores the differences between benzimides and fluorescent stains regarding cell membrane permeability, as well as the fluorescence characteristics of propidium iodide and Hoechst stains. Additionally, it covers key considerations for using ethidium bromide and the preparation methods for electron microscopy specimens.

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